Dataset DOI: 10.5061/dryad.6m905qgfp

Description of the data and file structure

Source data files for figures are included below. These include immunofluorescent imaging on fixed cells in .nd2 format; raw tif images of western blot for each antibody; Seahorse glycolytic stress test files reporting normalized ECAR and OCR in excel format; DIC timelapse of chemotaxing MDA-MB-231 cells on μ-slide chemotaxis chambers (Ibidi USA, Inc.) in .nd2 format; cell tracking data using Manual Tracking ImageJ plugin in .csv format.

Files and variables

Files related to Figure 1

File: Fig1A-antiPFKL.tif

Description: Western blot of non-transfected, siRNA control, or PFKL siRNA-transfected MDA-MB-231 cells for 48, 72, or 96 hours. Probed with antibodies raised against PFKL (Abcam, Waltham, MA, USA; ab181064; 1:1000) and goat anti-rabbit-HRP (Jackson ImmunoResearch, West Grove, PA, USA; 711-035-152; 1:10,000). Recombinant human PFKL was used as a positive control. 

File: Fig_1A-antiGAPDH.tif

Description: Western blot of non-transfected, siRNA control, or PFKL siRNA-transfected MDA-MB-231 cells for 48, 72, or 96 hours. Probed with antibodies raised against GAPDH (Cell Signaling Technologies, Danvers, MA, USA; 5147; 1:2000) and goat anti-rabbit-HRP (Jackson ImmunoResearch, West Grove, PA, USA; 711-035-152; 1:10,000).

File: Fig_1B_siRNA_ECAR.csv

Description: Normalized extracellular acidification rate (ECAR) measured through sequential injection of 10 mM glucose, 1 µM oligomycin and 100 mM 2-deoxy-glucose (2-DG). The data were normalized to cell number measured by a CyQUANT Cell Proliferation Assay (Thermo Fisher Scientific, Rockfield, IL, USA; C7026).

File: Fig_1B_siRNA_OCR.csv

Description: Normalized oxygen consumption rate (OCR) measured through sequential injection of 10 mM glucose, 1 µM oligomycin and 100 mM 2-deoxy-glucose (2-DG). The data were normalized to cell number measured by a CyQUANT Cell Proliferation Assay (Thermo Fisher Scientific, Rockfield, IL, USA; C7026).

File: Fig_1D_and_E_siRNA_chemotaxis_tracking_NT.csv

Description: Quantification of single-cell migration from the Manual Tracking ImageJ plugin for input into the Ibidi Chemotaxis and Migration Tool ImageJ plugin for non-transfected (NT) MDA-MB-231 cells.

Variables

• Column 1: Row index
• Column 2: Track number. Identifies the cell being tracked across slices.
• Column 3: Slice number. Indicates the current frame of the timelapse movie.
• Column 4: X position (pixels). The X-coordinate of the tracked cell in the current slice.
• Column 5: Y position (Pixels). The Y-coordinate of the tracked cell in the current slice.
• Column 6: Distance traveled (µm). The distance the cell moved from the previous frame
• Column 7: Velocity (µm/sec). The velocity of movement from the previous slice.
• Column 8: Pixel value. Intensity or grey value of the selected pixel on the tracked object.

File: Fig_1D_and_E_siRNA_chemotaxis_tracking_siCTRL.csv

Description: Quantification of single-cell migration from the Manual Tracking ImageJ plugin for input into the Ibidi Chemotaxis and Migration Tool ImageJ plugin for control siRNA-transfected (siCTRL) MDA-MB-231 cells.

Variables

• Column 1: Row index
• Column 2: Track number. Identifies the cell being tracked across slices.
• Column 3: Slice number. Indicates the current frame of the timelapse movie.
• Column 4: X position (pixels). The X-coordinate of the tracked cell in the current slice.
• Column 5: Y position (Pixels). The Y-coordinate of the tracked cell in the current slice.
• Column 6: Distance traveled (µm). The distance the cell moved from the previous frame
• Column 7: Velocity (µm/sec). The velocity of movement from the previous slice.
• Column 8: Pixel value. Intensity or grey value of the selected pixel on the tracked object.

File: Fig_1D_and_E_siRNA_chemotaxis_tracking_siPFKL.csv

Description: Quantification of single-cell migration from the Manual Tracking ImageJ plugin for input into the Ibidi Chemotaxis and Migration Tool ImageJ plugin for PFKL siRNA-transfected (siPFKL) MDA-MB-231 cells.

Variables

• Column 1: Row index
• Column 2: Track number. Identifies the cell being tracked across slices.
• Column 3: Slice number. Indicates the current frame of the timelapse movie.
• Column 4: X position (pixels). The X-coordinate of the tracked cell in the current slice.
• Column 5: Y position (Pixels). The Y-coordinate of the tracked cell in the current slice.
• Column 6: Distance traveled (µm). The distance the cell moved from the previous frame
• Column 7: Velocity (µm/sec). The velocity of movement from the previous slice.
• Column 8: Pixel value. Intensity or grey value of the selected pixel on the tracked object.

File: Fig_1F_and_G_shRNA_chemotaxis_tracking_Parental.csv

Description: Quantification of single-cell migration from the Manual Tracking ImageJ plugin for input into the Ibidi Chemotaxis and Migration Tool ImageJ plugin for Parental (Par compiled) MDA-MB-231 cells.

Variables

• Column 1: Row index
• Column 2: Track number. Identifies the cell being tracked across slices.
• Column 3: Slice number. Indicates the current frame of the timelapse movie.
• Column 4: X position (pixels). The X-coordinate of the tracked cell in the current slice.
• Column 5: Y position (Pixels). The Y-coordinate of the tracked cell in the current slice.
• Column 6: Distance traveled (µm). The distance the cell moved from the previous frame
• Column 7: Velocity (µm/sec). The velocity of movement from the previous slice.
• Column 8: Pixel value. Intensity or grey value of the selected pixel on the tracked object.

File: Fig_1F_and_G_shRNA_chemotaxis_tracking_shScramble.csv

Description: Quantification of single-cell migration from the Manual Tracking ImageJ plugin for input into the Ibidi Chemotaxis and Migration Tool ImageJ plugin for scrambled shRNA transduced (Scr compiled) MDA-MB-231 cells.

Variables

• Column 1: Row index
• Column 2: Track number. Identifies the cell being tracked across slices.
• Column 3: Slice number. Indicates the current frame of the timelapse movie.
• Column 4: X position (pixels). The X-coordinate of the tracked cell in the current slice.
• Column 5: Y position (Pixels). The Y-coordinate of the tracked cell in the current slice.
• Column 6: Distance traveled (µm). The distance the cell moved from the previous frame
• Column 7: Velocity (µm/sec). The velocity of movement from the previous slice.
• Column 8: Pixel value. Intensity or grey value of the selected pixel on the tracked object.

File: Fig_1F_and_G_shRNA_chemotaxis_tracking_sh767.csv

Description: Quantification of single-cell migration from the Manual Tracking ImageJ plugin for input into the Ibidi Chemotaxis and Migration Tool ImageJ plugin for PFKL shRNA transduced (767 compiled) MDA-MB-231 cells.

Variables

• Column 1: Row index
• Column 2: Track number. Identifies the cell being tracked across slices.
• Column 3: Slice number. Indicates the current frame of the timelapse movie.
• Column 4: X position (pixels). The X-coordinate of the tracked cell in the current slice.
• Column 5: Y position (Pixels). The Y-coordinate of the tracked cell in the current slice.
• Column 6: Distance traveled (µm). The distance the cell moved from the previous frame
• Column 7: Velocity (µm/sec). The velocity of movement from the previous slice.
• Column 8: Pixel value. Intensity or grey value of the selected pixel on the tracked object.

File: siRNA_chemotaxis_n_1.nd2

Description: Single-cell migration of non-transfected, control siRNA-transfected, or PFKL siRNA-transfected MDA-MB-231 cells was evaluated using μ-slide chemotaxis chambers (Ibidi USA, Inc.) using 50 ng/ml EGF as a chemoattractant (left side of image). Brightfield images of 4–5 fields of view of each chamber were captured using a 20× objective of a Nikon TI2-E Inverted Microscope. Images were captured every 10 min for 16 hours. Replicate 1. 

File: siRNA_chemotaxis_n_2.nd2

Description: Single-cell migration of non-transfected, control siRNA-transfected, or PFKL siRNA-transfected MDA-MB-231 cells was evaluated using μ-slide chemotaxis chambers (Ibidi USA, Inc.) using 50 ng/ml EGF as a chemoattractant (left side of image). Brightfield images of 4–5 fields of view of each chamber were captured using a 20× objective of a Nikon TI2-E Inverted Microscope. Images were captured every 10 min for 16 hours. Replicate 2. 

File: siRNA_chemotaxis_n_3.nd2

Description: Single-cell migration of non-transfected, control siRNA-transfected, or PFKL siRNA-transfected MDA-MB-231 cells was evaluated using μ-slide chemotaxis chambers (Ibidi USA, Inc.) using 50 ng/ml EGF as a chemoattractant (left side of image). Brightfield images of 4–5 fields of view of each chamber were captured using a 20× objective of a Nikon TI2-E Inverted Microscope. Images were captured every 10 min for 16 hours. Replicate 3. 

File: shRNA_chemotaxis_n_1.nd2

Description: Single-cell migration of parental, scrambled shRNA, or PFKL shRNA transduced MDA-MB-231 cells was evaluated using μ-slide chemotaxis chambers (Ibidi USA, Inc.) using 50 ng/ml EGF as a chemoattractant (left side of image). Brightfield images of 4–5 fields of view of each chamber were captured using a 20× objective of a Nikon TI2-E Inverted Microscope. Images were captured every 10 min for 16 hours. Replicate 1. 

File: shRNA_chemotaxis_n_2.nd2

Description: Single-cell migration of parental, scrambled shRNA, or PFKL shRNA transduced MDA-MB-231 cells was evaluated using μ-slide chemotaxis chambers (Ibidi USA, Inc.) using 50 ng/ml EGF as a chemoattractant (left side of image). Brightfield images of 4–5 fields of view of each chamber were captured using a 20× objective of a Nikon TI2-E Inverted Microscope. Images were captured every 10 min for 16 hours. Replicate 2. 

File: shRNA_chemotaxis_n_3.nd2

Description: Single-cell migration of parental, scrambled shRNA, or PFKL shRNA transduced MDA-MB-231 cells was evaluated using μ-slide chemotaxis chambers (Ibidi USA, Inc.) using 50 ng/ml EGF as a chemoattractant (left side of image). Brightfield images of 4–5 fields of view of each chamber were captured using a 20× objective of a Nikon TI2-E Inverted Microscope. Images were captured every 10 min for 16 hours. Replicate 3. 

Files related to Figure 2

File: Par_2ndary002.nd2

Description: Immunofluorescence images of fixed parental MDA-MB-231 cells stained with goat anti-mouse Alexa Fluor-488 (Thermo Fisher Scientific, Rockfield IL, USA; A-11001; 1:500) and goat anti-rabbit Alexa Fluor-567 (Thermo Fisher Scientific, Rockfield IL, USA; A11031; 1:500) secondary antibodies. Coverslips were also stained with Hoechst 33342 (Thermo Fisher Scientific, Rockfield, IL, USA; H3570) and phalloidin–Alexa Fluor-647 (Thermo Fisher Scientific, Rockfield, IL, USA; A22287). Cells were imaged using a Nikon TI2-E Inverted Microscope with a CSU-W1 Confocal Scanner spinning disk with a 60× oil immersion objective.

File: FLAG-L___HK2.nd2

Description: Immunofluorescence images of fixed MDA-MB-231 cells stained with primary antibodies raised against FLAG-M2 (Sigma-Aldrich, St. Louis MO, USA; F1804; 1:1000) and hexokinase 2 (GeneTex, Irvine CA, USA; GTX111525; 1:1000) followed by goat anti-mouse Alexa Fluor-488 (Thermo Fisher Scientific, Rockfield IL, USA; A-11001; 1:500) and goat anti-rabbit Alexa Fluor-567 (Thermo Fisher Scientific, Rockfield IL, USA; A11031; 1:500) secondary antibodies. Coverslips were also stained with Hoechst 33342 (Thermo Fisher Scientific, Rockfield, IL, USA; H3570) and phalloidin–Alexa Fluor-647 (Thermo Fisher Scientific, Rockfield, IL, USA; A22287). Cells were imaged using a Nikon TI2-E Inverted Microscope with a CSU-W1 Confocal Scanner spinning disk with a 60× oil immersion objective.

File: 030823_FLAG_L_0ng_EGF_PKM2001.nd2

Description: Immunofluorescence images of fixed MDA-MB-231 cells expressing FLAG-PFKL stained with primary antibodies raised against FLAG-M2 (Sigma-Aldrich, St. Louis MO, USA; F1804; 1:1000) and pyruvate kinase M2 (Cell Signaling Technologies, Danvers MA, USA; 4053; 1:500) followed by goat anti-mouse Alexa Fluor-488 (Thermo Fisher Scientific, Rockfield IL, USA; A-11001; 1:500) and goat anti-rabbit Alexa Fluor-567 (Thermo Fisher Scientific, Rockfield IL, USA; A11031; 1:500) secondary antibodies. Coverslips were also stained with Hoechst 33342 (Thermo Fisher Scientific, Rockfield, IL, USA; H3570) and phalloidin–Alexa Fluor-647 (Thermo Fisher Scientific, Rockfield, IL, USA; A22287). Cells were imaged using a Nikon TI2-E Inverted Microscope with a CSU-W1 Confocal Scanner spinning disk with a 60× oil immersion objective.

Files related to Figure 3

File: Fig_3A-antiFLAG.tif

Description: Western blot of EGFP, FLAG-PFKL wild type, or FLAG-PFKL-H199Y transduced MDA-MB-231 cells. Probed with antibodies raised against FLAG (Sigma-Aldrich, St. Louis, MO, USA; F1804; 1:1000) and goat anti-mouse-HRP (Jackson ImmunoResearch, West Grove, PA, USA; 715-035-150; 1:20,000).

File: Fig_3A-antiPFKL.tif

Description: Western blot of EGFP, FLAG-PFKL wild type, or FLAG-PFKL-H199Y transduced MDA-MB-231 cells. Probed with antibodies raised against PFKL (Abcam, Waltham, MA, USA; ab181064; 1:1000).

File: Fig_3A-antiGAPDH.tif

Description: Western blot of EGFP, FLAG-PFKL wild type, or FLAG-PFKL-H199Y transduced MDA-MB-231 cells. Probed with antibodies raised against GAPDH (Cell Signaling Technologies, Danvers, MA, USA; 5147; 1:2000) and goat anti-rabbit-HRP (Jackson ImmunoResearch, West Grove, PA, USA; 711-035-152; 1:10,000).

File: Fig_3B_H199Y_ECAR.csv

Description: Normalized extracellular acidification rate (ECAR) measured through sequential injection of 10 mM glucose, 1 µM oligomycin and 100 mM 2-deoxy-glucose (2-DG). The data were normalized to cell number measured by a CyQUANT Cell Proliferation Assay (Thermo Fisher Scientific, Rockfield, IL, USA; C7026).

File: Fig_3B_H199Y_OCR.csv

Description: Normalized oxygen consumption rate (OCR) measured through sequential injection of 10 mM glucose, 1 µM oligomycin and 100 mM 2-deoxy-glucose (2-DG). The data were normalized to cell number measured by a CyQUANT Cell Proliferation Assay (Thermo Fisher Scientific, Rockfield, IL, USA; C7026).

File: FLAG-PFKL_FLAG_Tom20_-EGF.nd2

Description: Immunofluorescence images of fixed and stained EGF-stimulated MDA-MB-231 cells expressing FLAG-PFKL stained with primary antibodies raised against FLAG-M2 (Sigma-Aldrich, St. Louis MO, USA; F1804; 1:1000) and TOM20 (D8T4N; Cell Signaling Technology, Danvers MA, USA; 42406S; 1:250) followed by goat anti-mouse Alexa Fluor-488 (Thermo Fisher Scientific, Rockfield IL, USA; A-11001; 1:500) and goat anti-rabbit Alexa Fluor-567 (Thermo Fisher Scientific, Rockfield IL, USA; A11031; 1:500) secondary antibodies. Coverslips were also stained with Hoechst 33342 (Thermo Fisher Scientific, Rockfield, IL, USA; H3570) and phalloidin–Alexa Fluor-647 (Thermo Fisher Scientific, Rockfield, IL, USA; A22287). Cells were imaged using a Nikon TI2-E Inverted Microscope with a CSU-W1 Confocal Scanner spinning disk with a 60× oil immersion objective.

File: H199Y_FLAG_PKM2_-EGF.nd2

Description: Immunofluorescence images of fixed and stained MDA-MB-231 cells stained with primary antibodies raised against FLAG-M2 (Sigma-Aldrich, St. Louis MO, USA; F1804; 1:1000) and pyruvate kinase M2 (Cell Signaling Technologies, Danvers MA, USA; 4053; 1:500) followed by goat anti-mouse Alexa Fluor-488 (Thermo Fisher Scientific, Rockfield IL, USA; A-11001; 1:500) and goat anti-rabbit Alexa Fluor-567 (Thermo Fisher Scientific, Rockfield IL, USA; A11031; 1:500) secondary antibodies. Coverslips were also stained with Hoechst 33342 (Thermo Fisher Scientific, Rockfield, IL, USA; H3570) and phalloidin–Alexa Fluor-647 (Thermo Fisher Scientific, Rockfield, IL, USA; A22287). Cells were imaged using a Nikon TI2-E Inverted Microscope with a CSU-W1 Confocal Scanner spinning disk with a 60× oil immersion objective.

File: Fig_3D_and_E_chemotaxis_tracking_EGFP.csv

Description: Quantification of single-cell migration from the Manual Tracking ImageJ plugin for input into the Ibidi Chemotaxis and Migration Tool ImageJ plugin for EGFP-transduced MDA-MB-231 cells.

Variables

• Column 1: Row index
• Column 2: Track number. Identifies the cell being tracked across slices.
• Column 3: Slice number. Indicates the current frame of the timelapse movie.
• Column 4: X position (pixels). The X-coordinate of the tracked cell in the current slice.
• Column 5: Y position (Pixels). The Y-coordinate of the tracked cell in the current slice.
• Column 6: Distance traveled (µm). The distance the cell moved from the previous frame
• Column 7: Velocity (µm/sec). The velocity of movement from the previous slice.
• Column 8: Pixel value. Intensity or grey value of the selected pixel on the tracked object.

File: Fig_3D_and_E_chemotaxis_tracking_PFKL_WT.csv

Description: Quantification of single-cell migration from the Manual Tracking ImageJ plugin for input into the Ibidi Chemotaxis and Migration Tool ImageJ plugin for FLAG-PFKL transduced (LWT) MDA-MB-231 cells.

Variables

• Column 1: Row index
• Column 2: Track number. Identifies the cell being tracked across slices.
• Column 3: Slice number. Indicates the current frame of the timelapse movie.
• Column 4: X position (pixels). The X-coordinate of the tracked cell in the current slice.
• Column 5: Y position (Pixels). The Y-coordinate of the tracked cell in the current slice.
• Column 6: Distance traveled (µm). The distance the cell moved from the previous frame
• Column 7: Velocity (µm/sec). The velocity of movement from the previous slice.
• Column 8: Pixel value. Intensity or grey value of the selected pixel on the tracked object. 

File: Fig_3D_and_E_chemotaxis_tracking_PFKL_H199Y.csv

Description: Quantification of single-cell migration from the Manual Tracking ImageJ plugin for input into the Ibidi Chemotaxis and Migration Tool ImageJ plugin for FLAG-PFKL-H199Y transduced (H199Y) MDA-MB-231 cells.

Variables

• Column 1: Row index
• Column 2: Track number. Identifies the cell being tracked across slices.
• Column 3: Slice number. Indicates the current frame of the timelapse movie.
• Column 4: X position (pixels). The X-coordinate of the tracked cell in the current slice.
• Column 5: Y position (Pixels). The Y-coordinate of the tracked cell in the current slice.
• Column 6: Distance traveled (µm). The distance the cell moved from the previous frame
• Column 7: Velocity (µm/sec). The velocity of movement from the previous slice.
• Column 8: Pixel value. Intensity or grey value of the selected pixel on the tracked object.

File: H199Y_chemotaxis_n_1.nd2

Description: Single-cell migration of EGFP, FLAG-PFKL-WT, and FLAG-PFKL-H199Y transduced MDA-MB-231 cells was evaluated using μ-slide chemotaxis chambers (Ibidi USA, Inc.) using 50 ng/ml EGF as a chemoattractant (left side of image). Brightfield images of 4–5 fields of view of each chamber were captured using a 20× objective of a Nikon TI2-E Inverted Microscope. Images were captured every 10 min for 16 hours. Replicate 1. 

File: H199Y_chemotaxis_n_2.nd2

Description: Single-cell migration of EGFP, FLAG-PFKL-WT, and FLAG-PFKL-H199Y transduced MDA-MB-231 cells was evaluated using μ-slide chemotaxis chambers (Ibidi USA, Inc.) using 50 ng/ml EGF as a chemoattractant (left side of image). Brightfield images of 4–5 fields of view of each chamber were captured using a 20× objective of a Nikon TI2-E Inverted Microscope. Images were captured every 10 min for 16 hours. Replicate 2. 

File: H199Y_chemotaxis_n_3.nd2

Description: Single-cell migration of EGFP, FLAG-PFKL-WT, and FLAG-PFKL-H199Y transduced MDA-MB-231 cells was evaluated using μ-slide chemotaxis chambers (Ibidi USA, Inc.) using 50 ng/ml EGF as a chemoattractant (left side of image). Brightfield images of 4–5 fields of view of each chamber were captured using a 20× objective of a Nikon TI2-E Inverted Microscope. Images were captured every 10 min for 16 hours. Replicate 3. 

Files related to Figure 4

File: Fig_4B_and_C_PFK15_chemotaxis_tracking_DMSO.csv

Description: Quantification of single-cell migration from the Manual Tracking ImageJ plugin for input into the Ibidi Chemotaxis and Migration Tool ImageJ plugin for DMSO-treated MDA-MB-231 cells.

Variables

• Column 1: Row index
• Column 2: Track number. Identifies the cell being tracked across slices.
• Column 3: Slice number. Indicates the current frame of the timelapse movie.
• Column 4: X position (pixels). The X-coordinate of the tracked cell in the current slice.
• Column 5: Y position (Pixels). The Y-coordinate of the tracked cell in the current slice.
• Column 6: Distance traveled (µm). The distance the cell moved from the previous frame
• Column 7: Velocity (µm/sec). The velocity of movement from the previous slice.
• Column 8: Pixel value. Intensity or grey value of the selected pixel on the tracked object.

File: Fig_4B_and_C_PFK15_chemotaxis_tracking_PFK15.csv

Description: Quantification of single-cell migration from the Manual Tracking ImageJ plugin for input into the Ibidi Chemotaxis and Migration Tool ImageJ plugin for PFK15-treated MDA-MB-231 cells.

Variables

• Column 1: Row index
• Column 2: Track number. Identifies the cell being tracked across slices.
• Column 3: Slice number. Indicates the current frame of the timelapse movie.
• Column 4: X position (pixels). The X-coordinate of the tracked cell in the current slice.
• Column 5: Y position (Pixels). The Y-coordinate of the tracked cell in the current slice.
• Column 6: Distance traveled (µm). The distance the cell moved from the previous frame
• Column 7: Velocity (µm/sec). The velocity of movement from the previous slice.
• Column 8: Pixel value. Intensity or grey value of the selected pixel on the tracked object.

File: PFK15_chemotaxis_n_1.nd2

Description: Single-cell migration of MDA-MB-231 cells treated with DMSO control or 2 μM PFK15 was evaluated using μ-slide chemotaxis chambers (Ibidi USA, Inc.) using 50 ng/ml EGF as a chemoattractant (left side of image). Brightfield images of 4–5 fields of view of each chamber were captured using a 20× objective of a Nikon TI2-E Inverted Microscope. Images were captured every 10 min for 16 hours. Replicate 1. 

File: PFK15_chemotaxis_n_2.nd2

Description: Single-cell migration of MDA-MB-231 cells treated with DMSO control or 2 μM PFK15 was evaluated using μ-slide chemotaxis chambers (Ibidi USA, Inc.) using 50 ng/ml EGF as a chemoattractant (left side of image). Brightfield images of 4–5 fields of view of each chamber were captured using a 20× objective of a Nikon TI2-E Inverted Microscope. Images were captured every 10 min for 16 hours. Replicate 2. 

File: PFK15_chemotaxis_n_3.nd2

Description: Single-cell migration of MDA-MB-231 cells treated with DMSO control or 2 μM PFK15 was evaluated using μ-slide chemotaxis chambers (Ibidi USA, Inc.) using 50 ng/ml EGF as a chemoattractant (left side of image). Brightfield images of 4–5 fields of view of each chamber were captured using a 20× objective of a Nikon TI2-E Inverted Microscope. Images were captured every 10 min for 16 hours. Replicate 3. 

Files related to Figure 5

File: Fig_5A-antiFLAG.tif

Description: Western blot of EGFP, FLAG-PFKL wild type, or FLAG-PFKL-H199Y transduced MDA-MB-231 cells. Probed with antibodies raised against FLAG (Sigma-Aldrich, St. Louis, MO, USA; F1804; 1:1000) and goat anti-mouse-HRP (Jackson ImmunoResearch, West Grove, PA, USA; 715-035-150; 1:20,000).

File: Fig_5A-antiGAPDH.tif

Description: Western blot of EGFP, FLAG-PFKL wild type, or FLAG-PFKL-N702T transduced MDA-MB-231 cells. Probed with antibodies raised against GAPDH (Cell Signaling Technologies, Danvers, MA, USA; 5147; 1:2000) and goat anti-rabbit-HRP (Jackson ImmunoResearch, West Grove, PA, USA; 711-035-152; 1:10,000).

File: Fig_5B_N702T_ECAR.csv

Description: Normalized extracellular acidification rate (ECAR) measured through sequential injection of 10 mM glucose, 1 µM oligomycin and 100 mM 2-deoxy-glucose (2-DG). The data were normalized to cell number measured by a CyQUANT Cell Proliferation Assay (Thermo Fisher Scientific, Rockfield, IL, USA; C7026).

File: Fig_5B_N702T_OCR.csv

Description: Normalized oxygen consumption rate (OCR) measured through sequential injection of 10 mM glucose, 1 µM oligomycin and 100 mM 2-deoxy-glucose (2-DG). The data were normalized to cell number measured by a CyQUANT Cell Proliferation Assay (Thermo Fisher Scientific, Rockfield, IL, USA; C7026).

File: LWT_FLAG_tom20_EGF004.nd2

Description: Immunofluorescence images of fixed and stained EGF-stimulated MDA-MB-231 cells expressing FLAG-PFKL stained with primary antibodies raised against FLAG-M2 (Sigma-Aldrich, St. Louis MO, USA; F1804; 1:1000) and TOM20 (D8T4N; Cell Signaling Technology, Danvers MA, USA; 42406S; 1:250) followed by goat anti-mouse Alexa Fluor-488 (Thermo Fisher Scientific, Rockfield IL, USA; A-11001; 1:500) and goat anti-rabbit Alexa Fluor-567 (Thermo Fisher Scientific, Rockfield IL, USA; A11031; 1:500) secondary antibodies. Coverslips were also stained with Hoechst 33342 (Thermo Fisher Scientific, Rockfield, IL, USA; H3570) and phalloidin–Alexa Fluor-647 (Thermo Fisher Scientific, Rockfield, IL, USA; A22287). Cells were imaged using a Nikon TI2-E Inverted Microscope with a CSU-W1 Confocal Scanner spinning disk with a 60× oil immersion objective.

File: N702T_FLAG_tom20_EGF005.nd2

Description: Immunofluorescence images of fixed and stained EGF-stimulated MDA-MB-231 cells expressing FLAG-PFKL-N702T stained with primary antibodies raised against FLAG-M2 (Sigma-Aldrich, St. Louis MO, USA; F1804; 1:1000) and TOM20 (D8T4N; Cell Signaling Technology, Danvers MA, USA; 42406S; 1:250) followed by goat anti-mouse Alexa Fluor-488 (Thermo Fisher Scientific, Rockfield IL, USA; A-11001; 1:500) and goat anti-rabbit Alexa Fluor-567 (Thermo Fisher Scientific, Rockfield IL, USA; A11031; 1:500) secondary antibodies. Coverslips were also stained with Hoechst 33342 (Thermo Fisher Scientific, Rockfield, IL, USA; H3570) and phalloidin–Alexa Fluor-647 (Thermo Fisher Scientific, Rockfield, IL, USA; A22287). Cells were imaged using a Nikon TI2-E Inverted Microscope with a CSU-W1 Confocal Scanner spinning disk with a 60× oil immersion objective.

File: PFKL_WT_Intensity_quant.csv

Description: To estimate the abundance of FLAG–PFKL in the lamellipodia versus the cytoplasm, line scans of maximum-intensity projections were generated in FIJI ImageJ, and the ratio of lamellipodia to cytoplasm intensity was quantified in Microsoft Excel. Files were converted to CSV.

Variables:

• Cell number: identifies the cell being quantified.
• Lamellipodia peak position (µm): position of peak signal on the line scan.
• Lamellipodia peak intensity: intensity (grey value) of peak signal in lamellipodia.
• Cytoplasm peak position µm: position of peak signal on the line scan.
• Cytoplasm peak intensity: intensity (grey value) of peak signal in the cytoplasm.
• Peak intensity ratio: Ratio of lamellipodia peak and cytoplasmic peak signals.

File: PFKL_N702T_Intensity_quant.csv

Description: To estimate the abundance of FLAG–PFKL-N702T in the lamellipodia versus cytoplasm, line scans of maximum-intensity projections were generated in FIJI ImageJ, and the ratio of lamellipodia to cytoplasm intensity was quantified in Microsoft Excel. Files were converted to CSV.

Variables

• Cell number: identifies the cell being quantified
• Lamellipodia peak position (µm): position of peak signal on the line scan.
• Lamellipodia peak intensity: intensity (grey value) of peak signal in lamellipodia.
• Cytoplasm peak position µm: position of peak signal on the line scan.
• Cytoplasm peak intensity: intensity (grey value) of peak signal in the cytoplasm.
• Peak intensity ratio: Ratio of lamellipodia peak and cytoplasmic peak signals.

File: Fig_5E-antiFLAG.tif

Description: Western blot of lysate and of S100 and P100 fractions, separated by subcellular fractionation, from EGFP, FLAG-PFKL wild type, or FLAG-PFKL-EGFP transduced MDA-MB-231 cells. Probed with antibodies raised against FLAG (Sigma-Aldrich, St. Louis, MO, USA; F1804; 1:1000) and goat anti-mouse-HRP (Jackson ImmunoResearch, West Grove, PA, USA; 715-035-150; 1:20,000).

File: Fig_5E-antiEGFR.tif

Description: Western blot of lysate and of S100 and P100 fractions, separated by subcellular fractionation, from EGFP, FLAG-PFKL wild type, or FLAG-PFKL-EGFP transduced MDA-MB-231 cells. Probed with antibodies raised against EGFR (Invitrogen, Carlsbad, CA, USA; PA1-1110; 1:1000) and goat anti-rabbit-HRP (Jackson ImmunoResearch, West Grove, PA, USA; 711-035-152; 1:10,000).

File: Fig_5E-antiAKT.tif

Description: Western blot of lysate and of S100 and P100 fractions, separated by subcellular fractionation, from EGFP, FLAG-PFKL wild type, or FLAG-PFKL-EGFP transduced MDA-MB-231 cells. Probed with antibodies raised against pan-AKT (Thermo Fisher Scientific, Rockfield, IL, USA; 44-609G; 1:1000) and goat anti-rabbit-HRP (Jackson ImmunoResearch, West Grove, PA, USA; 711-035-152; 1:10,000).

File: Fig_5F_and_G_chemotaxis_tracking_EGFP.csv

Description: Quantification of single-cell migration from the Manual Tracking ImageJ plugin for input into the Ibidi Chemotaxis and Migration Tool ImageJ plugin for EGFP-transduced MDA-MB-231 cells.

Variables

• Column 1 Row index
• Column 2: Track number. Identifies the cell being tracked across slices.
• Column 3: Slice number. Indicates the current frame of the timelapse movie.
• Column 4: X position (pixels). The X-coordinate of the tracked cell in the current slice.
• Column 5: Y position (Pixels). The Y-coordinate of the tracked cell in the current slice.
• Column 6: Distance traveled (µm). The distance the cell moved from the previous frame
• Column 7: Velocity (µm/sec). The velocity of movement from the previous slice.
• Column 8: Pixel value. Intensity or grey value of the selected pixel on the tracked object.

File: Fig_5F_and_G_chemotaxis_tracking_PFKL_WT.csv

Description: Quantification of single-cell migration from the Manual Tracking ImageJ plugin for input into the Ibidi Chemotaxis and Migration Tool ImageJ plugin for FLAG-PFKL transduced (LWT) MDA-MB-231 cells.

Variables

• Column 1: Row index
• Column 2: Track number. Identifies the cell being tracked across slices.
• Column 3: Slice number. Indicates the current frame of the timelapse movie.
• Column 4: X position (pixels). The X-coordinate of the tracked cell in the current slice.
• Column 5: Y position (pixels). The Y-coordinate of the tracked cell in the current slice.
• Column 6: Distance traveled (µm). The distance the cell moved from the previous frame
• Column 7: Velocity (µm/sec). The velocity of movement from the previous slice.
• Column 8: Pixel value. Intensity or grey value of the selected pixel on the tracked object.

File: Fig_5F_and_G_chemotaxis_tracking_PFKL_N702T.csv

Description: Quantification of single-cell migration from the Manual Tracking ImageJ plugin for input into the Ibidi Chemotaxis and Migration Tool ImageJ plugin for FLAG-PFKL-N702T transduced (N702T) MDA-MB-231 cells.

Variables

• Column 1: Row index
• Column 2: Track number. Identifies the cell being tracked across slices.
• Column 3: Slice number. Indicates the current frame of the timelapse movie.
• Column 4: X position (pixels). The X-coordinate of the tracked cell in the current slice.
• Column 5: Y position (pixels). The Y-coordinate of the tracked cell in the current slice.
• Column 6: Distance traveled (µm). The distance the cell moved from the previous frame
• Column 7: Velocity (µm/sec). The velocity of movement from the previous slice.
• Column 8: Pixel value. Intensity or grey value of the selected pixel on the tracked object.

File: N702T_chemotaxis_n_1.nd2

Description: Single-cell migration of EGFP, FLAG-PFKL-WT, and FLAG-PFKL-N702T transduced MDA-MB-231 cells was evaluated using μ-slide chemotaxis chambers (Ibidi USA, Inc.) using 50 ng/ml EGF as a chemoattractant (left side of image). Brightfield images of 4–5 fields of view of each chamber were captured using a 20× objective of a Nikon TI2-E Inverted Microscope. Images were captured every 10 min for 16 hours. Replicate 1. 

File: N702T_chemotaxis_n_2.nd2

Description: Single-cell migration of EGFP, FLAG-PFKL-WT, and FLAG-PFKL-N702T transduced MDA-MB-231 cells was evaluated using μ-slide chemotaxis chambers (Ibidi USA, Inc.) using 50 ng/ml EGF as a chemoattractant (left side of image). Brightfield images of 4–5 fields of view of each chamber were captured using a 20× objective of a Nikon TI2-E Inverted Microscope. Images were captured every 10 min for 16 hours. Replicate 2. 

File: N702T_chemotaxis_n_3.nd2

Description: Single-cell migration of EGFP, FLAG-PFKL-WT, and FLAG-PFKL-N702T transduced MDA-MB-231 cells was evaluated using μ-slide chemotaxis chambers (Ibidi USA, Inc.) using 50 ng/ml EGF as a chemoattractant (left side of image). Brightfield images of 4–5 fields of view of each chamber were captured using a 20× objective of a Nikon TI2-E Inverted Microscope. Images were captured every 10 min for 16 hours. Replicate 3. 

File: Chemotaxis_file_renaming_key.csv

Description: List of current and original file names for associated chemotaxis assays. 

Code/software

Image files (ND2 and TIFF) - Fiji ImageJ (https://fiji.sc/)

Manual Tracking ImageJ plugin: https://imagej.net/ij/plugins/track/track.html