https://doi.org/10.5061/dryad.z34tmpgn8

Co-corresponding authors: Siddharth Dey, sdey@ucsb.edu; Angela Pitenis, apitenis@ucsb.edu

File List

A) Figure2_representative_3wt.%_indentation_curve.csv

B) Figure2_representative_5wt.%_indentation_curve.csv

C) Figure2_3wt%_average_modulus.csv

D) Figure2_5wt%_average_modulus.csv

E) Figure3a_data.csv

F) Figure3b_data.csv

G) Figure5_soft_6h_1.txt

H) Figure5_soft_6h_2.txt

I) Figure5_stiff_6h_1.txt

J) Figure5_stiff_6h_2.txt

K) Figure6_soft_to_soft_1.txt

L) Figure6_soft_to_soft_2.txt

M) Figure6_soft_to_soft_3.txt

N) Figure6_soft_to_soft_4.txt

O) Figure6_soft_to_stiff_1.txt

P) Figure6_soft_to_stiff_2.txt

Q) Figure6_soft_to_stiff_3.txt

R) Figure6_stiff_to_soft_1.txt

S) Figure6_stiff_to_soft_2.txt

T) Figure6_stiff_to_soft_3.txt

U) Figure6_stiff_to_soft_4.txt

V) Figure6_stiff_to_stiff_1.txt

W) Figure6_stiff_to_stiff_2.txt

X) Figure6_stiff_to_stiff_3.txt

Y) Figure6_stiff_to_stiff_4.txt

Z) Figure7a_soft_to_soft.tif

AA) Figure7a_soft_to_stiff.tif

AB) Figure7a_stiff_to_soft.tif

AC) Figure7a_stiff_to_stiff.tif

AD) Figure7b_calculations.csv

AE) Figure7c_soft_to_soft.tif

AF) Figure7c_soft_to_stiff.tif

AG) Figure7c_stiff_to_soft.tif

AH) Figure7c_stiff_to_stiff.tif

AI) Figure7d_calculations.csv

AJ) FigureS2_3wt.%diameter.csv

AK) FigureS2_5wt.%diameter.csv

AL) FigureS3_color_heatmap_raw_data.nd2

AM) FigureS4_3wt.%_Live_dead_stain_celltrackergreen488_propidiumiodidered.nd2

AN) FigureS4_5wt.%_Live_dead_stain_celltrackergreen488_propidiumiodidered_01.nd2

AO) FigureS4_5wt.%_Live_dead_stain_celltrackergreen488_propidiumiodidered_02.nd2

AP) FigureS7_3wt.%.nd2

AQ) FigureS7_5wt.%.nd2

AR) FigureS8_2D_1.txt

AS) FigureS8_2D_2.txt

AT) FigureS8_2D_3.txt

AU) FigureS10_3wt.%_radial_tubulin_intensity.csv

AV) FigureS10_5wt.%_radial_tubulin_intensity.csv

AW) Figure7b_raw_image_data.zip

AX) Figure7d_raw_image_data.zip

FIGURE 2: microtribometer

A) Figure2_representative_3wt.%_indentation_curve.csv: representative indentation curve of 3 wt.% bulk polyacrylamide hydrogel

• row 1: labels for column data with units

• column 1: time, in seconds, of indentation

• column 2: normal force, Fn, in micro-Newtons, or the y-axis in the indentation curve

• column 3: standard deviation in the normal force measurement, in micro-Newtons

• column 4: z-stage displacement in micrometers, or the x-axis in the indentation curve

• rows 2 and beyond: data for each of these columns

B) Figure2_representative_5wt.%_indentation_curve.csv: representative indentation curve of 5 wt.% bulk polyacrylamide hydrogel

• row 1: labels for column data with units

• column 1: time, in seconds, of indentation

• column 2: normal force, Fn, in micro-Newtons, or the y-axis in the indentation curve

• column 3: standard deviation in the normal force measurement, in micro-Newtons

• column 4: z-stage displacement in micrometers, or the x-axis in the indentation curve

• rows 2 and beyond: data for each of these columns

C) Figure2_3wt%_average_modulus.csv: average moduli values calculated from indentations on the micro tribometer for the 3 wt.% bulk polyacrylamide hydrogel

• row 1: column labels

• column 1: position number on a gel for a set of measurements

• column 2: cycle number per position on a gel

• column 3: maximum force obtained during measurement, in micro-Newtons

• column 4: pressure, in kPa

• column 5: contact radii, in micrometers

• column 6: measured E* elastic modulus using a Hertz fit, in kPa

D) Figure2_5wt%_average_modulus.csv: average moduli values calculated from indentations on the microtribometer for the 5 wt.% bulk polyacrylamide hydrogel

• row 1: column labels

• column 1: position number on a gel for a set of measurements

• column 2: cycle number per position on a gel

• column 3: maximum force obtained during measurement, in micro-Newtons

• column 4: pressure, in kPa

• column 5: contact radii, in micrometers

• column 6: measured E* elastic modulus using a Hertz fit, in kPa

FIGURE 3: rheology

E) Figure3a_data.csv: data for angular frequency sweep reporting storage moduli for both the 3 wt.% and 5 wt.% microgel particles

• row 1: column labels for angular frequency, storage modulus, and loss modulus

• row 2: units corresponding to each column

• row 3: replicate number for each sample

• row 4 and beyond: data for the frequency, storage, and loss moduli

• columns A-F: data for 5 wt.% polyacrylamide microgel

• columns G-L: data for 3 wt.% polyacrylamide microgel

F) Figure3b_data.csv: data for oscillatory strain sweep

• row 1: columns labels for oscillatory strain, storage, and loss moduli

• row 2: corresponding units for each column

• row 3: replicate number for each column

• row 4 and beyond: data for the oscillatory strain, storage, and loss moduli

• columns A-F: data for 5 wt.% polyacrylamide microgel

• columns G-L: data for 3 wt.% polyacrylamide microgel

FIGURE 5: cells cultured for 6 h in microgel; data analyzed with bulk-mRNA-seq R script

G) Figure5_soft_6h_1.txt: gene count data for replicate 1 of soft-microgel-cultured PDAC cells after 6 h

• row 1: list of column numbers

• column 1: list of gene names

• column 10: list of gene counts

• other columns: NA, 0-filled matrices

H) Figure5_soft_6h_2.txt: gene count data for replicate 2 of soft-microgel-cultured PDAC cells after 6 h

• row 1: list of column numbers

• column 1: list of gene names

• column 10: list of gene counts

• other columns: NA, 0-filled matrices

I) Figure5_stiff_6h_1.txt: gene count data for replicate 1 of stiff-microgel-cultured PDAC cells after 6 h

• row 1: list of column numbers

• column 1: list of gene names

• column 10: list of gene counts

• other columns: NA, 0-filled matrices

J) Figure5_stiff_6h_2.txt: gene count data for replicate 2 of stiff-microgel-cultured PDAC cells after 6 h

• row 1: list of column numbers

• column 1: list of gene names

• column 10: list of gene counts

• other columns: NA, 0-filled

FIGURE 6, S5, and S9: mechanical switch RNA-seq data, analyzed with bulk-mRNA-seq R script

K) Figure6_soft_to_soft_1.txt: gene count file for replicate 1 of the soft-to-soft condition shown in Figure 6b

• row 1: labels of each column

• column 1: list of gene names

• column 5: list of gene counts

• other columns: NA, 0-filled

L) Figure6_soft_to_soft_2.txt: gene count file for replicate 2 of the soft-to-soft condition shown in Figure 6b

• row 1: labels of each column

• column 1: list of gene names

• column 5: list of gene counts

• other columns: NA, 0-filled

M) Figure6_soft_to_soft_3.txt: gene count file for replicate 3 of the soft-to-soft condition shown in Figure 6b

• row 1: labels of each column

• column 1: list of gene names

• column 5: list of gene counts

• other columns: NA, 0-filled

N) Figure6_soft_to_soft_4.txt: gene count file for replicate 4 of the soft-to-soft condition shown in Figure 6b

• row 1: labels of each column

• column 1: list of gene names

• column 5: list of gene counts

• other columns: NA, 0-filled

O) Figure6_soft_to_stiff_1.txt: gene count file for replicate 1 of the soft-to-stiff condition shown in Figure 6b

• row 1: labels of each column

• column 1: list of gene names

• column 5: list of gene counts

• other columns: NA, 0-filled

P) Figure6_soft_to_stiff_2.txt: gene count file for replicate 2 of the soft-to-stiff condition shown in Figure 6b

• row 1: labels of each column

• column 1: list of gene names

• column 5: list of gene counts

• other columns: NA, 0-filled

Q) Figure6_soft_to_stiff_3.txt: gene count file for replicate 3 of the soft-to-stiff condition shown in Figure 6b

• row 1: labels of each column

• column 1: list of gene names

• column 5: list of gene counts

• other columns: NA, 0-filled

R) Figure6_stiff_to_soft_1.txt: gene count file for replicate 1 of the stiff-to-soft condition shown in Figure 6b

• row 1: labels of each column

• column 1: list of gene names

• column 5: list of gene counts

• other columns: NA, 0-filled

S) Figure6_stiff_to_soft_2.txt: gene count file for replicate 2 of the stiff-to-soft condition shown in Figure 6b

• row 1: labels of each column

• column 1: list of gene names

• column 5: list of gene counts

• other columns: NA, 0-filled

T) Figure6_stiff_to_soft_3.txt: gene count file for replicate 3 of the stiff-to-soft condition shown in Figure 6b

• row 1: labels of each column

• column 1: list of gene names

• column 5: list of gene counts

• other columns: NA, 0-filled

U) Figure6_stiff_to_soft_4.txt: gene count file for replicate 4 of the stiff-to-soft condition shown in Figure 6b

• row 1: labels of each column

• column 1: list of gene names

• column 5: list of gene counts

• other columns: NA, 0-filled

V) Figure6_stiff_to_stiff_1.txt: gene count file for replicate 1 of the stiff-to-stiff condition shown in Figure 6b

• row 1: labels of each column

• column 1: list of gene names

• column 5: list of gene counts

• other columns: NA, 0-filled

W) Figure6_stiff_to_stiff_2.txt: gene count file for replicate 2 of the stiff-to-stiff condition shown in Figure 6b

• row 1: labels of each column

• column 1: list of gene names

• column 5: list of gene counts

• other columns: NA, 0-filled

X) Figure6_stiff_to_stiff_3.txt: gene count file for replicate 3 of the stiff-to-stiff condition shown in Figure 6b

• row 1: labels of each column

• column 1: list of gene names

• column 5: list of gene counts

• other columns: NA, 0-filled

Y) Figure6_stiff_to_stiff_4.txt: gene count file for replicate 4 of the stiff-to-stiff condition shown in Figure 6b

• row 1: labels of each column

• column 1: list of gene names

• column 5: list of gene counts

• other columns: NA, 0-filled

FIGURE 7: immunofluorescence

Z) Figure7a_soft_to_soft.tif: raw image of soft-to-soft condition appearing in Figure 7a

• processed using FIJI into a SUM intensity projection over 4.65 micrometers (slices 9-14)

• green channel shows BCAR3 staining, and blue channel shows nuclear staining

AA) Figure7a_soft_to_stiff.tif: raw image of soft-to-stiff condition appearing in Figure 7a

• processed using FIJI into a SUM intensity projection over 4.65 micrometers (slices 9-14)

• green channel shows BCAR3 staining, and blue channel shows nuclear staining

AB) Figure7a_stiff_to_soft.tif: raw image of stiff-to-soft condition appearing in Figure 7a

• processed using FIJI into a SUM intensity projection over 4.65 micrometers (slices 8-13)

• green channel shows BCAR3 staining, and blue channel shows nuclear staining

AC) Figure7a_stiff_to_stiff.tif: raw image of stiff-to-stiff condition appearing in Figure 7a

• processed using FIJI into a SUM intensity projection over 4.65 micrometers (slices 10-15)

• green channel shows BCAR3 staining, and blue channel shows nuclear staining

AD) Figure7b_calculations.csv: intensity measurements for Figure 7b

• row 1: column labels

• column A: sample number

• column B: minimum intensity

• column C: maximum intensity

• column D: integrated fluorescent density

• column E: area measurement, in micrometers squared

• column F: average fluorescence intensity (integrated density/unit area; column D / column E), in fluorescence unit/micrometers squared

• row 2 and beyond: data

AE) Figure7c_soft_to_soft.tif: raw image of soft-to-soft condition appearing in Figure 7c

• processed using FIJI to create a SUM intensity z-projection across the entire stack

• green channel shows RAC3 staining, and blue channel shows nuclear staining

AF) Figure7c_soft_to_stiff.tif: raw image of soft-to-stiff condition appearing in Figure 7c

• processed using FIJI to create a SUM intensity z-projection across the entire stack

• green channel shows RAC3 staining, and blue channel shows nuclear staining

AG) Figure7c_stiff_to_soft.tif: raw image of stiff-to-soft condition appearing in Figure 7c

• processed using FIJI to create a SUM intensity z-projection across the entire stack

• green channel shows RAC3 staining, and blue channel shows nuclear staining

AH) Figure7c_stiff_to_stiff.tif: raw image of stiff-to-stiff condition appearing in Figure 7c

• processed using FIJI to create a SUM intensity z-projection across the entire stack

• green channel shows RAC3 staining, and blue channel shows nuclear staining

AI) Figure7d_calculations.csv:

• row 1: column labels

• column A: sample number

• column B: minimum intensity

• column C: maximum intensity

• column D: integrated fluorescent density

• column E: area measurement, in micrometers squared

• column F: average fluorescence intensity (integrated density/unit area; column D / column E), in fluorescence unit/micrometers squared

• row 2 and beyond: data

AW) Figure7b_raw_image_data.zip

• this compressed file has four folders for the four different conditions of our experiment: soft-to-soft, soft-to-stiff, stiff-to-soft, and stiff-to-stiff

• files in each of these subfolders are TIFF images of BCAR3 immunofluorescent staining

• channel 1 shows a nuclear stain

• channel 2 shows the BCAR3 stain

AX) Figure7d_raw_image_data.zip

• this compressed file has four folders for the four different conditions of our experiment: soft-to-soft, soft-to-stiff, stiff-to-soft, and stiff-to-stiff

• files in each of these subfolders are TIFF images of RAC3 immunofluorescent staining

• channel 1 shows a nuclear stain

• channel 2 shows the RAC3 stain

• channel 3 shows actin staining that was only used to identify the cell body

FIGURE S2: microgel size distribution

AJ) FigureS2_3wt.%diameter.csv: microgel size information for 3 wt.% polyacrylamide microgel particles

• row 1: column labels

• column 1: number of measurements from 1 - 250 for n = 250 samples counted

• column 2: radii measurements for each sample, in micrometers

• column 3: diameter measurements for each sample, in micrometers

AK) FigureS2_5wt.%diameter.csv: microgel size information for 5 wt.% polyacrylamide microgel particles

• row 1: column labels

• column 1: number of measurements from 1 - 250 for n = 250 samples counted

• column 2: radii measurements for each sample, in micrometers

• column 3: diameter measurements for each sample, in micrometers

FIGURE S3: color depth heatmap

AL) FigureS3_color_heatmap_raw_data.nd2: raw image processed in FIJI

• maximum intensity projection was created after using the Z-stack Depth Colorcode 0.0.2 plugin

FIGURE S4: cell viability

AM) FigureS4_3wt.%_Live_dead_stain_celltrackergreen488_propidiumiodidered.nd2:

• propidium iodide staining for cells cultured in 3 wt.% microgels for 24 h

• cell tracker green is in channel one for the identification of cells

• dead stain in the second channel identifies dead cells

AN) FigureS4_5wt.%_Live_dead_stain_celltrackergreen488_propidiumiodidered_01.nd2

• propidium iodide staining for cells cultured in 5 wt.% microgel for 24 h

• cell tracker green is in channel one for the identification of cells

• dead stain in the second channel identifies dead cells

AO) FigureS4_5wt.%_Live_dead_stain_celltrackergreen488_propidiumiodidered_02.nd2

• propidium iodide staining for cells cultured in 5 wt.% microgel for 24 h, second image

• cell tracker green is in channel one for the identification of cells

• dead stain in the second channel identifies dead cells

FIGURE S6 and S7: microgel packing

AP) FigureS7_3wt.%.nd2:

• image was processed in FIJI using the local thickness native analysis

• image was thresholded, and packing density was calculated as the percentage of fluorescence per slice

• local thickness values were extracted from FIJI, in micrometers

AQ) FigureS7_5wt.%.nd2:

• image was processed in FIJI using the local thickness native analysis

• image was thresholded, and packing density was calculated as the percentage of fluorescence per slice

• local thickness values were extracted from FIJI, in micrometers

FIGURE S8: 2D and 3D comparison, analyzed with bulk-mRNA-seq R script

AR) FigureS8_2D_1.txt: Gene counts of 2D tissue culture plastic-culture PDAC cells after 6 h

• gene count data is given in column 5

• row 1 shows column labels

• column 1 shows gene names

• data in all other columns are NA (shown as 0s)

• comparisons were made to files K-Y

AS) FigureS8_2D_2.txt: gene counts of 2D tissue culture plastic-culture PDAC cells after 6 h, replicate 2

• gene count data is given in column 5

• row 1 shows column labels

• column 1 shows gene names

• data in all other columns are NA (shown as 0s)

• comparisons were made to files K-Y

AT) FigureS8_2D_3.txt: gene counts of 2D tissue culture plastic-culture PDAC cells after 6 h, replicate 3

• gene count data is given in column 5

• row 1 shows column labels

• column 1 shows gene names

• data in all other columns are NA (shown as 0s)

• comparisons were made to files K-Y

FIGURE S10: radial tubulin expression

AU) FigureS10_3wt.%_radial_tubulin_intensity.csv: data on tubulin expression across a cell diameter for PDAC cells cultured in soft microgel for 24 h

• row 1: column labels for distance in microns (A, E, I, M), intensity (called gray value, B, F, J, N), normalized distance (from 0 to 1, C, G, K, O), and normalized intensity (gray value normalized, D, H, L, P) for each sample

• row 2: sample number information

• row 3 and below: data

• column Q maps distance values from 0 to 1 for the x-axis of the plots in Figure S10

• column R shows average intensity values calculated as averages of columns D, H, L, and P

• column S shows standard deviations in intensity values from columns D, H, L, and P

• sample 1: columns A-D

• sample 2: column E-H

• sample 3: columns I-L

• sample 4: columns M-P

AV) FigureS10_5wt.%_radial_tubulin_intensity.csv: data on tubulin expression across a cell diameter for PDAC cells cultured in stiff microgel for 24 h

• row 1: column labels for distance in microns (A, E, I, M), intensity (called gray value, B, F, J, N), normalized distance (from 0 to 1, C, G, K, O), and normalized intensity (gray value normalized, D, H, L, P) for each sample

• row 2: sample number information

• row 3 and below: data row 4 and below: data

• column Q maps distance values from 0 to 1 for the x-axis of the plots in Figure S10

• column R shows average intensity values calculated as averages of columns D, H, L, and P

• column S shows standard deviations in intensity values from columns D, H, L, and P

• sample 1: columns A-D

• sample 2: column E-H

• sample 3: columns I-L

• sample 4: columns M-P

---

R script provided: RNA-seq analysis R script

Functions utilized:

A) BiocManager version 3.14

B) DESeq2

C) plyr

D) apeglm

E) ggplot2

F) pheatmap

G) vsn

H) RColorBrewer

I) EnhancedVolcano

J) magrittr

K) ashr

L) pasilla

M) tidyverse

N) hrbrthemes

O) viridis

P) plotly

Q) heatmaply

R) ComplexHeatmap

S) psych

T) reshape2

ACCESSED DATA

Data for generating survival curves, based on mRNA data from the pancreas, appearing in Figures 5d and 6b used The Cancer Genome Atlas (TCGA) Data, Resources, and Materials originally published by the National Cancer Institute. Data was accessed through kmplot.com. The data obtained from TCGA can be published under CC0. The results published here are in part based upon data generated by the TCGA Research Network: https://www.cancer.gov/tcga

• Nagy A, Munkacsy G, Gyorffy B: Pancancer survival analysis of cancer hallmark genes, Sci Rep., 2021 Mar 15;11(1):6047, https://doi.org/10.1038/s41598-021-84787-5