Data and code from: Intra-cluster receptor density (IRD): A molecular switch for TNFR1 clusters’ signaling
Data files
Jul 09, 2026 version files 552.32 KB
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Code-for-Core_and-Rim_of_TNFR1-EGFP-cluster.ijm
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Code-for-TNFR1-EGFP-Cluster-anisotropy-calculation.m
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Figure_1_Source_Data.xlsx
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Figure_2_Source_Data.xlsx
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Figure_3_Source_Data.xlsx
44.67 KB
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Figure_4_Source_Data.xlsx
36.41 KB
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Figure_5_Source_Data.xlsx
37.16 KB
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Figure_6_Source_Data.xlsx
43.21 KB
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README.md
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registerImages.m
1.87 KB
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Supplimentary_Figure_S1_Source_Data.xlsx
101.10 KB
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Supplimentary_Figure_S2_Source_Data.xlsx
39.14 KB
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Supplimentary_Figure_S3_Source_Data.xlsx
17.13 KB
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Supplimentary_Figure_S4_Source_Data.xlsx
37.74 KB
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Supplimentary_Figure_S5_Source_Data.xlsx
41.07 KB
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Supplimentary_Figure_S6_Source_Data.xlsx
13.91 KB
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Supplimentary_Figure_S7_Source_Data.xlsx
30.10 KB
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Supplimentary_Figure_S8_Source_Data.xlsx
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Supplimentary_Figure_S9_Source_Data.xlsx
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Abstract
Tumor Necrosis Factor Receptor 1 (TNFR1) signaling regulates cell fate in inflammation, immune responses, and tumorigenesis. While TNF-α–mediated TNFR1 pathways are well known, the role of receptor clustering remains unclear. Utilizing homo-FRET using fluorescence anisotropy, we show that intra-cluster receptor density (IRD) governs TNFR1 signaling outcomes. Soluble TNF-α (sTNF-α) increases IRD at cluster cores but decreases it at rims via receptor reorganization. Reducing IRD through membrane tension, zafirlukast, actin depolymerization, or cholesterol depletion suppresses sTNF-α signaling, whereas increasing IRD by lowering membrane tension or exposing cells in a 3D gel-like microenvironment triggers ligand-independent activation. These findings reveal IRD as a key regulator of receptor signaling, with potential relevance across related receptor families and innovative strategies in modulating TNFR1 signaling.
Dataset DOI: 10.5061/dryad.qbzkh1904
Description of the data and file structure
This repository contains the datasets associated with the manuscript: "Intra-cluster receptor density (IRD): a molecular switch for TNFR1 clusters' signaling."
The data were collected to investigate how the intra-cluster receptor density (IRD) of TNFR1 regulates receptor cluster organization and downstream signaling. Multiple complementary imaging and biochemical approaches were employed to quantify TNFR1 clustering, receptor abundance, membrane mechanics, and NF-κB signaling under different experimental conditions.
This repository contains the source data underlying the figures and supplementary figures of the associated manuscript, together with the custom analysis code used to generate the reported measurements. Source data are provided as Microsoft Excel workbooks (.xlsx), one per main or supplementary figure, with individual sheets corresponding to specific figure panels. Analysis code is provided as FIJI/ImageJ macro (.ijm) and MATLAB (.m) scripts.
Unless otherwise noted:
- r denotes fluorescence anisotropy.
- r(i) / r(f) denote pre-bleach (initial) and post-bleach (final) mean anisotropy, respectively; Δr = r(f) − r(i).
- r(t) denotes mean anisotropy at time t; Δr/ri (%) denotes the percentage change in anisotropy relative to the initial (pre-bleach or t = 0) value.
- "Core" and "rim" refer to the two sub-regions used to segment TNFR1-EGFP nanoclusters for intensity- and anisotropy-based analysis.
- p65 N/C denotes the nuclear-to-cytoplasmic intensity ratio of NF-κB p65, used as a readout of NF-κB nuclear translocation/signaling.
- MFI denotes median fluorescence intensity (flow cytometry).
- SE / SD / Stdev denote standard error and standard deviation, as labeled per sheet.
- sTNF-α denotes soluble TNF-α; KD denotes siRNA-mediated knockdown; Scr/Sc-siRNA denotes scrambled (control) siRNA; CytoD denotes Cytochalasin D; MβCD denotes methyl-β-cyclodextrin; Zaf denotes zafirlukast; VitC denotes vitamin C; Utr/Ctrl/Control denote untreated conditions; Hypo denotes Hypotonic shock; Hyper denotes hypertonic shock.
Note: To quantify TNFR1 IRD, a C-terminal mEGFP-tagged TNFR1 construct (TNFR1-EGFP) was expressed in HeLa cells. IRD was determined using fluorescence anisotropy based on the principle of HomoFRET in a widefield fluorescence microscope. Because fluorescence anisotropy depends on the instrument-specific G-factor, absolute anisotropy values may vary between microscope systems.
Files and variables
File: Figure_1_Source_Data.xlsx
Description:
- Sheet 1 (Fig. 1F): Relative change in anisotropy with differential photobleaching. Columns: Bleaching % (photobleaching percentage); Core Δr/ri (relative change in anisotropy, core region) with se_core (standard error); Rim Δr/ri (relative change in anisotropy, rim region) with se_rim (standard error). Δr = r(f) − r(i).
- Sheet 2 (Fig. 1H): Intensity histogram of membrane-associated TNFR1 across conditions, with MFI inset. Columns: Sc-siRNA (scramble siRNA); KD 30nM and KD 60nM (TNFR1 siRNA at indicated concentrations); MFI (median fluorescence intensity).
- Sheet 3 (Fig. 1I): Left: TNFR1-EGFP cluster anisotropy (core/rim) in control vs. knockdown. Columns: Scr siRNA (core anisotropy); TNFR1 siRNA (core anisotropy); Scr siRNA (rim anisotropy); TNFR1 siRNA (rim anisotropy). Right: TNFR1-EGFP cluster anisotropy (core/rim) at increasing overexpression doses. Columns: Core anisotropy (0.1/0.5/1.0 µg TNFR1-EGFP overexpressed); Rim anisotropy (0.1/0.5/1.0 µg TNFR1-EGFP overexpressed).
- Sheet 4 (Fig. 1J): Cluster area (µm²) of endogenous TNFR1 (control vs. TNFR1-siRNA) and of TNFR1-EGFP at varying expression levels (Airyscan). Columns: Scr siRNA (Endogenous); TNFR1 siRNA (Endogenous); 0.1 µg / 0.5 µg / 1 µg (TNFR1-EGFP).
File: Figure_2_Source_Data.xlsx
Description:
- Sheet 1 (Fig. 2C): Relative % change in anisotropy over time, core region, live-cell TNFR1-EGFP clusters. Δr/ri _clus1 %: Δr = r(t) − r(i).
- Sheet 2 (Fig. 2D): As Sheet 1, fixed cells.
- Sheet 3 (Fig. 2E): FRAP of TNFR1-EGFP clusters, live HeLa cells. Columns: Time (sec); Recovery % (post-bleach recovery relative to pre-bleach); SE.
- Sheet 4 (Fig. 2F): Relative % change in anisotropy over time, rim region, live cells.
- Sheet 5 (Fig. 2G): As Sheet 4, fixed cells.
- Sheet 6 (Fig. 2H): Relative % change in fluorescence intensity over time, core region, live cells (Fcore(t)), relative to initial intensity.
- Sheet 7 (Fig. 2I): As Sheet 6, fixed cells.
File: Figure_3_Source_Data.xlsx
Description:
- Sheet 1 (Fig. 3B): Mean anisotropy, core/rim, untreated vs. sTNF-α-treated. Columns: Core Ani_Utr; Core Ani_TNFa (5 min sTNF-α); Rim ani_Utr; Rim ani_TNFa (5 min sTNF-α).
- Sheet 2 (Fig. 3D): Mean anisotropy, core/rim, untreated vs. zafirlukast-treated. Columns: Core Ani_Utr; Core Ani_Zaf; Rim ani_Utr; Rim ani_Zaf.
- Sheet 3 (Fig. 3E): Relative % change in anisotropy upon 50–60% photobleaching, core/rim, untreated/sTNF-α/zafirlukast. Δr/ri % with SE.
- Sheet 4 (Fig. 3F): Scatter: post-bleach relative anisotropy change vs. pre-bleach anisotropy, core region. Columns: Initial Core Ani_Utr, Norm Ani_Utr; Initial Core Ani_wTNFa, Norm Ani_wTNFa; Initial Core Ani_Zaf, Norm Ani_Zaf.
- Sheet 5 (Fig. 3G): As Sheet 4, rim region. Columns: Initial Rim Ani_Utr, Norm Ani_Utr; Initial Rim Ani_wTNFa, Norm Ani_wTNFa; Initial Rim Ani_Zaf, Norm Ani_Zaf.
- Sheet 6 (Fig. 3H): Endogenous p65 N/C ratio. Columns: Control p65 N/C; TNFa p65 N/C; Zaf p65 N/C; Zaf + TNFa p65 N/C; TNFR1 KD p65 N/C; TNFR1 KD +TNFa p65 N/C.
- Sheet 7 (Fig. 3I): Intensity histogram of membrane-associated TNFR1, with MFI inset. Columns: Control; TNFa_30min; Zaf; MFI.
- Sheet 8 (Fig. 3J): TNFR1-EGFP cluster area, untreated vs. zafirlukast-treated (TIRF). Columns: Control; Zaf.
File: Figure_4_Source_Data.xlsx
Description:
- Sheet 1 (Fig. 4C): Mean anisotropy, core/rim, untreated vs. hypoosmotic. Columns: Utr Core_r; Hypo Core_r; Utr Rim_r; Hypo Rim_r.
- Sheet 2 (Fig. 4D): As Sheet 1, hyperosmotic. Columns: Utr Core_r; Hyper Core_r; Utr Rim_r; Hyper Rim_r.
- Sheet 3 (Fig. 4E): Endogenous p65 N/C ratio. Columns: Iso; Hypo; Hyper; TNFR1-KD (isotonic); TNFR1 KD +Hyper.
- Sheet 4 (Fig. 4F): Intensity histogram of membrane-associated TNFR1, with MFI inset. Columns: Ctrl (isotonic); Hypo; Hyper; MFI.
- Sheet 5 (Fig. 4G): p65 N/C over 30 min post-sTNF-α, hypotonic (left) vs. hypertonic (right) vs. isotonic control. Left columns: Ctrl+sTNFa, SE_CtrlsTNFa; Hypo+sTNFa, SE_Hypo+sTNFa; Hypo, SE_Hypo. Right columns: Ctrl+sTNFa, SE; Hyper+sTNFa, SE; Hyper, SE.
- Sheet 6 (Fig. 4H): TNFR1 signaling efficacy under isotonic/hypotonic/hypertonic shock with sTNF-α, mean p65 fold change inset. Columns: w sTNFa (condition); Signaling Efficacy per TNFR1 (a.u.) × 10⁻³; Error; inset Mean p65 fold change; SE.
- Sheet 7 (Fig. 4I): TNFR1-EGFP cluster area, isotonic vs. hypertonic (TIRF). Columns: Isotonic; Hypertonic.
File: Figure_5_Source_Data.xlsx
Description:
- Sheet 1 (Fig. 5A): p65 N/C in 2D vs. 3D culture under scrambled-siRNA, TNFR1-siRNA, or zafirlukast treatment. Columns: 2D; 3D; 2D_TNFR1-siRNA; 3D_TNFR1-siRNA; 2D_Zaf; 3D_Zaf.
- Sheet 2 (Fig. 5B): % change in relative mean anisotropy (core/rim) under sTNF-α, hypotonic, hypertonic, and 3D conditions, normalized to untreated controls. Columns: Core_Mean Δr/ri %; Rim_Mean Δr/ri %.
- Sheet 3 (Fig. 5C): Mean anisotropy, core/rim, 2D vs. 3D, untreated vs. zafirlukast-treated. Columns: Core_2D_ani; Core_3D_ani; Rim_2D_ani; Rim_3D_ani; 2D Zaf Core Ani; 3D Zaf Core Ani; 2D Zaf Rim Ani; 3D Zaf Rim Ani.
- Sheet 4 (Fig. 5D): Mean anisotropy, core/rim, 2D vs. 3D at 4 h and 12 h post-transition. Columns: 2D_Core; 4h-3D_Core; 12h-3D_Core; 2D_Rim; 4h-3D_Rim; 12h-3D_Rim.
- Sheet 5 (Fig. 5E): Flipper-TR fluorescence lifetime (tau1), 2D vs. 3D culture. Columns: 2D_Fl LT; 3D_Fl LT.
- Sheet 6 (Fig. 5F): Mean anisotropy, core/rim, during 3D-to-2D transition. Columns: 3D (CORE, RIM); 2D_15min (CORE, RIM); 2D_30min (CORE, RIM).
- Sheet 7 (Fig. 5G): TNFR1-EGFP cluster area, 2D vs. 3D (4 h, 12 h). Columns: 2D; 3D-4h; 3D-12h.
- Sheet 8 (Fig. 5H): Cluster area vs. mean anisotropy (core), 2D vs. 3D. Left: 2D-Area, 2D-Core, 3D-Area-4h, 3D-4h-core. Right: 2D-Area, 2D-Core, 3D-Area-12h, 3D-12h-core.
File: Figure_6_Source_Data.xlsx
Description:
- Sheet 1 (Fig. 6A): Surface expression of endogenous TNFR1 (flow cytometry), control vs. CytoD vs. MβCD, with MFI inset. Columns: Ctrl; CytoD; MbCD; inset Ctrl_MFI, CytoD_MFI, MbCD_MFI.
- Sheet 2 (Fig. 6B): Mean anisotropy, core/rim, untreated vs. CytoD-treated. Columns: Core-Untreated; Core-CytoD; Rim-Untreated; Rim-CytoD.
- Sheet 3 (Fig. 6C): p65 N/C, effect of CytoD in control/TNFR1-KD/sTNF-α-stimulated cells. Columns: Control; CytoD; TNFR1-KD + CytoD; TNFa; CytoD + TNFa.
- Sheet 4 (Fig. 6D): Temporal p65 N/C, control vs. CytoD-treated, upon sTNF-α stimulation. Columns: Time(min); N/C p65_Control, SE; N/C p65_CytoD 1 h.
- Sheet 5 (Fig. 6E): Mean anisotropy, core/rim, untreated vs. MβCD-treated. Columns: Utr_Core_ani; MbCD_Core_ani; Utr_Rim_ani; MbCD_Rim_ani.
- Sheet 6 (Fig. 6F): SDspace, SDtime, and membrane tension maps (IRM), control vs. MβCD. Columns: SD_Space(nm); SD_time(nm); Tension (pN/µm).
- Sheet 7 (Fig. 6G): p65 N/C, control vs. MβCD, ± sTNF-α. Columns: Control; sTNF a; MBCD 2mM; MBCD 2mM +TNFa.
- Sheet 8 (Fig. 6H): TNFR1 signaling efficacy, control/CytoD/MβCD, with mean p65 fold change inset. Columns: w sTNFa (condition); Signaling Efficacy per TNFR1 (a.u.) × 10⁻³; SE; inset Mean p65 fold change.
File: Supplimentary_Figure_S1_Source_Data.xlsx
Description:
- Sheet 1 (fig. S1C): Mean intensity of a single TNFR1-EGFP cluster during photobleaching over time. Columns: Time (Second); Mean Fluorescence Intensity.
- Sheet 2 (fig. S1D): Fraction of plasma-membrane-associated TNFR1-EGFP clusters over time post-transfection. Columns: Membrane fraction; Stdev.
- Sheet 3 (fig. S1E): % change in relative mean anisotropy, mEGFP vs. 2EGFP-overexpressing cells, upon 50–60% photobleaching. Columns: Cell_type; Δr/ri (%); SE; inset HeLa mEGFP (r), HeLa 2GFP (r).
File: Supplimentary_Figure_S2_Source_Data.xlsx
Description:
- Sheet 1 (fig. S2A): Mean anisotropy, core/rim, TNFR1-EGFP constructs with 7/14/21 amino-acid linkers. Columns: 7/14/21 AA linker_r-core; 7/14/21 AA linker_r-rim.
- Sheet 2 (fig. S2C): Endogenous TNFR1 cluster area, non-transfected vs. TNFR1-EGFP-transfected, scrambled-siRNA vs. TNFR1-KD. Columns: WO Overexpression (Endo-TNFR1); TNFR1-EGFP 0.1/0.5/1.0 µg (Endo-TNFR1/Scr-siRNA); TNFR1-EGFP 0.1/0.5/1.0 µg (Endo-TNFR1/TNFR1-siRNA).
- Sheet 3 (fig. S2D): TNFR1-EGFP cluster area, scrambled-siRNA vs. TNFR1-KD, across expression doses. Columns: TNFR1-EGFP 0.1/0.5/1.0 µg (Scr siRNA); TNFR1-EGFP 0.1/0.5/1.0 µg (TNFR1 siRNA).
- Sheet 4 (fig. S2E): Cluster area vs. mean anisotropy (core, rim), across expression doses, scrambled-siRNA (left) vs. TNFR1-KD (right). Paired area/anisotropy columns per dose (0.1/0.5/1.0 µg) and region (core/rim), labeled by condition (e.g., TNFR1-EGFP 1.0 µg(TNFR1-EGFP Area/scr-siRNA), TNFR1-EGFP 1.0 µg(r_core/scr-siRNA), etc.).
File: Supplimentary_Figure_S3_Source_Data.xlsx
Description:
- Sheet 1 (fig. S3A): Relative % change in anisotropy over time, TNFR1-7aa-EGFP clusters, core/rim, live/fixed cells. Columns (per quadrant): Δr/ri % (core or rim, live or fixed) and Time(sec).
- Sheet 2 (fig. S3B): As Sheet 1, TNFR1-21aa-EGFP clusters.
File: Supplimentary_Figure_S4_Source_Data.xlsx
Description:
- Sheet 1 (fig. S4A): Left: p65 N/C vs. sTNF-α dose. Columns: sTNF-a (ng/mL); Mean p65 N/C; SE. Right: temporal p65 N/C at 20 ng/mL sTNF-α. Columns: Time; Avg p65 N/C; error.
- Sheet 2 (fig. S4B): TNFR1-EGFP cluster area, untreated vs. sTNF-α-treated. Columns: Control; sTNFa-30mins.
- Sheet 3 (fig. S4D): Left: p65 N/C across zafirlukast doses (25–200 µM) ± sTNF-α. Right: temporal p65 N/C, 100 µM zafirlukast ± sTNF-α, across 15–90 min timepoints. Columns labeled by dose/timepoint and treatment (see workbook headers).
- Sheet 4 (fig. S4E): p65 N/C, control vs. TNFR1 knockdown. Columns: Scr siRNA_p65 N/C; TNFR1 siRNA_p65 N/C.
File: Supplimentary_Figure_S5_Source_Data.xlsx
Description:
- Sheet 1 (fig. S5A): p65 N/C across TNFR1-EGFP (left) or EGFP (right) plasmid dose. Columns: Control_p65 N/C; 0.1/0.5/1.0 µg_TNFR1-EGFP (or EGFP).
- Sheet 2 (fig. S5B): TNFR1 signaling efficacy, control vs. zafirlukast, mean p65 fold change inset. Columns: Signaling Efficacy per TNFR1 (a.u.) × 10⁻³; SE; inset Mean p65 fold change.
- Sheet 3 (fig. S5C): Total TNFR1 abundance (western blot) across treatments. Columns: Treatment (Ctrl, sTNF-α, Zaf, CytoD, Noco, WFA, Hypo, Hyper); Fold change TNFR1/β-Actin.
- Sheet 4 (fig. S5D): Surface expression of endogenous TNFR1, control vs. sTNF-α at 5/15/30 min, with MFI inset. Columns: Ctrl; TNFa5; TNFa15; TNFa30; inset Ctrl_MFI, TNF5_MFI, TNF15_MFI, TNF30_MFI.
File: Supplimentary_Figure_S6_Source_Data.xlsx
Description:
- Sheet 1 (fig. S6A): Flipper-TR fluorescence lifetime over time, hypotonic treatment. Columns: Lifetime (tau1); SE.
- Sheet 2 (fig. S6B): As Sheet 1, hypertonic treatment.
- Sheet 3 (fig. S6D): % change in relative mean anisotropy (core/rim), zafirlukast/hypotonic/hypertonic, normalized to untreated controls. Columns: Core; Rim.
- Sheet 4 (fig. S6F): TNFR1-EGFP cluster area, isotonic condition over time (TIRF). Columns: Isotonic; Isotonic 2 mins.
- Sheet 5 (fig. S6G): TNFR1-EGFP cluster area, isotonic vs. hypertonic (TIRF). Columns: Isotonic; Hypertonic.
File: Supplimentary_Figure_S7_Source_Data.xlsx
Description:
- Sheet 1 (fig. S7B): p65-GFP N/C, 2D vs. 3D, ± zafirlukast. Columns: 2D; 3D; 2D +Zaf; 3D +Zaf.
- Sheet 2 (fig. S7D): Surface expression of endogenous TNFR1, 2D vs. 3D, with MFI inset. Columns: 2D; 3D; inset 2D_MFI, 3D_MFI.
- Sheet 3 (fig. S7E): Flipper-TR fluorescence lifetime, THP-1 cells, 2D vs. 3D. Columns: 2D_Fl lifetime; 3D_Fl lifetime.
- Sheet 4 (fig. S7F): Cluster area vs. mean anisotropy (rim), 2D vs. 3D. Left: 2D-Area, 2D-Rim, 3D-Area-4h, 3D-4h-Rim. Right: 2D-Area, 2D-rim, 3D-Area-12h, 3D-12h-Rim.
File: Supplimentary_Figure_S8_Source_Data.xlsx
Description:
- Sheet 1 (fig. S8B): H2DCFDA fluorescence intensity, control vs. CytoD, ± vitamin C. Columns: Fl H2DCFDA_Utr; Fl H2DCFDA_wVitC; SD_er/SD_error.
- Sheet 2 (fig. S8C): p65 N/C, control/CytoD/sTNF-α/CytoD+sTNF-α, ± vitamin C. Columns: Ctrl, CytoD, sTNF-α, CytoD + sTNF-α (each with/without VitC).
- Sheet 3 (fig. S8E): Mean anisotropy, core/rim, control vs. vitamin-C-treated. Columns: Utr (Core_r); VitC(Core_r); Utr (Rim_r); VitC (Rim_r).
File: Supplimentary_Figure_S9_Source_Data.xlsx
Description:
Sheet 1 (fig. S9A): TNFR1-EGFP cluster area, control vs. MβCD-treated (TIRF). Columns: Control; MbCD.
File: Code-for-Core_and-Rim_of_TNFR1-EGFP-cluster.ijm
Description: In-house FIJI/ImageJ macro for intensity-based segmentation and analysis of the core and rim regions of TNFR1-EGFP clusters.
File: Code-for-TNFR1-EGFP-Cluster-anisotropy-calculation.m
Description: In-house MATLAB script for calculating fluorescence anisotropy of TNFR1-EGFP clusters.
File: registerImages.m
Description: MATLAB function for monomodal, intensity-based image registration, used for TNFR1-EGFP image registration.
Code/software
Software requirements: The analysis scripts were developed using two platforms. The ImageJ/Fiji macro Code-for-Core_and-Rim_of_TNFR1-EGFP-cluster.ijmrequires Fiji/ImageJ, while the MATLAB scripts Code-for-TNFR1-EGFP-Cluster-anisotropy-calculation.m and registerImages.m require MATLAB. Usage instructions are provided within the individual scripts.