ECM dimensionality tunes actin tension to modulate endoplasmic reticulum function and spheroid phenotypes of mammary epithelial cells (2022)
Figure 2

Figure 2. Ligation of rBM in 3D alters ER function.

(A) Bar graph showing qPCR quantification of SEC61B level in MCF10A MECs ligated with rBM in 2D and 3D 12h post-plating (mean ± SEM; n=3 independent biological replicates). Statistical analysis by one-way ANOVA followed by Uncorrected Fisher's LSD, **P = 0.0022.

(B) MCF10A MECs expressing VSVGts045-EGFP were ligated to rBM in 2D or 3D and incubated at 40°C for 16h to trap VSVGts045-EGFP protein in the ER. Cells were shifted to 32°C for 2h to release VSVGts045-EGFP into the secretory pathway. Representative fluorescence microscopy images of MECs expressing VSVGts045-EGFP 2h post incubation at 32°C. Scale bar (whole cell), 10 μm; Scale bar (magnified), 2 μm.

(C) Scatter plot of the secretory trafficking efficiency of VSVGts045-EGFP based on the fluorescence of VSVGts045 at the plasma membrane versus total fluorescence measured in cells from Panel B (mean ± SEM; 2D, n=45; 3D, n=49 cells from three independent experiments). ****P < 0.0001 (Student's t test).

(D) Representative line graph showing the changes in cytosolic [Ca2+] levels of MCF10A MECs under different treatment. Fura-2-loaded MECs ligated to 2D (blue) or 3D rBM (red) were pre-incubated with EGTA to chelate extracellular Ca2+ in the bath solution, challenged with 2μM thapsigargin to induce ER Ca2+ release, and replenished with 4 mM Ca2+ in bath solution at the indicated time. F340/F380 values for each cell were quantified over the course of imaging (mean ± SD; 2D, n=7; 3D, n=7 cells from one experiment).

(E) Quantification of the amplitude of the Ca2+ response induced by thapsigargin (∆F=F-F0), where F0 is the basal fluorescence before thapsigargin treatment (mean ± SEM (2D, n=20; 3D, n=21 cells from three independent experiments). ***P = 0.0007 (Student's t test).

(F) Bar graph showing qPCR of the relative level of ATF3 mRNA in MCF10A MECs ligated with rBM in 2D and 3D 12h post-plating (mean ± SEM; n=3 independent biological replicates). Statistical analysis by one-way ANOVA followed by Uncorrected Fisher's LSD, **P = 0.0037.

(G) Representative immunoblots of phosphorylated EIF2a (pEIF2a), total EIF2a and alpha-tubulin in cell lysate from MCF10A MECs ligated to a rBM in 2D or 3D and corresponding quantification data (mean ± SEM; n=6 independent biological replicates). Statistical analysis by Student's t test, **P = 0.0049.

(H) Bar graph of qPCR data measuring the relative levels of CRACR2B mRNA in MCF10A MECs ligated with rBM in 2D and 3D 12h post-plating (mean ± SEM; n=4 independent biological replicates). *P = 0.0371 (Student's t test).

(I) Representative fluorescence microscopy images of the ER-plasma membrane contact site reporter GFP-MAPPER in MCF10A MECs ligated with rBM in 2D or 3D 18h post plating. Scale bar (whole cell), 10 μm; Scale bar (magnified), 1 μm.

(J) Graph of the levels of GFP-MAPPER at the plasma membrane relative total cellular GFP-MAPPER fluorescence in MCF10A MECs ligated with rBM in 2D or 3D in Panel I (mean ± SEM; 2D, n=52; 3D, n=57 cells from three independent experiments). **P = 0.0032 (Student's t test).

(K) Model of how ECM dimensionality affects ER structure/function.