Endoplasmic reticulum stress actively suppresses hepatic molecular identity in damaged liver (2020)
Figure 5

Figure 5. Loss of LIVER-ID TF protein expression is a primary event upon acute ERS.

(A) RT-qPCR analyses of 21 LIVER-ID genes (12 TFs and 9 non-TFs, listed in Table EV5) in MPH which were treated with 1µM thapsigargin (ERS) for 4h, 8h, 16h or 24h (4 independent experiments). For each gene, the timepoint showing maximal repression by ERS was recorded. The heatmap depicts the percentage of genes maximally repressed by ERS at each timepoint.

(B) RT-qPCR analyses of Hnf4a, Nr1h4 and Foxa2 expression in MPH treated with vehicle (Control) or 1µM thapsigargin (ERS) for 1h or 4h (4 independent experiments). The bar graph shows means ± SD (standard deviations). One-sample t-test with BH correction for multiple testing was used to determine if the mean Log2 FC ERS/Control is statistically different from 0, *P < 0.05.

(C) Densitometric quantification of the protein expression data shown in Fig.5D, Fig.5F and Fig.S18A-B for the 1h timepoint (average of 9 biological replicates) and Fig.2H for the 4h timepoint (average of 3 biological replicates). The bar graph shows means ± SD (standard deviations). One-sample t-test with BH correction for multiple testing was used to determine if the mean Log2 FC ERS/Control is statistically different from 0, *P < 0.05.

(D) Nuclear extracts from MPH pre-treated for 30min with 1µM ISRIB followed by addition of 1µM thapsigargin (ERS) for 1h were subjected to Western blot with antibodies against HNF4A, NR1H4, FOXA2 or DDIT3. LMNA was used as loading control. Results obtained from 3 independent biological replicates are shown.

(E) Densitometric quantification of the protein expression data shown in panel (D). Repression by ERS in the ISRIB condition (average of 3 biological replicates) is shown relative to repression by ERS in vehicle condition. The bar graph shows means ± SD (standard deviations). One-sample t-test with BH correction for multiple testing was used to determine if the mean relative repression is statistically different from 100%, *P < 0.05.

(F) Nuclear extracts from MPH pre-treated for 30min with 10µM MG132 or 10µM PP2 followed by addition of 1µM thapsigargin (ERS) for 1h were subjected to Western blot or Simple Western immunoassay with antibodies against HNF4A, NR1H4, FOXA2 or XBP1S. LMNA was used as loading control. Results obtained from 2 independent biological replicates are shown. Additional replicates are shown in Appendix Fig.S18A-B.

(G) Densitometric quantification of the protein expression data shown in panel (F) and Appendix Fig.S18A. Repression by ERS in MG132 condition (average of 4 biological replicates) is shown relative to repression by ERS in vehicle condition. The bar graph shows means ± SD (standard deviations). One-sample t-test with BH correction for multiple testing was used to determine if the mean relative repression is statistically different from 100%, *P < 0.05.

(H) (Left) Cytoplasmic extracts issued from the MPH used in panel (F) (2 biological replicates) were subjected to Western blot with antibodies against p-Y416-SRC or total SRC. ACTB was used as loading control. (Right) Densitometric quantification of the protein expression data. The bar graphs show means ± SD (standard deviations). Two-way ANOVA with Bonferroni's post-hoc test was used to assess statistical significance.

(I) Densitometric quantification of the protein expression data shown in panel F and Appendix Fig.S18B. Repression by ERS in the PP2 condition (average of 4 biological replicates) is shown relative to repression by ERS in vehicle condition. The bar graph shows means ± SD (standard deviations). One-sample t-test with BH correction for multiple testing was used to determine if the mean relative repression is statistically different from 100%.

(J) RT-qPCR analyses of selected ERS UP and LIVER-ID TF genes monitoring expression changes induced by 4h ERS in MPH pre-treated or not for 30min with 10µM PP2 (3 independent experiments). Mean Log2 FC ERS/Control are shown. The bar graph shows means ± SD (standard deviations). Two-way ANOVA with Bonferroni's post-hoc test was used to assess statistical significance, *P < 0.05.