Supplementary Figures 1-14 from Palmitate-Induced IRE1–XBP1–ZEB Signaling Represses Desmoplakin Expression and Promotes Cancer Cell Migration

Abstract

<p>S1A. Plasmids containing single guide RNAs targeted against the IRE1 locus. B. XBP1 splicing assay to assay IRE1 endoribonuclease activity in Hep3B and MDA MB 231 cells. S2. CRISPR clones obtained for MDA-MB-231 cells. S3. CRISPR clones obtained for Hep3B cells. S4. CRISPR clones obtained for HepG2 cells. S5. Representative brightfield images of Boyden's chamber migration assay. S6. Representative brightfield images of Boyden's chamber migration assay. S7. Representative brightfield images of Boyden's chamber migration assay. S8. Heatmap of standardized DSP, ZEB1 or ZEB2 expression in the TCGA PANCAN dataset. S9. Scatter plots showing the correlation between DSP methylation and the expression of DSP, ZEB1 or ZEB2 in the TCGA. S10A. Western blots showing expression levels of DSP, ZEB1, and GAPDH in MDA-MB-231 cells treated with scramble, no treatment, or siZEB1 at 100 nM (ORIGENE, cat # SR321982) for 48 hrs. The plots represent the quantification of the western blots. B-C. Barplots displaying relative gene expression levels measured using qRT-PCR of (B) ZEB1 and DSP (normalized to scramble) in MDA-MB-231 cells treated with siZEB1 at 100 nM 24 hrs, and (C) ZEB2 and DSP (normalized to scramble) in MDA-MB-231 cells treated with siZEB2 at 80 nM for 24 hrs. S11. A. Western blots showing expression levels of DSP, ZEB1, and GAPDH in Hep3B cells treated with scramble, no treatment, or siZEB1 at 100 nM (ORIGENE, cat # SR321982) for 48 hrs. The plots represent the quantification of the western blots. B-C. Barplots displaying relative gene expression levels measured using qRT-PCR of (B) ZEB1 and DSP (normalized to scramble) in Hep3B cells treated with siZEB1 at 100 nM 24 hrs, and (C) ZEB2 and DSP (normalized to scramble) in Hep3B cells treated with siZEB2 at 80 nM for 24 hrs. S12. Positive ChIP control for ZEB1 binding in Hep3B, MDA-MB-231 and HepG2 cells in the CDH1 promoter. S13. Relative ZEB1 protein expression in PA or BSA treated (48 hr) WT- or IRE1-KO MDA-MB-231 cells. S14. Relative ZEB1 protein expression in PA or BSA treated (72 hrs) WT- or IRE1-KO Hep3B cells.</p>