Thout KRAS induction (Figure 3B and D, Figure 3–figure supplement 4A, and Supplementary file 3). To rule out any possible clonal bias, we also performed RNA-seq on a second clone (clone #11). We observed that ALDH1A1 was also considerably upregulated within the second clone below each conditions (Figure 3–figure supplement 4B and Supplementary file 3). The upregulation of ALDH1A1 in ARID1A-KO cells was further verified by each qRT-PCR (Figure 3–figure supplement 4E) and western blot (Figure 3E). Considering that ALDH1A1 has been shown to take part in the clearance of ROS (Raha et al., 2014) and ROS are essential mediators of KRAS-induced senescence (Storz, 2017), we hypothesize that ALDH1A1 would be the gene that mediates the effect of ARID1A deficiency on KRAS-induced senescence. Next, we examined our PanIN- seq information to evaluate the expression of Aldh1a1 and also other members on the ALDH family members. Interestingly, we observed that Aldh3a1 is significantlyLiu, Cao, et al. eLife 2021;10:e64204. DOI: https://doi.org/10.7554/eLife.6 ofResearch articleCancer Biology | Chromosomes and Gene ExpressionA0.BDown-regulated Up-regulated Not significantCALDH1ANon-Induce 111 57 KRAS- InduceLeading logFC dim0.0.-log10FDR0.-0.6 -0.4 -0.Up-regulated genesKRAS-Wild Form KRAS-ARID1A-KOWild Type ARID1A-KONon-Induce 186 0 -5 0KRAS-Induce-1.-1.-0.0.0.1.1.Leading logFC dimlog2Fold-ChangeDown-regulated genesDALDH1A1 Expression (CPM)KRAS-InduceNon-InduceENon-target AR KO #2 AR KO #F100 80 60 40 20ALDH3AACTINAPMALDH1AKCAKCARKO WildTypeARKOWildTypeGALDH3AKCAKCHH-Score325 300 275 250 225AKCKCFigure three. ARID1A knockout GlyT1 drug upregulates aldehyde dehydrogenase (ALDH) expression. (A) Multidimensional scaling plot demonstrated clear separation between the transcriptome profiles of ARID1A-KO human pancreatic Nestin-expressing (HPNE) cells and wildtype cells with or without KRAS induction. RNA sequencing was performed with three biological repeats. (B) Volcano plot of differentially expressed genes between ARID1A knockout cells and wildtype cells with KRAS induction. (C) Venn diagram showing the upregulated genes (upper) and downregulated genes (bottom) that are shared Figure three continued on next pageLiu, Cao, et al. eLife 2021;ten:e64204. DOI: https://doi.org/10.7554/eLife.7 ofResearch report Figure 3 continuedCancer Biology | Chromosomes and Gene Expressionbetween cells with (gray) or without (blue) KRAS induction. (D) ALDH1A1 mRNA levels quantified by sequencing data are considerably different in between ARID1A-KO cells and wildtype cells with (left) or with no (correct) Kras induction. CPM: count per CXCR4 Species million reads. (E) Western blot for ALDH1A1 expression in ARID1A-KO cells and wildtype cells with KRAS induction. (F) mRNA level of Aldh3a1 in KC and AKC lesions determined by pancreatic intraepithelial neoplasia (PanIN)-seq information. APM: amplicon per million reads. (G) IHC staining against ALDH3A1 in KC and AKC lesions. Scale bars: 200 . (H) Comparison of ALDH3A1 levels among KC and AKC lesions depending on the intensity of staining in (G). H-score was calculated by counting the amount of lesions with distinct levels of staining intensity at 4 random fields below the microscope. Student’s t-test: p0.001; p0.0001. The on line version of this article contains the following figure supplement(s) for figure 3: Figure supplement 1. Gene set enrichment analysis on RNA-seq data. Figure supplement 2. ARID1A knockout impairs phosphorylation of ERK in human pancreatic Nestin-expressing (HPNE) cells upon KRAS i.
