Thout KRAS induction (Figure 3B and D, Figure 3–figure supplement 4A, and Supplementary file three). To rule out any possible clonal bias, we also performed RNA-seq on a second clone (clone #11). We observed that CCR4 custom synthesis ALDH1A1 was also significantly upregulated in the second clone below both conditions (Figure 3–figure supplement 4B and Supplementary file 3). The upregulation of ALDH1A1 in ARID1A-KO cells was further LTB4 Compound verified by each qRT-PCR (Figure 3–figure supplement 4E) and western blot (Figure 3E). Contemplating that ALDH1A1 has been shown to take part in the clearance of ROS (Raha et al., 2014) and ROS are crucial mediators of KRAS-induced senescence (Storz, 2017), we hypothesize that ALDH1A1 will be the gene that mediates the effect of ARID1A deficiency on KRAS-induced senescence. Subsequent, we examined our PanIN- seq data to evaluate the expression of Aldh1a1 along with other members from the ALDH loved ones. Interestingly, we observed that Aldh3a1 is significantlyLiu, Cao, et al. eLife 2021;10:e64204. DOI: https://doi.org/10.7554/eLife.six 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 Type KRAS-ARID1A-KOWild Sort 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 upregulates aldehyde dehydrogenase (ALDH) expression. (A) Multidimensional scaling plot demonstrated clear separation in between the transcriptome profiles of ARID1A-KO human pancreatic Nestin-expressing (HPNE) cells and wildtype cells with or without having KRAS induction. RNA sequencing was performed with 3 biological repeats. (B) Volcano plot of differentially expressed genes among ARID1A knockout cells and wildtype cells with KRAS induction. (C) Venn diagram displaying the upregulated genes (upper) and downregulated genes (bottom) that are shared Figure 3 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 information are substantially various among ARID1A-KO cells and wildtype cells with (left) or devoid of (ideal) Kras induction. CPM: count per million reads. (E) Western blot for ALDH1A1 expression in ARID1A-KO cells and wildtype cells with KRAS induction. (F) mRNA amount of Aldh3a1 in KC and AKC lesions according to pancreatic intraepithelial neoplasia (PanIN)-seq data. APM: amplicon per million reads. (G) IHC staining against ALDH3A1 in KC and AKC lesions. Scale bars: 200 . (H) Comparison of ALDH3A1 levels in between KC and AKC lesions based on the intensity of staining in (G). H-score was calculated by counting the number of lesions with different levels of staining intensity at four random fields under the microscope. Student’s t-test: p0.001; p0.0001. The on the web version of this article includes 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.
