Journal: Cell reports

Article Title: De novo sphingolipid biosynthesis necessitates detoxification in cancer cells

doi: 10.1016/j.celrep.2022.111415

Figure Lengend Snippet: (A) Heatmap of unsupervised hierarchical clustering analysis of the mRNA expression patterns of 2 KDSR KO-sensitive (DLD1 and NCIH838) and 1 -insensitive (HUH7) cancer cell line following CTRL or KDSR KO. Numbers at the end of each label are biological repeat identifier. Sensitive lines with KDSR KO are labeled in red. (B and C) Gene clusters that are upregulated (B, yellow) or downregulated (C, blue) specifically in DLD1 and NCIH838 KDSR KO (sensitive) cells. Tables list examples of Gene Ontology (GO) biological processes that are significantly enriched in each gene cluster. (D) Western blot showing protein levels of characteristic markers of ER stress in DLD1 cells subjected to CTRL or KDSR g1- or g2-mediated KO at 9 days after lentiviral transduction. DLD1 cells treated with 2 μM tunicamycin for 24 h were used as a positive control for ER stress induction. (E) Top row, representative electron microscope images of DLD1 CTRL and KDSR KO cells, depicting an overview of the aberrant morphology observed in KDSR KO cells. N indicates nucleus; scale bar: 3 μm. Bottom row, higher magnification images displaying specific features of the subcellular structures in KDSR KO cells. Red arrows point to areas where ribosomes line the edges of the structures. Left image scale bar: 0.2 μm, right image scale bar: 0.1 μm. (F) Immunofluorescent images of CTRL and KDSR KO cells stained for calnexin (ER membrane, red), phalloidin (actin, green), and Hoechst 33342 (nucleus, blue). Bottom rows show zoomed-in view of ER structures. Top row scale bar: 10 μm, bottom row scale bar: 4 μm. (G) Western blot of K48 ubiquitinated proteins in DLD1 cells treated with 1 or 10 μM of the proteasome inhibitor MG132 (18 h) or with KDSR KO with g1 or g2 (10 days) compared with vehicle and CTRL conditions, respectively. (H) Representative immunofluorescent images of DLD1 cells subjected to CTRL or KDSR KO or treated with vehicle or 3 μM MG132 and stained with aggresome dye (red), which binds beta-sheet structures found in protein aggregates. Nuclei were stained with DAPI (blue). Scale bar: 40 μm. (I) Fold change in protein levels of ER stress markers (IRE1a and BiP) and K48 ubiquitinated proteins in KDSR KO cells (average of g1 and g2) relative to CTRL cells in 3 non-cancer (GM05565, THLE3, CCD841CoN) and 3 sensitive cancer (DLD1, NCIH838, U251) cell lines. Each point represents an independent cell line as n = 3 replicates. Band intensities were quantified from westerns that are shown in (D) and (G) and and and were normalized to actin band intensity. For RNA sequencing, RNA was extracted from n = 3 biological replicates for each condition. GO enrichment analysis was performed using the PANTHER overrepresentation test. Data are shown as mean + SD. p values were calculated using two-tailed Student’s t test (*p < 0.05, **p < 0.01, ns = not significant).

Article Snippet: THLE3 (normal liver) cells were a kind gift of the Kwan Yong Choi Lab (POSTECH, Korea).

Techniques: Expressing, Labeling, Western Blot, Transduction, Positive Control, Microscopy, Staining, Membrane, RNA Sequencing, Two Tailed Test