Fig 1: TONSL and MMS22L mRNA expression and copy number variations. (A) TONSL mRNA expression in tumors and the adjacent normal tissues. The RNA seq data were derived from the TCGA Pan-Cancer Atlas study and analyzed by Q-omics. LUAD—Lung adenocarcinoma; LUSC—Lung squamous cell carcinoma; BRCA—Breast invasive carcinoma; STAD—Stomach adenocarcinoma. The p value was acquired by Student’s t-test. (B) Relation between TONSL and MMS22L mRNA expression level and the copy number variations (CNV) in tumors. The analysis was performed at cBioportal using the original dataset from the TCGA Pan-Cancer Atlas Study. AC—adenocarcinoma; SQCC—squamous cell carcinoma; Homdel—Homologous deletion; Amp—Amplification.
Fig 2: The loss of the stem cell population by TONSL depletion. (A) The limited dilution assay was performed using the secondary CSC spheres. The primary sphere generated by the seeding of BCC was split by accutase and reseeded into 96 wells at different cell density. After one week, the no-sphere forming wells were counted and plotted against the seeding of cells. (B) Aldehyde dehydrogenase (ALDH) activity assay was performed using the siRNA-treated secondary spheres of U87MG cells after 48 h of transfection on primary CSC spheres. siSCD1(26) was used as a positive control. (C) Cell senescence was induced by the loss of TONSL expression. The histogram of SA-ßgal positive cells in the control shRNA and shTONSL(#1)-treated OVCAR8 cells. The senescence cells increased much more in CSC than in the BCC by the shTONSL. **, p < 0.01. Student’s t-test vs. siNC or shNC. DEAB is the inhibitor of ALDH activity.
Fig 3: CSC requires TONSL to survive from Camptothecin (Cpt)-induced DNA damage (A) The BCC cell survival was not influenced by TONSL shRNA or Cpt (2 nM and 10 nM). (B) CSC requires the TONSL to recover from the toxicity by the Cpt (2 nM and 10 nM). *, p < 0.05; **, p < 0.01. Student’s t-test vs. shNC.
Fig 4: Loss of TONSL in colon cancer CSC (cancer stem cell-enriched culture) results in the DNA damage-induced apoptosis. (A) The biochemical pathways related to the DNA damage were activated by the shTONSL infection. The damage accumulated more in CSC than in BCC, and increased the apoptosis in CSC. Represented cell apoptosis flowcytometry after shRNA of TONSL was infected. (B) gamma H2AX accumulated more in CSC by shTONSL virus infection. NC—negative control sequence virus. (C) The homologous recombination frequency was measured by DR-GFP plasmid transfected U87MG cells after I-SCE1 endonuclease plasmid was transfected. The GFP-positive cells are counted as the HRR-positive cells. *, p < 0.05 Student’s t-test vs. siNC.
Fig 5: Requirement of TONSL expression in BCC (monolayer bulk cultured cells) and CSC (cancer stem-cell enriched cultured cells). (A) Cell survival/growth of BCC and sphere numbers of CSC after the knocking down of TONSL. Cell survival/growth was moderately or not limited by the knockdown of TONSL in the BCC, while it is critical in the CSC in two ovarian cancer cell lines (IGROV1 and OVCAR8), breast cancer cell lines (MDAMB231 and MDAMB468) and lung cancer cell lines (H460 and H1299). (B) Cell survival/growth of BCC and sphere numbers of CSC after the knocking down of TONSL. Cell survival/growth was moderately or not limited by the knockdown of TONSL in the BCC, while it is critical in the CSC in two colon cancer cell lines (HCT15 and HT29) and Glioblastomas (U87MG and LN229). (C) The representative images of CSC spheres. *, p < 0.05; **, p < 0.01. Student’s t-test vs. siNC or shNC.
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