Fig 1: CTSS is upregulated in glioblastoma, and the deletion of CTSS reduces GBC viability and increases TMZ sensitivity. (A) Boxed plots from the GEPIA database were used to compare the expression of CTSS in mormal tissues, low grade glioma (LGG) and GBM. p LGG/Normal<0.05, p GBM/Normal<0.05. (B, C) OS and DFS were compared between the low-CTSS group and high-CTSS group. (D) The expression of CTSS of human tissues was assessed by immunohistochemical staining. (E) The expression of CTSS in HAs and the U87, U251, and A172 cell lines was detected by western blotting. The level of CTSS was clearly increased in the A172, U251, and U87 cell lines compared with that in human astrocytes (HAs). (F–H) Cells were transfected with shCtrl or shCTSS lentivirus labeled with GFP. Scale bar, 50 μm. Western blotting showed that CTSS was obviously decreased in the shCTSS groups. (I, J) The U87 and U251 cell lines were treated with ZFL at the indicated concentrations (0, 2, 4, 8, 16, 32 μM) for 48 h. The CCK-8 assay showed that the viability of U251 and U87 cells was decreased in a dose-dependent manner. (K, L) U87 and U251 cells were transfected with shRNA. The shCtrl groups and shCTSS groups were separately treated with the indicated concentrations of TMZ (0, 10, 20, 40, 80, 160 μM) for 8 h, and cell viability was then detected by CCK-8 assay. (M, N) Cell death was detected by TUNEL assays after cells were transfected with shRNA for 48 h. Scale bar, 25 μm. Human normal astrocyte (HA) cell line was transfected with CTSS-KD virus or scramble virus. (O, P) Cell viability was tested by CCK-8 assay. (Q, R) Apoptosis-related proteins were tested by Western blot. The results showed that no significant change was observed between the shCtrl groups and the shCTSS groups. Data are represented as the means ± SEMs of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 versus the control group.
Fig 2: Inhibition of CTSS restores TGF-β-induced tight junction turnover. Cells were treated as above. (A-C) The expressions of the tight junction proteins, ZO-1 and Occludin, were detected by western blot. ZO-1 and Occludin were decreased in the TGF-β groups, while elevated in the TGF-β+ZFL groups. (D, E) Immunofluorescence staining of Claudin 1 and ZO-1 in different groups was also recorded. Scale bar, 25 µm. Data were represented as the means ± SEM of three independent experiments. *p<0.05, **p<0.01, ***p<0.001 versus control group.
Fig 3: Macrophage exosome-derived CTSS induced pyroptosis in vitro. Western blot analysis of caspase-1, GSDMD, pro-IL-1ß, IL-1ß, ASC, and caspase-8 in acinar cells, which are treated with EXO-CTSS or EXO-vector and transfected with scr or shGSDMD (A). Immunofluorescence staining for GSDMD, caspase-1, caspase-8, and ASC in acinar cells are treated with EXO-CTSS or EXO-vector, and transfected with scr or shGSDMD (B). The location and level of GSDMD, caspase-1, caspase-8, and ASC are shown by red fluorescence, and nucleiare stained with DAPI. QRT-PCR and western blot analysis (C) for the efficiency of GSDMD gene knockdown in acinar cells, scr as the control. Acinar cells viability over time in control, EXO-vector + scr, EXO-CTSS + scr, and EXO-CTSS + shGSDMD (D). TUNEL assay for apoptosis was performed in acinar cells after EXO-CTSS treatment; cells were transfected with scr or shGSDMD. (Scale bars = 100 µm). (E). Data are shown as the mean ± SEM, n = 3, ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001
Fig 4: Knockdown of CTSS inhibits tumor growth in vivo. (A–D) U87 cells with stable knockdown of CTSS were established and then used to inoculate nude mouse. The growth of tumors in the shCtrl group and shCTSS group was calculated as described above. The growth curves of tumors in the two groups were generated (n=6). (E, F) Immunohistochemical staining for Ki-67 was used to detect the tumor proliferation state. Scale bar, 50 µm. Ki-67-positive cells in six independent fields were quantified. (G) Tumor vascularity was visualized by immunofluorescence staining for CD31. Scale bar, 100 µm. Data are represented as the means ± SEMs of independent experiments. ***p < 0.001 versus the vehicle group.
Fig 5: CTSS deletion induces cytochrome c release from the mitochondria. Cells were treated as above. (A, B) The levels of cytochrome C in the mitochondria and cytoplasm were measured by western blotting. (C) Cells were first dyed with MitoTracker Red. After washing with PBS three times, the cells on slides were fixed with paraformaldehyde. The localization of cytochrome c was subsequently measured by immunofluorescence staining. As shown in this picture, mitochondria in the ZFL-treated groups tended to appear as particles. Moreover, the cytochrome c fluorescence was more dispersed in the ZFL-treated groups compared with the control groups and ZFL+siMCU groups. Scale bar, 10 µm. Data are represented as the means ± SEMs of three independent experiments. *p < 0.05 versus the control group.
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