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. 2021 Oct 26;13(21):5373. doi: 10.3390/cancers13215373

Figure 4.

Figure 4

Requirement of TSPAN5 for oncogene-induced senescence. (A) Quantification of senescence-associated ß-galactosidase-positive HuH7 cells transiently transfected with control siRNA (sictrl) and TSPAN5 siRNA (siTSPAN5). ß-galactosidase staining was performed 5 days after transfection and ß-gal-positive cells were counted in 100 cells per condition. Data are means ± SD (n = 3); ***, p < 0.001. (B) Immunofluorescence analysis of focal adhesions with α-paxillin staining and F-actin staining with phalloidin in HuH7 cells transfected with TSPAN5 siRNA (siTSPAN5) or negative control siRNA (sictrl). DAPI was used for nuclei staining. The focal adhesion length was measured manually in 50 cells in each of the 5 randomly selected fields by using the ImageJ software in three independent experiments. Scale bar, 10 µm. Data are means ± SD (n = 3); ***, p < 0.001. (C–F) Lysates of HuH7 cells transiently transfected with negative control siRNA (sictrl) or TSPAN5 siRNA (siTSPAN5) for 6 days were immunoblotted with anti-pERK1/2 and anti-ERK (C), anti-p16INK4a (quantitation on the left) (D), anti-pRb (E) and anti-H3K9me3 antibodies (F). HSP90 was used as a loading control and immunoblotted with anti-HSP90 antibodies. (G) HuH7 cells transfected with negative control (sictrl) or TSPAN5 siRNA (siTSPAN5) were subjected to qRT-PCR with the respective gene-specific primers and 18S rRNA primers for normalization in order to analyze CXCL10 and TNFSF10 mRNA expression. TSPAN5 knockdown efficiency is shown in Figure S3F. Values are means ± SD (n = 3); ***, p < 0.001. (H) Immunofluorescence staining with anti-PML antibodies and DAPI for nuclear counterstaining in HuH7 cells expressing control (sictr) or TSPAN5 siRNA (siTSPAN5) (left). Three independent experiments were performed. Scale bar, 5 µm. Quantification of PML nuclear body accumulation by counting the red spots in 50 cells per condition. DAPI was used for nuclei staining. Data are means ± SD (n = 3); ***, p < 0.001, **, p < 0.01. The whole Wester Blots are available at Figure S11.