Research Paper Volume 11, Issue 13 pp 4407—4437

Figure 3. Knockdown of HN1 induces cellular senescence in normal and cancer cells. (A–B) Validation of HN1 knockdown (KD) in HUVEC with two shRNAs (shHN1_#1, shHN1_#2), as quantified by qRT-PCR (A) and Western blot (B), respectively. GAPDH served as internal control for both mRNA and protein. (C) Cell proliferation rate evaluated by Cell Counting Kit-8 (CCK-8) assay in HN1-KD HUVEC cells with two shRNAs. *, ** and *** stand for p < 0.05, p < 0.01 and p < 0.001, respectively, based on t-test with three biological replicates. (D–E) Representative SA-β-Gal staining (D) and quantitative statistics (E) in HN1-KD and control HUVEC cells. *** represents p < 0.001 based on t-test with three independent countings. (F–G) EdU incorporation assay (F) and quantitative statistics (G) in HN1-KD and control HUVEC cells. Green and blue dots stand for incorporated EdU and DNA DAPI (4',6-diamidino-2-phenylindole) staining, respectively. * and *** stand for p < 0.05 and p < 0.001, respectively, based on t-test with three independent countings. (H–I) Validation of HN1 knockdown in A549 cells, as described in panel A-B. (J) Cell proliferation rate evaluated by CCK-8 assay in HN1-KD A549 cells, as described in panel C. (K–L) SA-β-Gal staining (K) and quantitative statistics (L) were shown in A549 cells, as described in panel D-E. ** represents p < 0.01 based on t-test with three independent countings. (M–N) EdU incorporation assay (M) and positive cell (EdU incorporated) statistics (N) were shown in A549 cells, as described in panel F-G. ** represents p < 0.01 based on t-test with three independent countings. (O–Q) Over-expression of HN1 partially rescued HN1-KD induced SA-β-Gal activity in HUVECs. qRT-PCR confirmed overexpression of HN1 in HN1-KD HUVECs (O). *** represents p < 0.001 based on t-test with three qPCR reactions. SA-β-Gal staining (P), and positive staining cell statistics (Q) in control (+MOCK) and overexpression (+HN1) HUVECs. *** represents p < 0.001 based on t-test with three independent countings. (R–T) Over-expression of HN1 partially rescued HN1-KD induced SA-β-Gal activity in A549 cells. qRT-PCR confirmed overexpression of HN1 in HN1-KD A549 cells (R). SA-β-Gal staining (S) and positive staining cell statistics (T) in control (+MOCK) and overexpression (+HN1) A549 cells. *** represents p < 0.001, as described in panel O-Q.