Figure 4.

Effects of NR6 treatment in CDKN2A silenced MSTO-211H MCSs. (A) Representative phase contrast images of MSTO-211H MCSs transfected with non-specific siRNA (NS siRNA) or specific CDKN2A siRNA (CDKN2A siRNA) treated or not with NR6, for 72 h. Scale bar = 100 µm. (B) Bar graph shows the number of viable cells in NS siRNA and in CDKN2A siRNA MCSs treated or not with NR6, for 72 h, represented as percentage versus untreated NS siRNA. (C) Representative Western blot analysis of γ-H2AX expression in NS siRNA and CDKN2A siRNA MCSs treated or not with NR6, for 72 h. H2AX was used as loading control. (D) Representative Western blot analysis of PARP-1 expression/cleavage in NS siRNA and CDKN2A siRNA MCSs treated or not with NR6, for 72 h. Tubulin was used as loading control. (E) Bar graph shows CDKN2A, IL6, CXCL8, BBC3 and BCL2L11 mRNA expression in NS siRNA and CDKN2A siRNA MCSs treated with NR6, for 72 h, expressed as fold changes versus untreated NS siRNA. 18S rRNA was used as housekeeping gene. (F) Bar graph shows the levels (pg/mL) of IL-8 released in the medium by MSTO-211H MCSs transfected with NS siRNA or specific CDKN2A siRNA and treated with NR6, for 72 h. IL-8 levels are expressed as percentage versus untreated NS siRNA. (G) Representative phase contrast images of MSTO-211H MCSs transfected with NS siRNA or specific CDKN2A siRNA treated or not with NR6, for 48 h, and then co-cultured with neutrophils for additional 24 h. Scale bar = 100 µm. In all graphs reported in Figure 4, each bar represents mean of three independent experiments ± s.d., * p ≤ 0.05, ** p ≤ 0.01.