mock controls wildtype mefs  (Cell Signaling Technology Inc)

Journal: PLOS Genetics

Article Title: STIL overexpression shortens lifespan and reduces tumor formation in mice

doi: 10.1371/journal.pgen.1011460

Figure Lengend Snippet: ( A ) Proliferation of B6-STIL control, CMV-STIL +/- , CMV-STIL +/+ , CMV-p53-R172H +/- and CMV-STIL +/- /p53-R172H +/- MEFs. Cells were enumerated every 24 h for a total of 5 days. Data are means ± SEM from three independent experiments. ( B ) Immunoblotting showing p S18 -p53 and p53 protein expression levels in protein extracts from p2-6 CMV-STIL +/- and B6-STIL control MEFs. β-actin served as a loading control; DMSO- and etoposide-treated NIH3T3 cell lysate extracts as controls for p53 increase/phosphorylation after DNA damage. The right panel shows the changes in p S18 -p53 and p53 expression compared to passage 2 (p2) of B6-STIL control MEFs. ( C ) Representative images of B6-STIL control, CMV-STIL +/- and CMV-STIL +/+ MEFs, stained for β-galactosidase-cleaved x-Gal (lower panel). In the upper panel differential contrast interference (DIC) images of the cells are shown. Scale bar, 1 μm. ( D ) Percentage of β-galactosidase-positive, senescent B6-STIL control, CMV-STIL +/- , CMV-STIL +/+ , CMV-p53-R172H +/- and CMV-STIL +/- /p53-R172H +/- MEFs. Data are means ± Clopper-Pearson 95%-CI. Comparisons were made using the two-sided Fisher’s exact test. For space reasons, only statistically significant differences are displayed. * p <0.05, ** p <0.01, *** p <0.001. ( E ) Immunoblotting showing p16 INK4A protein expression levels in protein extracts from p2-6 CMV-STIL +/- and B6-STIL control MEFs. β-actin served as a loading control. The right panel shows the changes in p16 INK4A expression compared to passage 2 (p2) of B6-STIL control MEFs. ( F ) RNA sequencing showing p16 INK4A mRNA levels in CMV-STIL +/- and CMV-STIL +/+ relative to B6-STIL control MEFs. ( G , H) Percentage of ( G ) Apo-15-positive and ( H ) sub-G 1 phase, apoptotic B6-STIL control, CMV-STIL +/- , CMV-STIL +/+ , CMV-p53-R172H +/- and CMV-STIL +/- /p53-R172H +/- MEFs. Data are means ± SEM from three independent experiments. The differences between groups were tested using the one-way ANOVA with post-hoc Tukey test for multiple comparison. No statistical significance was found. For space reasons, only statistically significant differences are displayed. ( I ) Genotyping of p3-5 CMV-STIL +/- and CMV-STIL +/+ MEFs. All passages harbor the STIL transgene. ( J ) RNA sequencing showing STIL mRNA levels in p3-5 B6-STIL control, CMV-STIL +/- and CMV-STIL +/+ MEFs. ( K ) Immunoblotting showing STIL protein expression levels in p1-5 of B6-STIL control and CMV-STIL +/- MEFs. The STIL band at 170 kDa is marked with an asterisk. β-actin served as a loading control. The lower panel shows the changes in STIL expression compared to p1 of CMV-STIL +/- MEFs. ( L ) Percentage of p3 and p6 B6-STIL control, CMV-STIL +/- , CMV-STIL +/+ , CMV-p53-R172H +/- and CMV-STIL +/- /p53-R172H +/- MEFs with supernumerary centrioles. Comparisons were made using the two-sided Fisher’s exact test. Note : for reasons of space, only statistically significant differences were marked. * p <0.05, ** p <0.01, *** p <0.001.

Article Snippet: As positive and mock controls wildtype MEFs were treated with 100 nM paclitaxel and DMSO, respectively for 48 h. Thereafter, MEFs were stained using the Senescence β-Galactosidase Staining Kit (Cell Signaling) according to manufacturer’s instructions and eosin as cytoplasmic counter staining to detect senescent cells.

Techniques: Control, Western Blot, Expressing, Phospho-proteomics, Staining, RNA Sequencing, Comparison