Fig. 3

TIS7 regulates ICln protein and RNA levels. a Quantitative analyses of ICln protein expression in proliferating (PM; n = 4 biological replicates) and differentiating (DM; n = 3 independent experiments) MSCs. Equal amounts of total cell lysates were probed by immunoblotting with anti-ICln antibodies and normalized by tubulin. b TIS7 ectopic expression rescued ICln protein levels in TIS7 KO MSCs. Proliferating TIS7 KO MSCs were transfected with GFP plasmid as a control or with GFP-TIS7 plasmid constructs and total cell lysates were probed with the indicated antibodies. Quantification (n = 3 biological replicates) was based on tubulin signal. Representative western blots are shown. c Quantitative PCR analysis of ICln was normalized to GAPDH expression (n = 3 biological replicates). d TIS7 overexpression rescued ICln RNA levels in TIS7 KO MSCs. Proliferating TIS7 KO MSCs were transfected with indicated plasmids and ICln quantitative PCR was performed as in panel c. e TIS7 transcriptionally regulates ICln levels. Proliferating WT MSCs were transiently co-transfected with full length ICln promoter-luciferase reporter construct, β-galactosidase expression plasmid, and GFP plasmid as a control or GFP-TIS7 plasmid constructs. Luciferase values were normalized to β-galactosidase values. In all graphs, values represent the mean ± SD and resulting data were analyzed via two-tailed type 2 Student’s t-test, *** P < 0.001, ** P < 0.01, and * P < 0.05