Fig 2: Ncx1 inhibition/inactivation increases the expression of key regulators of ß-cell function and regulates the activity of proteins controlling cell proliferation. (a) Gene expression of Ins1, Ins2, and Pdx1 assessed by quantitative RT-PCR is shown in islets from 12-week-old Ncx1+/+ and Ncx1+/- mice or (b) in INS-1E treated or not during 24 hours by KB-R7943. Means ± SEM are shown of four individual experiments in each case (*P < 0.05; **P < 0.01; ***P < 0.001). (c & d) The level of phosphorylated pRb assessed by immunofluorescence is shown in insulin-positive cells in islets from 12-week-old Ncx1+/- and Ncx1+/+ mice. (c) Representative image of four 12-week-old pancreas sections in each case. Arrows are showing pRb-positive cells. (d) Quantification of the number of insulin-positive cells where pRb phosphorylation is observed. Means ± SEM are shown of four individual experiments in each case (*P < 0.05). (e) Relative luciferase activities of E2F, p53, MEF2, and NFAT transcription factors in INS-1E cells treated with KB-R7943 or transfected with SiNcx1 are shown as means ± SEM of three independent experiments (*P < 0.05; **P < 0.01). DAPI, 4',6-diamidino-2-phenylindole; Luc, luciferase.

Fig 3: USP5 promotes cell cycle progression and proliferation in non-small cell lung cancer (NSCLC) cells. (A,B) A549 and H1299 cells were transfected with siUSP5 or siNC (150 nM) for 48 hours. Cell cycle phases were then analyzed by flow cytometry. Representative images of flow cytometric cell cycle analysis (A) and the percentage of cells in each cell cycle phase distribution (B) are shown. G1 or S in A549: G1, **, P<0.01 or S, ***, P<0.001; G1 or S in H1299: *, P<0.05, Student’s t-test. (C) In A549 and H1299 cells, the levels of proteins involved in the CCND1-CDK4-Rb signaling pathway after knockdown of USP5 were assessed by immunoblotting (IB) assay using the indicated antibodies (Abs) [primary Ab: anti-CCND1, 1:1,000, anti-CCND2, 1:1,000, anti-CCND3, 1:1,000, anti-CDK4, 1:1,000, anti-CDK6, 1:1,000, anti-pRb (Ser780), 1:1,000, or anti-Rb, 1:1,000; secondary Ab: horseradish peroxidase-labeled goat anti-rabbit or anti-mouse IgG, 1:2,000]. (D,E) Myc-USP5- and siUSP5-transfected A549 and H1299 cells along with the respective control cells were cultured for different time periods (24–72 hours), and cell viability was then measured by Cell Counting Kit-8 assay. **, P<0.01 or ***, P<0.001 compared with their respective control at the same time point, Student’s t-test. (F,G) Colony formation assay was performed using siUSP5-tranfected A549 and H1299 cells and their respective control cells. Representative images of colony formation assay (F) and the number of colonies (G) are shown. *, P<0.05, **, P<0.01, or ***, P<0.001 compared with the respective control, Student’s t-test.