Fig. 6: SKP2 silencing reduces stemness and tumorigenicity in vitro and suppresses growth in vivo. | Nature Communications

Fig. 6: SKP2 silencing reduces stemness and tumorigenicity in vitro and suppresses growth in vivo.

From: MYOD-SKP2 axis boosts tumorigenesis in fusion negative rhabdomyosarcoma by preventing differentiation through p57Kip2 targeting

Fig. 6

a Representative western blot (n = 3 independent experiments) of the indicated proteins on RD and JR1 cells infected with either Scrambled (shSCR), SKP2.1 (shSKP2.1) or SKP2.2 (shSKP2.2) lentiviral shRNA at 72 h post-selection. Vinculin is the loading control. b Representative light microscopy pictures of soft agar colony formation assay on RD and JR1 cells treated as in (a) and grown for 2 weeks. Scale bar = 50 μm. c Histogram depicts the quantification of soft agar colony numbers per field. n = 3 (RD) and n = 4 (JR1) independent experiments, data presented as mean values ± SD, one-way ANOVA. d Representative light microscopy pictures of single cell colony formation assay on RD and JR1 cells treated as in (a) and grown for 2 weeks. Scale bar = 50 μm. e Histogram depicts the quantification of colony numbers per field. n = 3 independent experiments, data presented as mean values ± SD, one-way ANOVA. f Representative light microscopy pictures of β-Galactosidase staining of RD and JR1 cells transfected with either SCR or SKP2 siRNA. Scale bar = 100 μm. g Histogram depicts the quantification of the percentage of senescent cells per field. n = 3 independent experiments, data presented as mean values ± SD, Student’s two-tailed t-test. h Images of JR1 shSCR, shSKP2.1, and shSKP2.2 tumors explanted from mice post euthanasia at 48 days post-inoculation. i Tumor volume of shSCR (n = 11), shSKP2.1 (n = 5) and shSKP2.2 (n = 6) JR1 xenografts assessed by caliper measurement represented in mm3 followed for 48 days post-inoculation. Data presented as mean values ± SD, two-way ANOVA. j Tumor weight of shSCR (n = 11), shSKP2.1 (n = 5) and shSKP2.2 (n = 6) JR1 xenografts. Box plots show 25th to 75th quartiles, black bar shows the median, and whiskers go down to the smallest value and up to the largest. One-way ANOVA k Representative images (n = 3 independent experiments) of H&E, p27Kip1, p57Kip2, MYOG, MyHC, and Ki67 immunohistochemistry of tumor sections from JR1 xenografts expressing either shSCR or shSKP2.2. Scale Bars = 100 μm. Source data are provided as a Source Data file.

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