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. 2023 Jun 8;16(3):369–383. doi: 10.1016/j.jcmgh.2023.06.002

Figure 4.

Figure 4

Restoration of reduced ERK activation by rhIGF1 mitigates Trp53-induced ICC-SC loss in vitro. (A) rhIGF1 (100 ng/mL) mitigated nutlin 3a–induced (30 μmol/L) reduced ERK phosphorylation (upper left panel), and DNA damage accumulation (upper right panel), reduced CDKN1B (lower left panel), and reduced CCND1B (lower right panel) in ICC-SC line D2211B previously established in Dr Ordog’s laboratory (Rochester MN) and well characterized11,17 (n = 10–15/group). Trp53 was induced with the murine double minute 2 antagonist nutlin 3a (30 μmol/L), with the inactive enantiomer nutlin 3b (30 μmol/L) serving as a control. ACTB (actin beta) was used as a loading control. Statistical significance was determined using Kruskal–Wallis 1-way analysis of variance (ANOVA on ranks). (B) rhIGF1 treatment restored nutlin 3a–induced reduction of ICC-SC cell viability by MTS assay (n = 16/group). Statistical significance was determined using Kruskal–Wallis 1-way ANOVA (ANOVA on ranks). (C) rhIGF1 treatment prevented nutlin 3a–induced G2/M cell-cycle arrest of ICC-SC. Cell-cycle analysis by combined Alexa Fluor 647–EdU incorporation and PI labeling. Left panels: Representative Alexa Fluor 647 EdU vs PI (area) projections of events gated for single cells with at least diploid DNA content. Right panels: Cell frequencies in the cell-cycle phases identified in the quadrants (n = 8/group). Horizontal lines indicate mean frequencies. Statistical significance was determined using Kruskal–Wallis 1-way ANOVA (ANOVA on ranks). P-ERK1, P-extracellular signal-regulated kinase 1; T-ERK1, T-extracellular signal-regulated kinase 1.