Fig. 3

DDR1 inhibition suppresses the properties of cancer stem-like cells through lowering SOX2 in UM cells. a, b 92.1 and Mel270 cells were treated with 10 μM 7rh for 48 h, the ALDH activity was detected by flow cytometry. Representative flow cytometry (a) and quantitative analysis of ALDH⁺ cells (b) from three independent experiments are shown. Data are shown as the mean ± SD (n = 3). c, d 92.1 and Mel270 cells were treated with 10 μM 7rh for 48 h, and then drug-freely cultured for three rounds of melanosphere assay for 14 days. Data are shown as the mean ± SD (n = 3). Scale bar: 200 µm. e 92.1 and Mel270 cells stably transduced with lentiviral Scramble or shDDR1 were assayed by flow cytometry for the proportion of ALDH⁺ cells. Quantitative analysis of ALDH⁺ cells from three independent experiments is shown. Data are shown as the mean ± SD (n = 3). f 92.1 cells stably transduced with lentiviral Scramble or shDDR1 were plated in the stem cell culture medium. Melanospheres were counted on day 14. The cells were harvested and replated for the secondary and tertiary rounds of evaluation, respectively. Data are shown as the mean ± SD (n = 3). ***P < 0.001, one-way ANOVA, post hoc comparisons, Tukey’s test. g Ectopic expression of wild-type DDR1 or mutant DDR1 (DDR1 P529*) in 92.1 and Mel270 cells were determined by western blotting analysis. h Overexpression of wild-type DDR1 but not mutant DDR1 (DDR1 P529*) increased the percentage of ALDH⁺ cells as detected by flow cytometry in 92.1 and Mel270 cells. ns no significant. Data are shown as the mean ± SD (n = 3). i Ectopic expression of wild-type DDR1 rather than mutant DDR1 (DDR1 P529*) potentiated self-renewal capacity as evaluated by melanosphere growth and serially-replating assay in 92.1 and Mel270 cells. ns no significant. Data are shown as the mean ± SD (n = 3). j, k Silencing DDR1 decreased in vivo frequency of CSCs in UM cells. Omm1 cells stably transduced with lentiviral Scramble or shDDR1 were subjected to limiting dilution assay in NOD-SCID mice. Representative image of tumors removed from the mice of each group (n = 6) are shown. The frequency of CSCs was calculated by L-Cal software. l DDR1 deletion decreased the expression of SOX2. The protein levels of stemness-related proteins were detected by western blotting in 92.1 and Mel270 cells stably transduced with Scramble or shDDR1 lentivirus. m DDR1 deletion downregulated the mRNA levels of SOX2. The mRNA levels of SOX2 in 92.1 and Mel270 cells stably transduced with Scramble or shDDR lentivirus were determined by qRT-PCR. Data are shown as the mean ± SD (n = 3). n, o The percentage of ALDH⁺ cells and melanosphere assay were performed in Mel270 cells stably transduced with Scramble or shSOX2 lentivirus with or without 7rh. Data are shown as the mean ± SD (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001, Student’s t test for results in (b, d). *P < 0.05; **P < 0.01; ***P < 0.001, one-way ANOVA, post hoc comparisons, Tukey’s test for results in (e, f, h, i and m–o)