Fig. 5. DDR2 regulates cell proliferation and YAP/TAZ activity.

(A-B) The clonogenic assay showed Ddr2 knockdown reduced the number of colony formation. Recurrent tumor cells transduced with control or Ddr2 shRNAs were plated on 6 well plates (250 cells/well) and incubated for 10 days. The cells were then fixed by paraformaldehyde and stained with crystal violet for direct counting of colony numbers as quantified in (B). (C) Recurrent tumor cells with Ddr2 knockdown showed a reduction in cell proliferation under image-based monitoring of cell numbers. Recurrent tumor cells stably expressing histone H2B-mCherry were knocked down by control or Ddr2 shRNAs and plated in a 96-well plate. The image of each well was taken every 8 hours using Incucyte S3 for the quantification of cell numbers. (D-E) Ddr2 knockdown decreased the nuclear localization of YAP(D)/TAZ(E). Recurrent tumor cells were labeled by YAP(D) and TAZ(E) specific antibodies with a counterstain of DAPI for nuclei. Quantification was performed using line analysis by Image J. Nuclear (DAPI positive) and nuclear (DAPI negative) intensity were determined by the average of 5 cells for each sample and 3 independent repeats. Scale bar, 10μm. (F) RT-PCR validated the downregulation of Ctgf and Cyr61, two canonical YAP/TAZ target genes, upon Ddr2 knockdown in recurrent tumor cells. (G) Nuclear/cytosol fractionation showed reduced nuclear YAP/TAZ upon Ddr2 knockdown in recurrent tumor cells. GAPDH: cytosolic marker; Lamin A/C: nuclear marker. Relative nuclear/cytosolic YAP and TAZ ratio was determined by ImageJ. (H) Pharmacological suppression YAP by verteporfin (2 μM) mitigated erastin induced ferroptosis in recurrent tumor cells as determined by CellTiter Glo assay. (B) One-way ANOVA, ****p < 0.0001 Tukey’s multiple comparisons. (G) *p<0.05, ***p<0.001, two-tailed Student’s t-test. (C, H) n=3 biological replicates. Two-way ANOVA, *p < 0.05, **p < 0.01, Dunnett’s multiple comparisons. Bars show S.E.M..