Fig. 5: Caspase-3 sustains melanoma cell motility by regulating CORO1B activity. | Cell Death & Disease

Fig. 5: Caspase-3 sustains melanoma cell motility by regulating CORO1B activity.

From: Atypical contribution of caspase-3 to melanoma cancer cell motility by regulation of coronin 1B activity

Fig. 5

A Summary table with the most frequent CASP3 putative interacting protein partners identified in WM793 cells following CASP3-GFP pulldown and proximity biotinylation in melanoma cells expressing CASP3 fused with BioID2, in either N-ter or C-ter. White circle: absence of hit; green circle: presence of hit. B Analysis of CASP3-GFP and CORO1B/P-CORO1B interaction after immunoprecipitation (IP) of GFP protein complexes in GFP- or CASP3-GFP in WM793 melanoma cells. C Analysis of the proximity between endogenous CASP3 and CORO1B proteins in control and CASP3/CORO1B-knockdown WM793 using a Proximity Ligation Assay (PLA). D Quantification of PLA signal in control, CASP3- and CORO1B-knockdown WM793 cells (unpaired t test; Statistical significance: ns - P > 0.05; * - P ≤ 0.05; ** - P ≤ 0.01; *** - P ≤ 0.001; **** - P ≤ 0.0001). E Left panel: Analysis of CASP3, CORO1B and F-actin localization by immunofluorescence in control and CASP3-knockdown WM793 cells. Right panel: Signal intensity measurement of CASP3, CORO1B and F-actin along the indicated corresponding line in control and CASP3-knockdown WM793 in the left panel. F Quantification of CORO1B immunostaining intensity in proximity to plasma membrane and in cytosol, between control and CASP3-depleted cells (n = 3, paired t test; Statistical significance: ns - P > 0.05; * - P ≤ 0.05; ** - P ≤ 0.01; *** - P ≤ 0.001; **** - P ≤ 0.0001). G Measurement of cell invasion through Matrigel in control and CORO1B-knockdown WM793 (n = 3, one representative experiment shown, two-way ANOVA statistical test; Statistical significance: ns - P > 0.05; * - P ≤ 0.05; ** - P ≤ 0.01; *** - P ≤ 0.001; **** - P ≤ 0.0001). H Analysis by immunoblotting of P-CORO1B, CORO1B, P-PKCα, PKCα, ARP2/3 and CASP3 in WM793 transfected with two different siRNAs for CASP3 and one for CORO1B. HSC70 serves as a loading control. I Analysis by immunoblotting of P-CORO1B, CORO1B, ARP2/3 and CASP3 in control and CASP3-knockdown WM793 cells that were serum-starved for 24 h and treated with PDGF (20 ng/mL) for the indicated time. HSC70 serves as a loading control. J Left panel: Analysis of ARP2/3 and F-actin localization by immunofluorescence in control and CASP3-knockdown WM793 cells. Right panel: Signal intensity measurement of ARP2/3 and F-actin along the indicated corresponding line in control and CASP3-knockdown WM793 in the left panel. K Quantification of ARP2/3 immunostaining intensity in proximity to plasma membrane and in cytosol, between control and CASP3-depleted cells (n = 3, paired t test; Statistical significance: ns - P > 0.05; * - P ≤ 0.05; ** - P ≤ 0.01; *** - P ≤ 0.001; **** - P ≤ 0.0001).

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