Journal: bioRxiv

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

doi: 10.1101/2024.06.27.601010

Figure Lengend Snippet: (A) Analysis of CASP3 expression in normal human tissues obtained from the GTEx (the Adult Genotype Tissue Expression) Portal. Skin tissue is highlighted. (B) Analysis of the most frequent mutations occurring in melanoma in the TCGA-SKCM dataset. CASP3 mutation rate is highlighted in orange. (C) Analysis of caspase expression in 39 melanoma cell lines from the Cancer Cell Line Encyclopedia (CCLE). (D) Comparison of CASP3 expression in primary (n = 104) and metastatic melanoma (n = 368) in the TCGA-SKCM dataset (p=2.36 x 10 -7 ). (E) Clustering based on gene ontology (GO)-based classification of CASP3 interacting proteins identified by mass spectrometry after immunoprecipitation of CASP3-GFP (compared to GFP only precipitation) in WM852 cells. (F) Analysis of the type of protein domains contained in caspase-3 interacting partners in WM852. (G) Clustering based on the GO-based classification of CASP3-interacting proteins identified after immunoprecipitation of CASP3-GFP in WM793 and WM852 cells.

Article Snippet: CASP3-GFP interacting partners were characterized using GFP-Trap® Magnetic Agarose (20 reactions, Euromedex, CR-gtma-20) following the manufacturer’s instructions.

Techniques: Expressing, Mutagenesis, Comparison, Mass Spectrometry, Immunoprecipitation

Journal: bioRxiv

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

doi: 10.1101/2024.06.27.601010

Figure Lengend Snippet: (A) Upper panel: Western blot analysis of CASP3 in WM793, WM852 and A375 cells transfected with CASP3 -targeting siRNA. HSC70 serves as a loading control. Lower panel: Densitometry analysis of CASP3 expression, relative to HSC70 expression. (B) Clustering of the most differentially-expressed genes (DEGs) between parental and CASP3-deficient WM793 cells, identifying a DEG signature constituted of 310 genes (FDR<0.05 & logFC>|2|). (C) Volcano plot illustrating the two gene signatures constituted of DEGs that are the most significantly upregulated or downregulated following CASP3-depletion in WM793. (D) Analysis of the overall survival in patients with melanoma from the TCGA dataset displaying a high or low (relative to the median) GSVA score for the signature of genes increased when caspase-3 is knocked-down (50 genes the most upregulated upon C3 knocked-down) (log rank test; p-value = 0.015). (E) The protocol used for identifying interaction protein partners for CASP3 using immunoprecipitation of CASP3-GFP with GFP Traps ® , which are ready-to-use pull-down reagent consisting of an anti-GFP nanobody coupled to agarose beads. (F-G) Validation by immunoblotting of GFP and CASP3-GFP immunoprecipitation using GFP Traps ® in GFP- and CASP3-GFP-expressing WM793 ( F ) and WM852 cells ( G ). (H) Summary of key steps of the proximity labelling protocol for identifying protein interacting partners of CASP3 fused with BioID2 through the immunoprecipitation of biotinylated proteins with streptavidin beads, following biotin treatment. (I-J) Validation of myc tagged-BioID2, -BioID2-CASP3 and -CASP3-BioID2 conditional overexpression following doxycycline treatment (1 µg/mL for 24 h) using anti-Myc antibody in WM793 cells ( I ). Analysis of biotinylated proteins following biotin treatment (50 µM, 12 h) of BioID2, BioID2-CASP3 and CASP3-BioID2-expressing WM793 cells ( J ). ( K-L ) Same as in I-J, for WM852 cells.

Article Snippet: CASP3-GFP interacting partners were characterized using GFP-Trap® Magnetic Agarose (20 reactions, Euromedex, CR-gtma-20) following the manufacturer’s instructions.

Techniques: Western Blot, Transfection, Control, Expressing, Immunoprecipitation, Over Expression

Journal: bioRxiv

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

doi: 10.1101/2024.06.27.601010

Figure Lengend Snippet: (A) Workflow for the generation of primary zebrafish melanomas upon microinjection of the transposon-based MiniCoopR vector into one-cell stage embryos. (B) Western blot analysis showing the expression levels of Caspase-3 (CASP3) and Green Fluorescent Protein (GFP/Ctl) in primary zebrafish melanoma tumors. (C) Tumor-free survival curves of Tg (mitfa:BRAF V600E ), tp53 −/− , mitfa −/− zebrafish injected with vectors for the overexpression of CASP3 or GFP (Ctl). Statistical analysis was performed using the log-rank test. (D) Workflow for the transplantation of primary zebrafish melanomas into the dorsal cavity of secondary recipient Casper fish. (E) Representative images of Casper fish transplanted with primary zebrafish melanomas overexpressing CASP3 or GFP. Arrows indicate the location of disseminated melanoma cells. (F) Quantification of the percentage of secondary recipients with tumor spreading, comparing control GFP (n = 6) and CASP3 (n = 7) overexpressing tumors. Data are presented as mean ± standard deviation (S.D.); statistical analysis was performed using two-tailed t-test.

Article Snippet: CASP3-GFP interacting partners were characterized using GFP-Trap® Magnetic Agarose (20 reactions, Euromedex, CR-gtma-20) following the manufacturer’s instructions.

Techniques: Microinjection, Plasmid Preparation, Western Blot, Expressing, Injection, Over Expression, Transplantation Assay, Control, Standard Deviation, Two Tailed Test

Journal: bioRxiv

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

doi: 10.1101/2024.06.27.601010

Figure Lengend Snippet: (A) Summary table with the most frequent CASP3 putative interacting protein partners identified following CASP3-GFP pulldown and proximity biotinylation in melanoma cells expressing CASP3 fused with BioID2, in either Nter or Cter. (B) Analysis of CASP3-GFP and 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-deficient WM793 using a Proximity Ligation Assay (PLA). (D) Quantification of PLA signal in control (n = 35 cells) and CASP3 (n = 31 cells) deficient WM793 cells. (E) Left panel: Analysis of CASP3, CORO1B and F-actin localization by immunofluorescence in control and CASP3-deficient WM793 cells. Right panel: Signal intensity measurement of CASP3, CORO1B and F-actin along the indicated corresponding line in control and CASP3-deficient WM793 in the left panel. (F) Measurement of cell invasion through Matrigel in control and CORO1B-deficient WM793 (data represent mean with SD of a representative experiment). (G) Analysis by immunoblotting of P-CORO1B, CORO1B, P-PKCα, PKCα, CASP3 in WM793 transfected with two different siRNA for CASP3 and CORO1B. HSC70 serves as a loading control. (H) Analysis by immunoblotting of P-CORO1B, CORO1B, ARP2/3 and CASP3 in control and CASP3-deficient 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. (I) Left panel: Analysis of ARP2/3 and F-actin localization by immunofluorescence in control and CASP3-deficient WM793 cells. Right panel: Signal intensity measurement of ARP2/3 and F-actin along the indicated corresponding line in control and CASP3-deficient WM793 in the left panel.

Article Snippet: CASP3-GFP interacting partners were characterized using GFP-Trap® Magnetic Agarose (20 reactions, Euromedex, CR-gtma-20) following the manufacturer’s instructions.

Techniques: Expressing, Immunoprecipitation, Control, Proximity Ligation Assay, Immunofluorescence, Western Blot, Transfection