rac1 cdc42 activation assay kit  (Cytoskeleton Inc)

Journal: Advanced Science

Article Title: Low YTHDC1 Expression Upregulates FSCN1 to Promote Nuclear F‐Actin Formation and Facilitate Double‐strand DNA Breaks Repair in TMZ‐Resistant Glioblastoma

doi: 10.1002/advs.202513632

Figure Lengend Snippet: FSCN1 activates the CDC42/N‐WASP/Arp2/3 axis by recruiting FGD1. (A) Western blot analysis of CDC42 after pulldown by PAK‐PBD in the nucleus of LN229 and 229R cells. (B) Left: Western blot analysis in the nucleus of LN229 and 229R GBM cells with N‐WASP antibody, Arp2 antibody and Lamin B1 antibody. Right: Immunoprecipitation in the nucleus of LN229 and 229R GBM cells with an antibody against N‐WASP; the cells were subsequently labeled with the anti‐Arp2 antibody. (C) Western blot analysis of CDC42 after pulldown by PAK‐PBD in the nucleus of 229R shNC and 229R shFSCN1 GBM cells. (D) Upper: Western blot analysis in the nucleus of 229R shNC and 229R shFSCN1 GBM cells with N‐WASP antibody, Arp2 antibody and Lamin B1 antibody. Lower: Immunoprecipitation in the nucleus of 229R shNC and 229R shFSCN1 cells with an antibody against N‐WASP; the cells were subsequently labeled with the anti‐Arp2 antibody. (E) Normalized timecourse of pyrene‐labelled actin assembly in 229R shNC and 229R shFSCN1 cell nuclear extracts. (F) Western blot analysis of FGD1 in parental and TMZ‐resistant GBM cell nuclear extracts. (G) Western blot analysis of FGD1 in 229R shNC and 229R shFSCN1 GBM cell nuclear extracts. (H) Upper: Immunoprecipitation of 229R GBM cells with an antibody against FSCN1; the cells were subsequently labeled with the anti‐FGD1 antibody. Lower: Immunoprecipitation of 229R GBM cells with an antibody against FGD1; the cells were subsequently labeled with the anti‐FSCN1 antibody. (I) Western blot analysis in the nucleus of LN229 OE‐NC and LN229 OE‐FSCN1 GBM cells with FSCN1 antibody, FGD1 antibody and Lamin B1 antibody. (J) Normalized timecourse of pyrene‐labelled actin assembly in LN229 OE‐NC and LN229 OE‐FSCN1 GBM cell nuclear extracts. (K) Western blot analysis in the nucleus of LN229 OE‐FSCN1 shNC and LN229 OE‐FSCN1 shFGD1 GBM cells with FSCN1 antibody, FGD1 antibody and Lamin B1 antibody. (L) Normalized timecourse of pyrene‐labelled actin assembly in LN229 OE‐FSCN1 shNC and LN229 OE‐FSCN1 shFGD1 GBM cell nuclear extracts. (M) Immunofluorescence analysis of 229R cells treated with Lifeact‐EGFP‐2xNLS plasmids after treatment with DMSO or ML141 (10 µM, 37°C, 1 h). Scale bar = 5µm. (N) Immunofluorescence analysis of 229R cells treated with Lifeact‐EGFP‐2xNLS plasmids after treatment with DMSO or CK‐666 (100 µM, 37°C, 6 h). Scale bar = 5µm. (O) Immunofluorescence of γH2AX foci in FSCN1 knockdown TMZ‐resistant GBM cells with TMZ treatment show γH2AX foci in DAPIbright heterochromatin. Scale bar = 5µm. (P) Immunofluorescence of γH2AX foci in TMZ‐resistant GBM cells with TMZ or TMZ + ML141 (10 µM, 37°C, 1 h) treatment show γH2AX foci in DAPIbright heterochromatin. Scale bar = 5µm. (Q) Immunofluorescence of γH2AX foci in TMZ‐resistant GBM cells with TMZ or TMZ + CK‐666 (100 µM, 37°C, 6 h) treatment show γH2AX foci in DAPIbright heterochromatin. Scale bar = 5µm. (R) Detection of HR activity using the DR‐GFP reporter system. Different combinations of DR‐GFP reporter plasmids were co‐transfected into LN229 shNC and shFSCN1 cells, as well as 229R OE‐NC and OE‐FSCN1 cells. Cells were treated with 100µM TMZ for 24 h, and the percentage of GFP‐positive cells was determined by flow cytometry (n = 3). For M, N, O, P and Q scale bars, 5µm. Data were analyzed using Student's t‐test (R). Significant results were presented as NS non‐significant, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The Cdc42 activation assay was conducted using the Cdc42 Activation Biochem Kit (BK034, Cytoskeleton) following the manufacturer's instructions.

Techniques: Western Blot, Immunoprecipitation, Labeling, Immunofluorescence, Knockdown, Activity Assay, Transfection, Flow Cytometry