Journal: Developmental cell

Article Title: Cell-Intrinsic Adaptation Arising from Chronic Ablation of a Key Rho GTPase Regulator.

doi: 10.1016/j.devcel.2016.08.020

Figure Lengend Snippet: Figure 3. Reduction of CDC42 Activity Caused by Acute DOCK6 Depletion Is Compensated for by Cell-Intrinsic Adaptation for Proper Mitotic Progression (A) HeLa cells stably expressing H2B-mCherry and GFP-tubulin were transfected with NSC or DOCK6 siRNAs, synchronized with thymidine, and analyzed by time-lapse microscopy 9 hr after release. Asterisks denote misaligned chromosomes. Arrowheads point to abnormal membrane blebs. Scale bars, 10 mm. (B) Prometaphase, metaphase, and non-arrested cell numbers quantified from time-lapse microscopy in (A). Values represent mean ± SEM from 100 NSC or DOCK6 siRNA transfected cells (n = 2). (legend continued on next page)

Article Snippet: Measurement of GTPase Activities RHOA, RAC1, and CDC42 activity levels were determined using a G-LISA assay kit (Cytoskeleton, BK135).

Techniques: Activity Assay, Stable Transfection, Expressing, Transfection, Time-lapse Microscopy, Membrane

Journal: Cell Death & Disease

Article Title: ASAP1 activates the IQGAP1/CDC42 pathway to promote tumor progression and chemotherapy resistance in gastric cancer

doi: 10.1038/s41419-023-05648-9

Figure Lengend Snippet: A The RNA-seq of ASAP1-depletedGC cell and a volcano plot showing the differential genes. B KEGG pathway analysis showing enrichment of differential gene. C The whole cell lysates were extracted from SGC-7901 cells expressing ASAP1-HA or from ASAP1 +/− SG-7901 cells to measure CDC42, RAC1, RALA and RhoA GTPase activity by G-LISA activation assay. D The whole cell lysates prepared from either SGC-7901 or MGC803 cell expressing ASAP1-HA were used for CDC42 activation assay followed by western blotting. E Combined phalloidin staining for F-actin (red) and ASAP1 (green) immunofluorescence. Nuclei were counterstained with DAPI (blue). In ASAP1 +/− SGC-7901 cells, the filamentous pseudopodia disappeared and cytoskeleton depolymerized. F SGC-7901 cells were treated with CDC42 inhibitor ZCL278 and combined phalloidin staining for F-actin (red). The filamentous pseudopodia disappeared and cytoskeleton depolymerized. G SGC-7901 cells were treated with CDC42 inhibitor ZCL278 and the whole cell lysates were extracted to measure CDC42 GTPase activity by G-LISA activation assay. H The proliferation of SGC-7901 cells transfected with pcDNA3.1, SGC-7901 cells transfected with pcDNA3.1-ASAP1-HA, SGC-7901 cells treated with ZCL278 or SGC-7901 cells transfected with pcDNA3.1-ASAP1-HA followed by treated with ZCL278 was determined by CCK-8. I The migration of various types of cells described in H was determined by Transwell assays.

Article Snippet: The levels of activated Rho GTPases were detection with The Cdc42/RhoA/Rac1 G-LISA kit (Cytoskeleton.

Techniques: RNA Sequencing Assay, Expressing, Activity Assay, Activation Assay, Western Blot, Staining, Immunofluorescence, Transfection, CCK-8 Assay, Migration

Journal: Cell Death & Disease

Article Title: ASAP1 activates the IQGAP1/CDC42 pathway to promote tumor progression and chemotherapy resistance in gastric cancer

doi: 10.1038/s41419-023-05648-9

Figure Lengend Snippet: A The ASAP-associated interactomes were determined by immunoprecipitation and mass spectrometry (IP-MS). Venn diagrams show the number of proteins that may interact listed in the tables. B Correlation analysis of ASAP1 and IQGAP1 RNA expression in TCGA STAD database. C Images showing the immunofluorescence of ASAP1 (red) and IQGAP1 (green). Nuclei were counterstained with DAPI (blue). C SGC-7901 was transfected with pcDNA3.1 or pcDNA3.1-ASAP1-HA. 24 h after transfection, HA-tag fused proteins were immunoprecipitated with their interacting proteins. Western blot was used to detect indicated proteins. D Western blot showing that endogenous ASAP1 co-immunoprecitated with IQGAP1 from SGC-7901 cells. An anti-ASAP1 antibody was used for IP. Normal mouse IgG was used as a control. E Western blot showing that HA-tagged ASAP1 coimmunoprecipitated with IQGAP1 from SGC-7901 cells expressing HA-tagged ASAP1, but not from control SGC-7901 cells. An anti-HA antibody was used for IP. F Molecular docking pattern diagram of ASAP1 and IQGAP1. G IQGAP1 mRNA level of MGC-803 and SGC-7901 after ASAP1 overexpression. H IQGAP1 protein level of MGC-803 and SGC-7901 after ASAP1 overexpression. I SGC-7901 and SGC-ASAP1 +/− cells were incubated with MG132 (+) or DMSO (−) for 4 h. Equal amounts of protein lysates were loaded directly onto gels or immunoprecipitated (IP) with an anti-IQGAP1 antibody. Proteins were analyzed by SDS–PAGE and immunoblotting (IB) using anti-ubiquitin and anti-IQGAP1 antibodies, respectively. J Western blot showing that IQGAP was depleted by two different siRNA oligos in SGC-7901 cells. K The proliferation of various types of SGC-7901 cells was determined via CCK-8, including cells transfected with pcDNA3.1, cells transfected with pcDNA3.1-ASAP1-HA, cells transfected with pcDNA3,1 followed by transfection with a siRNA oligo against IQGAP1, and cells transfected with pcDNA3.1-ASAP1-HA followed by transfection with a siRNA oligo against IQGAP1. M The migration of various types of SGC-7901 cells described in L was determined was determined by transwell assays. N The who cell lysates were extracted from various types of SGC-7901 cells to measure CDC42 GTPase activity by G-LISA activation assay, including cells transfected with pcDNA3.1, cells transfected with pcDNA3.1-ASAP1-HA, and cells transfected with pcDNA3.1-ASAP1-HA followed by transfection with a siRNA oligo against IQGAP1.

Article Snippet: The levels of activated Rho GTPases were detection with The Cdc42/RhoA/Rac1 G-LISA kit (Cytoskeleton.

Techniques: Immunoprecipitation, Mass Spectrometry, RNA Expression, Immunofluorescence, Transfection, Western Blot, Expressing, Over Expression, Incubation, SDS Page, CCK-8 Assay, Migration, Activity Assay, Activation Assay

Journal: Cell Death & Disease

Article Title: ASAP1 activates the IQGAP1/CDC42 pathway to promote tumor progression and chemotherapy resistance in gastric cancer

doi: 10.1038/s41419-023-05648-9

Figure Lengend Snippet: A The effect of ZCL278 on the viability of WT SGC-7901 cells in response to oxaliplatin. Cell viability was measured by CCK-8. IC50 values in two different drug treatments are shown ( n = 3). B WB analysis of EGFR-MAPK pathway in SGC-7901 cells expressing ASAP1-HA and partially knocked out of ASAP1. C Phalloidin staining for F-actin (red) and EBFR (green) immunofluorescence. Nuclei were counterstained with DAPI (blue). In ASAP1 ± SGC-7901 cells, the EGFR was not located on the membrane and the cytoskeleton depolymerized. In WT SGC-7901 cells treated with ZCL278, the EGFR was not located on the membrane and the cytoskeleton depolymerized. D The effect of IQGAP1 on the viability of SGC-7901-ASAP1 ± cells in response to oxaliplatin. Cell viability was measured by CCK-8. IC50 values in two different drug treatments are shown ( n = 3). E The effect of EGFR on the viability of SGC-7901 cells expressing ASAP1-HA or not in response to oxaliplatin. Cell viability was measured by CCK-8. IC50 values in two different drug treatments are shown ( n = 3). ns-not significant; * p < 0.05; ** p < 0.01; *** p < 0.001. F A working model of ASAP1 summarizing this study. ASAP1 was highly expressed in gastric cancer, inhibits ubiquitin-mediated degradation of IQGAP1 and promotes CDC42 activation through interaction with IQGAP1. Activated CDC42 contributes to the growth and metastasis of gastric cancer cells. In addition, activated CDC42 upregulates the EGFR pathway and causes oxaliplatin resistance in gastric cancer.

Article Snippet: The levels of activated Rho GTPases were detection with The Cdc42/RhoA/Rac1 G-LISA kit (Cytoskeleton.

Techniques: CCK-8 Assay, Expressing, Staining, Immunofluorescence, Activation Assay