Journal: Nature Communications

Article Title: A resilient formin-derived cortical actin meshwork in the rear drives actomyosin-based motility in 2D confinement

doi: 10.1038/ncomms9496

Figure Lengend Snippet: ( a ) B16-F1 melanoma cells, ectopically expressing EGFP-tagged IQGAP1, were plated on laminin-coated glass coverslips, fixed and labelled with Alexa488-conjugated nanobodies (green) and rhodamine phalloidin to visualize filamentous actin (red). Note the enrichment of IQGAP1 in the rear. ( b ) Subcellular localization of ectopically expressed EGFP-tagged active mDia1 in B16-F1 cells migrating on laminin. The cells were processed as described in a , but were additionally stained for myosin IIA. Active mDia1 was excluded from the leading edge and other actin-rich structures, with the exception of its prominent cortex localization in the trailing edge, where it colocalized together with cortical F-actin and myosin IIA. ( c ) mDia1 dynamics in repolarizing cells. Relocalization of active mDia1 in repolarizing B16-F1 cells to the new prospective end is indicated by white arrowheads and comparable to active ForA in Dictyostelium cells. Still images from time-lapse movies are shown. Time corresponds to min:s. Scale bars, 20 μm.

Article Snippet: EGFP-tagged IQGAP1 was provided by Addgene (Plasmid #30112).

Techniques: Expressing, Staining

Journal: Oncotarget

Article Title: Reactivation of epigenetically silenced miR-124 reverses the epithelial-to-mesenchymal transition and inhibits invasion in endometrial cancer cells via the direct repression of IQGAP1 expression

doi: 10.18632/oncotarget.7754

Figure Lengend Snippet: A. Reverse transcription quantitative PCR (qPCR) analysis of IQGAP1 mRNA in the immortalized human endometrial cell line EM and the EC cells HEC-1, HEC-50, and HEC-50-HI (HI). The results are presented as the fold-change in expression compared to the EM cells. B. Western blot analysis of the IQGAP1 protein in EC cells. C, E. The expression of IQGAP1, E-cadherin, and N-cadherin proteins in HI cells transfected with control (Ctr) or IQGAP1 siRNA (C) and in HEC-1 cells expressing either the control or IQGAP1 vector (E). D, F. Phase-contrast microscopy shows the morphology of HI cells transfected with control or IQGAP1 siRNA (D) and HEC-1 cells transfected with the control or IQGAP1 vector (F). G-I. Detection of migration (G), invasion (H), and proliferation (I) in HI and HEC-1 cells after the indicated transfection. J, K. qPCR analysis of ZO-1 , CK-18 , and Vimentin expression in HI (J) and HEC-1 (K) cells, transfected as indicated. L. Representative images from the invasion assays.

Article Snippet: The Renilla luciferase-reporter plasmids containing human IQGAP1 mRNA [ ] with either wild-type (Wt; #14503) or mutant (Mut; #14504) miR-124 binding sites (875–882 bp and 1172–1178 bp from the start site of the 3′-UTR) were obtained from Addgene (Cambridge, MA, USA).

Techniques: Reverse Transcription, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Transfection, Control, Plasmid Preparation, Microscopy, Migration

Journal: Oncotarget

Article Title: Reactivation of epigenetically silenced miR-124 reverses the epithelial-to-mesenchymal transition and inhibits invasion in endometrial cancer cells via the direct repression of IQGAP1 expression

doi: 10.18632/oncotarget.7754

Figure Lengend Snippet: A. Profiling data of down-regulated microRNAs in highly invasive HEC-50-HI (HI) cells. B. Two putative conserved miR-124-binding sites in the IQGAP1 3′ untranslated region (3′-UTR). C. Relative expression of miR-124 in immortalized human endometrial epithelial and EC cells, assessed by quantitative PCR assays. D. Expression of miR-124 in HI or HEC-1 cells transfected with a miR-124 mimic, miR-124 inhibitor, or their respective negative controls. E. Expression of the indicated mRNA and proteins in HI and HEC-1 cells after the overexpression or knockdown of miR-124. F, G. HI (F) and HEC-1 (G) cells were cotransfected with reporter plasmids containing wild-type IQGAP1 or a mutant IQGAP1 3′-UTR together with a miR-124 mimic, miR-124 inhibitor, or respective negative control. The relative luciferase activity was assayed.

Article Snippet: The Renilla luciferase-reporter plasmids containing human IQGAP1 mRNA [ ] with either wild-type (Wt; #14503) or mutant (Mut; #14504) miR-124 binding sites (875–882 bp and 1172–1178 bp from the start site of the 3′-UTR) were obtained from Addgene (Cambridge, MA, USA).

Techniques: Binding Assay, Expressing, Real-time Polymerase Chain Reaction, Transfection, Over Expression, Knockdown, Mutagenesis, Negative Control, Luciferase, Activity Assay

Journal: Oncotarget

Article Title: Reactivation of epigenetically silenced miR-124 reverses the epithelial-to-mesenchymal transition and inhibits invasion in endometrial cancer cells via the direct repression of IQGAP1 expression

doi: 10.18632/oncotarget.7754

Figure Lengend Snippet: A. Overexpression of miR-124 in HEC-50-HI (HI) cells with fibroblastic morphology converts them to an epithelial-like morphology. B. HEC-1 cells transfected with miR-124 inhibitor exhibit more mesenchymal morphology than the control cells. (A and B, phase-contrast microscopy). C-E. Migration (C), invasion (D), and proliferation (E) of HI and HEC-1 cells after the overexpression or knockdown of miR-124. F, G. Quantitative PCR analysis of the indicated genes in HI (F) and HEC-1 (G) cells after the overexpression or knockdown of miR-124, as indicated. H-J. A miR-124 mimic or its control was transfected into HI cells along with a control vector or the IQGAP1 cDNA vector lacking the 3′-UTR region. The cells were assayed for cell migration (H), invasion (I), and proliferation (J). ** P < 0.01.

Article Snippet: The Renilla luciferase-reporter plasmids containing human IQGAP1 mRNA [ ] with either wild-type (Wt; #14503) or mutant (Mut; #14504) miR-124 binding sites (875–882 bp and 1172–1178 bp from the start site of the 3′-UTR) were obtained from Addgene (Cambridge, MA, USA).

Techniques: Over Expression, Transfection, Control, Microscopy, Migration, Knockdown, Real-time Polymerase Chain Reaction, Plasmid Preparation

Journal: Oncotarget

Article Title: Reactivation of epigenetically silenced miR-124 reverses the epithelial-to-mesenchymal transition and inhibits invasion in endometrial cancer cells via the direct repression of IQGAP1 expression

doi: 10.18632/oncotarget.7754

Figure Lengend Snippet: A-D. The expression levels of miR-124 (A), IQGAP1 (B), E-cadherin (C), and Vimentin (D) were assessed by a quantitative PCR analysis of 20 paired cancerous and normal tissue samples from endometrial cancer patients. E. A Kaplan-Meier survival curve of 309 TCGA (Cancer Genome Atlas database) endometrial cancer samples was created using the SurvMicro database based on the low or high risk for a poor outcome. F. Box plots demonstrating significantly lower levels of miR-124 expression in the high-risk patients.

Article Snippet: The Renilla luciferase-reporter plasmids containing human IQGAP1 mRNA [ ] with either wild-type (Wt; #14503) or mutant (Mut; #14504) miR-124 binding sites (875–882 bp and 1172–1178 bp from the start site of the 3′-UTR) were obtained from Addgene (Cambridge, MA, USA).

Techniques: Expressing, Real-time Polymerase Chain Reaction

Journal: Oncotarget

Article Title: Reactivation of epigenetically silenced miR-124 reverses the epithelial-to-mesenchymal transition and inhibits invasion in endometrial cancer cells via the direct repression of IQGAP1 expression

doi: 10.18632/oncotarget.7754

Figure Lengend Snippet: A, B. HI and HEC-1 cells were treated with 5-aza-2′-deoxycytidine (5-AZA), Trichostatin A, or both, after which, quantitative PCR was used to measure the expression levels of miR-124 (A) and miR-34b (B). C, D. IQGAP1 protein levels (C), invasion, and proliferation (D) in endometrial cancer cells after treating with 5-AZA.

Article Snippet: The Renilla luciferase-reporter plasmids containing human IQGAP1 mRNA [ ] with either wild-type (Wt; #14503) or mutant (Mut; #14504) miR-124 binding sites (875–882 bp and 1172–1178 bp from the start site of the 3′-UTR) were obtained from Addgene (Cambridge, MA, USA).

Techniques: Real-time Polymerase Chain Reaction, Expressing

Journal: eLife

Article Title: Localization of KRAS downstream target ARL4C to invasive pseudopods accelerates pancreatic cancer cell invasion

doi: 10.7554/eLife.66721

Figure Lengend Snippet: ( A ) The ARL4C-interacting proteins in X293T cells were analyzed by mass spectrometry. The results are listed in and . Arrowheads indicate the identified proteins, including IQGAP1 (red). ( B,C ) Lysates of S2-CP8 cells expressing ARL4C-GFP ( B ) or S2-CP8 WT cells ( C ) were immunoprecipitated with anti-GFP antibody ( B ) or anti-ARL4C antibody ( C ), and the immunoprecipitates were probed with the indicated antibodies. ( D ) Lysates of X293T cells expressing the indicated proteins were immunoprecipitated with anti-GFP antibody, and the immunoprecipitates were probed with the indicated antibodies. ( E ) S2-CP8 cells were stained with the indicated antibodies. Images of ARL4C and IQGAP1 were merged. ( F ) S2-CP8 cells expressing ARL4C-tdTomato were subjected to a 3D collagen I gel invasion assay and were stained with the indicated antibodies. Images of ARL4C and IQGAP1 were merged. ( G,H ) S2-CP8 cells expressing GFP or GFP-IQGAP1 were transfected with the indicated siRNAs and subjected to migration ( G ) and invasion ( H ) assays. Migratory and invasive abilities are expressed as the percentage of the same cells transfected with control siRNA. ( I ) S2-CP8 cells depleted of the indicated proteins were subjected to an invasion assay. Invasive activities are expressed as the percentage of control cells. ( J ) PDAC tissues were stained with anti-IQGAP1 antibody and hematoxylin. ( K ) The relationship between overall survival and IQGAP1 expression in PDAC patients was analyzed. ( L ) Scatter plot showing the correlation between the mRNA expression levels of ARL4C (X-axis) and IQGAP1 (Y-axis) in pancreatic cancer patients obtained from TCGA datasets using the R2: Genomics Analysis and Visualization Platform. r indicates the Pearson’s correlation coefficient. ( G-I ) Data are shown as the mean ± s.d. of three biological replicates. p Values were calculated using a two-tailed Student’s t-test ( G,H ) or one-way ANOVA followed by Bonferroni post hoc test ( I ). ( K ) The data were analyzed by Kaplan–Meier survival curves, and a log-rank test was used for statistical analysis. ( E,F ) The regions in the yellow dashed squares are shown enlarged in the left bottom images. The right bottom images are shown with a false color representation of fluorescence intensity. More than 50 cells were imaged and the representative image is shown. False color representations were color-coded on the spectrum. Scale bars in ( E ) 10 μm; (F ) 20 μm; ( J ) 50 µm. KD, knockdown. RFI, relative fluorescence intensity. n.s., not significant. *, p < 0.05; **, p < 0.01. See . Figure 3—source data 1. Excel file containing quantitative data for .

Article Snippet: Antibody , IQGAP1 (mouse monoclonal) , Santa Cruz Santa Cruz Biotechnology , #sc-376021 , (WB 1:1000, IHC 1:800, ICC 1:100).

Techniques: Mass Spectrometry, Expressing, Immunoprecipitation, Staining, Invasion Assay, Transfection, Migration, Control, Two Tailed Test, Fluorescence, Knockdown

Journal: eLife

Article Title: Localization of KRAS downstream target ARL4C to invasive pseudopods accelerates pancreatic cancer cell invasion

doi: 10.7554/eLife.66721

Figure Lengend Snippet: ( A ) Lysates from S2-CP8 WT or ARL4C KO cells were probed with the indicated antibodies. ( B ) S2-CP8 WT or ARL4C KO cells were stained with anti-ARL4C antibody and phalloidin. ( C ) PANC-1 cells were stained with the indicated antibodies. Images of ARL4C and IQGAP1 were merged. Enlarged images of the regions in the yellow dashed squares are shown in a false color representation of fluorescence intensity on the bottom right. False color representations were color-coded on the spectrum. ( D ) PANC-1 cells transfected with control or IQGAP1 siRNAs were subjected to migration and invasion assays. Migratory and invasive abilities are expressed as the percentage of control cells. Data are shown as the mean ± s.d. of three biological replicates. p Values were calculated using one-way ANOVA followed by Bonferroni post hoc test. ( E ) Lysates were prepared from S2-CP8 cells transfected with control or IQGAP1 siRNA, and S2-CP8 WT or IQGAP1 KO cells. Lysates were probed with the indicated antibodies. ( F ) S2-CP8 WT or IQGAP1 KO cells were stained with anti-IQGAP1 antibody and phalloidin. ( G ) PDAC tissues were stained with or without anti-IQGAP1 antibody as the primary antibody and hematoxylin. ( H ) IQGAP1 mRNA levels in pancreatic adenocarcinoma and normal tissues of the pancreas were analyzed using TCGA and GTEx datasets. The results are shown as scatter plots with the mean ± s.e.m. p Values were calculated using a two-tailed Student’s t-test. ( I ) TCGA RNA sequencing and clinical outcome data for pancreatic cancer were analyzed. ( J ), The relationship between overall survival and IQGAP1 expression in PDAC patients with high ARL4C expression was analyzed. ( K ) The relationship between ARL4C and IQGAP1 expression on patient survival using TCGA dataset was analyzed. ( I–K ) The data were analyzed by Kaplan–Meier survival curves, and a log-rank test ( I,J ) or log-rank trend test ( K ). Scale bars in ( B,C,F ) 10 μm; ( G ) 50 μm. KO, knockout. RFI, relative fluorescence intensity. *, p < 0.05; **, p < 0.01. See . Figure 3—figure supplement 2—source data 1. Excel file containing quantitative data for .

Article Snippet: Antibody , IQGAP1 (mouse monoclonal) , Santa Cruz Santa Cruz Biotechnology , #sc-376021 , (WB 1:1000, IHC 1:800, ICC 1:100).

Techniques: Staining, Fluorescence, Transfection, Control, Migration, Two Tailed Test, RNA Sequencing, Expressing, Knock-Out

Journal: eLife

Article Title: Localization of KRAS downstream target ARL4C to invasive pseudopods accelerates pancreatic cancer cell invasion

doi: 10.7554/eLife.66721

Figure Lengend Snippet: ( A ) A schematic representation of four ARL4C-GFP mutants is shown. ( B ) S2-CP8 cells were transfected with the indicated mutants of ARL4C-GFP. The percentages of cells with ARL4C-GFP mutant accumulated at invasive pseudopods compared with the total number of cells were calculated. ( C,D ) Lysates of X293T cells expressing the indicated proteins were immunoprecipitated with anti-GFP antibody and the immunoprecipitates were probed with anti-HA and anti-GFP antibodies. ( E ) S2-CP8 WT or ARL4C KO cells transfected with control or the indicated mutants of ARL4C-GFP were stained with anti-IQGAP1 antibody and phalloidin. The percentages of cells with IQGAP1 accumulated at invasive pseudopods compared with the total number of cells were calculated. ( F ) S2-CP8 WT or IQGAP1 KO cells were transfected with ARL4C-GFP. The percentages of cells with ARL4C-GFP accumulated at invasive pseudopods compared with the total number of cells were calculated. ( G ) S2-CP8 cells stably expressing GFP or the indicated mutants of ARL4C-GFP were transfected with control or ARL4C ASO and subjected to invasion assays. Invasive ability is expressed as the percentage of the same cells transfected with control ASO. ( B,E–G ) Data are shown as the mean ± s.d. of three biological replicates. p Values were calculated using a two-tailed Student’s t-test ( F,G ) or one-way ANOVA followed by Bonferroni post hoc test ( B,E ). ( B,E,F ) The regions in the yellow dashed squares are shown enlarged in the left bottom images. The right bottom images are shown in a false color representation of fluorescence intensity. False color representations were color-coded on the spectrum. Scale bars in ( B,E,F ) 10 μm. KO, knockout. RFI, relative fluorescence intensity. n.s., not significant. *, p < 0.05; **, p < 0.01. See . Figure 4—source data 1. Excel file containing quantitative data for .

Article Snippet: Antibody , IQGAP1 (mouse monoclonal) , Santa Cruz Santa Cruz Biotechnology , #sc-376021 , (WB 1:1000, IHC 1:800, ICC 1:100).

Techniques: Transfection, Mutagenesis, Expressing, Immunoprecipitation, Control, Staining, Stable Transfection, Two Tailed Test, Fluorescence, Knock-Out

Journal: eLife

Article Title: Localization of KRAS downstream target ARL4C to invasive pseudopods accelerates pancreatic cancer cell invasion

doi: 10.7554/eLife.66721

Figure Lengend Snippet: ( A-C ) S2-CP8 cells were transfected with the indicated mutants of ARL4C-GFP and stained with the indicated antibodies. The percentages of cells with invasive pseudopods compared with the total number of cells were calculated ( B ). Cells were classified according to the number of pseudopods as indicated ( C ). ( D ) S2-CP8 WT or ARL4C KO cells were stained with anti-IQGAP1 antibody and phalloidin. Circles of 2 μm diameter were placed at the edge of pseudopods and mean intensity of each circle was measured. The distribution of the data was depicted as a violin plot and the center lines show the medians. More than 100 pseudopods were calculated for each condition. p Values were calculated using a two-tailed Student’s t-test. ( E-G ) S2-CP8 WT or IQGAP1 KO cells were stained with the indicated antibodies and the same assays as in ( B ) and ( C ) were performed for ( F ) and ( G ), respectively. ( H ) S2-CP8 WT cells transfected with control ASO or ARL4C ASO-1316 were stained with anti-IQGAP1 antibody and phalloidin. The percentages of cells with IQGAP1 accumulated at invasive pseudopods compared with the total number of cells were calculated. ( I ) S2-CP8 cells expressing GFP or the indicated mutants of ARL4C-GFP were transfected with control or ARL4C ASO-1316. Lysates were probed with the indicated antibodies. ( A,D,E,H ) The regions in the yellow dashed squares are shown enlarged in the left bottom images. The right bottom images are shown in a false color representation of fluorescence intensity. False color representations were color-coded on the spectrum. ( B,F,H ) Data are shown as the mean ± s.d. of three biological replicates. p Values were calculated using a two-tailed Student’s t-test ( F,H ) or one-way ANOVA followed by Bonferroni post hoc test ( B ). Scale bars in ( A,D,E,H ) 10 μm. KO, knockout. RFI, relative fluorescence intensity. n.s., not significant. **, p < 0.01. See . Figure 4—figure supplement 1—source data 1. Excel file containing quantitative data for .

Article Snippet: Antibody , IQGAP1 (mouse monoclonal) , Santa Cruz Santa Cruz Biotechnology , #sc-376021 , (WB 1:1000, IHC 1:800, ICC 1:100).

Techniques: Transfection, Staining, Two Tailed Test, Control, Expressing, Fluorescence, Knock-Out

Journal: eLife

Article Title: Localization of KRAS downstream target ARL4C to invasive pseudopods accelerates pancreatic cancer cell invasion

doi: 10.7554/eLife.66721

Figure Lengend Snippet: ( A ) S2-CP8 cells were transfected with GFP-PLCδ PH or GFP-GRP1 PH . The percentages of cells with GFP-PLCδ PH or GFP-GRP1 PH accumulated at invasive pseudopods compared with the total number of cells were calculated. ( B ) S2-CP8 cells expressing FRB-CFP, mRFP-FKBP-5-ptase domain, and ARL4C-FLAG-HA were treated with or without rapamycin or LY294002 and stained with anti-HA and anti-IQGAP1 antibodies. The percentages of cells with IQGAP1 or ARL4C-FLAG-HA accumulated at invasive pseudopods compared with the total number of cells were calculated. ( C ) S2-CP8 cells expressing FRB-CFP and mRFP-FKBP-5-ptase domain were treated with or without rapamycin or LY294002 and subjected to an invasion assay. Invasive abilities are expressed as the percentage of control cells. ( D ) S2-CP8 cells expressing ARL4C-mCherry and GFP-GRP1 PH were stained with anti-IQGAP1 antibody. Images of GFP-GRP1 PH , ARL4C-mCherry, and IQGAP1 were merged. ( E ) A schematic representation of ARL4C-GFP mutants is shown. ( F ) S2-CP8 cells were transfected with the indicated mutants of ARL4C-GFP. The percentages of cells with ARL4C-GFP mutant accumulated at invasive pseudopods compared with the total number of cells were calculated. ( G ) ARL4C KO cells expressing control or the indicated mutants of ARL4C-GFP were stained with anti-IQGAP1 antibody. Quantification was performed as in ( B ). ( H ) S2-CP8 cells stably expressing GFP or the indicated mutants of ARL4C-GFP were transfected with control or ARL4C ASO and subjected to an invasion assay. Invasive abilities are expressed as the percentage of the same cells transfected with control ASO. ( A,F ) Enlarged images of the regions in the yellow dashed squares and a false color representation of fluorescence intensity are shown on the right. (a) and (c) show the pseudopods, and (b) and (d) show the cell body. ( B,D,G ) The regions in the yellow dashed squares are shown enlarged in the left bottom images. The right bottom images are shown in a false color representation of fluorescence intensity. ( A–C,F–H ) Data are shown as the mean ± s.d. of three biological replicates. p Values were calculated using a two-tailed Student’s t-test ( A,H ) or one-way ANOVA followed by Bonferroni post hoc test ( B,C,F,G ). ( A,B,D,F,G ) False color representations were color-coded on the spectrum. Scale bars in ( A,B,D,F,G ) 10 μm. KO, knockout. RFI, relative fluorescence intensity. n.s., not significant. **, p < 0.01. See . Figure 5—source data 1. Excel file containing quantitative data for .

Article Snippet: Antibody , IQGAP1 (mouse monoclonal) , Santa Cruz Santa Cruz Biotechnology , #sc-376021 , (WB 1:1000, IHC 1:800, ICC 1:100).

Techniques: Transfection, Expressing, Staining, Invasion Assay, Control, Mutagenesis, Stable Transfection, Fluorescence, Two Tailed Test, Knock-Out

Journal: eLife

Article Title: Localization of KRAS downstream target ARL4C to invasive pseudopods accelerates pancreatic cancer cell invasion

doi: 10.7554/eLife.66721

Figure Lengend Snippet: ( A ) Lysates of X293T cells expressing the indicated mutants of ARL4C-FLAG-HA and GFP or GFP-IQGAP1 proteins were immunoprecipitated with anti-GFP antibody, and the immunoprecipitates were probed with the indicated antibodies. ( B ) S2-CP8 cells expressing GFP or the indicated mutants of ARL4C-GFP were transfected with control or ARL4C ASO-1316. Lysates were probed with the indicated antibodies.

Article Snippet: Antibody , IQGAP1 (mouse monoclonal) , Santa Cruz Santa Cruz Biotechnology , #sc-376021 , (WB 1:1000, IHC 1:800, ICC 1:100).

Techniques: Expressing, Immunoprecipitation, Transfection, Control

Journal: eLife

Article Title: Localization of KRAS downstream target ARL4C to invasive pseudopods accelerates pancreatic cancer cell invasion

doi: 10.7554/eLife.66721

Figure Lengend Snippet: ( A ) Scatter plot showing the correlation between the mRNA expression levels of ARL4C or IQGAP1 (X-axis) and MMP14 (Y-axis) in pancreatic cancer patients obtained from TCGA datasets using the R2: Genomics Analysis and Visualization Platform. r indicates the Pearson’s correlation coefficient. ( B ) TCGA RNA sequencing and clinical outcome data for pancreatic cancer were analyzed. The data were analyzed by Kaplan–Meier survival curves, and a log-rank test was used for statistical analysis. ( C ) S2-CP8 WT or ARL4C KO cells expressing MMP14-GFP were stained with anti-MMP14 antibody without permeabilization and then labeled with phalloidin. ( D ) S2-CP8 WT cells expressing MMP14-GFP were transfected with control ASO or ARL4C ASO-1316. Cells were stained with anti-MMP14 antibody without permeabilization and then labeled with phalloidin. The percentages of cells with MMP14 accumulated at invasive pseudopods compared with the total number of cells were calculated. ( E ) Lysates of S2-CP8 cells transfected with control or MMP14 siRNA were probed with anti-MMP14 and anti-Clathrin antibodies. ( F ) S2-CP8 cells transfected with control or MMP14 siRNAs were subjected to invasion assay. Invasive abilities are expressed as the percentage of control cells. ( G ) S2-CP8 cells or ARL4C KO S2-CP8 cells expressing MMP14-mCherry or MMP14ΔC-mCherry were stained with anti-MMP14 antibody without permeabilization. The percentages of cells with MMP14 accumulated at invasive pseudopods compared with the total number of cells were calculated. ( H ) S2-CP8 cells stably expressing MMP14-mCherry and MMP14ΔC-mCherry were transfected with control or ARL4C ASO-1316 and were then subjected to a 3D collagen I gel invasion assay with DQ collagen I. The cells were stained with phalloidin. Three representative images for each condition are shown. Percentages of cells with DQ collagen I-positive pseudopods compared with the total number of cells were calculated. ( I ) S2-CP8 cells stably expressing MMP14-mCherry and MMP14ΔC-mCherry were transfected with control or ARL4C ASO-1316. Lysates were probed with the indicated antibodies. ( J ) PDAC tissues were stained with the indicated antibodies. The regions in the yellow dashed squares are shown enlarged in the bottom. Images of ARL4C, IQGAP1, and MMP14 were merged as the right bottom panel shows. Nine patient samples were imaged and the representative images are shown. ( D,F-H ) Data are shown as the mean ± s.d. of three biological replicates. p Values were calculated using a two-tailed Student’s t-test ( D,H ) or one-way ANOVA followed by Bonferroni post hoc test ( F,G ). ( C,D,G ) The regions in the yellow dashed squares are shown enlarged in the left bottom images. The right bottom images are shown in a false color representation of fluorescence intensity. False color representations were color-coded on the spectrum. Scale bars in ( C,D,G,H ) 10 μm; ( J ) 20 μm. KO, knockout. RFI, relative fluorescence intensity. n.s., not significant. *, p < 0.05; **, p < 0.01. See . Figure 6—figure supplement 1—source data 1. Excel file containing quantitative data for .

Article Snippet: Antibody , IQGAP1 (mouse monoclonal) , Santa Cruz Santa Cruz Biotechnology , #sc-376021 , (WB 1:1000, IHC 1:800, ICC 1:100).

Techniques: Expressing, RNA Sequencing, Staining, Labeling, Transfection, Control, Invasion Assay, Stable Transfection, Two Tailed Test, Fluorescence, Knock-Out

Journal: eLife

Article Title: Localization of KRAS downstream target ARL4C to invasive pseudopods accelerates pancreatic cancer cell invasion

doi: 10.7554/eLife.66721

Figure Lengend Snippet: ( A ) S2-CP8 cells expressing MMP14-GFP and ARL4C-FLAG-HA were stained with anti-MMP14 without permeabilization, followed by permeabilization and staining with anti-HA and anti-IQGAP1 antibodies. ( B ) S2-CP8 WT or ARL4C KO cells expressing MMP14-GFP and the indicated mutants of ARL4C-FLAG-HA were stained with anti-MMP14 without permeabilization. The percentages of cells with MMP14 accumulated at invasive pseudopods compared with the total number of cells were calculated. ( C ) The same assay as in ( B ) was performed except with S2-CP8 WT or IQGAP1 KO cells expressing MMP14-GFP and FLAG-HA-IQGAP1. ( D ) S2-CP8 cells expressing MMP14-GFP, FRB-CFP, and mRFP-FKBP-5-ptase domain were treated with 100 nM rapamycin or 50 µM LY294002 for 30 min. Staining and quantification were performed as in ( B ). ( E ) S2-CP8 cells depleted of the indicated proteins were subjected to an invasion assay. Invasive activities are expressed as the percentage of control cells. ( F–H ) S2-CP8 cells ( F,G ) or S2-CP8 cells expressing MMP14-mCherry or MMP14ΔC-mCherry ( H ) depleted of the indicated proteins were subjected to a 3D collagen I gel invasion assay with DQ collagen I. The distances from the edge of the gel interface of all cells that invaded into the gel were measured ( F,H ). The percentages of cells with DQ collagen I-positive pseudopods compared with the total number of cells were calculated ( G ). ( I ) PDAC tissues were stained with the indicated antibodies and hematoxylin. The regions in the black dashed squares are shown enlarged in the solid squares. Nine patient samples were imaged and the representative images are shown. ( A–D ) The regions in the yellow dashed squares are shown enlarged in left bottom and a false color representation of fluorescence intensity is shown in right bottom. False color representations were color-coded on the spectrum. ( B–H ) Data are shown as the mean ± s.d. of three biological replicates. p Values were calculated using a two-tailed Student’s t-test ( H ) or one-way ANOVA followed by Bonferroni post hoc test ( B–G ). Scale bars in ( A–D ) 10 μm; ( F,H ) 100 µm; ( G ) 5 µm; ( I ) 100 µm. KO, knockout; KD, knockdown. RFI, relative fluorescence intensity. n.s., not significant. *, p < 0.05; **, p < 0.01. See . Figure 6—source data 1. Excel file containing quantitative data for .

Article Snippet: Antibody , IQGAP1 (mouse monoclonal) , Santa Cruz Santa Cruz Biotechnology , #sc-376021 , (WB 1:1000, IHC 1:800, ICC 1:100).

Techniques: Expressing, Staining, Invasion Assay, Control, Fluorescence, Two Tailed Test, Knock-Out, Knockdown

Journal: eLife

Article Title: Localization of KRAS downstream target ARL4C to invasive pseudopods accelerates pancreatic cancer cell invasion

doi: 10.7554/eLife.66721

Figure Lengend Snippet: ( A ) S2-CP8/Luciferase cells were implanted into the pancreas of nude mice, and control ASO (n = 6) or ARL4C ASO-1316 (n = 7) was subcutaneously administered. ( B ) Bioluminescence images of the intraperitoneal tumors are presented (left) and quantification of the tumor burden is shown (right). The data are presented as the mean ± s.e.m. of the fold change in luminescent intensity relative to that of week 1 treated with control ASO. ( C ) Sections from the pancreatic tumors from control ASO- or ARL4C ASO-1316-treated tumor-bearing mice were stained with anti-ARL4C antibody and hematoxylin. ( D ) Representative images of the tumors in the pancreas (left bottom) and metastatic mesenteric lymph nodes (top and right bottom) are shown. ( E ) Primary tumor weight (left) and metastatic mesenteric lymph node number are presented (right). Data are shown as the mean ± s.e.m. ( F,G ) Four hr after subcutaneous injection of 6-FAM-ARL4C ASO-1316 into tumor-bearing mice, the fluorescence intensities of various organs were measured ( F ), and the sections prepared from the pancreas were stained with the indicated antibodies ( G ). Area indicated by yellow dashed square is enlarged on the right panel ( F ). ( H ) Total RNA was extracted from tumors of control ASO- or ARL4C ASO-1316-treated tumor-bearing mice. Tumor block was cut into three pieces from each tumor of 2 mice per group. ARL4C mRNA levels were measured by quantitative real-time PCR. Relative ARL4C mRNA levels were normalized to those of B2M and expressed as fold changes compared with the levels in one of the control samples. ( I ) Sections from the pancreatic tumors were stained with anti-IQGAP1 antibody and hematoxylin. The two panels on the right show enlarged images of the yellow dashed squares. Positive staining of IQGAP1 is color-coded as yellow (weakly positive) or red (strongly positive). The percentage of the strongly positive IQGAP1 area was calculated. Data are shown as the mean ± s.e.m. Twenty fields were analyzed from 3 mice per group. ( J ) Sections from the pancreatic tumor were stained with the indicated antibodies. The numbers of tumor cells in the lymphatic vessels (indicated with yellow arrowheads) were counted. Data are shown as the mean ± s.e.m. Thirty lymphatic vessels were analyzed from 3 mice per group. ( K,L ) RNA sequencing was performed for S2-CP8-derived primary tumors, and the results of principal component analysis ( K ) and hierarchical clustering ( L ) are shown. ( M ) Differentially expressed genes were subjected to Ingenuity Pathway Analysis (IPA). The top five disease or function annotations of the positive and negative Z-score groups are shown. Bars indicate the -log10 (p value). Inhibited pathways are represented by blue-colored bars while activated pathways are shown by red-colored bars. ( B,E,H-J ) p Values were calculated using a two-tailed Student’s t-test. Scale bars in ( D ) 5 mm; ( C,G,I ) 50 µm; ( J ) 20 µm. n.s., not significant. *, p < 0.05; **, p < 0.01. See and . Figure 7—source data 1. Excel file containing quantitative data for . Figure 7—source data 2. Excel file containing quantitative data for .

Article Snippet: Antibody , IQGAP1 (mouse monoclonal) , Santa Cruz Santa Cruz Biotechnology , #sc-376021 , (WB 1:1000, IHC 1:800, ICC 1:100).

Techniques: Luciferase, Control, Staining, Injection, Fluorescence, Blocking Assay, Real-time Polymerase Chain Reaction, RNA Sequencing, Derivative Assay, Two Tailed Test

Journal: eLife

Article Title: Localization of KRAS downstream target ARL4C to invasive pseudopods accelerates pancreatic cancer cell invasion

doi: 10.7554/eLife.66721

Figure Lengend Snippet:

Article Snippet: Antibody , IQGAP1 (mouse monoclonal) , Santa Cruz Santa Cruz Biotechnology , #sc-376021 , (WB 1:1000, IHC 1:800, ICC 1:100).

Techniques: Transfection, Construct, Recombinant, Plasmid Preparation, Sequencing, Control, Cloning, Blocking Assay, Cell Culture, Invadopodia Assay, Membrane, Silver Staining, Mass Spectrometry, Bicinchoninic Acid Protein Assay, Software, Laser-Scanning Microscopy, Imaging

Journal: Journal of cell science

Article Title: A dual role for IQGAP1 in regulating exocytosis.

doi: 10.1242/jcs.016881

Figure Lengend Snippet: Fig. 1. Association of IQGAP1 with the exocyst-septin complex is regulated by CDC42. (A) IQGAPs are conserved proteins. Domain structure of mammalian IQGAP1 (top) and yeast Iqg1p (bottom) with percent identity indicated on top of each domain. The sequences downstream of the GRD are 24% identical and are well-conserved across species. (B) IQGAP1, but not IQGAP2 (or IQGAP3, not shown), co-immunoprecipitates with the EXO70-SEPT2 complex. Antibodies against EXO70 and SEPT2 were used as indicated on the top of each lane to immunoprecipitate equal amounts of protein from pancreatic TC-6 cells, and western blots were stained with monoclonal antibodies specific for IQGAP2 (middle panel) or IQGAP1 (right panel). Left panels: 5% of the input probed with the indicated antibodies is shown. ‘Mock’ is a negative control using anti-Myc. (C) CDC42 disrupts the interaction between IQGAP1 and the exocyst-septin complex. TC-6 stably expressing wild-type CDC42 (Cdc42WT) or the vector control were either treated with mastoparan (Mp +) to activate CDC42 or with vehicle alone as a control (Mp –), and their lysates were used for co-immunoprecipitation with anti-EXO70 monoclonal antibodies. Right panel: 5% of the proteins used for the IP is shown. The blots represent four experiments with identical results. CHD, calponin homology domain; IR, specific IQGAP repeats; WW, domain resembling the SH3 protein-interacting domains, not well-conserved in yeast; IQ, eight IQ motifs that bind calmodulin; GRD, rasGAP-related domain containing sequences that bind CDC42 and RAC1.

Article Snippet: Antibodies for IQGAP1 and IQGAP2 were from Upstate Biotech, for IQGAP3 were from Novus Biologicals, and those for EXO70 and SEPT2 were previously described (Vega and Hsu, 2001; Vega and Hsu, 2003).

Techniques: Western Blot, Staining, Bioprocessing, Negative Control, Stable Transfection, Expressing, Plasmid Preparation, Control, Immunoprecipitation

Journal: Journal of cell science

Article Title: A dual role for IQGAP1 in regulating exocytosis.

doi: 10.1242/jcs.016881

Figure Lengend Snippet: Fig. 2. Active CDC42 alleles diminish the association of IQGAP1 with the exocyst. (A) Effects of different alleles of CDC42 on the co-immunoprecipitation of IQGAP1 with EXO70. Lysates from pancreatic -cells transiently expressing equal amounts of different HA-tagged CDC42 alleles (bottom) were used to co- immunoprecipitate IQGAP1 (middle panel) with EXO70 (top panel) monoclonal antibodies. IQGAP1 was blotted from 5% of the input protein (lower panel) as a loading control. Cells expressing the vector alone were used as a positive control and mock is a negative control with anti-Myc antibodies. (B) Quantitation of IQGAP1 association with EXO70 in the presence of CDC42 mutants. The intensity of the IQGAP1 bands co-precipitated with EXO70 in the presence of the CDC42 alleles were quantified by densitometry using Quantity One software and a ChemiDoc XRS system (Bio-Rad), and are expressed as the mean ± s.d. for n=3. (C) Binding of CDC42 to IQGAP1 is necessary for the negative effects of CDC42 on the co-immunoprecipitation of IQGAP1 and EXO70. Co- immunoprecipitation of IQGAP1 and EXO70 was performed from cells expressing either a HA-CDC42-F28LC37A double mutant that diminishes CDC42 binding to IQGAP1 (see D, top), or expressing the mutant V5-IQGAP1-FMK, which lacks the 24 amino acids of the CDC42-binding region (see D, bottom), and from their parental F28L or V5-F1 proteins. The histogram represents the mean ± s.d. for n=3 blots. (D) Confirmation of the binding abilities of the mutants used in C. The double-mutant HA-CDC42-F28LC37A, the deletion-mutant V5-IQGAP1-FMK and their parental constructs transiently expressed in TC-6 cells were used for IP with antibodies for the respective tags as shown on the figure and probed for co-immunoprecipitation of IQGAP1 (top) or CDC42 (bottom). The input is presented in the lower part of each panel. WB, western blot; WCL, whole cell lysate.

Article Snippet: Antibodies for IQGAP1 and IQGAP2 were from Upstate Biotech, for IQGAP3 were from Novus Biologicals, and those for EXO70 and SEPT2 were previously described (Vega and Hsu, 2001; Vega and Hsu, 2003).

Techniques: Immunoprecipitation, Expressing, Bioprocessing, Control, Plasmid Preparation, Positive Control, Negative Control, Quantitation Assay, Software, Binding Assay, Mutagenesis, Construct, Western Blot

Journal: Journal of cell science

Article Title: A dual role for IQGAP1 in regulating exocytosis.

doi: 10.1242/jcs.016881

Figure Lengend Snippet: Fig. 3. The N-terminus of IQGAP1 interacts with EXO70 and SEPT2. (A) Schematic representation of the V5-His-tagged IQGAP1 constructs cloned into the pcDNA3.1 Topo vector and used for transient and stable expressions in this study: IQGAP1 full-length (F1), N-terminal (N1), C-terminal (C2) and the IR-WW domains. For abbreviations of the domains, see legend to Fig. 1. (B) Endogenous or exogenous IQGAP1 can co-immunoprecipitate two exocyst subunits and SEPT2 from different cell lines. Left: anti-IQGAP1 antibodies were used to co-immunoprecipitate SEC8, EXO70 and SEPT2 subunits from total lysate of the indicated cell lines and 5% of representative input lysate was blotted as a loading control; Mock is a negative control immunoprecipitate. Right: V5 antibodies for the recombinant IQGAP1 constructs expressed in COS7 cells shown in C were used for IP, and western blots (WB) were probed with antibodies for SEC8, EXO70 and SEPT2. Cells expressing the vector alone were included as negative controls (last lane). (C) Pull-down of the recombinant IQGAP1 proteins with GST-SEPT2 or GST-EXO70. Left: expression of the IQGAP1 constructs resolved on a 10-20% gradient SDS-PAGE and blotted with V5 antibodies. Right: the lysates shown on the left were incubated with immobilized GST alone (lysate from N1 cells is represented in the first lane), GST-SEPT2 (upper panel) or GST-EXO70 (lower panel) and processed as described in the Materials and Methods. The blots were probed with V5 antibodies to detect the recombinant IQGAP1 proteins. The bottom section of each panel demonstrates equal input of GST-SEPT2 (upper panel) or GST-EXO70 (lower panel). (D) Pull-down of GST-SEPT2 and GST-EXO70 with 6His-IQGAP1-N. Proteins from cells expressing V5-His-IQGAP1-N or V5-His vector alone were bound to the TALON cobalt resin, incubated with the bacterial lysates of GST-SEPT2 (upper panel) or GST-EXO70 (middle panel) and blotted with SEPT2 or EXO70 antibodies, respectively. 5% of the purified IQGAP1-N or bacterial lysate of GST-EXO70 or GST-SEPT2 were blotted in the lower panel. Data represent three independent experiments with identical results.

Article Snippet: Antibodies for IQGAP1 and IQGAP2 were from Upstate Biotech, for IQGAP3 were from Novus Biologicals, and those for EXO70 and SEPT2 were previously described (Vega and Hsu, 2001; Vega and Hsu, 2003).

Techniques: Construct, Clone Assay, Plasmid Preparation, Control, Negative Control, Recombinant, Western Blot, Expressing, SDS Page, Incubation, Purification

Journal: Journal of cell science

Article Title: A dual role for IQGAP1 in regulating exocytosis.

doi: 10.1242/jcs.016881

Figure Lengend Snippet: Fig. 4. Confocal images of IQGAP1 co-localization with EXO70. Upper panels: fixed HeLa cells were double-stained with antibodies for IQGAP1 (Texas red) and EXO70 (Alexa- Fluor-488, green) and examined with a confocal microscope. Lower panels: Left, confocal slice (~0.2 m) of the perinuclear area. Middle, thin confocal slice (~0.18 m). Right, high- resolution image of the merged section in the upper panel for the cytoplasmic tubular structures (arrow) of IQGAP1-EXO70. No post-image-acquisition processing was carried out.

Article Snippet: Antibodies for IQGAP1 and IQGAP2 were from Upstate Biotech, for IQGAP3 were from Novus Biologicals, and those for EXO70 and SEPT2 were previously described (Vega and Hsu, 2001; Vega and Hsu, 2003).

Techniques: Staining, Microscopy

Journal: Journal of cell science

Article Title: A dual role for IQGAP1 in regulating exocytosis.

doi: 10.1242/jcs.016881

Figure Lengend Snippet: Fig. 5. Effects of IQGAP1 depletion on EXO70. (A) Depletion of IQGAP1 with targeted RNAi. A representative western blot with the indicated antibodies from equal amounts of total protein from HeLa cells transfected at 100 nM with either control RNAi (first lane) or IQGAP1-RNAi (second lane). (B) Effects of IQGAP1 depletion on EXO70 localization. Upper panels: co- localization of IQGAP1 with EXO70 in control HeLa cells. Fixed cells were double-stained with antibodies for IQGAP1 (Texas red) and EXO70 (Alexa-Fluor-488, green). Lower panels: localization of EXO70 (right panel) in HeLa cells that were depleted of IQGAP1 (left panel).

Article Snippet: Antibodies for IQGAP1 and IQGAP2 were from Upstate Biotech, for IQGAP3 were from Novus Biologicals, and those for EXO70 and SEPT2 were previously described (Vega and Hsu, 2001; Vega and Hsu, 2003).

Techniques: Western Blot, Transfection, Control, Staining

Journal: Journal of cell science

Article Title: A dual role for IQGAP1 in regulating exocytosis.

doi: 10.1242/jcs.016881

Figure Lengend Snippet: Fig. 6. Co-localization of IQGAP1 and SEPT2, and the effects of IQGAP1 depletion or overexpression on SEPT2 organization. (A) Stable expression of the IR- WW domain induces septin-filament disorganization. Immunofluorescence was performed in vector-control (upper panels) and IR-WW stable (lower panels) HeLa cells that were grown in dual-chamber slides with antibodies against SEPT2. (B) Localization of SEPT2 in IQGAP1-depleted cells. HeLa cells depleted for IQGAP1 at 100 nM and 40 nM were double-stained with anti-SEPT2 (bottom, green) and anti-IQGAP1 (top, red) antibodies.

Article Snippet: Antibodies for IQGAP1 and IQGAP2 were from Upstate Biotech, for IQGAP3 were from Novus Biologicals, and those for EXO70 and SEPT2 were previously described (Vega and Hsu, 2001; Vega and Hsu, 2003).

Techniques: Over Expression, Expressing, Immunofluorescence, Plasmid Preparation, Control, Staining

Journal: Journal of cell science

Article Title: A dual role for IQGAP1 in regulating exocytosis.

doi: 10.1242/jcs.016881

Figure Lengend Snippet: Fig. 7. IQGAP1 influences protein synthesis and exocytosis. (A) Differential effects of IQGAP1 domains on insulin exocytosis. Early passages of TC-6 cells (Beta) stably expressing similar levels of the V5-IQGAP1 constructs (on the x-axis) were selected to measure insulin exocytosis (ng/ml) in duplicate. Means ± s.d. for n=6 are shown. Values were normalized to cells that incorporated the vector (Beta-V), which were used as a control. For each construct, the left column represents the basal and the right column the glucose-induced insulin exocytosis. Statistical significance: P<0.001. (B) IQGAP1 influences protein synthesis. Early passages of TC-6 clones stably expressing the indicated IQGAP1 domains were pulsed and chased/stimulated with glucose, as described in the Materials and methods. Upper panels: insulin antibodies were used to immunoprecipitate insulin from the labeled (left) and the chased (right) sets, resolved on a 20% SDS- PAGE and evaluated with a phosphorimager. Lower panel: western blot (WB) of 5% of the lysate from the 1-hour chased cells. The autoradiograph and the blot represent three experiments with identical results. (C) Immunofluorescence of insulin in cells stably expressing the IQGAP1 domains. Fixed TC-6 cells stably expressing equal amounts of the indicated IQGAP1 domains were stimulated for 1 hour, fixed and stained with insulin guinea-pig polyclonal antibodies and Alexa-Fluor-488 goat anti-guinea-pig secondary antibodies, and the images were acquired at a 500-msec exposure time without post-image-acquisition processing.

Article Snippet: Antibodies for IQGAP1 and IQGAP2 were from Upstate Biotech, for IQGAP3 were from Novus Biologicals, and those for EXO70 and SEPT2 were previously described (Vega and Hsu, 2001; Vega and Hsu, 2003).

Techniques: Stable Transfection, Expressing, Construct, Plasmid Preparation, Control, Clone Assay, Labeling, SDS Page, Western Blot, Autoradiography, Immunofluorescence, Staining

Journal: Journal of cell science

Article Title: A dual role for IQGAP1 in regulating exocytosis.

doi: 10.1242/jcs.016881

Figure Lengend Snippet: Fig. 8. Localization and association of IQGAP1 with sites of protein synthesis and exocytosis. (A) Confocal slice (0.16 m) showing the co-localization of IQGAP1 (red) with the ER marker calnexin (green) in HeLa cells. Arrows indicate localization spots. (B) Co-localization of IQGAP1 (red) with the ER resident marker IP3R3 (green) in NIH3T3 cells. (C) Co-localization of IQGAP1 (red) with the t-SNARE syntaxin 1A (green) in HeLa cells. No post-image- acquisition processing was performed. Arrows indicate plasma membrane leading edge or ruffles. (D) Top: co-immunoprecipitation of IQGAP1 with the ER- translocon subunit Sec61. Monoclonal antibodies for Sec61 or IQGAP1 were used conversely for IP. The immune complexes were resolved on a 15% SDS-PAGE and conversely blotted with anti-IQGAP1 or -Sec61 antibodies. Bottom: co-immunoprecipitation of IQGAP1 and syntaxin 1A (Synt1A). Anti- Synt1A or -IQGAP1 antibodies were used for IP, and the immune complexes resolved on a 10% SDS-PAGE and conversely blotted with the indicated antibodies.

Article Snippet: Antibodies for IQGAP1 and IQGAP2 were from Upstate Biotech, for IQGAP3 were from Novus Biologicals, and those for EXO70 and SEPT2 were previously described (Vega and Hsu, 2001; Vega and Hsu, 2003).

Techniques: Marker, Clinical Proteomics, Membrane, Immunoprecipitation, Bioprocessing, SDS Page

Journal: Journal of cell science

Article Title: A dual role for IQGAP1 in regulating exocytosis.

doi: 10.1242/jcs.016881

Figure Lengend Snippet: Fig. 9. Mechanism of the role of IQGAP1 in regulating secretion. (A) CDC42 negatively regulates IQGAP1 secretion and is abrogated by IQGAP1-N1 expression. Pancreatic -cells stably co-expressing CDC42WT (depicted as 42) and a IQGAP1 domain (on the x-axis) were assayed for insulin exocytosis as described in the Materials and Methods. CDC42WT stable cells expressing the V5 vector were used for control (42-V). Means ± s.d. for n=6 are shown. Values were normalized to cells that incorporated the vector (42-V) as a control. (B) IQGAP1 increases cellular CDC42-GTP. Left: the expression level of the V5-IQGAP1 constructs the cells used for the pull-down. Middle: positive (GTPS) and negative (GDP) controls for the pull-down experiment. Right: GST construct expressing the CDC42-binding (CRIB) domain of PAK (PBD) was used to pull-down active CDC42 (GTP-CDC42) from -cells expressing IQGAP1 (F1), IQGAP1-C (C2), IQGAP1-N (N1) or vector control (V). Bottom: western blot of 10% of the total protein used for IP demonstrating equal input of cellular CDC42. (C) A model for the mechanism of the role of IQGAP1 in secretion as a conformational switch. In protein synthesis/exocytosis, IQGAP1 operates in a closed form generated by folding of the C- terminus. External or internal signals lead to the phosphorylation of IQGAP1 at a C-terminus serine (pS) and to the binding and activation of CDC42, perhaps to switch effector molecules in order to regulate exocytosis (on or off) or influence alternate cellular functions, such as cell division.

Article Snippet: Antibodies for IQGAP1 and IQGAP2 were from Upstate Biotech, for IQGAP3 were from Novus Biologicals, and those for EXO70 and SEPT2 were previously described (Vega and Hsu, 2001; Vega and Hsu, 2003).

Techniques: Expressing, Stable Transfection, Plasmid Preparation, Control, Construct, Binding Assay, Western Blot, Generated, Phospho-proteomics, Activation Assay

Journal: Asian Pacific Journal of Cancer Prevention : APJCP

Article Title: Novel Approach Using shRNA of IQGAP1 for Colon Cancer Therapy: HCT116 as a Surrogate Model Colorectal Carcinoma

doi: 10.31557/APJCP.2022.23.7.2387

Figure Lengend Snippet: The Sequence of Human IQGAP1 siRNA and Primers Used in the Study. Note: all sequences are in 5′ → 3′ orientation

Article Snippet: This plasmid encoding variant 1 of the human IQGAP1 was obtained from Santa Cruz Biotechnology Company, CA, USA (Cat. No.sc-35700).

Techniques: Sequencing

Journal: Asian Pacific Journal of Cancer Prevention : APJCP

Article Title: Novel Approach Using shRNA of IQGAP1 for Colon Cancer Therapy: HCT116 as a Surrogate Model Colorectal Carcinoma

doi: 10.31557/APJCP.2022.23.7.2387

Figure Lengend Snippet: Showing Cell Proliferation by MTT Assay. HCT166 cells were incubated with 5μM of IQGAP1 shRNA vector for 24 48, 72 and 72 h

Article Snippet: This plasmid encoding variant 1 of the human IQGAP1 was obtained from Santa Cruz Biotechnology Company, CA, USA (Cat. No.sc-35700).

Techniques: MTT Assay, Incubation, shRNA, Plasmid Preparation

Journal: Asian Pacific Journal of Cancer Prevention : APJCP

Article Title: Novel Approach Using shRNA of IQGAP1 for Colon Cancer Therapy: HCT116 as a Surrogate Model Colorectal Carcinoma

doi: 10.31557/APJCP.2022.23.7.2387

Figure Lengend Snippet: Healthy Population of HCT166 Cells was Counted Using Flow Cytometry up on Treatment with 5μM of IQGAP1 shRNA Vector for 24 and 48 h. The experiment was repeated 3 independent times (n=3).

Article Snippet: This plasmid encoding variant 1 of the human IQGAP1 was obtained from Santa Cruz Biotechnology Company, CA, USA (Cat. No.sc-35700).

Techniques: Flow Cytometry, shRNA, Plasmid Preparation

Journal: Asian Pacific Journal of Cancer Prevention : APJCP

Article Title: Novel Approach Using shRNA of IQGAP1 for Colon Cancer Therapy: HCT116 as a Surrogate Model Colorectal Carcinoma

doi: 10.31557/APJCP.2022.23.7.2387

Figure Lengend Snippet: Expressions of mRNA for Definite Genes after HCT166 Cell Treatment with IQGAP1 shRNA Vector. (a-f) * indicated significantly differences between means at P <0.05 and error bars represents standard error of mean (SEM). Means comparisons were performed by using Duncan’s multiple range test

Article Snippet: This plasmid encoding variant 1 of the human IQGAP1 was obtained from Santa Cruz Biotechnology Company, CA, USA (Cat. No.sc-35700).

Techniques: shRNA, Plasmid Preparation

Journal: Asian Pacific Journal of Cancer Prevention : APJCP

Article Title: Novel Approach Using shRNA of IQGAP1 for Colon Cancer Therapy: HCT116 as a Surrogate Model Colorectal Carcinoma

doi: 10.31557/APJCP.2022.23.7.2387

Figure Lengend Snippet: Protein Expression of IQGAP1, KRAS, BAX, casp-9 and cleaved casp-3 Using Western Blot Analysis

Article Snippet: This plasmid encoding variant 1 of the human IQGAP1 was obtained from Santa Cruz Biotechnology Company, CA, USA (Cat. No.sc-35700).

Techniques: Expressing, Western Blot

Journal: Nature Communications

Article Title: Inhibition of polar actin assembly by astral microtubules is required for cytokinesis

doi: 10.1038/s41467-021-22677-0

Figure Lengend Snippet: a Dividing HeLa cells fixed and stained with antibodies recognizing β-actin, γ-actin, DIAPH1, DIAPH3, and IQGAP1. b Quantitation of the number of multinucleated cells in the presence and absence of DIAPH1, IQGAP1, and DIAPH1-I862A that cannot nucleate actin. Bars denote the average, whiskers denote ±SD. * p = 0.0001, ** p < 0.0001 to control using nonparametric two-tailed Mann-Whitney t -tests for three experimental repeats, n = 600 cells examined over three independent experiments. c β-actin or γ-actin localization in HeLa cells treated with control siRNA or DIAPH1 siRNA in the presence and absence of exogenously expressed GFP-DIAPH1 or GFP-DIAPH1-I862A. d Frames from time-lapse phase contrast movies demonstrating that depletion of DIAPH1 caused an increase in plasma membrane blebbing during anaphase. White arrows denote, blebs, black arrows point to the cytokinetic furrow. e Quantitation of the number of plasma membrane blebs in fixed metaphase and anaphase cells. Bars denote the average, boxes represent 25–75 percentile and whiskers denote extremes. * p = 0.014, ** p = 0.0007, *** p = 0.0006, **** p = 0.0005, ***** p = 0.0004 difference to control using nonparametric two-tailed Mann-Whitney t -tests for three experimental repeats, n = 300 cells examined over three independent experiments. CT = C terminus; DIA1 = DIAPH1; DIA3 = DIAPH3; IQ1 = IQGAP1. f Quantitation of the rate of cell elongation from 2–8 min after anaphase onset. Bars denote the average, whiskers denote ±SD. * p = 0.029, ** p = 0.005, *** p = 0.0007, **** p = 0.0003, ***** p < 0.0001 to control, using nonparametric two-tailed Mann-Whitney t- tests for three experimen t al repeats, n = 10 cells examined in each live-cell imaging group. CT = C terminus; DIA1 = DIAPH1; DIA3 = DIAPH3. Scale bars are 10 μm.

Article Snippet: The PCR fragments were mixed and reamplified using 5’ and 3’ oligos of DIAPH1. cDNAs of DIAPH1-CT were generated and fused to phospholipase Cδ1-PH (PLCδ1-PH) using analogues strategy to generated cDNAs of PLCδ1-PH-DIAPH1-CT. Alternatively, PCR fragments of DIAPH1-CT were cloned using the TOPO Gateway system (Life Technologies) being first cloned into the entry plasmid vector pCR8/GW/TOPO, then moved into the destination vectors pKM596 (Addgene plasmid 8837) to generate MBP fusion proteins. cDNAs of full-length human IQGAP1 (1–1657aa), CLIP170 (1–1320aa), or fragments of IQGAP1-DBR (1500–1657aa), CLIP170-NT (1–350aa), CLIP170-CT (500–1320aa) were amplified using the i-Max II DNA polymerase (Froggalab) using oligonucleotide primers listed in Supplementary Table .

Techniques: Staining, Quantitation Assay, Two Tailed Test, MANN-WHITNEY, Live Cell Imaging

Journal: Nature Communications

Article Title: Inhibition of polar actin assembly by astral microtubules is required for cytokinesis

doi: 10.1038/s41467-021-22677-0

Figure Lengend Snippet: a HeLa cell fixed and probed with an antibody recognizing RhoA, white and DAPI stained chromosomes in blue. b Preformed complexes of His 6 -DIAPH1-NT with GST-RhoA, GST-IQGAP1 or both prevent MBP-DIAPH1-CT binding to His 6 -DIAPH1-NT. Schematic depicts that DIAPH1 deactivation requires the removal of both RhoA and IQGAP1. CT = C-terminus.

Article Snippet: The PCR fragments were mixed and reamplified using 5’ and 3’ oligos of DIAPH1. cDNAs of DIAPH1-CT were generated and fused to phospholipase Cδ1-PH (PLCδ1-PH) using analogues strategy to generated cDNAs of PLCδ1-PH-DIAPH1-CT. Alternatively, PCR fragments of DIAPH1-CT were cloned using the TOPO Gateway system (Life Technologies) being first cloned into the entry plasmid vector pCR8/GW/TOPO, then moved into the destination vectors pKM596 (Addgene plasmid 8837) to generate MBP fusion proteins. cDNAs of full-length human IQGAP1 (1–1657aa), CLIP170 (1–1320aa), or fragments of IQGAP1-DBR (1500–1657aa), CLIP170-NT (1–350aa), CLIP170-CT (500–1320aa) were amplified using the i-Max II DNA polymerase (Froggalab) using oligonucleotide primers listed in Supplementary Table .

Techniques: Staining, Binding Assay

Journal: Nature Communications

Article Title: Inhibition of polar actin assembly by astral microtubules is required for cytokinesis

doi: 10.1038/s41467-021-22677-0

Figure Lengend Snippet: a Preassembled GST-IQGAP1-DBR and His 6 -DIAPH1-NT complexes were incubated with MBP-CLIP170-NT. In parallel preassembled GST-IQGAP1-DBR and MBP-CLIP170-NT complexes were incubated with His 6 -DIAPH1-NT. After reisolating GST-IQGAP1-DBR, the amount of co-purifying MBP-CLIP170-NT or His 6 -DIAPH1-NT was determined. NT = N-terminus, DBR = Diaphanous Binding Region. b Schematic representing inferred IQGAP1 interaction dynamics.

Article Snippet: The PCR fragments were mixed and reamplified using 5’ and 3’ oligos of DIAPH1. cDNAs of DIAPH1-CT were generated and fused to phospholipase Cδ1-PH (PLCδ1-PH) using analogues strategy to generated cDNAs of PLCδ1-PH-DIAPH1-CT. Alternatively, PCR fragments of DIAPH1-CT were cloned using the TOPO Gateway system (Life Technologies) being first cloned into the entry plasmid vector pCR8/GW/TOPO, then moved into the destination vectors pKM596 (Addgene plasmid 8837) to generate MBP fusion proteins. cDNAs of full-length human IQGAP1 (1–1657aa), CLIP170 (1–1320aa), or fragments of IQGAP1-DBR (1500–1657aa), CLIP170-NT (1–350aa), CLIP170-CT (500–1320aa) were amplified using the i-Max II DNA polymerase (Froggalab) using oligonucleotide primers listed in Supplementary Table .

Techniques: Incubation, Binding Assay

Journal: Nature Communications

Article Title: Inhibition of polar actin assembly by astral microtubules is required for cytokinesis

doi: 10.1038/s41467-021-22677-0

Figure Lengend Snippet: a Time-lapse images of GFP-CLIP170 during cell division. b Proximity ligation assays (PLA) mapping the proximity DIAPH1 to IQGAP1 (top), IQGAP1 to CLIP170 (middle) and CLIP170 to DIAPH1 (bottom) during mitotic progression. c Quantitation of PLA foci in metaphase and anaphase * p < 0.0001 using a nonparametric two-tailed Mann-Whitney t -tests for three experimental repeats, n = 50 cells examined over three independent experiments. d Proximity ligation assays (PLA) mapping the proximity DIAPH1 to IQGAP1 and IQGAP1 to CLIP170 in Centrinone B-treated cells. e Quantitation of PLA foci in d . At cell poles lacking astral microtubules, DIAPH1 and IQGAP1 are in close proximity but IQGAP1 and CLIP170 are not. * p = 0.0006, ** p < 0.0001 using nonparametric two-tailed Mann-Whitney t -tests for three experimen t al repeats, n = 50 cells examined over three independent experiments. Scale bars are 10 μm.

Article Snippet: The PCR fragments were mixed and reamplified using 5’ and 3’ oligos of DIAPH1. cDNAs of DIAPH1-CT were generated and fused to phospholipase Cδ1-PH (PLCδ1-PH) using analogues strategy to generated cDNAs of PLCδ1-PH-DIAPH1-CT. Alternatively, PCR fragments of DIAPH1-CT were cloned using the TOPO Gateway system (Life Technologies) being first cloned into the entry plasmid vector pCR8/GW/TOPO, then moved into the destination vectors pKM596 (Addgene plasmid 8837) to generate MBP fusion proteins. cDNAs of full-length human IQGAP1 (1–1657aa), CLIP170 (1–1320aa), or fragments of IQGAP1-DBR (1500–1657aa), CLIP170-NT (1–350aa), CLIP170-CT (500–1320aa) were amplified using the i-Max II DNA polymerase (Froggalab) using oligonucleotide primers listed in Supplementary Table .

Techniques: Ligation, Quantitation Assay, Two Tailed Test, MANN-WHITNEY