Journal: Kidney international

Article Title: Adipose tissue explants and MDCK cells reciprocally regulate their morphogenesis in coculture.

doi: 10.1038/ki.2010.68

Figure Lengend Snippet: Figure 5 | Zonula occludens-1 (ZO-1), atypical protein kinase C (aPKC), protease-activated receptor 3 (Par3), phosphatase and tensin homolog deleted from chromosome 10 (PTEN), cell division cycle 42 (Cdc42), and pendrin expression of Madin–Darby canine kidney (MDCK) cells with or without adipose tissue fragments (ATFs) at 7 days by immunofluorescence. MDCK cells with ATFs express the tight junction protein, ZO-1, at the apical end of their cell–cell contact regions (arrowheads) more effectively than MDCK cells cultured alone. MDCK cells with ATFs express the polarity-related molecules, aPKC (arrowheads) and Cdc42, more effectively than MDCK cells cultured alone, whereas Par3 and PTEN expressions of MDCK cells with ATFs are similar to those of MDCK cells cultured alone. The aPKC and Par3 are detected at the apical tight junction regions of the cells. Cdc42 is detected at the apical end to basolateral regions of the cells. MDCK cells with ATFs express the chloride/iodide transporter, pendrin, at the apical surface more prominently than MDCK cells cultured alone.

Article Snippet: The sheet was incubated for 1 h at room temperature with each of ZO-1 (Zymed Laboratories and SC8146, Santa Cruz Biotechnology), aPKC (BD Transduction Laboratories, San Jose, CA, USA and SC-12894-R, Santa Cruz Biotechnology), Par3 (Zymed Laboratories and SC-22914, Santa Cruz Biotechnology), PTEN (Cell Signaling Technology and SC-7974, Santa Cruz Biotechnology), Cdc42 (SC-8401, Santa Cruz Biotechnology), and pendrin (kindly provided by Nakao et al.22 and SC16894, Santa Cruz Biotechnology) antibodies.

Techniques: Expressing, Immunofluorescence, Cell Culture

Journal: Kidney international

Article Title: Adipose tissue explants and MDCK cells reciprocally regulate their morphogenesis in coculture.

doi: 10.1038/ki.2010.68

Figure Lengend Snippet: Figure 6 | Western blot and corresponding densitometry analyses. Western blotting (a) of zonula occludens-1 (ZO-1), atypical protein kinase C (aPKC), protease-activated receptor 3 (Par3), phosphatase and tensin homolog deleted from chromosome 10 (PTEN), cell division cycle 42 (Cdc42), and pendrin, and corresponding densitometry analyses (b) in Madin–Darby canine kidney (MDCK) cells cultured with or without adipose tissue fragments (ATFs) at 7 days. MDCK cells with ATFs express ZO-1, aPKC, Cdc42, and pendrin more prominently than MDCK cells cultured alone, whereas Par3 and PTEN expressions of MDCK cells with ATFs are similar to those of MDCK cells cultured alone. This supports the immunofluoresence results of Figure 5.

Article Snippet: The sheet was incubated for 1 h at room temperature with each of ZO-1 (Zymed Laboratories and SC8146, Santa Cruz Biotechnology), aPKC (BD Transduction Laboratories, San Jose, CA, USA and SC-12894-R, Santa Cruz Biotechnology), Par3 (Zymed Laboratories and SC-22914, Santa Cruz Biotechnology), PTEN (Cell Signaling Technology and SC-7974, Santa Cruz Biotechnology), Cdc42 (SC-8401, Santa Cruz Biotechnology), and pendrin (kindly provided by Nakao et al.22 and SC16894, Santa Cruz Biotechnology) antibodies.

Techniques: Western Blot, Cell Culture

Journal: Kidney international

Article Title: Adipose tissue explants and MDCK cells reciprocally regulate their morphogenesis in coculture.

doi: 10.1038/ki.2010.68

Figure Lengend Snippet: Figure 7 | Expression of zonula occludens-1 (ZO-1), protease- activated receptor 3 (Par3), phosphatase and tensin homolog deleted from chromosome 10 (PTEN), cell division cycle 42 (Cdc42), and pendrin mRNAs in Madin–Darby canine kidney (MDCK) cells with or without adipose tissue fragments (ATFs) at 7 days in culture by real-time reverse transcriptase PCR. ZO-1, Cdc42, and PTEN mRNA expressions of MDCK cells with ATFs are significantly lower than those of MDCK cells cultured alone (*Po0.001). There is no significant difference in Par3 and pendrin mRNA expressions between MDCK cells with and without ATFs.

Article Snippet: The sheet was incubated for 1 h at room temperature with each of ZO-1 (Zymed Laboratories and SC8146, Santa Cruz Biotechnology), aPKC (BD Transduction Laboratories, San Jose, CA, USA and SC-12894-R, Santa Cruz Biotechnology), Par3 (Zymed Laboratories and SC-22914, Santa Cruz Biotechnology), PTEN (Cell Signaling Technology and SC-7974, Santa Cruz Biotechnology), Cdc42 (SC-8401, Santa Cruz Biotechnology), and pendrin (kindly provided by Nakao et al.22 and SC16894, Santa Cruz Biotechnology) antibodies.

Techniques: Expressing, Reverse Transcription, Cell Culture

Journal: Molecular cancer research : MCR

Article Title: Mechanical stress signaling in pancreatic cancer cells triggers p38 MAPK- and JNK-dependent cytoskeleton remodeling and promotes cell migration via Rac1/Cdc42/Myosin II

doi: 10.1158/1541-7786.MCR-21-0266

Figure Lengend Snippet: A-B, Phalloidin staining was performed to monitor cell shape, actin cytoskeleton organization of control (0 mmHg) and compressed (4 mmHg) MIA PaCa-2 (A) and PANC-1 (B) cancer cells. Scale bar 1 mm. C-D, Staining for phospho-Myosin II (Ser1943) was performed to show actomyosin contractility in control and compressed MIA PaCa-2 (C) and PANC-1 (D) cells. Scale bar 1mm. E, PANC-1 cells were compressed by 4.0 mmHg in low-serum medium and then subjected to a scratch wound healing assay for 16 hours. Control cells (0 mmHg) were compressed by an agarose cushion only. Scale bar: 0.2 mm. F, Graph showing the average percentage of wound closure ±SE as quantified using ImageJ software. Statistically significant difference in wound closure of compressed PANC-1 cells compared to control cells is indicated with an asterisk (*) (n≥10; 3 biological replicates; p<0.05 in student’s t test). G, Relative mRNA expression of EMT markers Snail, Twist, Slug as quantified by qPCR in MIA PaCa-2 and PANC-1 cells. Each bar indicates the mean fold change ±SE of three independent experiments (n=9). Asterisk (*) indicates a statistically significant difference (p<0.05 in student’s t test). H, G-LISA was performed to analyze activation of cdc42- and Rac-1- small GTPases in compressed (4.0 mmHg) cells at different time points. Assay was performed in triplicates and graphs represent the average fold change ±SE of each protein in compressed relative to uncompressed (control) cells. Asterisk (*) indicates a statistically significant difference (p<0.05 in student’s t test).

Article Snippet: G-LISA assays To examine whether Rac1 and cdc42 small GTPases are activated in response to mechanical stress, the Rac1- and cdc42 Small GTPase Activation Assay kits (G-LISA) were used (027BK127-S, 027BK128-S, Cytoskeleton, Inc) following the company’s guidelines.

Techniques: Staining, Control, Wound Healing Assay, Software, Expressing, Activation Assay

Journal: Molecular cancer research : MCR

Article Title: Mechanical stress signaling in pancreatic cancer cells triggers p38 MAPK- and JNK-dependent cytoskeleton remodeling and promotes cell migration via Rac1/Cdc42/Myosin II

doi: 10.1158/1541-7786.MCR-21-0266

Figure Lengend Snippet: A-B, Rac1 (A) and cdc42 (B) mRNA expression was quantified by qPCR in both MIA PaCa-2 and PANC-1. Each bar indicates the mean fold change ±SE of two biological replicates (n=6). Asterisk (*) indicates a statistically significant difference (p<0.05 in one-way ANOVA analysis). C, MIA PaCa-2 and PANC-1 pancreatic cancer cells were treated with siRNA against Rac1 (siRac1) or cdc42 (sicdc42) and then subjected to a scratch wound healing assay for 16 hours under 0.0 or 4.0 mmHg of compression in 2 % FBS containing DMEM. Control cells were treated with stealth siRNA (siCTRL). Scale bar: 0.1 mm. D-E, Graphs showing the percentage ±SE wound closure of MIA PaCa-2 (D) and PANC-1 (E) as quantified using ImageJ software. Statistically significant difference in wound closure of compressed siRac1- or sicdc42-treated cells compared to siCTRL-treated cells is indicated with an asterisk (*) (2 biological replicates; n≥6; p<0.05 in one-way ANOVA analysis). F, Graph showing the average ±SE Ki67 area fraction in MIA PaCa-2 and PANC-1 control and compressed cells treated with siCTRL, siRac1 or sicdc42 from at least 5 different fields/ condition from two biological replicates as quantified automatically using an in-house code in MATLAB. No statistically significant changes were observed.

Article Snippet: G-LISA assays To examine whether Rac1 and cdc42 small GTPases are activated in response to mechanical stress, the Rac1- and cdc42 Small GTPase Activation Assay kits (G-LISA) were used (027BK127-S, 027BK128-S, Cytoskeleton, Inc) following the company’s guidelines.

Techniques: Expressing, Wound Healing Assay, Control, Software

Journal: Molecular cancer research : MCR

Article Title: Mechanical stress signaling in pancreatic cancer cells triggers p38 MAPK- and JNK-dependent cytoskeleton remodeling and promotes cell migration via Rac1/Cdc42/Myosin II

doi: 10.1158/1541-7786.MCR-21-0266

Figure Lengend Snippet: Mechanical compressive forces are generated during pancreatic tumor growth in the restricted environment of a host tissue. These forces transmit the respective solid stress intracellularly, by activation of JNK/c-Jun, p38 MAPK/HSP27, Rac1 and cdc42. The activation of p38 MAPK/HSP27 and JNK/c-Jun signaling axes can regulate cell adaptation to the new environment by driving their proliferation under compression. Rac1 and cdc42 are activated in turn, and can regulate actin cytoskeleton remodeling for the formation of cell protrusions changing progressively the cell shape. Rac1 and cdc42 also mediate actomyosin contractility, to eventually promote pancreatic cancer cell migration under compression.

Article Snippet: G-LISA assays To examine whether Rac1 and cdc42 small GTPases are activated in response to mechanical stress, the Rac1- and cdc42 Small GTPase Activation Assay kits (G-LISA) were used (027BK127-S, 027BK128-S, Cytoskeleton, Inc) following the company’s guidelines.

Techniques: Generated, Activation Assay, Migration

Journal: Cell reports

Article Title: NELL2-cdc42 signaling regulates BAF complexes and Ewing sarcoma cell growth

doi: 10.1016/j.celrep.2021.109254

Figure Lengend Snippet: (A) CN04 treatment increases the abundance of actin in the BAF complexes in A-673 cells. A-673 cells were treated or not with 1 μg/mL CN04 for 4 h, and the nuclear extract was immunoprecipitated with anti-BRG1 antibody or control immunoglobulin G (IgG). The abundance of indicated BAF subunits in the BAF complexes was examined by immunoblotting. (B) CN04 treatment increases the phalloidin reactivity of BAF complexes in A-673 cells. The BAF complexes immunopurified as in (A) were analyzed for the binding to Biotin-XX-Phalloidin by dot blot. (C) CN04 treatment increases the abundance of actin in the BAF complexes in 293T cells. 293T cells were transfected with FLAG-BRG1 or FLAG-vector and were treated or not with 1 μg/mL CN04 for 4 h, as indicated. FLAG-BRG1-containing BAF complexes were isolated by anti-FLAG immunoprecipitation, and the abundance of indicated BAF subunits was examined by immunoblotting. (D) CN04 treatment increases the phalloidin reactivity of BAF complexes in 293T cells. The BAF complexes immunopurified as in C were analyzed for the binding to Biotin-XX-Phalloidin by dot blot. (E) The amount of BRG1 and BRM pulled down from A-673 nuclear extract with Biotin-XX-Phalloidin increases with CN04 treatment (1 μg/mL for 4 h, top), NELL2 silencing (center), and Jasplakinolide treatment (100 nM for 1 h, bottom). (F) NELL2 signaling reversibly regulates the assembly of BAF complexes in A-673 cells. Nuclear extract of A-673 cells treated as indicated was analyzed by gel filtration chromatography and immunoblotting. (G) CN04 treatment disassembles the BAF complexes in A-673 cells.

Article Snippet: Rho/Rac/Cdc42 Activator 1 (CN04) , Cytoskeleton , CN04-A.

Techniques: Immunoprecipitation, Control, Western Blot, Binding Assay, Dot Blot, Transfection, Plasmid Preparation, Isolation, Filtration, Chromatography

Journal: Cell reports

Article Title: NELL2-cdc42 signaling regulates BAF complexes and Ewing sarcoma cell growth

doi: 10.1016/j.celrep.2021.109254

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rho/Rac/Cdc42 Activator 1 (CN04) , Cytoskeleton , CN04-A.

Techniques: Control, Virus, Recombinant, Transfection, Enzyme-linked Immunosorbent Assay, Mass Spectrometry, Plasmid Preparation