Journal: International Journal of Molecular Sciences

Article Title: Simulated Microgravity Reduces Focal Adhesions and Alters Cytoskeleton and Nuclear Positioning Leading to Enhanced Apoptosis via Suppressing FAK/RhoA-Mediated mTORC1/NF-κB and ERK1/2 Pathways

doi: 10.3390/ijms19071994

Figure Lengend Snippet: Simulated microgravity reduces focal adhesions and inhibits FAK and RhoA signaling. ( A ) BL6-10 cells were cultured in medium of chamber slides for one day under ground conditions (1 g) or SMG (µg). The cells were stained with anti-paxillin (green) and anti-vinculin (red) antibodies followed by observation under a fluorescence microscope using 40× objectives (formation of cellular focal adhesions (a,c), arrows); ( B ) Western blotting analysis. Lysates were harvested from BL6-10 cells cultured for three days under 1 g or µg and subjected to SDS-PAGE analysis. Proteins were transferred onto PVDF membranes. Blots were stained with various antibodies and analyzed by chemiluminescence. Bands were qualified using Imaging Lab software (Bio-Rad). Densitometric values were normalized to the GAPDH control; ( C ) RhoA activity analysis. BL6-10 cells (three days) under 1 g and µg were subjected to RhoA activity assay by using a G-LISA RhoA Activation Assay Biochem kit. Data represent the mean ± SD of three independent experiments. * p < 0.05 versus different groups. One representative experiment of two is shown.

Article Snippet: To measure RhoA activity and cell adhesion, we performed in vitro experiments using a G-LISA RhoA Activation Assay Biochem kit (Cytoskeleton Inc., Denver, CO, USA) and a CytoSelect™ 24-Well Cell Adhesion Assay kit, according to the manufacturers’ manuals [ ].

Techniques: Cell Culture, Staining, Fluorescence, Microscopy, Western Blot, SDS Page, Imaging, Software, Activity Assay, Activation Assay

Journal: International Journal of Molecular Sciences

Article Title: Simulated Microgravity Reduces Focal Adhesions and Alters Cytoskeleton and Nuclear Positioning Leading to Enhanced Apoptosis via Suppressing FAK/RhoA-Mediated mTORC1/NF-κB and ERK1/2 Pathways

doi: 10.3390/ijms19071994

Figure Lengend Snippet: CNF1 restores focal adhesions and NEPCs and activates FAK/RhoA, mTORC1/NF-κB, and ERK1/2 pathways leading to apoptosis reduction in cells under SMG. ( A ) BL6-10 cells were cultured in chamber slides for one day at 1 g, µg, and µg + CNF1, and cells were stained with anti-paxillin, and paxillin spots were counted for each cell. Approximately 20 cells were analyzed per experimental condition; ( B ) Lysates were harvested from BL6-10 cells cultured for two days under 1 g or µg and subjected to SDS-PAGE analysis. Proteins were transferred onto PVDF membranes. Blots were stained with various antibodies and analyzed by chemiluminescence. Bands were qualified using Imaging Lab software (Bio-Rad). Densitometric values were normalized to the GAPDH control; ( C ) RhoA activity analysis. BL6-10 cells were cultured for three days at 1 g, µg, and µg + CNF1 and were subjected to RhoA activity assay by using G-LISA RhoA Activation Assay Biochem kit. Data represent the mean ± SD of three independent experiments; ( D ) BL6-10 cells cultured for three days at 1 g or µg in a Lab-Tek1 II Chamber Slide TM System were fixed with paraformaldehyde, and subsequently incubated with anti-p-NF-κB (S337) (green) antibody and then incubated with FITC-labeled goat-anti-rabbit secondary antibody. Slides were covered using Prolong Gold Antifade Reagent with DAPI (blue) and observed by confocal microscopy. Panels ( a – c ) using 20× magnification; panels ( d – f ) using 50× magnification; ( E ) BL6-10 tumor cells cultured under ground conditions (1 g) or SMG (µg) and SMG (µg) + CNF1 for one day were stained with Annexin V-FITC and propidium iodide (PI), and then analyzed by flow cytometry. * p < 0.05 versus different groups. One representative experiment of three is shown.

Article Snippet: To measure RhoA activity and cell adhesion, we performed in vitro experiments using a G-LISA RhoA Activation Assay Biochem kit (Cytoskeleton Inc., Denver, CO, USA) and a CytoSelect™ 24-Well Cell Adhesion Assay kit, according to the manufacturers’ manuals [ ].

Techniques: Cell Culture, Staining, SDS Page, Imaging, Software, Activity Assay, Activation Assay, Incubation, Labeling, Confocal Microscopy, Flow Cytometry

Journal: Nature Communications

Article Title: FAT1 mutations cause a glomerulotubular nephropathy

doi: 10.1038/ncomms10822

Figure Lengend Snippet: ( a ) Cell lysates from fibroblasts of individuals A4623 were collected and protein level of FAT1 was analysed by western blotting with FAT1–GST antibody. FAT1–GST antibody recognizes the intracellular domain of FAT1 (C-terminal 385 aa of mouse FAT1), thus demonstrating the deficiency of the truncated FAT1 protein (p.P1032Cfs*11) resulting from c.3093_3096del FAT1 mutation in A4623. ( b ) Cell migration assay using the xCELLigence system. Fibroblasts from A4623 show decreased migration compared with control. Note that the decrease in migration of A4623 fibroblasts was partially rescued by the RAC/CDC42 activator II. Each cell index value corresponds to the average of more than triplicates and s.d. is in only one direction for clarity. ( c ) Bar graphs represent the area under curves of b and data respresent the mean+s.d. of three independent experiments. * P <0.05, t -test. ( d ) Effect of FAT1 knockdown on podocyte migration. Differentiated cultured human podocytes transfected with FAT1 siRNA exhibited decreased migration (red line) compared with scrambled siRNA controls (black line). Decreased podocyte migration due to FAT1 knockdown was partially rescued by RAC/CDC42 activator II (green line). Each cell index value corresponds to the average of more than triplicates and s.d. is in only one direction for clarity. ( e ) Bar graphs represent the area under curves of d and data respresent the mean+s.d. of three independent experiments. * P <0.05, t -test. ( f ) Cell–cell adhesion assay using calcein AM demonstrated that decreased cell–cell adhesion in fibroblasts from individual A4623 compared with control fibroblasts. Data respresent the mean±s.d. of more than five independent experiments in f and g . * P <0.05; t -test. ( g ) Knockdown of FAT1 by two different siRNAs in differentiated podocytes resulted in decreased cell–cell adhesion in the calcein AM assay.

Article Snippet: About 1.6 pmol zFat1 MO was injected either alone or together with either 1 μg ml −1 of Rho/Rac/Cdc42 Activator I or 0.5 U ml −1 of Rac/Cdc42 activator II (Cytoskeleton).

Techniques: Western Blot, Mutagenesis, Cell Migration Assay, Migration, Cell Culture, Transfection, Cell Adhesion Assay, Calcein AM Assay

Journal: Nature Communications

Article Title: FAT1 mutations cause a glomerulotubular nephropathy

doi: 10.1038/ncomms10822

Figure Lengend Snippet: ( a ) IMCD3 cells ciliated apically and formed a spheroid containing a central lumen (asterisk) when grown in 3D matrigel culture for 3 days; lumens were perturbed on Fat1 knockdown (shRNA#1 and shRNA#3). Cells were stained for acetylated α-tubulin (red), β-catenin (green) and DAPI (blue). Scale bars, 10 μm. ( b ) Percentage of abnormal spheroids. More than 50 spheroids were examined in each experiment. Data represent the mean+s.d. of three independent experiments in b , c . * P <0.05, t -test. ( c ) The effect of Fat1 knockdown on cell migration. Bar graphs represent the area under curves of d . ( d ) The effect of Fat1 knockdown on cell migration. Compared with baseline (dashed black line), addition of serum strongly increases migration rate in IMCD3 cells with scrambled shRNA (Scr) (solid black line). In contrast, IMCD3 cells stably transfected with Fat1 shRNAs #1 (red continuous line) or #3 (red dashed line) exhibited slower rate of migration. Each cell index value corresponds to the average of more than triplicates and s.d. is in only one direction for clarity. ( e ) Active GTP-bound RHOA precipitated from IMCD3. Cells transfected with scrambled control siRNA versus Fat1 shRNA exhibited no significant difference in relative RHOA activity. This is representative of three experiments. ( f ) Active GTP-bound CDC42 or RAC1 using a GST–PAK1 (CRIB) pull-down assay. Note that Fat1 knockdown leads to a significant decrease in relative CDC42 and RAC1 (31% and 44%, respectively) compared with Scr control cells. This is representative of four experiments. Quantification of e and f is presented in . ( g – h ) fat1 morpholino-oligonucelotide (MO) was injected to Wt1b::GFP transgenic zebrafish. Zebrafish injected with control MO did not produce any phenotype. Depletion of fat1 by a fat1 MO targeting the translation initiation site of zebrafish fat1 caused pronephric cysts (asterisks) in 78 of 325 zebrafish embryos (24%). Scale bars, 100 μm. ( i ) Activator I (Rho/Rac/Cdc42 activator I) reduced cyst formation to 9.7% (26 of 268 embryos), and activator II (Rac/Cdc42 activator II) to 12.0% (59 of 490 embryos). * P <0.001, χ 2 -test.

Article Snippet: About 1.6 pmol zFat1 MO was injected either alone or together with either 1 μg ml −1 of Rho/Rac/Cdc42 Activator I or 0.5 U ml −1 of Rac/Cdc42 activator II (Cytoskeleton).

Techniques: shRNA, Staining, Migration, Stable Transfection, Transfection, Activity Assay, Pull Down Assay, Injection, Transgenic Assay

Journal: Nature Communications

Article Title: FAT1 mutations cause a glomerulotubular nephropathy

doi: 10.1038/ncomms10822

Figure Lengend Snippet: ( a ) Cell lysates from fibroblasts of individuals A4623 were collected and protein level of FAT1 was analysed by western blotting with FAT1–GST antibody. FAT1–GST antibody recognizes the intracellular domain of FAT1 (C-terminal 385 aa of mouse FAT1), thus demonstrating the deficiency of the truncated FAT1 protein (p.P1032Cfs*11) resulting from c.3093_3096del FAT1 mutation in A4623. ( b ) Cell migration assay using the xCELLigence system. Fibroblasts from A4623 show decreased migration compared with control. Note that the decrease in migration of A4623 fibroblasts was partially rescued by the RAC/CDC42 activator II. Each cell index value corresponds to the average of more than triplicates and s.d. is in only one direction for clarity. ( c ) Bar graphs represent the area under curves of b and data respresent the mean+s.d. of three independent experiments. * P <0.05, t -test. ( d ) Effect of FAT1 knockdown on podocyte migration. Differentiated cultured human podocytes transfected with FAT1 siRNA exhibited decreased migration (red line) compared with scrambled siRNA controls (black line). Decreased podocyte migration due to FAT1 knockdown was partially rescued by RAC/CDC42 activator II (green line). Each cell index value corresponds to the average of more than triplicates and s.d. is in only one direction for clarity. ( e ) Bar graphs represent the area under curves of d and data respresent the mean+s.d. of three independent experiments. * P <0.05, t -test. ( f ) Cell–cell adhesion assay using calcein AM demonstrated that decreased cell–cell adhesion in fibroblasts from individual A4623 compared with control fibroblasts. Data respresent the mean±s.d. of more than five independent experiments in f and g . * P <0.05; t -test. ( g ) Knockdown of FAT1 by two different siRNAs in differentiated podocytes resulted in decreased cell–cell adhesion in the calcein AM assay.

Article Snippet: Rho/Rac/Cdc42 activator I and Rac/Cdc42 activator II were purchased from the Cytoskeleton, Inc.

Techniques: Western Blot, Mutagenesis, Cell Migration Assay, Migration, Cell Culture, Transfection, Cell Adhesion Assay, Calcein AM Assay

Journal: Nature Communications

Article Title: FAT1 mutations cause a glomerulotubular nephropathy

doi: 10.1038/ncomms10822

Figure Lengend Snippet: ( a ) IMCD3 cells ciliated apically and formed a spheroid containing a central lumen (asterisk) when grown in 3D matrigel culture for 3 days; lumens were perturbed on Fat1 knockdown (shRNA#1 and shRNA#3). Cells were stained for acetylated α-tubulin (red), β-catenin (green) and DAPI (blue). Scale bars, 10 μm. ( b ) Percentage of abnormal spheroids. More than 50 spheroids were examined in each experiment. Data represent the mean+s.d. of three independent experiments in b , c . * P <0.05, t -test. ( c ) The effect of Fat1 knockdown on cell migration. Bar graphs represent the area under curves of d . ( d ) The effect of Fat1 knockdown on cell migration. Compared with baseline (dashed black line), addition of serum strongly increases migration rate in IMCD3 cells with scrambled shRNA (Scr) (solid black line). In contrast, IMCD3 cells stably transfected with Fat1 shRNAs #1 (red continuous line) or #3 (red dashed line) exhibited slower rate of migration. Each cell index value corresponds to the average of more than triplicates and s.d. is in only one direction for clarity. ( e ) Active GTP-bound RHOA precipitated from IMCD3. Cells transfected with scrambled control siRNA versus Fat1 shRNA exhibited no significant difference in relative RHOA activity. This is representative of three experiments. ( f ) Active GTP-bound CDC42 or RAC1 using a GST–PAK1 (CRIB) pull-down assay. Note that Fat1 knockdown leads to a significant decrease in relative CDC42 and RAC1 (31% and 44%, respectively) compared with Scr control cells. This is representative of four experiments. Quantification of e and f is presented in . ( g – h ) fat1 morpholino-oligonucelotide (MO) was injected to Wt1b::GFP transgenic zebrafish. Zebrafish injected with control MO did not produce any phenotype. Depletion of fat1 by a fat1 MO targeting the translation initiation site of zebrafish fat1 caused pronephric cysts (asterisks) in 78 of 325 zebrafish embryos (24%). Scale bars, 100 μm. ( i ) Activator I (Rho/Rac/Cdc42 activator I) reduced cyst formation to 9.7% (26 of 268 embryos), and activator II (Rac/Cdc42 activator II) to 12.0% (59 of 490 embryos). * P <0.001, χ 2 -test.

Article Snippet: Rho/Rac/Cdc42 activator I and Rac/Cdc42 activator II were purchased from the Cytoskeleton, Inc.

Techniques: shRNA, Staining, Migration, Stable Transfection, Transfection, Activity Assay, Pull Down Assay, Injection, Transgenic Assay

Journal: Archives of Toxicology

Article Title: Combustion-derived particles from biomass sources differently promote epithelial-to-mesenchymal transition on A549 cells

doi: 10.1007/s00204-021-02983-8

Figure Lengend Snippet: Immunofluorescence of the EMT epithelial marker E-cadherin on A549 cells after 72 h of exposure to BCDPs (2.5 µg/cm 2 ). Nuclei are stained with DAPI (blue); E-cadherin with rabbit anti-E-cadherin (green) antibody and F-actin with rhodamine-phalloidin (red). Scale bar = 50 µm (color figure online)

Article Snippet: Cells stained with E-cadherin were also stained for 20 min with rhodamine phalloidin (Cytoskeleton Inc., Denver, CO, US) for the evaluation of actin changes.

Techniques: Immunofluorescence, Marker, Staining