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    ATCC transfections
    Transfections, supplied by ATCC, used in various techniques. Bioz Stars score: 96/100, based on 900 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ATCC 5637 bladder epithelial cells
    A gain-of-function screen for in vitro pod formation. ( A ) Schematic of the screen. Orange shapes represent individual <t>bladder</t> <t>epithelial</t> <t>cells,</t> light blue represents nuclei, and green represents aggregates of fluorescent E. coli . In the actual screen, <t>5637</t> cells at ∼10 6 cells per well in a 6- or 12-well plate are infected with E. coli (either a transconjugant library or individual strains) at an MOI of 10 for 2 h, then a high-dose (100 μg/mL for 2 h) and a low-dose (10 μg/mL overnight) gentamycin treatment is used to kill extracellular bacteria; then, gentamycin is removed, 0.025% saponin is added for 30 min then washed away, and cells are observed for pod formation. A representative image of green fluorescence ( E. coli ) 6 h after saponin treatment and removal is shown at the bottom right of the panel. (Scale bar, 100 μm.) ( B ) Whole-genome sequencing of 5 pod-forming transconjugants (red) and 18 additional transconjugants (magenta and black; see text for details) ( y axis). The transferred regions for each clone are shown in red or magenta (pod-forming transconjugants) or black (non-pod-forming transconjugants), based on the MG1655 coordinates ( x axis). The left-most and right-most vertical dotted lines indicate the common region of UTI89 transferred to the clones depicted in red. The middle vertical dotted line delimits the left border of the common region of UTI89 transferred to all pod-forming transconjugants. ( C ) Genetic map of the common overlapping region for all pod-forming transconjugants. Notations are as in Fig. 1 C . Magenta line between the UTI89 and MG1655 coordinate axes at the bottom denotes the common overlapping region of all pod-forming clones. ( D ) Representative images of the pod-forming assay. Each strain is represented by three images; the smaller images on the top are the DAPI (blue) and wheat germ agglutinin (orange) channels ( Left ) and GFP (green) ( Right ). The larger image is a merge of all channels. White arrows indicate identified large pods. Scale bar shown at the bottom is 50 μm for larger images. Scale bar (50 μm) for smaller images is in white in the MG1655/pChu GFP image. ( E ) Box-and-whisker plot of area (in pixels) for all intracellular structures identified for UTI89 strains. Log (base 10) of the area is indicated on the x axis, strain genotype on the y axis. Center line for each plot represents the median, boxes indicate the first and third quartiles, and whiskers are set at 1.5× the interquartile range. Open circles represent outliers outside the range of the whiskers. A violin plot is superimposed on each box, depicting the distribution of all the data. The vertical dotted line indicates 10,000 pixels. ns, P > 0.05; * P
    5637 Bladder Epithelial Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    A gain-of-function screen for in vitro pod formation. ( A ) Schematic of the screen. Orange shapes represent individual bladder epithelial cells, light blue represents nuclei, and green represents aggregates of fluorescent E. coli . In the actual screen, 5637 cells at ∼10 6 cells per well in a 6- or 12-well plate are infected with E. coli (either a transconjugant library or individual strains) at an MOI of 10 for 2 h, then a high-dose (100 μg/mL for 2 h) and a low-dose (10 μg/mL overnight) gentamycin treatment is used to kill extracellular bacteria; then, gentamycin is removed, 0.025% saponin is added for 30 min then washed away, and cells are observed for pod formation. A representative image of green fluorescence ( E. coli ) 6 h after saponin treatment and removal is shown at the bottom right of the panel. (Scale bar, 100 μm.) ( B ) Whole-genome sequencing of 5 pod-forming transconjugants (red) and 18 additional transconjugants (magenta and black; see text for details) ( y axis). The transferred regions for each clone are shown in red or magenta (pod-forming transconjugants) or black (non-pod-forming transconjugants), based on the MG1655 coordinates ( x axis). The left-most and right-most vertical dotted lines indicate the common region of UTI89 transferred to the clones depicted in red. The middle vertical dotted line delimits the left border of the common region of UTI89 transferred to all pod-forming transconjugants. ( C ) Genetic map of the common overlapping region for all pod-forming transconjugants. Notations are as in Fig. 1 C . Magenta line between the UTI89 and MG1655 coordinate axes at the bottom denotes the common overlapping region of all pod-forming clones. ( D ) Representative images of the pod-forming assay. Each strain is represented by three images; the smaller images on the top are the DAPI (blue) and wheat germ agglutinin (orange) channels ( Left ) and GFP (green) ( Right ). The larger image is a merge of all channels. White arrows indicate identified large pods. Scale bar shown at the bottom is 50 μm for larger images. Scale bar (50 μm) for smaller images is in white in the MG1655/pChu GFP image. ( E ) Box-and-whisker plot of area (in pixels) for all intracellular structures identified for UTI89 strains. Log (base 10) of the area is indicated on the x axis, strain genotype on the y axis. Center line for each plot represents the median, boxes indicate the first and third quartiles, and whiskers are set at 1.5× the interquartile range. Open circles represent outliers outside the range of the whiskers. A violin plot is superimposed on each box, depicting the distribution of all the data. The vertical dotted line indicates 10,000 pixels. ns, P > 0.05; * P

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Development of mass allelic exchange, a technique to enable sexual genetics in Escherichia coli

    doi: 10.1073/pnas.2105458119

    Figure Lengend Snippet: A gain-of-function screen for in vitro pod formation. ( A ) Schematic of the screen. Orange shapes represent individual bladder epithelial cells, light blue represents nuclei, and green represents aggregates of fluorescent E. coli . In the actual screen, 5637 cells at ∼10 6 cells per well in a 6- or 12-well plate are infected with E. coli (either a transconjugant library or individual strains) at an MOI of 10 for 2 h, then a high-dose (100 μg/mL for 2 h) and a low-dose (10 μg/mL overnight) gentamycin treatment is used to kill extracellular bacteria; then, gentamycin is removed, 0.025% saponin is added for 30 min then washed away, and cells are observed for pod formation. A representative image of green fluorescence ( E. coli ) 6 h after saponin treatment and removal is shown at the bottom right of the panel. (Scale bar, 100 μm.) ( B ) Whole-genome sequencing of 5 pod-forming transconjugants (red) and 18 additional transconjugants (magenta and black; see text for details) ( y axis). The transferred regions for each clone are shown in red or magenta (pod-forming transconjugants) or black (non-pod-forming transconjugants), based on the MG1655 coordinates ( x axis). The left-most and right-most vertical dotted lines indicate the common region of UTI89 transferred to the clones depicted in red. The middle vertical dotted line delimits the left border of the common region of UTI89 transferred to all pod-forming transconjugants. ( C ) Genetic map of the common overlapping region for all pod-forming transconjugants. Notations are as in Fig. 1 C . Magenta line between the UTI89 and MG1655 coordinate axes at the bottom denotes the common overlapping region of all pod-forming clones. ( D ) Representative images of the pod-forming assay. Each strain is represented by three images; the smaller images on the top are the DAPI (blue) and wheat germ agglutinin (orange) channels ( Left ) and GFP (green) ( Right ). The larger image is a merge of all channels. White arrows indicate identified large pods. Scale bar shown at the bottom is 50 μm for larger images. Scale bar (50 μm) for smaller images is in white in the MG1655/pChu GFP image. ( E ) Box-and-whisker plot of area (in pixels) for all intracellular structures identified for UTI89 strains. Log (base 10) of the area is indicated on the x axis, strain genotype on the y axis. Center line for each plot represents the median, boxes indicate the first and third quartiles, and whiskers are set at 1.5× the interquartile range. Open circles represent outliers outside the range of the whiskers. A violin plot is superimposed on each box, depicting the distribution of all the data. The vertical dotted line indicates 10,000 pixels. ns, P > 0.05; * P

    Article Snippet: The 5637 bladder epithelial cells were obtained from ATCC (product HTB-9).

    Techniques: In Vitro, Infection, Fluorescence, Sequencing, Clone Assay, Whisker Assay

    Mice transplanted with mAIM2-overexpressed MBT-2 cells show prolonged survival and delayed tumor progression. (A) Experimental schematic. Mice were transplanted with 1.5 × 10 4 mAIM2-overexpressed MB49 or MBT-2 bladder carcinoma cells and the indicated control cells on day 0 and sacrificed on day 14 or 12, respectively. (B) Images of bladders from the indicated groups. (C) Bladder weights of orthotopic tumors from the indicated groups. (D) Representative pictures of H and E staining of orthotopic MB49 and MBT-2 transplanted BLCA model. Inflammatory cells significantly infiltrate mAIM2-overexpressed tumors. Black arrows indicate inflammatory cells. (E) Representative pictures of immunohistochemistry (IHC) staining of CD11b for MBT-2 xenografts. CD11b + cells were significantly enriched in mAIM2-overexpressed tumors. Red arrows indicate inflammatory cells. (F) Survival curves of mice transplanted with mAIM2-overexpressed MBT-2 cells or the control. (G) Fluorescence of mAIM2-overexpressed MBT-2 cells or the control cells after orthotopic transplantation were monitored by IVIS at indicated days. Data in (C) are presented as the means ± SD. p -values in (C) were calculated using the student’s t -test. Survival rate (F) is compared by Log-Rank (Mantel-Cox) test. * indicates p

    Journal: Frontiers in Pharmacology

    Article Title: AIM2 inflammasome activation benefits the therapeutic effect of BCG in bladder carcinoma

    doi: 10.3389/fphar.2022.1050774

    Figure Lengend Snippet: Mice transplanted with mAIM2-overexpressed MBT-2 cells show prolonged survival and delayed tumor progression. (A) Experimental schematic. Mice were transplanted with 1.5 × 10 4 mAIM2-overexpressed MB49 or MBT-2 bladder carcinoma cells and the indicated control cells on day 0 and sacrificed on day 14 or 12, respectively. (B) Images of bladders from the indicated groups. (C) Bladder weights of orthotopic tumors from the indicated groups. (D) Representative pictures of H and E staining of orthotopic MB49 and MBT-2 transplanted BLCA model. Inflammatory cells significantly infiltrate mAIM2-overexpressed tumors. Black arrows indicate inflammatory cells. (E) Representative pictures of immunohistochemistry (IHC) staining of CD11b for MBT-2 xenografts. CD11b + cells were significantly enriched in mAIM2-overexpressed tumors. Red arrows indicate inflammatory cells. (F) Survival curves of mice transplanted with mAIM2-overexpressed MBT-2 cells or the control. (G) Fluorescence of mAIM2-overexpressed MBT-2 cells or the control cells after orthotopic transplantation were monitored by IVIS at indicated days. Data in (C) are presented as the means ± SD. p -values in (C) were calculated using the student’s t -test. Survival rate (F) is compared by Log-Rank (Mantel-Cox) test. * indicates p

    Article Snippet: The human BLCA cell line 5637 and MGH-U3 were purchased from the American Type Culture Collection (ATCC) and Leibniz-Institut Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), respectively.

    Techniques: Mouse Assay, Staining, Immunohistochemistry, Fluorescence, Transplantation Assay

    A schematic model that AIM2 inflammasome promotes the sensitivity of BCG therapy in BLCA. During intravesical BCG instillation of mouse bladders, the DNA fragments of BCG and dead tumor cells gain access to the cytosol and are sensed by AIM2. Overexpressed AIM2 assembles more ASC-caspase-1 inflammasome, which activates more IL-1β, IL-18, and GSDMD. The activated GSDMD (GSDMD-N) forms pores on the plasma membrane, causing pyroptosis. Inflammation activation elevates the recruitment of CD11b + cells (myeloid marker, including neutrophils, monocytes, macrophages, NK cells, and so forth) to inhibit the tumor progression. However, cells expressed low level of AIM2 fails to secrete sufficient cytokines to recruit immune cells, leading to tumor progression.

    Journal: Frontiers in Pharmacology

    Article Title: AIM2 inflammasome activation benefits the therapeutic effect of BCG in bladder carcinoma

    doi: 10.3389/fphar.2022.1050774

    Figure Lengend Snippet: A schematic model that AIM2 inflammasome promotes the sensitivity of BCG therapy in BLCA. During intravesical BCG instillation of mouse bladders, the DNA fragments of BCG and dead tumor cells gain access to the cytosol and are sensed by AIM2. Overexpressed AIM2 assembles more ASC-caspase-1 inflammasome, which activates more IL-1β, IL-18, and GSDMD. The activated GSDMD (GSDMD-N) forms pores on the plasma membrane, causing pyroptosis. Inflammation activation elevates the recruitment of CD11b + cells (myeloid marker, including neutrophils, monocytes, macrophages, NK cells, and so forth) to inhibit the tumor progression. However, cells expressed low level of AIM2 fails to secrete sufficient cytokines to recruit immune cells, leading to tumor progression.

    Article Snippet: The human BLCA cell line 5637 and MGH-U3 were purchased from the American Type Culture Collection (ATCC) and Leibniz-Institut Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), respectively.

    Techniques: Activation Assay, Marker

    The innate immune sensor AIM2 expression level is positively correlated with the prognosis of BLCA patients. (A) Kaplan-Meier curves showing the overall survival (OS) time of patients with BLCA in the TCGA cohort. (B) Kaplan-Meier curves presenting the progression-free survival (PFS) of patients with BLCA in the TCGA cohort. (C) H and E staining of human high- and low-grade BLCA samples. (D) The protein level of AIM2, EpCAM (representing the epithelial component of bladder cancer), and Vimentin (the biomarker of the stromal component) by 4-color multiplex immunofluorescence (MIHC) staining assays. Survival rate (A,B) is compared by Log-Rank (Mantel-Cox) test.

    Journal: Frontiers in Pharmacology

    Article Title: AIM2 inflammasome activation benefits the therapeutic effect of BCG in bladder carcinoma

    doi: 10.3389/fphar.2022.1050774

    Figure Lengend Snippet: The innate immune sensor AIM2 expression level is positively correlated with the prognosis of BLCA patients. (A) Kaplan-Meier curves showing the overall survival (OS) time of patients with BLCA in the TCGA cohort. (B) Kaplan-Meier curves presenting the progression-free survival (PFS) of patients with BLCA in the TCGA cohort. (C) H and E staining of human high- and low-grade BLCA samples. (D) The protein level of AIM2, EpCAM (representing the epithelial component of bladder cancer), and Vimentin (the biomarker of the stromal component) by 4-color multiplex immunofluorescence (MIHC) staining assays. Survival rate (A,B) is compared by Log-Rank (Mantel-Cox) test.

    Article Snippet: The human BLCA cell line 5637 and MGH-U3 were purchased from the American Type Culture Collection (ATCC) and Leibniz-Institut Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), respectively.

    Techniques: Expressing, Staining, Biomarker Assay, Multiplex Assay, Immunofluorescence

    OPTN regulates cell adhesion to the extracellular matrix. a Confocal sections showing enhanced formation of integrin alpha-5-positive (ITGA5) and paxillin-positive focal adhesions (FA) in siOPTN transfected HUVEC plated 2 h on fibronectin-coated coverslips in defined medium. Bar = 10 μm. Representative experiment, n = 3. b Number and mean area of FA structures positive for paxillin in siCtrl or siOPTN transfected HUVEC were quantified. Results are expressed as fold change to siCtrl condition, set to 1 (boxplots). n = 30 SiCtrl cells and 27 SiOPTN cells, three independent experiments. Wilcoxon rank sum test (two-tailed p value): * p = 0.0286, ** p = 0.0043. c Adhesion of siCtrl or siOPTN transfected HUVEC on 1.25, 2.5 and 5 µg/ml of fibronectin was assessed upon short-term plating of cells in triplicate in 96-well plates. Bars represent the extent of adhesion to fibronectin as function of absorbance upon elution of the crystal violet dye. Means ± SD, n = 3 independent experiments (single dots). Two-way ANOVA with Sidak’s multiple comparison test p value: siCtrl-siOPTN 1.25 * p = 0.0080; siCtrl-siOPTN 2.5 * p = 0.0018; siCtrl-siOPTN 5 *** p = 0.0001. d Cortical elastic modulus determined by AFM on cells plated for 1 h 30 on Fibronectin plates in defined medium, boxplots. siCtrl ( n = 21 cells), siOPTN ( n = 29 cells) and siOPTN cells treated with CK-666 (250 μM) to inhibit Arp2/3 activity ( n = 29 cells). One-way ANOVA with Dunnett’s correction test for multiple comparisons p value: **** p ≤ 0.0001. e Quantification of filamentous actin levels (F-Actin) to total actin (F + G-actin) in control and OPTN knocked down cells. Mean values ± SD, n = 4 independent experiments (single dots). Wilcoxon rank sum test (two-tailed): * p = 0.0286. f HUVEC transfected with siCtrl or siOPTN were G0 Synchronized and then plated at low confluence in defined medium on fibronectin-coated hydrogels of indicated stiffness (kPa) for 1 h. Immunoblots anti-phospho-FAK-tyr397, anti-phospho-Src-tyr416, anti-phospho-Paxilin-tyr118 and anti-phospho-p130CAS-tyr410, together with Anti-FAK, Src, Paxillin, p130CAS and GAPDH as control were performed. Graphs represent quantification of phospho-FAK(tyr-397), phospho-Src(tyr-416), phospho-Paxillin(tyr-118) and phospho-p130CAS(tyr-410) signals to total proteins and normalized to GAPDH. Densitometry was normalized to value on 0.2 kPa ECM, set to 1. Bars represent means ± SD, n = 3 independent experiments (single dots). Wilcoxon rank sum test (two-tailed p value): p-FAK ns, p = 0.1508; p-Src ** p = 0.0079; p-PAX * p = 0.0317; p-P130Cas ** p = 0.0079.

    Journal: Nature Communications

    Article Title: Optineurin links Hace1-dependent Rac ubiquitylation to integrin-mediated mechanotransduction to control bacterial invasion and cell division

    doi: 10.1038/s41467-022-33803-x

    Figure Lengend Snippet: OPTN regulates cell adhesion to the extracellular matrix. a Confocal sections showing enhanced formation of integrin alpha-5-positive (ITGA5) and paxillin-positive focal adhesions (FA) in siOPTN transfected HUVEC plated 2 h on fibronectin-coated coverslips in defined medium. Bar = 10 μm. Representative experiment, n = 3. b Number and mean area of FA structures positive for paxillin in siCtrl or siOPTN transfected HUVEC were quantified. Results are expressed as fold change to siCtrl condition, set to 1 (boxplots). n = 30 SiCtrl cells and 27 SiOPTN cells, three independent experiments. Wilcoxon rank sum test (two-tailed p value): * p = 0.0286, ** p = 0.0043. c Adhesion of siCtrl or siOPTN transfected HUVEC on 1.25, 2.5 and 5 µg/ml of fibronectin was assessed upon short-term plating of cells in triplicate in 96-well plates. Bars represent the extent of adhesion to fibronectin as function of absorbance upon elution of the crystal violet dye. Means ± SD, n = 3 independent experiments (single dots). Two-way ANOVA with Sidak’s multiple comparison test p value: siCtrl-siOPTN 1.25 * p = 0.0080; siCtrl-siOPTN 2.5 * p = 0.0018; siCtrl-siOPTN 5 *** p = 0.0001. d Cortical elastic modulus determined by AFM on cells plated for 1 h 30 on Fibronectin plates in defined medium, boxplots. siCtrl ( n = 21 cells), siOPTN ( n = 29 cells) and siOPTN cells treated with CK-666 (250 μM) to inhibit Arp2/3 activity ( n = 29 cells). One-way ANOVA with Dunnett’s correction test for multiple comparisons p value: **** p ≤ 0.0001. e Quantification of filamentous actin levels (F-Actin) to total actin (F + G-actin) in control and OPTN knocked down cells. Mean values ± SD, n = 4 independent experiments (single dots). Wilcoxon rank sum test (two-tailed): * p = 0.0286. f HUVEC transfected with siCtrl or siOPTN were G0 Synchronized and then plated at low confluence in defined medium on fibronectin-coated hydrogels of indicated stiffness (kPa) for 1 h. Immunoblots anti-phospho-FAK-tyr397, anti-phospho-Src-tyr416, anti-phospho-Paxilin-tyr118 and anti-phospho-p130CAS-tyr410, together with Anti-FAK, Src, Paxillin, p130CAS and GAPDH as control were performed. Graphs represent quantification of phospho-FAK(tyr-397), phospho-Src(tyr-416), phospho-Paxillin(tyr-118) and phospho-p130CAS(tyr-410) signals to total proteins and normalized to GAPDH. Densitometry was normalized to value on 0.2 kPa ECM, set to 1. Bars represent means ± SD, n = 3 independent experiments (single dots). Wilcoxon rank sum test (two-tailed p value): p-FAK ns, p = 0.1508; p-Src ** p = 0.0079; p-PAX * p = 0.0317; p-P130Cas ** p = 0.0079.

    Article Snippet: Unless for experiments shown in Supplementary Fig. , where 5637 cells (ATCC HTB-9) were used, all experiments were performed on human vein endothelial cells (HUVECs) before passage 5, that are primary cells, an important feature to keep normal ubiquitin-mediated Rac regulation.

    Techniques: Transfection, Two Tailed Test, Activity Assay, Western Blot

    OPTN controls cellular invasion by Ec and impairs CNF1 and FimH-mediated mechanochemical signaling of integrins. a Quantification of internalized Ec 30 min after gentamicin treatment in siCTRL (control) or siOPTN (optineurin) HUVEC cells on plastic treated with 1 nM CNF1. Graph displays absolute CFU/ml. Boxplots of n = 5 independent experiments and three replicates per condition. Wilcoxon rank sum test (two-tailed): *** p = 0.004. b Rac1 activation in HUVEC transfected with siCtrl or siOPTN on plastic and either untreated (UT) or treated 2 h with 1 nM CNF1 (CNF1), measured by GST-PAK affinity purification. Rac1-GTP levels were quantified and reported to total Rac1 and GAPDH. Fold change to untreated siCtrl condition, set to 1. Mean ± SD, n = 3 independent experiments (single dots). One-way ANOVA with Dunnett’s test for multiple comparisons: * p = 0.0460. c Cortical elastic modulus determined by AFM on cells cultured overnight on plastic in complete medium and either untreated or treated 2 h with 1 nM CNF1. SiCtrl ( n = 45 cells), SiCtrl + CNF1 ( n = 44 cells) and SiOPTN + CNF1 ( n = 41 cells). Boxplots of n = 5 independent experiments and three replicates per condition. One-way ANOVA with Dunnett’s correction test for multiple comparisons p value: *** p = 0.0005; **** p ≤ 0.0001. d Quantification of phospho-protein levels for phospho-FAK (tyr-397), phospho-Paxillin (tyr-118), phospho-Src (tyr-416) and phospho-p130CAS (tyr-410) in siCTRL (white bars) and siOPTN (hatched bars) cells on plastic, either non-treated (−) or after 30 min of Ec infection in presence of 1 nM CNF1. Bars display levels of phospho-proteins relative to GAPDH determined by immunoblotting and normalized to the siCTRL non-treated condition. Mean ± SD of n = 3 (FAK and p130CAS) or n = 4 (SRC and PAX) independent experiments (single dots). Two-way ANOVA with Tukey’s correction test for multiple comparisons: ** p = 0.0021 ≤ 0.01. Total levels of the protein do not change in the experimental conditions. e Quantification of internalized Ec in HUVECs transfected with empty vector (pGFP) or Talin-Head expressing vector (pTLN-head-GFP), after 30 min of gentamicin treatment and under 1 nM CNF1 treatment, MOI:100. Boxplots display absolute number of CFU/ml of n = 3 biologically independent experiments and three replicates per condition. Wilcoxon rank sum test (two-tailed): **** p ≤ 0.0001.

    Journal: Nature Communications

    Article Title: Optineurin links Hace1-dependent Rac ubiquitylation to integrin-mediated mechanotransduction to control bacterial invasion and cell division

    doi: 10.1038/s41467-022-33803-x

    Figure Lengend Snippet: OPTN controls cellular invasion by Ec and impairs CNF1 and FimH-mediated mechanochemical signaling of integrins. a Quantification of internalized Ec 30 min after gentamicin treatment in siCTRL (control) or siOPTN (optineurin) HUVEC cells on plastic treated with 1 nM CNF1. Graph displays absolute CFU/ml. Boxplots of n = 5 independent experiments and three replicates per condition. Wilcoxon rank sum test (two-tailed): *** p = 0.004. b Rac1 activation in HUVEC transfected with siCtrl or siOPTN on plastic and either untreated (UT) or treated 2 h with 1 nM CNF1 (CNF1), measured by GST-PAK affinity purification. Rac1-GTP levels were quantified and reported to total Rac1 and GAPDH. Fold change to untreated siCtrl condition, set to 1. Mean ± SD, n = 3 independent experiments (single dots). One-way ANOVA with Dunnett’s test for multiple comparisons: * p = 0.0460. c Cortical elastic modulus determined by AFM on cells cultured overnight on plastic in complete medium and either untreated or treated 2 h with 1 nM CNF1. SiCtrl ( n = 45 cells), SiCtrl + CNF1 ( n = 44 cells) and SiOPTN + CNF1 ( n = 41 cells). Boxplots of n = 5 independent experiments and three replicates per condition. One-way ANOVA with Dunnett’s correction test for multiple comparisons p value: *** p = 0.0005; **** p ≤ 0.0001. d Quantification of phospho-protein levels for phospho-FAK (tyr-397), phospho-Paxillin (tyr-118), phospho-Src (tyr-416) and phospho-p130CAS (tyr-410) in siCTRL (white bars) and siOPTN (hatched bars) cells on plastic, either non-treated (−) or after 30 min of Ec infection in presence of 1 nM CNF1. Bars display levels of phospho-proteins relative to GAPDH determined by immunoblotting and normalized to the siCTRL non-treated condition. Mean ± SD of n = 3 (FAK and p130CAS) or n = 4 (SRC and PAX) independent experiments (single dots). Two-way ANOVA with Tukey’s correction test for multiple comparisons: ** p = 0.0021 ≤ 0.01. Total levels of the protein do not change in the experimental conditions. e Quantification of internalized Ec in HUVECs transfected with empty vector (pGFP) or Talin-Head expressing vector (pTLN-head-GFP), after 30 min of gentamicin treatment and under 1 nM CNF1 treatment, MOI:100. Boxplots display absolute number of CFU/ml of n = 3 biologically independent experiments and three replicates per condition. Wilcoxon rank sum test (two-tailed): **** p ≤ 0.0001.

    Article Snippet: Unless for experiments shown in Supplementary Fig. , where 5637 cells (ATCC HTB-9) were used, all experiments were performed on human vein endothelial cells (HUVECs) before passage 5, that are primary cells, an important feature to keep normal ubiquitin-mediated Rac regulation.

    Techniques: Two Tailed Test, Activation Assay, Transfection, Affinity Purification, Cell Culture, Infection, Plasmid Preparation, Expressing

    ECM stiffness and integrin mechanoactivation control cnf1-producing uropathogenic E. coli invasion. a Internalized Ec or FimH-deleted Ec (EcΔFimH) 30 min after gentamicin treatment in HUVEC cultured on ECM of increasing stiffness (8, 25, 50 kPa) in complete medium. CNF1 was added at 1 nM where indicated (+). An MOI of 100 was used. Boxplots displays absolute number of CFU/ml, n = 3 independent experiments and at least three replicates per condition. One-way ANOVA with Dunnett’s correction test for multiple comparisons: ns, p > 0.999 not significant; * p = 0.0147; ** p = 0.0028; **** p ≤ 0.0001. b Representative immunoblots of phospho-protein levels for phospho-FAK(tyr-397), phospho-Paxillin(tyr-118), phospho-Src (tyr-416) and phospho-p130CAS(tyr-410) in response to HUVEC infection for 30 or 60 min by Ec or EcΔFimH (MOI 100), in presence of 1 nM CNF1 toxin when indicated. GAPDH was used as loading control. c Quantification of phospho-protein levels for phospho-FAK(tyr-397), phospho-Paxillin(tyr-118), phospho-Src(tyr-416) and phospho-p130CAS(tyr-410) relative to levels of GAPDH and normalized to the non-treated condition. Bars represent means ± SD, n3 independent experiments (single dots). Two-way ANOVA with Tukey’s correction test for multiple comparisons: ** p = 0.0016. Expression of FAK, PAX, Src and p130CAS are not modified in our experimental conditions. d Quantification of internalized Ec in HUVECs 30 min after gentamicin treatment in HUVECs knocked down for Vinculin(siVCL), Talin(siTLN) or Icap-1(siICAP), in presence of 1 nM CNF1. Boxplots display the quantification of CFU/ml relative to the control condition (siCTRL) from n = 3(siVCL, siTLN) and n = 5(siICAP) independent experiments and three replicates per condition. One-way ANOVA with Dunnett’s correction test for multiple comparisons: ***, siCtrl-siICAP1 p = 0.0005, siCtrl-siVCL p = 0.0007; ****, siCtrl-siTLN p ≤ 0.0001. e Quantification of internalized Ec in HUVECs either non-treated or treated with blebbistatin (20 μM) or CK666 (250 μM) 30 min after gentamicin treatment. Boxplots displays absolute number of CFU/ml, n = 4 independent experiment and three replicates per condition. One-way ANOVA with Dunnett’s correction test for multiple comparisons: **** p ≤ 0.0001.

    Journal: Nature Communications

    Article Title: Optineurin links Hace1-dependent Rac ubiquitylation to integrin-mediated mechanotransduction to control bacterial invasion and cell division

    doi: 10.1038/s41467-022-33803-x

    Figure Lengend Snippet: ECM stiffness and integrin mechanoactivation control cnf1-producing uropathogenic E. coli invasion. a Internalized Ec or FimH-deleted Ec (EcΔFimH) 30 min after gentamicin treatment in HUVEC cultured on ECM of increasing stiffness (8, 25, 50 kPa) in complete medium. CNF1 was added at 1 nM where indicated (+). An MOI of 100 was used. Boxplots displays absolute number of CFU/ml, n = 3 independent experiments and at least three replicates per condition. One-way ANOVA with Dunnett’s correction test for multiple comparisons: ns, p > 0.999 not significant; * p = 0.0147; ** p = 0.0028; **** p ≤ 0.0001. b Representative immunoblots of phospho-protein levels for phospho-FAK(tyr-397), phospho-Paxillin(tyr-118), phospho-Src (tyr-416) and phospho-p130CAS(tyr-410) in response to HUVEC infection for 30 or 60 min by Ec or EcΔFimH (MOI 100), in presence of 1 nM CNF1 toxin when indicated. GAPDH was used as loading control. c Quantification of phospho-protein levels for phospho-FAK(tyr-397), phospho-Paxillin(tyr-118), phospho-Src(tyr-416) and phospho-p130CAS(tyr-410) relative to levels of GAPDH and normalized to the non-treated condition. Bars represent means ± SD, n3 independent experiments (single dots). Two-way ANOVA with Tukey’s correction test for multiple comparisons: ** p = 0.0016. Expression of FAK, PAX, Src and p130CAS are not modified in our experimental conditions. d Quantification of internalized Ec in HUVECs 30 min after gentamicin treatment in HUVECs knocked down for Vinculin(siVCL), Talin(siTLN) or Icap-1(siICAP), in presence of 1 nM CNF1. Boxplots display the quantification of CFU/ml relative to the control condition (siCTRL) from n = 3(siVCL, siTLN) and n = 5(siICAP) independent experiments and three replicates per condition. One-way ANOVA with Dunnett’s correction test for multiple comparisons: ***, siCtrl-siICAP1 p = 0.0005, siCtrl-siVCL p = 0.0007; ****, siCtrl-siTLN p ≤ 0.0001. e Quantification of internalized Ec in HUVECs either non-treated or treated with blebbistatin (20 μM) or CK666 (250 μM) 30 min after gentamicin treatment. Boxplots displays absolute number of CFU/ml, n = 4 independent experiment and three replicates per condition. One-way ANOVA with Dunnett’s correction test for multiple comparisons: **** p ≤ 0.0001.

    Article Snippet: Unless for experiments shown in Supplementary Fig. , where 5637 cells (ATCC HTB-9) were used, all experiments were performed on human vein endothelial cells (HUVECs) before passage 5, that are primary cells, an important feature to keep normal ubiquitin-mediated Rac regulation.

    Techniques: Cell Culture, Western Blot, Infection, Expressing, Modification

    Proteome analysis of extracellular matrix stiffness-induced changes unveils regulation of OPTN expression. a Heatmap plot of the Z -scored LFQ intensities from significantly regulated proteins after non-supervised hierarchical clustering. The list of proteins can be found in Supplementary Data 3 . b Graphical representation of the functional enrichment analysis performed using DAVID for the list of significantly upregulated proteins. A 0.1 EASE score cut-off allowed to gather a group of enriched UP_KEYWORDS (in yellow) and KEGG pathways (in green). Results from non-CNF1-treated cells (NT) and CNF1 toxin-treated cells (CNF1) are analyzed separately. c Network of association between the HACE1 protein interactome and the five sets of proteins belonging to common enriched UP_Keywords retrieved from the non-CNF1-treated cells (NT) and CNF1 toxin-treated cells (CNF1) analysis. Rhomboid boxes represent common UP_Keywords. White nodes in the network represent proteins detected in the dataset that are not significantly upregulated. Yellow nodes correspond to upregulated proteins in any of the pairwise NT comparisons (50 vs. 1, 4 vs. 1 or 50 vs. 4), a dark yellow edge of the node represents upregulation also for the CNF1 samples in any of the pairwise comparisons. Blue nodes correspond to the Hace1 interactome. Larger protein nodes are those that belong both to the protein-protein interaction network and to the UP_Keywords network. d Representative immunoblots anti-OPTN and anti-HACE1 showing endogenous protein levels in HUVECs plated 15 h on fibronectin-coated hydrogels of the indicated stiffness (kPa) in complete medium. Immunoblot anti-GAPDH shows control of protein loading. e Quantification of Optineurin (OPTN) protein level from western blot analysis by densitometry, cells treated as in d . The OPTN signal was normalized to that of GAPDH expressed as fold relative to the signal on 1 kPa ECM, set to 1. Bars represent means ± SD, n = 5 independent experiments (single dots). One-way ANOVA with Dunnett’s correction test for multiple comparisons: *, 1 vs. 4 p = 0.0137; 1 vs. 8 p = 0.0255; 1 vs. 25 p = 0.0255; 1 vs. 50 p = 0.0255.

    Journal: Nature Communications

    Article Title: Optineurin links Hace1-dependent Rac ubiquitylation to integrin-mediated mechanotransduction to control bacterial invasion and cell division

    doi: 10.1038/s41467-022-33803-x

    Figure Lengend Snippet: Proteome analysis of extracellular matrix stiffness-induced changes unveils regulation of OPTN expression. a Heatmap plot of the Z -scored LFQ intensities from significantly regulated proteins after non-supervised hierarchical clustering. The list of proteins can be found in Supplementary Data 3 . b Graphical representation of the functional enrichment analysis performed using DAVID for the list of significantly upregulated proteins. A 0.1 EASE score cut-off allowed to gather a group of enriched UP_KEYWORDS (in yellow) and KEGG pathways (in green). Results from non-CNF1-treated cells (NT) and CNF1 toxin-treated cells (CNF1) are analyzed separately. c Network of association between the HACE1 protein interactome and the five sets of proteins belonging to common enriched UP_Keywords retrieved from the non-CNF1-treated cells (NT) and CNF1 toxin-treated cells (CNF1) analysis. Rhomboid boxes represent common UP_Keywords. White nodes in the network represent proteins detected in the dataset that are not significantly upregulated. Yellow nodes correspond to upregulated proteins in any of the pairwise NT comparisons (50 vs. 1, 4 vs. 1 or 50 vs. 4), a dark yellow edge of the node represents upregulation also for the CNF1 samples in any of the pairwise comparisons. Blue nodes correspond to the Hace1 interactome. Larger protein nodes are those that belong both to the protein-protein interaction network and to the UP_Keywords network. d Representative immunoblots anti-OPTN and anti-HACE1 showing endogenous protein levels in HUVECs plated 15 h on fibronectin-coated hydrogels of the indicated stiffness (kPa) in complete medium. Immunoblot anti-GAPDH shows control of protein loading. e Quantification of Optineurin (OPTN) protein level from western blot analysis by densitometry, cells treated as in d . The OPTN signal was normalized to that of GAPDH expressed as fold relative to the signal on 1 kPa ECM, set to 1. Bars represent means ± SD, n = 5 independent experiments (single dots). One-way ANOVA with Dunnett’s correction test for multiple comparisons: *, 1 vs. 4 p = 0.0137; 1 vs. 8 p = 0.0255; 1 vs. 25 p = 0.0255; 1 vs. 50 p = 0.0255.

    Article Snippet: Unless for experiments shown in Supplementary Fig. , where 5637 cells (ATCC HTB-9) were used, all experiments were performed on human vein endothelial cells (HUVECs) before passage 5, that are primary cells, an important feature to keep normal ubiquitin-mediated Rac regulation.

    Techniques: Expressing, Functional Assay, Western Blot

    OPTN regulates adhesion-mediated cell cycle progression and translation of cyclin D1. a Graph representing the percentage of proliferating cells (S and G2/M phases) assessed by FACS analysis of DNA content. HUVEC transfected with siCtrl or siOPTN were G0 Synchronized(sync) and plated at low confluence on fibronectin-coated hydrogels of indicated stiffness (kPa) for 18 h before cell cycle analysis by FACS. Means ± SD, n = 4 independent experiments (single dots). One-way ANOVA with Tukey’s multiple comparison test: ns, siCtrl8kPa vs. siCtrl 1 kPa p = 0.1501; siOPTN 8 kPa vs. siOPTN 1 kPa p = 0.9997 not significant; Two-way ANOVA with Sidak’s multiple comparison test: siCtrl-siOPTN 1 kPa p = 0.0030. b Immunoblots showing cyclin D1 expression in cells treated as in a . Representative experiment, n = 3. c Level of cyclin D1 mRNA and protein expression in siCtrl or siOPTN transfected cells. HUVECs were G0 synchronized (sync) and then plated on 15 µg/ml fibronectin-coated plates and 20% serum for 18 h (Stim). Cyclin D1 mRNA levels were assessed by RT-qPCR and normalized to siCtrl synchronized cells condition, set to 1. Mean ± SD, n = 3 independent experiments (single dots). Two-way ANOVA with Tukey’s correction for multiple comparisons: ns, p = 0.6364 not significant; ** p = 0.0047. In parallel, cyclin D1 protein levels were determined by immunoblots, GAPDH was used as loading control. d HUVEC transfected with siCtrl or siOPTN were G0 Synchronized (sync) and then plated on 15 µg/ml fibronectin-coated plates and 20% serum for 18 h (Stim), together with Cycloheximide (CHX) at 10 nM. Cells were harvested after 15, 30, 60 min treatment and processed for anti-cyclin D1 immunoblotting. Immunoblots anti-OPTN and anti-GAPDH were performed to control OPTN knockdown and protein loading, respectively. e Quantification of cyclin D1 half-life measured in d . Values are expressed as fold compared to level in untreated condition (time 0), set to 1. Mean ± SD, n = 3. Dashed lines show similar cyclin D1 half-life in siCtrl and siOPTN cells. f Immunoblots showing neo synthesized cyclin D1 upon metabolic labeling. HUVEC transfected with siCtrl or siOPTN were G0 Synchronized and then plated on 15 µg/ml fibronectin-coated plates in defined medium for 18 h. Cells were pulsed with AHA during the last hour. Cell lysates were subjected to Click-iT reaction with Biotin Alkyne followed by streptavidin pulldown. Cyclin D1 and GAPDH neosynthesis was then assessed upon immunoblotting the resolved streptavidin-pulled-down reaction product. Total neosynthesis of proteins was assessed upon immunoblotting the resolved total Click-iT reaction product with streptavidin-HRP (AHA-Proteins). Representative experiment, n = 3.

    Journal: Nature Communications

    Article Title: Optineurin links Hace1-dependent Rac ubiquitylation to integrin-mediated mechanotransduction to control bacterial invasion and cell division

    doi: 10.1038/s41467-022-33803-x

    Figure Lengend Snippet: OPTN regulates adhesion-mediated cell cycle progression and translation of cyclin D1. a Graph representing the percentage of proliferating cells (S and G2/M phases) assessed by FACS analysis of DNA content. HUVEC transfected with siCtrl or siOPTN were G0 Synchronized(sync) and plated at low confluence on fibronectin-coated hydrogels of indicated stiffness (kPa) for 18 h before cell cycle analysis by FACS. Means ± SD, n = 4 independent experiments (single dots). One-way ANOVA with Tukey’s multiple comparison test: ns, siCtrl8kPa vs. siCtrl 1 kPa p = 0.1501; siOPTN 8 kPa vs. siOPTN 1 kPa p = 0.9997 not significant; Two-way ANOVA with Sidak’s multiple comparison test: siCtrl-siOPTN 1 kPa p = 0.0030. b Immunoblots showing cyclin D1 expression in cells treated as in a . Representative experiment, n = 3. c Level of cyclin D1 mRNA and protein expression in siCtrl or siOPTN transfected cells. HUVECs were G0 synchronized (sync) and then plated on 15 µg/ml fibronectin-coated plates and 20% serum for 18 h (Stim). Cyclin D1 mRNA levels were assessed by RT-qPCR and normalized to siCtrl synchronized cells condition, set to 1. Mean ± SD, n = 3 independent experiments (single dots). Two-way ANOVA with Tukey’s correction for multiple comparisons: ns, p = 0.6364 not significant; ** p = 0.0047. In parallel, cyclin D1 protein levels were determined by immunoblots, GAPDH was used as loading control. d HUVEC transfected with siCtrl or siOPTN were G0 Synchronized (sync) and then plated on 15 µg/ml fibronectin-coated plates and 20% serum for 18 h (Stim), together with Cycloheximide (CHX) at 10 nM. Cells were harvested after 15, 30, 60 min treatment and processed for anti-cyclin D1 immunoblotting. Immunoblots anti-OPTN and anti-GAPDH were performed to control OPTN knockdown and protein loading, respectively. e Quantification of cyclin D1 half-life measured in d . Values are expressed as fold compared to level in untreated condition (time 0), set to 1. Mean ± SD, n = 3. Dashed lines show similar cyclin D1 half-life in siCtrl and siOPTN cells. f Immunoblots showing neo synthesized cyclin D1 upon metabolic labeling. HUVEC transfected with siCtrl or siOPTN were G0 Synchronized and then plated on 15 µg/ml fibronectin-coated plates in defined medium for 18 h. Cells were pulsed with AHA during the last hour. Cell lysates were subjected to Click-iT reaction with Biotin Alkyne followed by streptavidin pulldown. Cyclin D1 and GAPDH neosynthesis was then assessed upon immunoblotting the resolved streptavidin-pulled-down reaction product. Total neosynthesis of proteins was assessed upon immunoblotting the resolved total Click-iT reaction product with streptavidin-HRP (AHA-Proteins). Representative experiment, n = 3.

    Article Snippet: Unless for experiments shown in Supplementary Fig. , where 5637 cells (ATCC HTB-9) were used, all experiments were performed on human vein endothelial cells (HUVECs) before passage 5, that are primary cells, an important feature to keep normal ubiquitin-mediated Rac regulation.

    Techniques: FACS, Transfection, Cell Cycle Assay, Western Blot, Expressing, Quantitative RT-PCR, Synthesized, Labeling

    OPTN controls the HACE1-dependent ubiquitylation of Rac1. a Coimmunoprecipitation of Flag-OPTN with HA-HACE1(IP FLAG) expressed in HUVEC cultured on plastic. Input: immunoblots on 0.5% total lysate to control protein levels. Representative experiment, n = 3. b Immunoblot showing Rac1 ubiquitylation profile. HUVECs were transfected with siCtrl or siOPTN and expression vectors for Histidine-tagged ubiquitin, HA-Rac1Q61L and Myc-HACE1. His-Ub covalently bound to Rac1Q61L were purified(His-P) and the ubiquitylation profile of Rac1Q61L was revealed by anti-HA immunoblotting(HA). Input:immunoblots on 0.5% lysate to control protein expression or knockdown. Representative experiment, n = 3. c Immunoblots showing Rac1 ubiquitylation profile. Cells were transfected with expression vectors for Histidine-tagged ubiquitin and HA-Rac1Q61L and plated overnight on fibronectin-coated hydrogels of indicated stiffness (kPa). Covalently bound His-Ubiquitinated proteins were purified(HIS-P) and resolved on 12% SDS-PAGE before anti-HA immunoblotting. Input: immunoblots on 1% lysate to control protein expression. Representative experiment, n = 3. d Immunoblots showing Rac1 ubiquitylation profile. Cells were transfected with expression vectors for Histidine-tagged ubiquitin and HA-Rac1Q61L, with or without FLAG-OPTN and plated overnight on 1 kPa fibronectin-coated hydrogels. Covalently bound His-Ubiquitinated proteins were purified(HIS-P) and resolved on 12% SDS-PAGE before anti-HA immunoblotting. Input:immunoblots on 1% lysate to control protein expression. Representative experiment n = 3. e Coimmunoprecipitation of HA-HACE1 with Myc-Rac1Q61L(IP Myc) in siCtrl or siOPTN transfected HUVEC cells cultured on plastic. Input:immunoblots on 0.5% lysate, to control protein expression or knockdown. Representative experiment, n = 3. f Quantification of Rac1-GTP levels in HUVEC transfected with siCtrl or siOPTN and stimulated by adhesion on fibronectin. Cells were G0 synchronized and active Rac1 level was assessed by GST-PAK pulldown on lysates from non-stimulated cells (NS) or cells plated on 15 µg/ml fibronectin plates for 1 h. Results represent fold change to non-stimulated siCtrl condition, set to 1. Means ± SD, n = 3 independent experiments (single dots). Two-way ANOVA: * p = 0.0121. g Accumulation of Rac1 in Flotilin (FLOT1) enriched fractions. HUVECs were transfected with siCtrl or siOPTN and G0 synchronized before plating on fibronectin 15 µg/ml plates for 1 h. Cell lysates were fractioned on a sucrose gradient (40, 30, 0%). The 8 fractions recovered were analyzed by SDS-PAGE and immunoblotted anti-Rac, anti-Flotilin 1(FLOT1) and anti-Transferrin Receptor(Tfr). Immunoblots anti-Tfr show detergent-soluble fractions (6–8) and anti-FLOT1 show detergent-resistant Fractions (2–3). Representative experiment, n = 3.

    Journal: Nature Communications

    Article Title: Optineurin links Hace1-dependent Rac ubiquitylation to integrin-mediated mechanotransduction to control bacterial invasion and cell division

    doi: 10.1038/s41467-022-33803-x

    Figure Lengend Snippet: OPTN controls the HACE1-dependent ubiquitylation of Rac1. a Coimmunoprecipitation of Flag-OPTN with HA-HACE1(IP FLAG) expressed in HUVEC cultured on plastic. Input: immunoblots on 0.5% total lysate to control protein levels. Representative experiment, n = 3. b Immunoblot showing Rac1 ubiquitylation profile. HUVECs were transfected with siCtrl or siOPTN and expression vectors for Histidine-tagged ubiquitin, HA-Rac1Q61L and Myc-HACE1. His-Ub covalently bound to Rac1Q61L were purified(His-P) and the ubiquitylation profile of Rac1Q61L was revealed by anti-HA immunoblotting(HA). Input:immunoblots on 0.5% lysate to control protein expression or knockdown. Representative experiment, n = 3. c Immunoblots showing Rac1 ubiquitylation profile. Cells were transfected with expression vectors for Histidine-tagged ubiquitin and HA-Rac1Q61L and plated overnight on fibronectin-coated hydrogels of indicated stiffness (kPa). Covalently bound His-Ubiquitinated proteins were purified(HIS-P) and resolved on 12% SDS-PAGE before anti-HA immunoblotting. Input: immunoblots on 1% lysate to control protein expression. Representative experiment, n = 3. d Immunoblots showing Rac1 ubiquitylation profile. Cells were transfected with expression vectors for Histidine-tagged ubiquitin and HA-Rac1Q61L, with or without FLAG-OPTN and plated overnight on 1 kPa fibronectin-coated hydrogels. Covalently bound His-Ubiquitinated proteins were purified(HIS-P) and resolved on 12% SDS-PAGE before anti-HA immunoblotting. Input:immunoblots on 1% lysate to control protein expression. Representative experiment n = 3. e Coimmunoprecipitation of HA-HACE1 with Myc-Rac1Q61L(IP Myc) in siCtrl or siOPTN transfected HUVEC cells cultured on plastic. Input:immunoblots on 0.5% lysate, to control protein expression or knockdown. Representative experiment, n = 3. f Quantification of Rac1-GTP levels in HUVEC transfected with siCtrl or siOPTN and stimulated by adhesion on fibronectin. Cells were G0 synchronized and active Rac1 level was assessed by GST-PAK pulldown on lysates from non-stimulated cells (NS) or cells plated on 15 µg/ml fibronectin plates for 1 h. Results represent fold change to non-stimulated siCtrl condition, set to 1. Means ± SD, n = 3 independent experiments (single dots). Two-way ANOVA: * p = 0.0121. g Accumulation of Rac1 in Flotilin (FLOT1) enriched fractions. HUVECs were transfected with siCtrl or siOPTN and G0 synchronized before plating on fibronectin 15 µg/ml plates for 1 h. Cell lysates were fractioned on a sucrose gradient (40, 30, 0%). The 8 fractions recovered were analyzed by SDS-PAGE and immunoblotted anti-Rac, anti-Flotilin 1(FLOT1) and anti-Transferrin Receptor(Tfr). Immunoblots anti-Tfr show detergent-soluble fractions (6–8) and anti-FLOT1 show detergent-resistant Fractions (2–3). Representative experiment, n = 3.

    Article Snippet: Unless for experiments shown in Supplementary Fig. , where 5637 cells (ATCC HTB-9) were used, all experiments were performed on human vein endothelial cells (HUVECs) before passage 5, that are primary cells, an important feature to keep normal ubiquitin-mediated Rac regulation.

    Techniques: Cell Culture, Western Blot, Transfection, Expressing, Purification, SDS Page