Journal: Journal of Biological Chemistry

Article Title: Anti-GPR56 monoclonal antibody potentiates GPR56-mediated Src-Fak signaling to modulate cell adhesion

doi: 10.1016/j.jbc.2021.100261

Figure Lengend Snippet: Figure 2. 10C7 potentiates GPR56 activation of RhoA-mediated SRF signaling independent of receptor cleavage, but requires the STP domain. A, transient overexpression of GPR56 WT, ΔSTP, and T383A in 293T cells significantly enhances SRF-RE activity, which is blocked by pretreatment with Rho inhibitor I (2 μg/ml) for 3 h. Statistical significance determined by ANOVA, ****p < 0.0001. Error bars, S.D. B, 10C7 induces a dose-dependent potentiation of SRF-RE in WT, T383A, and ΔPLL transfected cells. Error bars, S.E. C, western blot of GPR56 WT, T383A, and ΔPLL overexpression. D, 10C7 does not potentiate in ΔSTP transfected cells. Error bars, S.E. E, western blot of GPR56 WT and ΔSTP overexpression. F, effects of Rho inhibitor I and Src inhibitor (saracatinib) on 10C7 potentiation of GPR56-meditated SRF signaling. Error bars, S.E. All data represent at least three independent experiments.

Article Snippet: Saracatinib and defactinib were from Selleck Chemicals. hIgG1 isotype control was from Fisher Scientific.

Techniques: Activation Assay, Over Expression, Activity Assay, Transfection, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Anti-GPR56 monoclonal antibody potentiates GPR56-mediated Src-Fak signaling to modulate cell adhesion

doi: 10.1016/j.jbc.2021.100261

Figure Lengend Snippet: Figure 5. GPR56 regulates Src–Fak phosphorylation and adhesion in colorectal cancer cells. A, confocal microscopy images and quantification of ICC showing 10C7 (15 μg/ml or 100 nM) binds GPR56 and internalizes in DLD-1 cells after 1 h at 37 C. No binding was detected using nontargeting hIgG1 isotype control. B, 10C7 binds shRNA control (shCTL), but not GPR56 shRNA knockdown DLD-1 cells (shGPR56-2). Statistical significance determined by Student’s t-test, ***p < 0.001 Error bars, S.D. C, western blot showing time-dependent effects of 10C7 (20 μg/ml or 130 nM) treatment on phosphorylation of Src, Fak, and paxillin in serum starved DLD-1 and HT-29 cells. D, GPR56 knockdown decreases phosphorylation of Src, Fak, paxillin and E and F, reduces collagen adhesion of E, DLD-1 and F, HT-29 cells. Statistical significance determined by two-way ANOVA. For DLD-1 cells, *p < 0.05, and ***p < 0.001 compared with shCTL and **p < 0.01 for shGPR56-2 compared with parental cells. For HT-29 cells, **p < 0.01, and ***p < 0.001 compared with shCTL and parental cells. Error bars, S.E. G, 10C7 (20 μg/ml or 130 nM) increases adhesion of HT-29 shCTL cells, but not GPR56 knockdown cells after 30 min. H, Src inhibitor, saracatinib (10 μM), decreases 10C7-induced adhesion in HT-29 cells after 30 min. Statistical significance for G and H determined by one-way ANOVA, ***p < 0.001. Error bars, S.D. All data represent at least three independent experiments.

Article Snippet: Saracatinib and defactinib were from Selleck Chemicals. hIgG1 isotype control was from Fisher Scientific.

Techniques: Phospho-proteomics, Confocal Microscopy, Binding Assay, Control, shRNA, Knockdown, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Anti-GPR56 monoclonal antibody potentiates GPR56-mediated Src-Fak signaling to modulate cell adhesion

doi: 10.1016/j.jbc.2021.100261

Figure Lengend Snippet: Figure 6. GPR56-mediated phosphorylation of Src occurs upstream of Fak and is independent of RhoA activation. Western blots showing knock- down of A, G⍺13 and B, G⍺12 or G⍺q does not significantly affect GPR56-mediated Src/Fak signaling in 293T cells. C and D, western blots showing effect of inhibitors of C, RhoA (Rho Inhibitor I, 2 μg/ml), Src (saracatinib, 10 μM), and D, Fak (defactinib, 10 μM) on constitutive and 10C7-induced phosphorylation in 293T-GPR56 stable cells. Cells were pretreated with inhibitors for 3 h, then treated with 10C7 (3 μg/ml or 20 nM) for 45 min. E, quantification of at least three independent experiments as shown in C and D. Statistical significance for western blots was performed by one-way ANOVA, *p < 0.05, **p < 0.01, and ***p < 0.001. Error bars, S.D.

Article Snippet: Saracatinib and defactinib were from Selleck Chemicals. hIgG1 isotype control was from Fisher Scientific.

Techniques: Phospho-proteomics, Activation Assay, Western Blot, Knockdown

Journal: Immunity

Article Title: The Short Chain Fatty Acid Butyrate Imprints an Antimicrobial Program in Macrophages

doi: 10.1016/j.immuni.2018.12.018

Figure Lengend Snippet: Increase of Antimicrobial LC3-Associated Immune Defense in Butyrate Macrophages (A) Extracellular acidification rate (ECAR) measured at steady state in control and butyrate macrophages. Data represent the mean of nine biological replicates from three independent experiments. (B–D) Quantification of glycolysis (B), glycolytic capacity (C) and glycolytic reserve (D). (E) Heatmap of metabolites that were significantly higher or lower in control and butyrate macrophages as detected by mass-spectrometry (left panel). Results are from five healthy donors. Right panel: Fold change of all significantly higher or lower metabolites in control macrophages (black closed circles) and butyrate macrophages (red closed circles). (F) AMPK phosphorylation (Thr172) measured by ELISA (n = 4 individual donors). (G) Percentage of pS6 (left) and representative blot of phosphorylation and quantification (right) of the ribosomal protein S6 and β-actin in control and butyrate macrophages at steady state. (H) Gentamicin protection assay performed on control macrophages, butyrate macrophages, and butyrate macrophages treated for 2 h with the mTOR activator MHY1485 (20 mM). (I) Representative immunoblot of the expression of LC3-II, P62, and β-actin at steady state or after 2 h infection with Salmonella . (J and K) Protein quantification performed by ImageJ of LC3-II (I) and P62 (J) compared to β-actin. (L) Degradation of GFP- Salmonella and LC3 induction was assessed by confocal microscopy. Representative images and quantification of GFP fluorescence and LC3 accumulation as outlined in Figure S7 C. Data from six independent donors in two independent experiments. Scale bar 5 μm. Each dot represents one donor. Statistical significance was determined using Mann-Whitney U test ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001. Please also see and .

Article Snippet: Proteins were detected using primary antibodies against LC3B (D11, Cell signaling), β-actin (13E5, Cell signaling), phospho-S6 ribosomal protein (Ser235 and/or 236) (D57.2.2E, Cell signaling), S100A8 (EPR3554, Abcam), acetylated-H3 (ab47915, Abcam) and acetylated-H4 (EPR16606, Abcam), P62 lck (clone 3/P62, BD bioscience) and detected using HRP-conjugated secondary antibodies.

Techniques: Mass Spectrometry, Enzyme-linked Immunosorbent Assay, Western Blot, Expressing, Infection, Confocal Microscopy, Fluorescence, MANN-WHITNEY

Journal: Immunity

Article Title: The Short Chain Fatty Acid Butyrate Imprints an Antimicrobial Program in Macrophages

doi: 10.1016/j.immuni.2018.12.018

Figure Lengend Snippet: Induction of Antimicrobial Activity by Butyrate in Macrophages In Vivo (A) WT mice received sodium butyrate in drinking water (150 mM final concentration) or PBS control for 7 days. At day 7, colonic segments were digested, macrophages were isolated by flow cytometry sorting and a gentamicin protection assay was performed. Each dot represents macrophages pooled from ten mice. Four independent experiments are shown. (B) Mouse bone marrow progenitor cells from mice gavaged with sodium butyrate or with PBS were differentiated into macrophages in the presence of M-CSF or with M-CSF with butyrate as a positive control. A gentamycin assay was performed at day 7 of differentiation. (C and D) Mice received butyrate or PBS 5 days prior to oral infection with Salmonella typhymurium def aroA (1 × 10 9 bacteria/mouse). 2 days post-infection bacterial dissemination was assessed in MLN, spleen, liver (C), and caecum (D). Each dot represents a mouse. (E) Colitis score of control and butyrate-treated mice either uninfected or infected with Salmonella . (F) Representative H&E stained colon sections from control and butyrate-treated mice either uninfected or infected with Salmonella (original magnification 100x). (G and H) Mice were treated with 150 mM sodium butyrate or with PBS 3 days prior and every other day after oral infection with Citrobacter rodentium (1 × 10 9 bacteria/mouse). Mice were weighed daily. Lines represents mean of 3 mice (G). At day 7 post infection bacterial dissemination was assessed in the spleen and in the liver (H). Each dot represents a mouse. Statistical significance was determined using Mann-Whitney U test ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001. Please also see Figure S6 .

Article Snippet: Proteins were detected using primary antibodies against LC3B (D11, Cell signaling), β-actin (13E5, Cell signaling), phospho-S6 ribosomal protein (Ser235 and/or 236) (D57.2.2E, Cell signaling), S100A8 (EPR3554, Abcam), acetylated-H3 (ab47915, Abcam) and acetylated-H4 (EPR16606, Abcam), P62 lck (clone 3/P62, BD bioscience) and detected using HRP-conjugated secondary antibodies.

Techniques: Activity Assay, In Vivo, Concentration Assay, Isolation, Flow Cytometry, Positive Control, Infection, Staining, MANN-WHITNEY

Journal: Immunity

Article Title: The Short Chain Fatty Acid Butyrate Imprints an Antimicrobial Program in Macrophages

doi: 10.1016/j.immuni.2018.12.018

Figure Lengend Snippet:

Article Snippet: Proteins were detected using primary antibodies against LC3B (D11, Cell signaling), β-actin (13E5, Cell signaling), phospho-S6 ribosomal protein (Ser235 and/or 236) (D57.2.2E, Cell signaling), S100A8 (EPR3554, Abcam), acetylated-H3 (ab47915, Abcam) and acetylated-H4 (EPR16606, Abcam), P62 lck (clone 3/P62, BD bioscience) and detected using HRP-conjugated secondary antibodies.

Techniques: Enzyme-linked Immunosorbent Assay, Generated, Expressing

Journal: Cell Death Discovery

Article Title: YAP nuclear translocation induced by HIF-1α prevents DNA damage under hypoxic conditions

doi: 10.1038/s41420-023-01687-5

Figure Lengend Snippet: A , B MDCK-parental cells were treated with vehicle (Veh) or 10 μM verteporfin (VP) under 1% O 2 . MDCK-shHIF-1α cells were treated with Veh or 10 μM XMU-MP-1 (XMU) under 1% O 2 . After 24 h, MDCK-parental and MDCK-shHIF-1α cells were immunostaining with anti-γH2AX antibody. Scale bar: 40 μm. B γH2AX intensity was analyzed and presented as mean ± SEM. *** p < 0.001. C , D MDCK-shHIF-1α cells were treated with Veh or XMU under normoxia or 1% O 2 condition for 36 h, followed by Annexin V staining. Scale bar: 20 μm. D Annexin V positive cell number was analyzed and presented as mean ± SEM. ** p < 0.01. All images were obtained using confocal microscope.

Article Snippet: The ERK inhibitor SCH772984, AKT inhibitor LY294002, YAP inhibitor verteporfin, and YAP activator XMU-MP-1, were purchased from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Immunostaining, Staining, Microscopy

Journal: Journal of Biological Chemistry

Article Title: Uropathogenic Escherichia coli invades bladder epithelial cells by activating kinase networks in host cells

doi: 10.1074/jbc.ra118.003499

Figure Lengend Snippet: Figure 3. EGFR facilitates the UPEC invasion. (A) UTI89 and UTI89∆FimH activate EGFR. BECs were treated with bacterial CM for the indicated times and cell lysates were examined for EGFR phosphorylation (p-EGFR) using anti-pEGFR (Y1068) antibody. Total EGFR (t-EGFR) served as protein loading control. (B) Afatinib and AG1478 specifically inhibit UTI89-induced p-EGFR (Y1068). BECs were pretreated with the inhibitors LY294002 (5 µM; PI3K and mTOR), wortmannin (0.2 µM; PI3K), Bay 80-6946 (0.4 µM; PI3K), MK-2206 (4 µM, Akt), rapamycin (0.1 µM; mTORC1), AZD8055 (10 µM; mTORC1/2), Afatinib (1.5 µM; EGFR) or AG1478 (1 µM; EGFR) for 1 hr followed by exposure to UTI89 CM for 10 min. Cell lysates were analyzed by Western blot for p-EGFR (Y1068). Total EGFR and GAPDH served as protein loading control. (C) EGFR mediates the UTI89 CM-induced Akt phosphorylation. BECs were treated with Afatinib or AG1478 as above then exposed to UTI89 CM. Cell lysates were analyzed by Western blot for EGFR and Akt phosphorylation. GAPDH served as protein loading control. (D) Afatinib and AG1478 inhibit the UTI89 invasion of BEC as determined with gentamicin protection assay. Results are shown as fold change from control non-treated (NT) cells. Error bars represent SEM. Statistical analysis was performed by Student’s t-test compared to the non-treated control; *P < 0.05, n=4. (E) Knockdown of EGFR gene was achieved with two targeting shRNA sequences in lentivirus. The EGFR gene expression was measured with qRT-PCR and results are presented as fold difference relative to shCon sample. Statistical analysis was performed by Student’s t-test compared to the LKO lentivirus control; ***P < 0.001, n=3. Inset represent EGFR protein knockdown. (F) Knockdown of EGFR expression obliterates UTI89 invasion. Bacterial invasion was determined using the gentamicin protection assay and results are shown as fold change from control shCon cells. Error bars represent SEM. Statistical analysis was performed by Student’s t-test compared to the LKO lentivirus control; ***P < 0.001, n=3. (G) Effect of EGFR knockdown on the UTI89 attachment to BECs. GFP-UTI89 attachment to host BECs was quantified under fluorescence microscopy. Results show the number of attached UTI89 per cell and 50 randomly-selected cells were counted from each group. Statistical analysis was performed by Student’s t-test compared to the LKO lentivirus control.

Article Snippet: Reagents were obtained as follows: Lipopolysaccharide (LPS) from E. coli 055:B5 and complete protease and phosphatase inhibitors from Sigma Aldrich (St. Louis, MO); CLI095 from InvivoGen (San Diego, CA); wortmannin, LY294002, and rapamycin from Cell Signaling Technology (Boston, MA); AZD8055 from Santa Cruz Biotechnology (Santa Cruz, CA); BAY 80- 6946, Afatinib and MK-2206 from Selleckchem (Houston, TX); AG1478 from Cayman Chemical (Ann Arbor, MI); and gentamicin from Gibco (Grans Island, NY).

Techniques: Phospho-proteomics, Control, Western Blot, Knockdown, shRNA, Gene Expression, Quantitative RT-PCR, Expressing, Fluorescence, Microscopy

Journal: Journal of Biological Chemistry

Article Title: Uropathogenic Escherichia coli invades bladder epithelial cells by activating kinase networks in host cells

doi: 10.1074/jbc.ra118.003499

Figure Lengend Snippet: Figure 5. Role of EGFR, Akt and mTORCs in UTI89 invasion of mouse bladder. (A, B) Strips of mouse transitional epithelial tissue were individually placed in wells (12-well plates) and treated, or not, with the indicated inhibitor for 1 hr at 37°C. UPEC (5 x 107/ml) were added for additional 2 hr and tissue strips were washed thrice with PBS. The tissue strips were lysed in PBS containing 0.1% (v/v) Triton X-100. For one part (A), cell lysates were analyzed for EGFR and Akt phosphorylation by Western blot using the indicated antibodies. GAPDH was used as a protein loading control. For the second part (B), dilutions of lysates were plated on LB-agar plates to allow colony growth. Colonies were counted and data are presented as the fold change of control values. Statistical analysis was performed by Student’s t-test compared to the non-treated control; *P < 0.05, **P < 0.01, n=3. (C) Afatinib and AZD8055 inhibit UTI89 invasion of mouse bladder in vivo. C3H/HeJ female mice were challenged with 108 cfu UTI89 after pretreatment of mice with Afatinib or AZD8055 for 1 hr. At 24 hr post infection, bladders (n = 7 mice per experimental group) were harvested and analyzed for bacterial invasion. Statistical analysis was performed by Student’s t-test compared to the non-treated control; *P < 0.05, n=7.

Article Snippet: Reagents were obtained as follows: Lipopolysaccharide (LPS) from E. coli 055:B5 and complete protease and phosphatase inhibitors from Sigma Aldrich (St. Louis, MO); CLI095 from InvivoGen (San Diego, CA); wortmannin, LY294002, and rapamycin from Cell Signaling Technology (Boston, MA); AZD8055 from Santa Cruz Biotechnology (Santa Cruz, CA); BAY 80- 6946, Afatinib and MK-2206 from Selleckchem (Houston, TX); AG1478 from Cayman Chemical (Ann Arbor, MI); and gentamicin from Gibco (Grans Island, NY).

Techniques: Phospho-proteomics, Western Blot, Control, In Vivo, Infection