Journal: Frontiers in Immunology

Article Title: Sialic Acid-Siglec-E Interactions During Pseudomonas aeruginosa Infection of Macrophages Interferes With Phagosome Maturation by Altering Intracellular Calcium Concentrations

doi: 10.3389/fimmu.2020.00332

Figure Lengend Snippet: Siglec-E silenced macrophages show enhanced intracellular calcium levels, calcium-related, MAPK and NF-κB signaling in response to PA +Sia . (A) Mock and siglec-E siRNA transfected J774A.1 cells were stained with Fluo-3-AM followed by PA +Sia infection for 15 min at 37°C. Cells were visualized by confocal microscopy as before. Microscopy images were representative of three independent experiments. Scale bar = 10 μm. (B) Intracellular calcium levels were detected by flow cytometric analysis of Fluo-3-AM stained macrophages following 15 min of 10 MOI PA +Sia infection at 37°C. MFI data from three independent experiments represented as mean ± s.e.m. (C) Expression of MAPK, ERK, JNK pathway molecules signaling molecules and calmodulin, calmodulin-dependent kinase type II was detected in lysates from PA +Sia -infected mock or siglec-E siRNA transfected macrophages by western blotting. (D) Similarly, infected macrophages were separated into cytosolic and nuclear fractions. Proteins were resolved in SDS-PAGE and probed for the expression of NF-κB signaling pathway to detect nuclear translocation of p65 subunit. Western blots (C,D) were representative of three independent experiments. Densitometric values reported are normalized with respect to β-actin band intensities and such values from atleast three independent experiments are used to calculate the mean band intensities which are presented as bar diagrams alongwith statistical significance of observed changes. Significance represented by * p ≤ 0.05 and ** p ≤ 0.01.

Article Snippet: The primary antibodies used includes—calmodulin (#4830), calcineurin A (#2614), PI3K-p85 (#4257), PI3K-p110γ (#4252), p-p38 MAPK (#4511), p38 MAPK (#9212), p-Akt Ser473 (#4060), pan-Akt (#4691), p-ERK1/2 (#4377), ERK1/2 (#4695), p-JNK/SAPK (#4668), JNK/SAPK (#9252), β-Actin (#4970), Lamin B (#13435), NF-κB Pathway Sampler Kit (#9936) from Cell Signaling Technology; biotinylated-anti phosphotyrosine (309304) from Biolegend (San Diego, CA, USA); calmodulin-dependent kinase type II (611293), SHP-1 (610126), SHP-2 (610622) from BD Biosciences; siglec-E (sc-377477) from Santacruz Biotechnology.

Techniques: Transfection, Staining, Infection, Confocal Microscopy, Microscopy, Expressing, Western Blot, SDS Page, Translocation Assay

Journal: American Journal of Physiology - Renal Physiology

Article Title: High-content screening assay-based discovery of paullones as novel podocyte-protective agents

doi: 10.1152/ajprenal.00338.2017

Figure Lengend Snippet: Paullone derivatives protect podocytes from injury. A: chemical structures of paullone, kenpaullone, 1-azakenpaullone, and alsterpaullone. B: graphs showing that paullone derivatives protect podocytes from PAN injury in a dose-dependent fashion. Podocytes were cultured in 96-well optical plates and were treated with PAN (30 μg/ml) and an increasing concentration of the various compounds, as indicated, at 37°C for 48 h. Change in various cellular phenotypes was assessed using Opera HCS system after staining cells with CellMask Blue (to measure cell morphology), fluorescently labeled phalloidin (for the quantification of F-actin fibers), and anti-paxillin antibody (for the quantification of focal adhesions). Cell roundness and F-actin fiber number were determined using the Columbus software. Dose-response curves show the relative effects of increasing concentration of the various compounds on two different cellular parameters [cell morphology, as defined by cell roundness (left graph), and the number of F-actin fibers per cell (right graph) as compared with healthy podocytes, as well as PAN-injured podocytes]. X-axis represents increasing compound concentration, and y-axis shows relative quantification of each of the two parameters at a defined dose of PAN (30 μg/ml). Dose-response curves show that kenpaullone, 1-azakenpaullone, and alsterpaullone protect podocytes from PAN injury with varying degree of efficacy, with alsterpaullone being the most efficacious. Five hundred to one thousand cells were quantified in each assay well. Data shown are means ± SE (n = 4). Dashed blue lines represent the average relative values for the two parameters from podocytes treated with vehicle DMSO alone (control, healthy cells). The dashed red lines represent the average relative values for the two parameters from podocytes treated with PAN (30 μg/ml) (injured cells). C. paullone derivatives protect podocytes from PAN injury mediated F-actin reorganization. Representative fluorescence confocal microscopy images of mouse podocytes treated either with 15 µg/ml PAN alone (b, injured cells), or cotreated with 15µg/ml PAN and 10 µM paullone (c), 1 µM alsterpaullone (d), 5 µM kenpaullone (e), 10 µM 1-azakenpaullone (f), 1 µM pyrintegrin (g), 5 µg/ml MZR (h), 10 µM SB216763 (i, GSK3i), or 20 µM SB203580 (j, p38i) for 48 h. Control vehicle DMSO-treated cells are shown in panel a. Cells were stained with CellMask (blue), Alexa Fluor 488 phalloidin (green), and anti-paxillin antibody (magenta). Scale bar = 20 μm. GSK3i, GSK3 inhibitor; p38i, p38 inhibitor.

Article Snippet: For Western blotting, the anti-p38 (cat. no. 9212), anti-phospho-p38 (Thr180/Tyr182) (cat. no. 9215), anti-GAPDH (cat. no. 2118), anti-JNK (cat. no. 9258), and the anti-phospho-JNK (Thr183/Tyr185) (cat. no. 4668) antibodies were obtained from Cell Signaling Technology (Danvers, MA); the anti-phospho-p38 (cat. no. sc-7972) and the anti-pJNK (cat. no. sc-6254) antibodies were obtained from Santa Cruz Biotechnology (Dallas, TX); the anti-acetylated tubulin antibody (cat. no. T7451) was purchased from Sigma (St. Louis, MO); the anti-β-catenin antibody (cat. no. 610154) was obtained from BD Transduction (San Jose, CA); and the anti-GAPDH antibody (cat. no. MAB374) was obtained from Millipore (Billerica, MA).

Techniques: Cell Culture, Concentration Assay, Staining, Labeling, Software, Quantitative Proteomics, Control, Fluorescence, Confocal Microscopy

Journal: American Journal of Physiology - Renal Physiology

Article Title: High-content screening assay-based discovery of paullones as novel podocyte-protective agents

doi: 10.1152/ajprenal.00338.2017

Figure Lengend Snippet: Paullone derivatives protect podocytes from PAN injury-mediated loss in microtubules. Representative fluorescence confocal microscopy images show acetylated tubulin expression in mouse podocytes treated either with 30 µg/ml PAN and DMSO vehicle control (B), or cotreated with 30 µg/ml PAN and 1 µM pyrintegrin (C), 5 µg/ml mizoribine (D), 10 µM paullone (E), 5 µM kenpaullone (F), 10 µM 1-azakenpaullone (G), 1 µM alsterpaullone (H), 10 µM SB216763 (I, GSK3i), or 20 µM SB203580 (J, p38i) for 48 h. Control vehicle DMSO-treated cells are shown in A. Cells were stained with CellMask and anti-acetylated tubulin antibody. Scale bar represents 100 μm and applies to all panels (A–J). GSK3i, GSK3 inhibitor; p38i, p38 inhibitor. Quantification of normalized acetylated tubulin signal intensity around nuclear membrane from each of these conditions is presented in K. Signal intensity was quantified using the Columbus software. The intensity values were normalized using the mean values from control cells. One-way ANOVA with Fisher’s LSD multiple comparison test was used. *P < 0.05; **P < 0.01; ***P < 0.005; ns = not significant as compared with the PAN-only (PAN) treated samples. Pyr, pyrintegrin; Mzr, mizoribine; P, paullone; KP, kenpaullone; AP, alsterpaullone; GSK3i, GSK3 inhibitor; p38i, p38 inhibitor.

Article Snippet: For Western blotting, the anti-p38 (cat. no. 9212), anti-phospho-p38 (Thr180/Tyr182) (cat. no. 9215), anti-GAPDH (cat. no. 2118), anti-JNK (cat. no. 9258), and the anti-phospho-JNK (Thr183/Tyr185) (cat. no. 4668) antibodies were obtained from Cell Signaling Technology (Danvers, MA); the anti-phospho-p38 (cat. no. sc-7972) and the anti-pJNK (cat. no. sc-6254) antibodies were obtained from Santa Cruz Biotechnology (Dallas, TX); the anti-acetylated tubulin antibody (cat. no. T7451) was purchased from Sigma (St. Louis, MO); the anti-β-catenin antibody (cat. no. 610154) was obtained from BD Transduction (San Jose, CA); and the anti-GAPDH antibody (cat. no. MAB374) was obtained from Millipore (Billerica, MA).

Techniques: Fluorescence, Confocal Microscopy, Expressing, Control, Staining, Membrane, Software, Comparison

Journal: American Journal of Physiology - Renal Physiology

Article Title: High-content screening assay-based discovery of paullones as novel podocyte-protective agents

doi: 10.1152/ajprenal.00338.2017

Figure Lengend Snippet: Alsterpaullone functions as a GSK-3β inhibitor and reduces JNK and p38 phosphorylation. A: alsterpaullone protects against PAN injury-mediated loss of β-catenin expression in mouse podocytes. Representative fluorescence confocal microscopy images of mouse podocytes treated with vehicle (DMSO, control), 30 µg/ml PAN (PAN+DMSO), cotreated with 30 µg/ml PAN and 1 µM alsterpaullone (PAN+Alsterpaullone), or cotreated with 30 µg/ml PAN and 10 µM SB216763 (PAN+GSK3i) for 48 h. Cells were immunostained with anti-β-catenin antibody and imaged using HCS Opera system and Zeiss 700 LSM confocal microscope. The images taken by Zeiss 700 LSM are shown as representative images. Scale bar = 20 μm. With the images taken by HCS Opera system, β-catenin expression was quantified by measuring fluorescence intensity of anti-β-catenin antibody staining per cell in each condition from 500 to 1,000 cells per well and is presented as a graph (right). Data are means ± SE (n = 4). One-way ANOVA with Dunnett’s multiple comparison test was used. ***P < 0.005; ****P < 0.001. B: alsterpaullone (AP) and GSK3β inhibitor SB216763 (GSK3i) protect against increase in phosphorylation of JNK (Thr183/Tyr185) and p38 (Thr180/Tyr182) in PAN-injured cells. Mouse podocytes were treated with 30 µg/ml PAN in the absence (Cont) or presence of 1 µM alsterpaullone or 10 µM SB216763 (GSK3bi), and cell lysates were analyzed by Western blot analysis for relative level of phosphorylated and total JNK and p38 proteins. Representative Western blot images are shown. Bar graphs show ratio of phosphorylated to total protein in the lysates. Data are means ± SE (n = 3). One-way ANOVA with Dunnett’s multiple comparison test was used. *P < 0.05; **P < 0.01.

Article Snippet: For Western blotting, the anti-p38 (cat. no. 9212), anti-phospho-p38 (Thr180/Tyr182) (cat. no. 9215), anti-GAPDH (cat. no. 2118), anti-JNK (cat. no. 9258), and the anti-phospho-JNK (Thr183/Tyr185) (cat. no. 4668) antibodies were obtained from Cell Signaling Technology (Danvers, MA); the anti-phospho-p38 (cat. no. sc-7972) and the anti-pJNK (cat. no. sc-6254) antibodies were obtained from Santa Cruz Biotechnology (Dallas, TX); the anti-acetylated tubulin antibody (cat. no. T7451) was purchased from Sigma (St. Louis, MO); the anti-β-catenin antibody (cat. no. 610154) was obtained from BD Transduction (San Jose, CA); and the anti-GAPDH antibody (cat. no. MAB374) was obtained from Millipore (Billerica, MA).

Techniques: Phospho-proteomics, Expressing, Fluorescence, Confocal Microscopy, Control, Microscopy, Staining, Comparison, Western Blot

Journal: American Journal of Physiology - Renal Physiology

Article Title: High-content screening assay-based discovery of paullones as novel podocyte-protective agents

doi: 10.1152/ajprenal.00338.2017

Figure Lengend Snippet: Alsterpaullone reduces pericardial edema and glomerular injury in zebrafish. A: schematic representation of the zebrafish embryo-based in vivo assay to test efficacy of the novel compounds. Adriamycin (ADR) treatment induces pericardial edema and renal injury in zebrafish embryos, which can be studied by examining body curvature and the size of cardiac region. Compounds that protect renal function reduce the in vivo damage as defined by these measurements. B–C: representative images (left) and graphs showing quantification of level of body curvature (B) and edema (C) in zebrafish embryos treated as described in materials and methods. Control animals (Cont) were kept in the E3 media. Glomerular injury was induced by treatment with ADR, which resulted in significant increase in body curvature and edema (expressed as a ratio of the cardiac area over total body area). Treatment with alsterpaullone (0.25 µM), 1-azakenpaullone (0.25 µM), GSK3 inhibitor SB216763 (0.5 µM), or p38 inhibitor SB203580 (1 µM) reduced body curvature and edema/cardiac area per body area. Graph in B presents relative number of embryos displaying normal vs. curved bodies in each treatment group (n = 18–36/group). Graph in C shows 10–90 percentiles per treatment group, with mean and 95% confidence interval in the graph (n = 18–36/group). One-way ANOVA with Dunnett’s multiple comparison test was used. **P < 0.01; ***P < 0.005; ****P < 0.001.

Article Snippet: For Western blotting, the anti-p38 (cat. no. 9212), anti-phospho-p38 (Thr180/Tyr182) (cat. no. 9215), anti-GAPDH (cat. no. 2118), anti-JNK (cat. no. 9258), and the anti-phospho-JNK (Thr183/Tyr185) (cat. no. 4668) antibodies were obtained from Cell Signaling Technology (Danvers, MA); the anti-phospho-p38 (cat. no. sc-7972) and the anti-pJNK (cat. no. sc-6254) antibodies were obtained from Santa Cruz Biotechnology (Dallas, TX); the anti-acetylated tubulin antibody (cat. no. T7451) was purchased from Sigma (St. Louis, MO); the anti-β-catenin antibody (cat. no. 610154) was obtained from BD Transduction (San Jose, CA); and the anti-GAPDH antibody (cat. no. MAB374) was obtained from Millipore (Billerica, MA).

Techniques: In Vivo, Control, Comparison

Journal: American Journal of Physiology - Renal Physiology

Article Title: High-content screening assay-based discovery of paullones as novel podocyte-protective agents

doi: 10.1152/ajprenal.00338.2017

Figure Lengend Snippet: Working model: a schematic representation of the potential mechanisms behind alsterpaullone-mediated protection of podocytes from injury. Podocyte injury due to agents, such as puromycin aminonucleotide (PAN), results in stress-mediated activation of p38 MAP kinase and GSK3β. It also induces a reorganization of the cellular filamentous network comprising of F-actin and microtubules that affect the mechanical stability of the cell. Activation of p38 MAPK leads to induction of caspase 3/7-mediated apoptosis and podocyte loss. Activation of GSK-3β results in reduced β-catenin levels, further affecting the integrity of the podocyte cytoskeleton and increasing podocyte dynamics and dysfunction. Alsterpaullone (and other paullone derivatives) suppress signaling downstream of GSK-3β and p38, thereby reducing progressive loss in podocyte function and ameliorating glomerular dysfunction.

Article Snippet: For Western blotting, the anti-p38 (cat. no. 9212), anti-phospho-p38 (Thr180/Tyr182) (cat. no. 9215), anti-GAPDH (cat. no. 2118), anti-JNK (cat. no. 9258), and the anti-phospho-JNK (Thr183/Tyr185) (cat. no. 4668) antibodies were obtained from Cell Signaling Technology (Danvers, MA); the anti-phospho-p38 (cat. no. sc-7972) and the anti-pJNK (cat. no. sc-6254) antibodies were obtained from Santa Cruz Biotechnology (Dallas, TX); the anti-acetylated tubulin antibody (cat. no. T7451) was purchased from Sigma (St. Louis, MO); the anti-β-catenin antibody (cat. no. 610154) was obtained from BD Transduction (San Jose, CA); and the anti-GAPDH antibody (cat. no. MAB374) was obtained from Millipore (Billerica, MA).

Techniques: Activation Assay

Journal: Molecular Biology of the Cell

Article Title: TOR complex 2–Ypk1 signaling regulates actin polarization via reactive oxygen species

doi: 10.1091/mbc.E14-06-1122

Figure Lengend Snippet: Regulation of ROS by Pkc1 is downstream of Ypk1 and Fpk1. (A) Ypk1-WT (PLY1083), Ypk1-AS (PLY1098), Ypk1-AS + PKC1 R398P (1531), Ypk1-AS rho 0 + PKC1 R398P (PLY1532), Ypk1-AS fpk1Δ (PLY1533), and Ypk1-AS fpk1Δ + PKC1 R398P (PLY1538) were grown in either SCD-Ura or SCD-Ura/-Leu medium and treated with 0.5 μM 2,3-DMB-PPI for 1 h. ROS was determined and quantified as in . p values were calculated using Student's t test; * p between 0.05 and 0.01; ** p ≤ 0.01. (B) Quantification of actin polarization after fixing and rhodamine-phalloidin labeling in the same strains as in A, with at least 100 cells counted for each sample. (C) Ypk1-WT (PLY1083), Ypk1-AS (PLY1098), and Ypk1-AS fpk1Δ (PLY1533) were grown in either SCD-Ura or SCD-Ura + 50 mM MES, pH 6.2, and treated with 0.5 μM 2,3-DMB-PPI for 1 h. Cells were harvested and lysed, and the resulting protein extracts were resolved by SDS–PAGE and immunoblotted with anti–phospho-p44/42 MAPK (for p-Mpk1), anti-Mpk1, and anti-G6PDH antibodies. Quantification below the blot describes the difference relative to Ypk1-WT after normalizing to the anti-p44/p42 MAPK signal. (D) WT (PLY062) and fpk1Δ (PLY1440) were grown in SCD medium and treated with 1.25 μM myriocin (Myr) for 1 h as noted and then processed as in C. (E) WT (PLY062) and WT + PKC1 R398P (PLY1550) were grown in SCD or SCD-Leu medium and treated as in D. ROS was detected and quantified as in . p values were calculated using Student's t test; ** p ≤ 0.01. (F) Quantification of actin polarization after fixing and rhodamine-phalloidin labeling in the same strains as in E, with least 100 cells counted for each sample.

Article Snippet: Membranes were probed with anti–phospho-p44/42 MAPK (1:1000; Cell Signaling Technology), anti-Mpk1 (1:1000; Santa Cruz Biotechnology), and anti-G6PDH (1:100,000; Sigma-Aldrich) primary antibodies and visualized using the appropriate secondary antibodies conjugated to IRDye (1:5000; LI-COR Biosciences) on the Odyssey Infrared Imaging System (LI-COR Biosciences).

Techniques: Labeling, SDS Page

Journal: Molecular Biology of the Cell

Article Title: TOR complex 2–Ypk1 signaling regulates actin polarization via reactive oxygen species

doi: 10.1091/mbc.E14-06-1122

Figure Lengend Snippet: Regulation of Pkc1/MAPK activity requires Fpk1- and sphingolipid-dependent PM localization of Rom2. (A) Ypk1-WT rom2Δ + ROM2-GFP (PLY1563), Ypk1-AS rom2Δ + ROM2-GFP (PLY1564), Ypk1-AS lcb4Δ rom2Δ + ROM2-GFP (PLY1566), and Ypk1-AS fpk1Δ rom2Δ + ROM2-GFP (PLY1565) cells were grown in SCD-Ura/-Leu medium, with 4 μM PHS or 20 mM NAC where noted, and treated with 0.5 μM 2,3-DMB-PPI for 1 h. Single focal plane images were collected by confocal microscopy. Quantification represents percentage of small-budded cells labeled with GFP, with 30–50 cells counted for each sample. Scale bar, 5 μm. (B) Ypk1-WT (PLY1083), Ypk1-AS (PLY1098), Ypk1-AS fpk1Δ (PLY1533), and Ypk1-AS fpk1Δ rom2Δ (PLY1561) were grown in SCD-Ura medium and treated with 0.5 μM 2,3-DMB-PPI for 1 h. Cells were harvested and lysed, and the resulting protein extracts were resolved by SDS/PAGE and immunoblotted with anti-phospho-p44/42 MAPK (for p-Mpk1), anti-Mpk1, and anti-G6PDH antibodies. Quantification below the blot describes the difference relative to Ypk1-WT after normalizing to the anti-p44/p42 MAPK signal. (C) Ypk1-WT (PLY1083), Ypk1-AS (PLY1098), and Ypk1-AS + Rom2-HA (PLY1568) were grown in SCD-Ura medium, treated with 0.5 μM 2,3-DMB-PPI for 1 h, and then processed as in B. (D) Ypk1-WT (PLY1083), Ypk1-AS (PLY1098), Ypk1-AS fpk1Δ (PLY1533), Ypk1-AS fpk1Δ rom2Δ (PLY1561), and Ypk1-AS + ROM2-HA (PLY1568) were grown in SCD-Ura medium and treated with 0.5 μM 2,3-DMB-PPI for 1 h. ROS was determined and quantified as in . p values were calculated using Student's t test; ** p ≤ 0.01. (E) Quantification of actin polarization after fixing and rhodamine-phalloidin labeling in the same strains as in D, with at least 100 cells counted for each sample.

Article Snippet: Membranes were probed with anti–phospho-p44/42 MAPK (1:1000; Cell Signaling Technology), anti-Mpk1 (1:1000; Santa Cruz Biotechnology), and anti-G6PDH (1:100,000; Sigma-Aldrich) primary antibodies and visualized using the appropriate secondary antibodies conjugated to IRDye (1:5000; LI-COR Biosciences) on the Odyssey Infrared Imaging System (LI-COR Biosciences).

Techniques: Activity Assay, Confocal Microscopy, Labeling, SDS Page

Journal: Diabetes, Metabolic Syndrome and Obesity: Targets and Therapy

Article Title: High Glucose Downregulates Connexin 43 Expression and Its Gap Junction and Hemichannel Function in Osteocyte-like MLO-Y4 Cells Through Activation of the p38MAPK/ERK Signal Pathway

doi: 10.2147/DMSO.S239892

Figure Lengend Snippet: Membrane Cx43 protein expression decreased and pERK1/2 and p38 MAPK were activated in MLO-Y4 cells in response to 20 mM glucose treatment. ( A ) Membrane Cx43 protein expression in MLO-Y4 cells was decreased by 60% after glucose treatment. A histogram representation of the data is shown on the right. ( B ) Phosphorylated EKK1/2, total ERK1/2, and p38 MAPK were activated in MLO-Y4 cells following glucose treatment. *** P <0.001 vs Ctrl. * P <0.05 vs Ctrl. Data are presented as the mean ± standard deviation of three independent experiments.

Article Snippet: The membranes were blocked with 5% non-fat milk (w/v) for 2 h at room temperature, then incubated with the following primary antibodies overnight at 4°C: rabbit polyclonal anti-Cx43 (1:1000, #3512, Cell Signaling Technology [CST], Danvers, MA, USA), rabbit polyclonal anti-p38 (1:1000, #8690, CST), rabbit polyclonal anti-ERK (1:1000, #9102, CST), anti-β-tubulin (WL01931, Wanleibio, Shenyang, China), anti-Na-K-ATP (1:1000, ab205967, Abcam, Cambridge, UK).

Techniques: Membrane, Expressing, Standard Deviation

Journal: Diabetes, Metabolic Syndrome and Obesity: Targets and Therapy

Article Title: High Glucose Downregulates Connexin 43 Expression and Its Gap Junction and Hemichannel Function in Osteocyte-like MLO-Y4 Cells Through Activation of the p38MAPK/ERK Signal Pathway

doi: 10.2147/DMSO.S239892

Figure Lengend Snippet: PD98059, an inhibitor of p38 MAPK and U0126, and an inhibitor of ERK1/2 partially reversed the glucose-mediated downregulation of total Cx43 protein. ( A ) The relative expression levels of Cx43 in cells treated with 20 mM glucose increased from 62.3% to 84.1% following PD98059 treatment as compared to untreated cells. ( B ) Immunofluorescence microscopy for Cx43. Expression was higher in the PD98059+20 mM glucose group compared to the 20 mM glucose group. A histogram representation of the data is shown on the right. ( C ) The relative expression of Cx43 in cells treated with 20 mM glucose increased from 61.51% to 82.98% following U0126 treatment as compared to untreated cells. ( D ) Immunofluorescence microscopy for Cx43. Expression was higher in the PD98059+20 mM glucose group compared to the 20 mM glucose group. A histogram representation of the data is shown on the right. # P <0.01 vs Ctrl. ** P <0.01 vs 20 mM Glu. Data are presented as the mean ± standard deviation of three independent experiments.

Article Snippet: The membranes were blocked with 5% non-fat milk (w/v) for 2 h at room temperature, then incubated with the following primary antibodies overnight at 4°C: rabbit polyclonal anti-Cx43 (1:1000, #3512, Cell Signaling Technology [CST], Danvers, MA, USA), rabbit polyclonal anti-p38 (1:1000, #8690, CST), rabbit polyclonal anti-ERK (1:1000, #9102, CST), anti-β-tubulin (WL01931, Wanleibio, Shenyang, China), anti-Na-K-ATP (1:1000, ab205967, Abcam, Cambridge, UK).

Techniques: Expressing, Immunofluorescence, Microscopy, Standard Deviation

Journal: Neural regeneration research

Article Title: p38 MAPK inhibitor SB202190 suppresses ferroptosis in the glutamate-induced retinal excitotoxicity glaucoma model.

doi: 10.4103/1673-5374.391193

Figure Lengend Snippet: Figure 2 ｜ p38 MAPK inhibitor SB202190 protects R28 cells from glutamate-induced cell death. (A) The effect of different concentrations of glutamate on R28 cell viability over 24 hours (n = 5). (B) Light microscopy was used to examine the effect of SB202190 on 10 mM glutamate-treated R28 cells. (C) Effect of SB202190 on the cell viability of R28 cells treated with 10 mM glutamate for 24 hours (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, vs. control group (one-way analysis of variance followed by Tukey’s multiple comparison test). Data are expressed as the means ± SD. At least three independent experiments were performed. (D) The effect of SB202190 on the morphology of R28 cells was observed under a light microscope. Arrows indicate dead cells. Scale bars: 20 µm.

Article Snippet: The following primary antibodies were used for staining: p38 MAPK rabbit polyclonal antibody (1:200, Proteintech, Cat# 14064-1-AP, RRID: AB_2878007), FTL rabbit polyclonal antibody (1:200, Proteintech, Cat# 10727-1-AP, RRID: AB_2278673) and SAT1 rabbit polyclonal antibody (1:200, Proteintech, Cat# 10708- 1-AP, RRID: AB_2877739); phospho-p38 MAPK Thr180/ Tyr182 rabbit mAb (1:200, Cell Signaling Technology, Cat# 4511); rabbit recombinant anti-GPx4 antibody (1:200, Abcam, Cat# ab125066, RRID: AB_10973901); and SLC7A11 rabbit polyclonal antibody (1:200, Thermo Fisher Scientific, Cat# PA1-16893).

Techniques: Light Microscopy, Control, Comparison

Journal: Neural regeneration research

Article Title: p38 MAPK inhibitor SB202190 suppresses ferroptosis in the glutamate-induced retinal excitotoxicity glaucoma model.

doi: 10.4103/1673-5374.391193

Figure Lengend Snippet: Figure 3 ｜ p38 MAPK inhibitor SB202190 protects R28 cells from glutamate-induced ferroptosis. (A) Propidium iodide (PI; red) and Hoechst 33342 (blue) staining of R28 cells treated with glutamate, SB202190 and ferrostatin-1 as indicated for 24 hours. Scale bar: 100 µm. (B) Quantification of the results shown in A (n = 5). (C) Transmission electron microscope images of R28 cells treated with glutamate and SB202190. Scale bar: 1 µm. (D) R28 cells treated as indicated were stained with the fluorescent probe Mito -FerroOrange to determine ferrous ion levels. FerroOrange undergoes an irreversible reaction with intracellular Fe2+ in live cells, resulting in orange fluorescence emission. Ex: 543 nm and Em: 580 nm. Scale bar: 50 µm. (E) Quantification of mean density of FerroOrange fluorescence in the groups (n = 5). (F) R28 cells treated as indicated were stained with Liperfluo for measurement of lipid peroxides. Liperfluo is selectively oxidized by lipid peroxides, and the oxidized form of Liperfluo exhibits strong green fluorescence at the cell membrane. Scale bar: 50 µm. (G) Comparison of mean density of Liperfluor fluorescence in the groups (n = 5). (H, I) Malondialdehyde (MDA; H) and glutathione (GSH; I) levels in R28 cells after glutamate and SB202190 treatment (n = 3). **P < 0.01, ***P < 0.001, ****P < 0.0001 (one-way analysis of variance followed by Tukey’s multiple comparison test). Data are expressed as the means ± SD. At least three independent experiments were performed. MAPK: Mitogen-activated protein kinase.

Article Snippet: The following primary antibodies were used for staining: p38 MAPK rabbit polyclonal antibody (1:200, Proteintech, Cat# 14064-1-AP, RRID: AB_2878007), FTL rabbit polyclonal antibody (1:200, Proteintech, Cat# 10727-1-AP, RRID: AB_2278673) and SAT1 rabbit polyclonal antibody (1:200, Proteintech, Cat# 10708- 1-AP, RRID: AB_2877739); phospho-p38 MAPK Thr180/ Tyr182 rabbit mAb (1:200, Cell Signaling Technology, Cat# 4511); rabbit recombinant anti-GPx4 antibody (1:200, Abcam, Cat# ab125066, RRID: AB_10973901); and SLC7A11 rabbit polyclonal antibody (1:200, Thermo Fisher Scientific, Cat# PA1-16893).

Techniques: Staining, Transmission Assay, Microscopy, Fluorescence, Membrane, Comparison

Journal: Neural regeneration research

Article Title: p38 MAPK inhibitor SB202190 suppresses ferroptosis in the glutamate-induced retinal excitotoxicity glaucoma model.

doi: 10.4103/1673-5374.391193

Figure Lengend Snippet: Figure 4 ｜ p38 MAPK inhibitor SB202190 regulates ferroptosis in a glutamate R28 cell model by regulating FTL and SAT1. (A) p38 MAPK, p-p38 MAPK, FTL and SAT1 protein expression in R28 cells treated with glutamate and SB202190 for 24 hours (n = 3). (B–D) Quantification analysis of p-p38 MAPK/ p38 MAPK, FTL and SAT1 protein expression in R28 cells (n = 3). (E–I) Three days after intravitreal injection of NMDA and SB202190, paraffin sections were collected and processed for immunofluorescence experiments to measure the fluorescence intensity of p-p38 MAPK (E, F), FTL (G, H), and SAT1 (I, J) of the retinal sections in each group under a fluorescence microscope (n = 3). Scale bars: 10 µm. *P < 0.05, **P < 0.01, ***P < 0.001 (one-way analysis of variance followed by Tukey’s multiple comparison test). Data are expressed as the means ± SD. At least three independent experiments were repeated. FTL: Ferritin light chain; MAPK: mitogen activated protein kinases.

Article Snippet: The following primary antibodies were used for staining: p38 MAPK rabbit polyclonal antibody (1:200, Proteintech, Cat# 14064-1-AP, RRID: AB_2878007), FTL rabbit polyclonal antibody (1:200, Proteintech, Cat# 10727-1-AP, RRID: AB_2278673) and SAT1 rabbit polyclonal antibody (1:200, Proteintech, Cat# 10708- 1-AP, RRID: AB_2877739); phospho-p38 MAPK Thr180/ Tyr182 rabbit mAb (1:200, Cell Signaling Technology, Cat# 4511); rabbit recombinant anti-GPx4 antibody (1:200, Abcam, Cat# ab125066, RRID: AB_10973901); and SLC7A11 rabbit polyclonal antibody (1:200, Thermo Fisher Scientific, Cat# PA1-16893).

Techniques: Expressing, Injection, Immunofluorescence, Fluorescence, Microscopy, Comparison

Journal: Neural regeneration research

Article Title: p38 MAPK inhibitor SB202190 suppresses ferroptosis in the glutamate-induced retinal excitotoxicity glaucoma model.

doi: 10.4103/1673-5374.391193

Figure Lengend Snippet: Figure 6 ｜ p38 MAPK inhibitor SB202190 attenuates N-methyl-D-aspartate (NMDA)- induced damage in the rat retina. (A, B) Qualitative observation (A) and quantitative analysis (B) of the effects of SB202190 on NMDA-induced retinal ganglion cell (RGC) injury in rat retina (n = 5). Three days after intravitreal injection of NMDA and SB202190, retinal plating was performed. RGCs were fluorescently labeled with Brn3a antibody and surviving RGC exhibited strong green fluorescence under a fluorescence microscope. Scale bar: 50 μm. (C) Effects of SB202190 on retinal morphology in NMDA model rats. Hematoxylin and eosin staining was performed 3 days after intravitreal injection of NMDA and SB202190. Scale bar: 50 µm. (D) Effect of SB202190 on RGC density (cells/100 µm) in NMDA model rats 3 days after intravitreal injection (n = 4). (E, F) Thickness of the retinal ganglion cell body complex (GCC) at 500, 1000, 1500, and 2000 µm from the optic disc 3 days after intravitreal injection of NMDA and SB202190. (G–I) Flash visual evoked potentials of rats 3 days after intravitreal injection of NMDA and SB202190 (n = 4). GCL: Ganglion cell layer; INL: inner nuclear layer; IPL: inner plexiform layer; ONL: outer nuclear layer; RGC: retinal ganglion cell. Inner retina consists of GCL and IPL. **P < 0.01, ****P < 0.0001 (one-way analysis of variance followed by Tukey’s multiple comparison test). Data are expressed as the means ± SD. At least three independent experiments were performed. MAPK: Mitogen- activated protein kinase.

Article Snippet: The following primary antibodies were used for staining: p38 MAPK rabbit polyclonal antibody (1:200, Proteintech, Cat# 14064-1-AP, RRID: AB_2878007), FTL rabbit polyclonal antibody (1:200, Proteintech, Cat# 10727-1-AP, RRID: AB_2278673) and SAT1 rabbit polyclonal antibody (1:200, Proteintech, Cat# 10708- 1-AP, RRID: AB_2877739); phospho-p38 MAPK Thr180/ Tyr182 rabbit mAb (1:200, Cell Signaling Technology, Cat# 4511); rabbit recombinant anti-GPx4 antibody (1:200, Abcam, Cat# ab125066, RRID: AB_10973901); and SLC7A11 rabbit polyclonal antibody (1:200, Thermo Fisher Scientific, Cat# PA1-16893).

Techniques: Injection, Labeling, Fluorescence, Microscopy, Staining, Comparison

Journal: Neural regeneration research

Article Title: p38 MAPK inhibitor SB202190 suppresses ferroptosis in the glutamate-induced retinal excitotoxicity glaucoma model.

doi: 10.4103/1673-5374.391193

Figure Lengend Snippet: Figure 5 ｜ p38 MAPK inhibitor SB202190 alleviates lipid peroxidation by regulating the SLC7A11/GSH/GPx4 pathway. (A, B) Flow cytometry was used to detect the effect of SB202190 on lipid reactive oxygen species production in R28 cells treated as indicated for 24 hours (n = 4). (C–E) SLC7A11 and GPx4 protein expressions in R28 cells were detected by western blotting (n = 3). (F–I) Fluorescence microscopy was performed to evaluate the fluorescence intensity changes of and GPx4 (F, G) and SLC7A11 (H, I) in rat retinal sections after intraluminal injection of NMDA and SB202190 for 3 days (n = 3). Scale bars in F and H: 10 µm. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (one-way analysis of variance followed by Tukey’s multiple comparison test). Data are expressed as the means ± SD. At least three independent experiments were performed. GPx4: Glutathione peroxidase 4; GSH: glutathione; MAPK: mitogen-activated protein kinase.

Article Snippet: The following primary antibodies were used for staining: p38 MAPK rabbit polyclonal antibody (1:200, Proteintech, Cat# 14064-1-AP, RRID: AB_2878007), FTL rabbit polyclonal antibody (1:200, Proteintech, Cat# 10727-1-AP, RRID: AB_2278673) and SAT1 rabbit polyclonal antibody (1:200, Proteintech, Cat# 10708- 1-AP, RRID: AB_2877739); phospho-p38 MAPK Thr180/ Tyr182 rabbit mAb (1:200, Cell Signaling Technology, Cat# 4511); rabbit recombinant anti-GPx4 antibody (1:200, Abcam, Cat# ab125066, RRID: AB_10973901); and SLC7A11 rabbit polyclonal antibody (1:200, Thermo Fisher Scientific, Cat# PA1-16893).

Techniques: Flow Cytometry, Western Blot, Fluorescence, Microscopy, Injection, Comparison

Journal: Genes & Genomics

Article Title: Autophagy induced by H. pylori VacA regulated the survival mechanism of the SGC7901 human gastric cancer cell line

doi: 10.1007/s13258-021-01151-7

Figure Lengend Snippet: Expression of LC3II and p62 in SGC7901 cells treated with H. pylori culture supernatant at different concentrations and durations. A Western blotting was used to detect the expression of LC3. The results show that the transformation of LC3I into LC3II increased with increasing concentration and that the expression of LC3II/β-tubulin was enhanced. B Different concentrations of H. pylori supernatant were used to treat cells, and the expression of P62 gradually decreased. C Western blotting was used to detect the expression of LC3II. LC3II/ β -tubulin levels increased with time, and autophagy peaked at 8 h. D Cells treated with concentrated H. pylori culture supernatant for 0, 2, 4, 8 and 12 h, and the expression of p62 gradually decreased. * means that a difference was statistically significant compared with the control group, p < 0.05; ** p value < 0.01; NS, showed no significant difference compared with the control group, p > 0.05

Article Snippet: Antibodies against P62 (sc-25329), LC3II (sc-376404), VacA (sc-32746), and β-tubulin (sc-166729) were obtained from Santa Cruz Biotechnology.

Techniques: Expressing, Western Blot, Transformation Assay, Concentration Assay, Control

Journal: Genes & Genomics

Article Title: Autophagy induced by H. pylori VacA regulated the survival mechanism of the SGC7901 human gastric cancer cell line

doi: 10.1007/s13258-021-01151-7

Figure Lengend Snippet: Autophagy after treatment with H. pylori culture supernatant or the VacA protein in SGC7901 cells. A After SGC901 cells were treated with culture supernatant, LC3II expression was detected by Western blotting. The results showed that the conversion of LC3I to LC3II was decreased and the LC3II/β-tubulin ratio was decreased, b p < 0.05. B Western blotting was used to detect the expression of P62. The results showed that the P62/β-tubulin ratio was increased, b p < 0.05. C After cells were treated with H. pylori culture supernatant or the VacA protein for 8 h, the expression levels of BECN1, PI3KC3 and ATG7 were enhanced, a p < 0.05 and b p < 0.05. D When concentrated H. pylori culture supernatant containing the VacA protein was used to treat stable GFP-LC3-expressing cells, a significantly increased number of GFP-LC3 particles were observed. E After purified VacA protein was used to treat GFP-LC3-expressing cells, the number of GFP-LC3 particles was increased, a p < 0.05 and b p < 0.05

Article Snippet: Antibodies against P62 (sc-25329), LC3II (sc-376404), VacA (sc-32746), and β-tubulin (sc-166729) were obtained from Santa Cruz Biotechnology.

Techniques: Expressing, Western Blot, Purification

Journal: Genes & Genomics

Article Title: Autophagy induced by H. pylori VacA regulated the survival mechanism of the SGC7901 human gastric cancer cell line

doi: 10.1007/s13258-021-01151-7

Figure Lengend Snippet: After Atg7 silencing, LC3 expression in SGC7901 cells was detected . A After Atg7 silencing and 8 h of treatment with VacA, the expression of LC3 in SGC7901 cells was detected by Western blotting. The conversion of LC3I to LC3II was decreased, and the LC3II/β-tubulin ratio was decreased, p < 0.05. B After Atg7 silencing and 8 h of treatment with VacA, GFP-LC3 particles in stable transfer cell lines were decreased under a fluorescence microscope

Article Snippet: Antibodies against P62 (sc-25329), LC3II (sc-376404), VacA (sc-32746), and β-tubulin (sc-166729) were obtained from Santa Cruz Biotechnology.

Techniques: Expressing, Western Blot, Fluorescence, Microscopy

Journal: Genes & Genomics

Article Title: Autophagy induced by H. pylori VacA regulated the survival mechanism of the SGC7901 human gastric cancer cell line

doi: 10.1007/s13258-021-01151-7

Figure Lengend Snippet: H. pylori culture supernatant or VacA protein treatment increased ROS levels and autophagy in SGC7901 cells. A ROS levels in SGC7901 cells were detected by H2DCF-DA staining. B After the treatment of cells with concentrated H. pylori culture supernatant or the VacA protein, ROS levels were quantitatively detected by H2DCF-DA staining with a microplate reader. Levels of significance are indicated as follows: a p < 0.05 vs the control group, b p < 0.05 vs the control group, c p < 0.05 vs the control group, d p < 0.05 vs the CMCC group, e p < 0.05 vs the VacA group. C After VacA and NAC were added to cells and incubated for 8 h, LC3II expression was detected by Western blotting. The results showed that the conversion of LC3I to LC3II was decreased after the addition of NAC and that the LC3II/β-tubulin ratio was significantly decreased, p < 0.05. D The VacA protein was used to treat a stable GFP-LC3-expressing cell line for 8 h, and an increased number of GFP-LC3 particles was observed under a fluorescence microscope, while a decreased number of GFP-LC3 particles was observed after the addition of NAC. E After the VacA protein was used to treat SGC7901 cells, an MTS kit was used to detect cell proliferation. The results showed that after treated with VacA for 8 h, cell proliferation was inhibited. F The survival rate of SGC7901 cells was significantly decreased after 12 h of treatment, as shown by trypan blue staining, p < 0.05

Article Snippet: Antibodies against P62 (sc-25329), LC3II (sc-376404), VacA (sc-32746), and β-tubulin (sc-166729) were obtained from Santa Cruz Biotechnology.

Techniques: Staining, Control, Incubation, Expressing, Western Blot, Fluorescence, Microscopy

Journal: RSC Advances

Article Title: Modified hydrothermal method for synthesizing titanium dioxide-decorated multiwalled carbon nanotube nanocomposites for the solar-driven photocatalytic degradation of dyes †

doi: 10.1039/d4ra05899b

Figure Lengend Snippet: (a) TEM image, scale bar 50 nm. (b) High-magnification TEM image, scale bar 20 nm. (c) HR-TEM image of TiO 2 /MWCNT-2.0 nanocomposites. (d) Size-distribution histogram of TiO 2 nanoparticles on MWCNTs. (e) TEM-EDS elemental mapping for (D) titanium, (E) oxygen, and (F) carbon.

Article Snippet: The elemental distribution in the TiO 2 /MWCNT nanomaterials was assessed by TEM mapping with a JEOL JEM-2100 instrument (JEOL Ltd, Tokyo, Japan).

Techniques: