Journal: Experimental and Therapeutic Medicine

Article Title: Biphasic function of GSK3β in gefitinib‑resistant NSCLC with or without EGFR mutations

doi: 10.3892/etm.2023.12187

Figure Lengend Snippet: GSK3β is involved in gefitinib resistance in PC-9G cells. (A) Genes related to the PI3K-AKT signaling pathway were differentially expressed between PC-9 and PC-9G cells. (B) Heatmap clustering analysis of differentially expressed genes between PC-9 and PC-9G cells. (C) Protein expression levels of GSK3β, pGSK3β (Ser9), AKT, pAKT (Ser473), c-Myc, E-cadherin and vimentin were detected via western blot analysis in PC-9 and PC-9G cells treated with DMSO or 300 nmol/l gefitinib. (D) Immunohistochemical analysis showed that the expression of E-cadherin in PC-9 cells was higher than that in PC-9G cells, whereas the expression of vimentin in PC-9 cells was lower than that in PC-9G cells (magnification, x20). GSK3β, glycogen synthase kinase 3β; p, phosphorylated; PC-9G, gefitinib-resistant PC-9.

Article Snippet: Equal amounts of heat-denatured protein (30 µg/lane) were separated by SDS-PAGE on 10% gels and transferred onto nitrocellulose membranes, which were blocked with blocking buffer containing 5% non-fat milk in 1X TBS-0.1% Tween-20 for 2 h at room temperature and incubated with primary antibodies at 4˚C overnight, followed by incubation with an appropriate secondary antibody at 37˚C for 2 h. The following primary antibodies (1:1,000 dilution; all from Cell Signaling Technology, Inc.) were used: c-Myc (cat. no. 5605), E-cadherin (cat. no. 3195), vimentin (cat. no. 5741), GAPDH (cat. no. 5174), β-catenin (cat. no. 8480), Lamin A (cat. no. 86846), GSK3β (cat. no. 12456), phosphorylated (p)GSK3β (cat. no. 9322), AKT (cat. no. 4691), pAKT (cat. no. 4060), MET (cat. no. 8198), pMET (cat. no. 3077), and β-actin (cat. no. 8457).

Techniques: Expressing, Western Blot, Immunohistochemistry

Journal: Experimental and Therapeutic Medicine

Article Title: Biphasic function of GSK3β in gefitinib‑resistant NSCLC with or without EGFR mutations

doi: 10.3892/etm.2023.12187

Figure Lengend Snippet: Effects of GSK3β overexpression on the proliferation, migration and invasion of PC-9, PC-9G and H1975 cells. (A) Fluorescence microscopy showed that stable GSK3β overexpression was successfully achieved in PC-9, PC-9G and H1975 cells (magnification, x10). (B) mRNA expression levels of GSK3β were upregulated in PC-9, PC-9G and H1975 cell lines with stable GSK3β overexpression. (C) Protein expression levels of GSK3β, pGSK3β (Ser9), c-Myc, E-cadherin, vimentin and β-catenin was determined via western blot analysis in PC-9, PC-9G and H1975 cells following overexpression of GSK3β. (D) GSK3β overexpression enhanced the proliferation of PC-9 and H1975 cells, but not that of PC-9G cells. GSK3β overexpression reduced the (E) migration and (F) invasion of PC-9G cells, but enhanced the migration and invasion of PC-9 and H1975 cells (magnification, x20). Data are presented as the mean ± SD of three independent experiments. * P<0.05, ** P<0.01, *** P<0.001. GSK3β, glycogen synthase kinase 3β; p, phosphorylated; PC-9G, gefitinib-resistant PC-9.

Article Snippet: Equal amounts of heat-denatured protein (30 µg/lane) were separated by SDS-PAGE on 10% gels and transferred onto nitrocellulose membranes, which were blocked with blocking buffer containing 5% non-fat milk in 1X TBS-0.1% Tween-20 for 2 h at room temperature and incubated with primary antibodies at 4˚C overnight, followed by incubation with an appropriate secondary antibody at 37˚C for 2 h. The following primary antibodies (1:1,000 dilution; all from Cell Signaling Technology, Inc.) were used: c-Myc (cat. no. 5605), E-cadherin (cat. no. 3195), vimentin (cat. no. 5741), GAPDH (cat. no. 5174), β-catenin (cat. no. 8480), Lamin A (cat. no. 86846), GSK3β (cat. no. 12456), phosphorylated (p)GSK3β (cat. no. 9322), AKT (cat. no. 4691), pAKT (cat. no. 4060), MET (cat. no. 8198), pMET (cat. no. 3077), and β-actin (cat. no. 8457).

Techniques: Over Expression, Migration, Fluorescence, Microscopy, Expressing, Western Blot

Journal: Experimental and Therapeutic Medicine

Article Title: Biphasic function of GSK3β in gefitinib‑resistant NSCLC with or without EGFR mutations

doi: 10.3892/etm.2023.12187

Figure Lengend Snippet: Effects of an AKT inhibitor on the proliferation, apoptosis, migration and invasion of PC-9, PC-9G and H1975 cells. (A) Protein expression levels of GSK3β, pGSK3β (Ser9), AKT, pAKT (Ser473), c-Myc, E-cadherin and vimentin were determined via western blot analysis in PC-9, PC-9G and H1975 cells treated with DMSO or 5 nmol/l MK2206. (B) Apoptotic rate of PC-9, PC-9G and H1975 cells was increased after MK2206 treatment. (C) MK2206 treatment inhibited the proliferation of H1975 cells from day 2 to 5 and reduced the proliferation of PC9 cells at day 5. (D) Quantification of apoptosis results. MK2206 treatment inhibited the (E) migration and (F) invasion of PC-9, PC-9G and H1975 cells (magnification, x20). Data are presented as the mean ± SD of three independent experiments. * P<0.05, ** P<0.01, *** P<0.001. FITC, fluorescein isothiocyanate; GSK3β, glycogen synthase kinase 3β; p, phosphorylated; PC-9G, gefitinib-resistant PC-9; PI, propidium iodide.

Article Snippet: Equal amounts of heat-denatured protein (30 µg/lane) were separated by SDS-PAGE on 10% gels and transferred onto nitrocellulose membranes, which were blocked with blocking buffer containing 5% non-fat milk in 1X TBS-0.1% Tween-20 for 2 h at room temperature and incubated with primary antibodies at 4˚C overnight, followed by incubation with an appropriate secondary antibody at 37˚C for 2 h. The following primary antibodies (1:1,000 dilution; all from Cell Signaling Technology, Inc.) were used: c-Myc (cat. no. 5605), E-cadherin (cat. no. 3195), vimentin (cat. no. 5741), GAPDH (cat. no. 5174), β-catenin (cat. no. 8480), Lamin A (cat. no. 86846), GSK3β (cat. no. 12456), phosphorylated (p)GSK3β (cat. no. 9322), AKT (cat. no. 4691), pAKT (cat. no. 4060), MET (cat. no. 8198), pMET (cat. no. 3077), and β-actin (cat. no. 8457).

Techniques: Migration, Expressing, Western Blot

Journal: Experimental and Therapeutic Medicine

Article Title: Biphasic function of GSK3β in gefitinib‑resistant NSCLC with or without EGFR mutations

doi: 10.3892/etm.2023.12187

Figure Lengend Snippet: Effects of an AKT activator on the proliferation, apoptosis, migration and invasion of PC-9, PC-9G and H1975 cells. (A) Protein expression levels of GSK3β, pGSK3β (Ser9), AKT, pAKT (Ser473), c-Myc, E-cadherin and vimentin was determined via western blot analysis in PC-9, PC-9G and H1975 cells treated with DMSO or 5 nmol/l SC79. (B) Apoptotic rate of PC-9 cells was decreased after SC79 treatment. (C) SC79 treatment promoted the proliferation of PC-9 and H1975 cells. (D) Quantification of apoptosis results. SC79 treatment promoted the (E) migration and (F) invasion of PC-9, PC-9G and H1975 cells (magnification, x20). Data are presented as the mean ± SD of three independent experiments. * P<0.05, ** P<0.01. FITC, fluorescein isothiocyanate; GSK3β, glycogen synthase kinase 3β; p, phosphorylated; PC-9G, gefitinib-resistant PC-9; PI, propidium iodide.

Article Snippet: Equal amounts of heat-denatured protein (30 µg/lane) were separated by SDS-PAGE on 10% gels and transferred onto nitrocellulose membranes, which were blocked with blocking buffer containing 5% non-fat milk in 1X TBS-0.1% Tween-20 for 2 h at room temperature and incubated with primary antibodies at 4˚C overnight, followed by incubation with an appropriate secondary antibody at 37˚C for 2 h. The following primary antibodies (1:1,000 dilution; all from Cell Signaling Technology, Inc.) were used: c-Myc (cat. no. 5605), E-cadherin (cat. no. 3195), vimentin (cat. no. 5741), GAPDH (cat. no. 5174), β-catenin (cat. no. 8480), Lamin A (cat. no. 86846), GSK3β (cat. no. 12456), phosphorylated (p)GSK3β (cat. no. 9322), AKT (cat. no. 4691), pAKT (cat. no. 4060), MET (cat. no. 8198), pMET (cat. no. 3077), and β-actin (cat. no. 8457).

Techniques: Migration, Expressing, Western Blot

Journal: Experimental and Therapeutic Medicine

Article Title: FOXA2 attenuates lipopolysaccharide‑induced pneumonia by inhibiting the inflammatory response, oxidative stress and apoptosis through blocking of p38/STAT3 signaling

doi: 10.3892/etm.2023.12168

Figure Lengend Snippet: FOXA2 decreases ERS by blocking p38/STAT3 signaling. (A) Expression levels of p38/STAT3 signaling-related proteins were examined using western blotting. *** P<0.001 vs. Control; ### P<0.001 vs. LPS + oe-NC. (B) FOXA2-overexpressing WI-38 cells were pretreated with U46619, an activator of p38 signaling, and then induced by LPS. The expression levels of ERS-related proteins were detected using western blotting. *** P<0.001 vs. Control; ### P<0.001 vs. LPS; + P<0.05, ++ P<0.01 and +++ P<0.001 vs. LPS + oe-FOXA2. ATF6, activating transcription factor 6; eIF2α, eukaryotic translation initiation factor 2 subunit α; ERS, endoplasmic reticulum stress; FOXA2, forkhead box protein A2; GRP78, glucose-regulated protein 78; LPS, lipopolysaccharide; oe-FOXA2, FOXA2 overexpression vector; oe-NC, overexpression negative control; p-, phosphorylated; t-, total; XBP1, X-box binding protein 1.

Article Snippet: The membranes were blocked with 5% skimmed milk for 2 h at room temperature, followed by probing with primary antibodies against FOXA2 (cat. no. ab23630; 1:1,000; Abcam), cyclooxygenase-2 (Cox2; cat. no. ab179800; 1:1,000; Abcam), inducible nitric oxide synthase (iNOS; cat. no. ab178945; 1:1,000; Abcam), NADPH oxidase (Nox)2 (cat. no. ab129068; 1:5,000; Abcam), Nox4 (cat. no. ab154244; 1:1,000; Abcam), Bcl-2 (cat. no. ab32124; 1:1,000; Abcam), Bax (cat. no. ab32503; 1:1,000; Abcam), Cleaved-poly(ADP-ribose) polymerase 1 (PARP1; cat. no. ab32064; 1:1,000; Abcam), PARP1 (cat. no. ab227244; 1:1,000; Abcam), phosphorylated (p)-p38 (cat. no. 9215; 1:1,000; Cell Signaling Technology, Inc.), p38 (cat. no. 9212; 1:1,000; Cell Signaling Technology, Inc.), p-STAT3 (cat. no. 94994; 1:1,000; Cell Signaling Technology, Inc.), STAT3 (cat. no. ab109085; 1:1,000; Abcam), glucose-regulated protein 78 (GRP78; cat. no. ab21685; 1:1,000; Abcam), CHOP (cat. no. 5554; 1:1,000; Cell Signaling Technology, Inc.), X-box binding protein 1 (XBP1; cat. no. ab37152; 1:1,000; Abcam), activating transcription factor 6 (ATF6; cat. no. orb381900; 1:1,000; Biorbyt, Ltd.), p-eukaryotic translation initiation factor 2 subunit α (eIF2α; cat. no. orb15000; 1:1,000; Biorbyt, Ltd.), eIF2α (cat. no. orb480065; 1:1,000; Biorbyt, Ltd.) and GAPDH (cat. no. ab9485; 1:2,500; Abcam) at 4˚C overnight.

Techniques: Blocking Assay, Expressing, Western Blot, Over Expression, Plasmid Preparation, Negative Control, Binding Assay

Journal: Experimental and Therapeutic Medicine

Article Title: FOXA2 attenuates lipopolysaccharide‑induced pneumonia by inhibiting the inflammatory response, oxidative stress and apoptosis through blocking of p38/STAT3 signaling

doi: 10.3892/etm.2023.12168

Figure Lengend Snippet: FOXA2 protects WI-38 cells against LPS-induced oxidative stress and inflammation via inactivation of p38/STAT3 signaling. (A) Production of pro-inflammatory cytokines, including TNF-α, IL-6 and IL-1β, was measured using ELISA. (B) Cellular reactive oxygen species levels were detected using the 2',7'-dichlorofluorescein diacetate method. Magnification, x200. Scale bar, 50 µM. (C) MDA levels, SOD activity and CAT activity were assessed using their corresponding kits. (D) Protein expression levels of Cox2, iNOS, Nox2 and Nox4 were examined using western blotting. *** P<0.001 vs. Control; ## P<0.01 and ### P<0.001 vs. LPS; + P<0.05, ++ P<0.01 and +++ P<0.001 vs. LPS + oe-FOXA2. CAT, catalase; Cox2, cyclooxygenase-2; FOXA2, forkhead box protein A2; iNOS, inducible nitric oxide synthase; LPS, lipopolysaccharide; MDA, malondialdehyde; Nox, NADPH oxidase; oe-FOXA2, FOXA2 overexpression vector; SOD, superoxide dismutase.

Article Snippet: The membranes were blocked with 5% skimmed milk for 2 h at room temperature, followed by probing with primary antibodies against FOXA2 (cat. no. ab23630; 1:1,000; Abcam), cyclooxygenase-2 (Cox2; cat. no. ab179800; 1:1,000; Abcam), inducible nitric oxide synthase (iNOS; cat. no. ab178945; 1:1,000; Abcam), NADPH oxidase (Nox)2 (cat. no. ab129068; 1:5,000; Abcam), Nox4 (cat. no. ab154244; 1:1,000; Abcam), Bcl-2 (cat. no. ab32124; 1:1,000; Abcam), Bax (cat. no. ab32503; 1:1,000; Abcam), Cleaved-poly(ADP-ribose) polymerase 1 (PARP1; cat. no. ab32064; 1:1,000; Abcam), PARP1 (cat. no. ab227244; 1:1,000; Abcam), phosphorylated (p)-p38 (cat. no. 9215; 1:1,000; Cell Signaling Technology, Inc.), p38 (cat. no. 9212; 1:1,000; Cell Signaling Technology, Inc.), p-STAT3 (cat. no. 94994; 1:1,000; Cell Signaling Technology, Inc.), STAT3 (cat. no. ab109085; 1:1,000; Abcam), glucose-regulated protein 78 (GRP78; cat. no. ab21685; 1:1,000; Abcam), CHOP (cat. no. 5554; 1:1,000; Cell Signaling Technology, Inc.), X-box binding protein 1 (XBP1; cat. no. ab37152; 1:1,000; Abcam), activating transcription factor 6 (ATF6; cat. no. orb381900; 1:1,000; Biorbyt, Ltd.), p-eukaryotic translation initiation factor 2 subunit α (eIF2α; cat. no. orb15000; 1:1,000; Biorbyt, Ltd.), eIF2α (cat. no. orb480065; 1:1,000; Biorbyt, Ltd.) and GAPDH (cat. no. ab9485; 1:2,500; Abcam) at 4˚C overnight.

Techniques: Enzyme-linked Immunosorbent Assay, Activity Assay, Expressing, Western Blot, Over Expression, Plasmid Preparation

Journal: Experimental and Therapeutic Medicine

Article Title: FOXA2 attenuates lipopolysaccharide‑induced pneumonia by inhibiting the inflammatory response, oxidative stress and apoptosis through blocking of p38/STAT3 signaling

doi: 10.3892/etm.2023.12168

Figure Lengend Snippet: FOXA2 protects WI-38 cells against LPS-induced apoptosis via inactivation of p38/STAT3 signaling. (A) Flow cytometry was performed to assess the cell apoptosis rate in each group and (B) quantified. (C) Caspase3 activity was determined using the related kit. (D) Expression levels of apoptosis-related proteins were examined using western blotting and (E) quantified. *** P<0.001 vs. Control; ### P<0.001 vs. LPS; ++ P<0.01 and +++ P<0.001 vs. LPS + oe-FOXA2. FOXA2, forkhead box protein A2; LPS, lipopolysaccharide; oe-FOXA2, FOXA2 overexpression vector; PARP1, poly(ADP-ribose) polymerase 1.

Article Snippet: The membranes were blocked with 5% skimmed milk for 2 h at room temperature, followed by probing with primary antibodies against FOXA2 (cat. no. ab23630; 1:1,000; Abcam), cyclooxygenase-2 (Cox2; cat. no. ab179800; 1:1,000; Abcam), inducible nitric oxide synthase (iNOS; cat. no. ab178945; 1:1,000; Abcam), NADPH oxidase (Nox)2 (cat. no. ab129068; 1:5,000; Abcam), Nox4 (cat. no. ab154244; 1:1,000; Abcam), Bcl-2 (cat. no. ab32124; 1:1,000; Abcam), Bax (cat. no. ab32503; 1:1,000; Abcam), Cleaved-poly(ADP-ribose) polymerase 1 (PARP1; cat. no. ab32064; 1:1,000; Abcam), PARP1 (cat. no. ab227244; 1:1,000; Abcam), phosphorylated (p)-p38 (cat. no. 9215; 1:1,000; Cell Signaling Technology, Inc.), p38 (cat. no. 9212; 1:1,000; Cell Signaling Technology, Inc.), p-STAT3 (cat. no. 94994; 1:1,000; Cell Signaling Technology, Inc.), STAT3 (cat. no. ab109085; 1:1,000; Abcam), glucose-regulated protein 78 (GRP78; cat. no. ab21685; 1:1,000; Abcam), CHOP (cat. no. 5554; 1:1,000; Cell Signaling Technology, Inc.), X-box binding protein 1 (XBP1; cat. no. ab37152; 1:1,000; Abcam), activating transcription factor 6 (ATF6; cat. no. orb381900; 1:1,000; Biorbyt, Ltd.), p-eukaryotic translation initiation factor 2 subunit α (eIF2α; cat. no. orb15000; 1:1,000; Biorbyt, Ltd.), eIF2α (cat. no. orb480065; 1:1,000; Biorbyt, Ltd.) and GAPDH (cat. no. ab9485; 1:2,500; Abcam) at 4˚C overnight.

Techniques: Flow Cytometry, Activity Assay, Expressing, Western Blot, Over Expression, Plasmid Preparation

Journal: bioRxiv

Article Title: Age-related STAT3 signaling regulates severity of respiratory syncytial viral infection in human bronchial epithelial cells

doi: 10.1101/2023.09.20.558606

Figure Lengend Snippet: (A) Representative Western blot analyses of p-STAT3 Y 705 and STAT3 levels at 2 dpi in neonatal and adult ALI cultures (n=3 BSC lines per age group). β-Actin was loading control. Each lane represents one BSC line. (B) Densitometry measurements of the relative level of p-STAT3 Y 705 normalized to β-Actin. **p<0.01 by two-way ANOVA followed by Sidak’s multiple comparison test. (C) Representative double staining for RSV F (fluorescence) and p-STAT3 Y 705 (chromogenic). Arrowhead marks RSV F + ciliated cells. Inserts show enlarged images of p-STAT3 Y 705 staining. Scale bar, 50 μm. (D) Schematic of RSV infection of a hybrid ALI culture. GFP-labelled neonatal BSCs (n=2 lines) were mixed with adult BSCs (n=2 lines) at a 1:1 ratio followed by differentiation in ALI. Hybrid ALI cultures were analyzed at 1, 2, and 4 dpi. (E) Representative staining for RSV F in hybrid ALI cultures. White arrows mark RSV F + GFP - (adult) cells and yellow arrows mark RSV F + GFP + (neonatal) cells. Scale bar, 50 μm. (F) The relative abundance of GFP + cells in hybrid ALI cultures.

Article Snippet: Primary antibodies include rabbit anti-phosphorylated STAT3 at Tyr705 (1:1000, Cat#9145S, Cell Signaling Technology), rabbit anti-STAT3 (1:1000, Cat#12640S, Cell Signaling Technology), rabbit anti-Cleaved Caspase-3 (Asp175) (1:1000, Cat#9661S, Cell Signaling Technology), and mouse anti-β-actin (1:2000, Cat#A5441, Sigma Aldrich).

Techniques: Western Blot, Comparison, Double Staining, Fluorescence, Staining, Infection

Journal: bioRxiv

Article Title: Age-related STAT3 signaling regulates severity of respiratory syncytial viral infection in human bronchial epithelial cells

doi: 10.1101/2023.09.20.558606

Figure Lengend Snippet: (A) Schematic of SARS-CoV-2 infection (MOI 3) of day 21 ALI cultures of neonatal and adult TA BSCs (n=2 BSC lines for each age group) followed by analyses at 1 and 3 dpi. (B) Representative double staining for AceTUB and SARS-CoV-2 N in cross sections of ALI cultures. (C) Representative whole-mount staining for SARS-CoV-2 N. (D) The relative abundance of SARS-CoV-2 N + cells in neonatal and adult ALI cultures. (E) Representative chromogenic double staining for SARS-CoV-2 N (cytoplasm) and p-STAT3 Y 705 (nucleus) at 3 dpi. Arrows point to SARS-CoV-2 N + ciliated cells. Arrowheads point to cells positive for nuclear p-STAT3 Y 705 . (F) Representative double staining for c-Casp-3 and AceTUB at 3 dpi. (G) Representative staining for p-STAT3 Y 705 , SARS-CoV-2 N, and c-Casp-3 using three adjacent lung sections (4µm apart) of post-mortem lung samples from fatal COVID-19 patients (n=5 donors). The dashed line marks basement membrane. Arrowheads mark SARS-CoV-2 N + ciliated cells. Asterisk marks a c-Casp-3 + cell in lung parenchyma that was negative for SARS-CoV-2 N. Each data point (D) represents quantifications of one 20X image of one BSC line. Bar graph represents mean ± SEM. ns, not significant by two-way ANOVA followed by Sidak’s multiple comparison test. Similar results were observed in 2 neonatal and 2 adult BSC lines. Scale bars, 50 μm.

Article Snippet: Primary antibodies include rabbit anti-phosphorylated STAT3 at Tyr705 (1:1000, Cat#9145S, Cell Signaling Technology), rabbit anti-STAT3 (1:1000, Cat#12640S, Cell Signaling Technology), rabbit anti-Cleaved Caspase-3 (Asp175) (1:1000, Cat#9661S, Cell Signaling Technology), and mouse anti-β-actin (1:2000, Cat#A5441, Sigma Aldrich).

Techniques: Infection, Double Staining, Staining, Membrane, Comparison

Journal: bioRxiv

Article Title: Age-related STAT3 signaling regulates severity of respiratory syncytial viral infection in human bronchial epithelial cells

doi: 10.1101/2023.09.20.558606

Figure Lengend Snippet: (A) Schematic of Stattic (20 μM) treatment. Stattic was applied in the bottom chamber of adult ALI cultures (n=3 BSC lines) 2 hours prior to RSV infection until 2 dpi. Results were shown in (B-D). (B) Representative double staining for RSV F and c-Casp-3. (C) The relative level of RSV L gene by RT-qPCR. (D) The relative abundance of RSV F + and RSV F + c-Casp-3 + cells. (E) Schematic of STAT3 knockdown assay using an inducible lenti-shRNA system. ALI cultures of lentivirus transduced adult BSCs (n=3 lines) were treated with Dox (500 ng/mL) in the bottom chamber from day 18 and infected with RSV at day 21. Assays were performed 2 dpi and results were shown in (F-H). (F) Representative Western blot analyses to assess STAT3 knockdown efficiency. β-actin was loading control. Each lane represents one BSC line. (G) Representative double staining for RSV F and c-Casp-3. (H) Quantification of the relative abundance of RSV F + cells and RSV F + c-Casp-3 + cells. Each dot represents one BSC line. **p<0.01 and ***p<0.001 calculated by Student’s t-test (two-tailed). Scale bars, 50 μm.

Article Snippet: Primary antibodies include rabbit anti-phosphorylated STAT3 at Tyr705 (1:1000, Cat#9145S, Cell Signaling Technology), rabbit anti-STAT3 (1:1000, Cat#12640S, Cell Signaling Technology), rabbit anti-Cleaved Caspase-3 (Asp175) (1:1000, Cat#9661S, Cell Signaling Technology), and mouse anti-β-actin (1:2000, Cat#A5441, Sigma Aldrich).

Techniques: Infection, Double Staining, Quantitative RT-PCR, shRNA, Western Blot, Two Tailed Test

Journal: bioRxiv

Article Title: Age-related STAT3 signaling regulates severity of respiratory syncytial viral infection in human bronchial epithelial cells

doi: 10.1101/2023.09.20.558606

Figure Lengend Snippet: (A) Schematic of IL6 treatment assay. Neonatal cultures were treated with IL6 (50 ng/mL) in the bottom chamber at D18 in ALI with and without Stattic (20 μM) applied 2 hours prior to RSV infection. ALI cultures were assayed at 6 hpi and 2 dpi. (B) Representative Western blot analyses for levels of p-STAT3 Y 705 and STAT3. β-actin was loading control. Each lane represents one neonatal BSC line. (C) Densitometry measurement of relative levels of p-STAT3 Y 705 and STAT3. (D) The relative level of RSV L gene at 6 hpi by RT-q-PCR. (E) Representative double staining for RSV F and c-Casp-3. (F) The relative abundance of RSV F + cells and RSV + c-Casp-3 + cells. Each dot represents one BSC line. Bar graphs show mean ± SEM. **p<0.01, ***p<0.001, and ns, not significant calculated by Student’s t-test (two-tailed) in (C and D) and **p<0.01 by one-way ANOVA followed Tukey’s multiple comparison test in (F). Scale bar, 50 μm.

Article Snippet: Primary antibodies include rabbit anti-phosphorylated STAT3 at Tyr705 (1:1000, Cat#9145S, Cell Signaling Technology), rabbit anti-STAT3 (1:1000, Cat#12640S, Cell Signaling Technology), rabbit anti-Cleaved Caspase-3 (Asp175) (1:1000, Cat#9661S, Cell Signaling Technology), and mouse anti-β-actin (1:2000, Cat#A5441, Sigma Aldrich).

Techniques: Infection, Western Blot, Double Staining, Two Tailed Test, Comparison

Journal: Scientific Reports

Article Title: Microglia-dependent neuroprotective effects of 4-octyl itaconate against rotenone-and MPP+-induced neurotoxicity in Parkinson’s disease

doi: 10.1038/s41598-023-42813-8

Figure Lengend Snippet: Regulation of HO-1/P62/Nf-κB pathway by OI in activated microglia. BV2 cells were exposed to different concentrations (5, 25, 50 μM) of OI ± 100 ng/ml LPS for 3 h and 24 h. HO-1 protein levels in the whole cell were tested by western blot at 3 h ( A ) and 24 h ( B ). β-actin was used as a loading control. ( C ) Western blot analysis compared HO-1 expression after being exposed to LPS with 50 μM OI at 3 h and 24 h. β actin was used as a loading control. ( D ) The bar graph represents the quantification of HO-1 expression normalized to β-actin in ( C ) (mean ± SEM, one-way ANOVA, * p < 0.05; n = 3 independent experiments. ( E ) Representative confocal images of Nf-κB fluorescence staining 24 h after treatment. Western blot analysis of phospho-P62 and total P62 protein levels 3 h ( F ) and 24 h ( G ) after treatment indicated. β-actin was used as a loading control. ( H ) Representative confocal images of p-P62 fluorescence staining 3 h after treatment.

Article Snippet: Primary antibodies used were Nrf-2 (1:250; Cell Signaling), Nf-kB (1:250; Cell Signaling), phosphorylated-P62 (1:500; Cell Signaling), and DAPI (1 μg/ml, Thermo Fisher).

Techniques: Western Blot, Expressing, Fluorescence, Staining