Journal: bioRxiv

Article Title: Transient cell-in-cell formation underlies tumor resistance to immunotherapy

doi: 10.1101/2020.09.10.287441

Figure Lengend Snippet: a. Representative images of cytosol-labeled B16F10 cell lines incubated overnight with gp100-reactive T cells. b. Representative images of B16F10 cell lines labeled with H2B-GFP and MyrPalm-TdTomato, and B16F10 cell lines labeled with H2B-TdTomato and lifeactin-GFP, following overnight incubation with gp100-reactive T cells. c. Representative images of β-catenin expression in B16F10 cells incubated with gp100-reactive T cells. d. Representative images of E-cadherin expression in B16F10 incubated with gp100-reactive T cells. e. Representative images of phosphorylated integrin-1 β expression on B16F10 cells incubated with activated T cells. f. Mean percentage of cell-in-cell of B16F10 cells treated with different inhibitors and incubated with T cells (n=3). g. Representative images of B16F10 cells treated with inhibitors and incubated overnight with gp100-reactive T cells. h. Relative expression of highly enriched genes in B16F10 cells isolated directly from relapsed tumors (Tumor) and after incubation with T-cell lysosomes (Lyso), compared to B16F10 control cells (WT) (n=3). i. Representative images of phospho-STAT3 in B16F10 incubated with gp100-reactive T cells. j. Representative images of phosphorylated EGFR of B16F10 incubated with gp100-reactive T cells k. Mean percentages of cell-in-cell formation following overnight incubation with T cell-derived STAT3 or EGFR inhibitors (n=3). l. Representative confocal images of B16F10 cells incubated with STAT3 or EGFR inhibitors and with T cell-derived lysosomes. Experiments were repeated independently at least three times. Statistical significance was calculated using ANOVA with Tukey’s correction for multiple comparisons (*** denotes p<0.001, **** denotes p<0.0001). Error bars represent standard error. Scale bars = 20 μm.

Article Snippet: We used the primary antibodies: anti-CD44 (clone IM7, BioLegend), anti-MHC class I (clone 28-8-6, BioLegend), anti-β-catenin (clone D2U8Y, Cell Signaling Technology), anti-phosphorylated STAT3 (clone D3A7, Cell Signaling Technology), antiactive integrin 1β (Cell Signaling Technology), anti-integrin 1β (clone 9EG7), anti-E-cadherin (clone 36, BD Biosciences), anti-TRP2 (clone C-9, Santa Cruz Biotechnology), anti-gp100 (clone EP4863(2)), and anti-phosphorylated EGFR (clone EP38Y Abcam).

Techniques: Labeling, Incubation, Expressing, Isolation, Control, Derivative Assay

Journal: bioRxiv

Article Title: Transient cell-in-cell formation underlies tumor resistance to immunotherapy

doi: 10.1101/2020.09.10.287441

Figure Lengend Snippet: a. Representative images of β-catenin expression in B16F10 incubated overnight with gp100-reactive T cells. b. Representative images of E-cadherin expression in B16F10 incubated overnight with gp100-reactive T cells. c. Representative images of integrin-1 β expression on B16F10 incubated overnight with gp100-reactive T cells. d. Representative images of phosphorylated integrin-1β expression on B16F10 incubated overnight with gp100-reactive T cells. e. STAT3 expression level in B16F10 cells activated for 4 hours with T cell-derived lysosomes. f . EGR1 expression levels in B16F10 cells following 4 hours’ activation with T cell-derived lysosomes. Scale bars = 20 μm. Center lines show the medians; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles, outliers are represented by dots. n = 3 sample points.

Article Snippet: We used the primary antibodies: anti-CD44 (clone IM7, BioLegend), anti-MHC class I (clone 28-8-6, BioLegend), anti-β-catenin (clone D2U8Y, Cell Signaling Technology), anti-phosphorylated STAT3 (clone D3A7, Cell Signaling Technology), antiactive integrin 1β (Cell Signaling Technology), anti-integrin 1β (clone 9EG7), anti-E-cadherin (clone 36, BD Biosciences), anti-TRP2 (clone C-9, Santa Cruz Biotechnology), anti-gp100 (clone EP4863(2)), and anti-phosphorylated EGFR (clone EP38Y Abcam).

Techniques: Expressing, Incubation, Derivative Assay, Activation Assay, Software

Journal: PLoS ONE

Article Title: Resistance to Bleomycin-Induced Lung Fibrosis in MMP-8 Deficient Mice Is Mediated by Interleukin-10

doi: 10.1371/journal.pone.0013242

Figure Lengend Snippet: The effects of MMP-8 on IL-10 activity were studied in cultured lung fibroblasts from wildtype and knockout mice (n = 4 per group). Intact IL-10 decreased in wildtype cells cultured in presence of bleomycin (Representative western blot: A ; quantification: B ). This was related to a decrease in phosphorylated STAT3 ( C ), demonstrating a decrease in the activity of IL-10 signaling pathway, and an increase in Type I collagen ( D ). In contrast, fibroblasts from knockout mice showed no IL-10 cleavage ( A–B ), an increase in STAT3 phosphorylation ( C ) and decreased collagen synthesis ( D ). Addition of a neutralizing antibody against IL-10 decreased STAT3 activation and increased collagen. Addition of a control IgG had no effects. Panel E shows representative western blots of these experiments. *p<0.05 compared against wildtype under the same culture conditions.

Article Snippet: To measure IL-10 activity, we quantified the phosphorylation balance of the transcription factor STAT-3 by western blot using antibodies against phosphorylated and non-phosphorylated forms of STAT3 (Signalway Antibody Co, USA).

Techniques: Activity Assay, Cell Culture, Knock-Out, Western Blot, Phospho-proteomics, Activation Assay, Control

Journal: Frontiers in Pharmacology

Article Title: Maresin 1 Mitigates Sepsis-Associated Acute Kidney Injury in Mice via Inhibition of the NF-κB/STAT3/MAPK Pathways

doi: 10.3389/fphar.2019.01323

Figure Lengend Snippet: MaR1 inhibits NF-κB/STAT3 pathway activation in the kidney of sepsis mice. The expressions of p-p65, p65 (A) and p-Stat3, Stat3 (B) in renal tissues of mice in each group were analyzed by Western blot. The p-p65 protein levels normalized by p65 (C) , p-p65 protein levels normalized by β-actin (D) , p-Stat3 protein levels normalized by Stat3 (E) . Data are shown as mean ± SD, n = 9. ** P 0.01 vs. sham group; # P 0.05, ## P 0.01 vs. CLP group; && P 0.01 vs LD-MaR1 group. MaR1, Maresin 1; NF-κB, nuclear factor-kappa B; STAT3, signal transducer and activator of transcriptor 3; CLP, cecal ligation and puncture; LD-MaR1, MaR1 low-dose group.

Article Snippet: The membranes were incubated with the following primary antibody at 4°C overnight: phosphorylation index (p-p65, p-Stat3, p-JNK, p-ERK, p-p38), non-phosphorylation index (p65, Stat3), and β-actin (Cell Signaling Technology, Danvers, MA, USA) at 1:1,000.

Techniques: Activation Assay, Western Blot, Ligation

Journal: Frontiers in Pharmacology

Article Title: Maresin 1 Mitigates Sepsis-Associated Acute Kidney Injury in Mice via Inhibition of the NF-κB/STAT3/MAPK Pathways

doi: 10.3389/fphar.2019.01323

Figure Lengend Snippet: MaR1 inhibits p65 nuclear translocation in the kidney of sepsis mice. Representative immunofluorescence staining of kidney tissue at 24 h after CLP in each group of mice, and white arrow indicates p65 into the nucleus, magnification 1,000×. MaR1, Maresin 1; CLP, cecal ligation and puncture.

Article Snippet: The membranes were incubated with the following primary antibody at 4°C overnight: phosphorylation index (p-p65, p-Stat3, p-JNK, p-ERK, p-p38), non-phosphorylation index (p65, Stat3), and β-actin (Cell Signaling Technology, Danvers, MA, USA) at 1:1,000.

Techniques: Translocation Assay, Immunofluorescence, Staining, Ligation

Journal: Frontiers in Pharmacology

Article Title: Maresin 1 Mitigates Sepsis-Associated Acute Kidney Injury in Mice via Inhibition of the NF-κB/STAT3/MAPK Pathways

doi: 10.3389/fphar.2019.01323

Figure Lengend Snippet: MaR1 inhibits MAPK pathway activation and p65 nuclear translocation in the kidney of sepsis mice. The expressions of p-JNK, p-ERK, and p-p38 in renal tissues of mice in each group were analyzed by Western blot (A) . The p-JNK (B) , p-ERK (C) , and p-p38 (D) protein levels normalized by β-actin. (E) Nuclear translocation of p65 count/field. Data are shown as mean ± SD, n = 9. ** P 0.01 vs. sham group; ## P 0.01 vs. CLP group; && P 0.01 vs. LD-MaR1 group. MaR1, Maresin 1; MAPK, mitogen-activated protein kinase; CLP, cecal ligation and puncture; LD-MaR1, MaR1 low-dose group.

Article Snippet: The membranes were incubated with the following primary antibody at 4°C overnight: phosphorylation index (p-p65, p-Stat3, p-JNK, p-ERK, p-p38), non-phosphorylation index (p65, Stat3), and β-actin (Cell Signaling Technology, Danvers, MA, USA) at 1:1,000.

Techniques: Activation Assay, Translocation Assay, Western Blot, Ligation

Journal: Frontiers in Pharmacology

Article Title: Maresin 1 Mitigates Sepsis-Associated Acute Kidney Injury in Mice via Inhibition of the NF-κB/STAT3/MAPK Pathways

doi: 10.3389/fphar.2019.01323

Figure Lengend Snippet: MaR1 inhibits NF-κB/STAT3 pathway activation in the kidney of sepsis mice. The expressions of p-p65, p65 (A) and p-Stat3, Stat3 (B) in renal tissues of mice in each group were analyzed by Western blot. The p-p65 protein levels normalized by p65 (C) , p-p65 protein levels normalized by β-actin (D) , p-Stat3 protein levels normalized by Stat3 (E) . Data are shown as mean ± SD, n = 9. ** P 0.01 vs. sham group; # P 0.05, ## P 0.01 vs. CLP group; && P 0.01 vs LD-MaR1 group. MaR1, Maresin 1; NF-κB, nuclear factor-kappa B; STAT3, signal transducer and activator of transcriptor 3; CLP, cecal ligation and puncture; LD-MaR1, MaR1 low-dose group.

Article Snippet: The membranes were incubated with the following primary antibody at 4°C overnight: phosphorylation index (p-p65, p-Stat3, p-JNK, p-ERK, p-p38), non-phosphorylation index (p65, Stat3), and β-actin (Cell Signaling Technology, Danvers, MA, USA) at 1:1,000.

Techniques: Activation Assay, Western Blot, Ligation

Journal: Frontiers in Pharmacology

Article Title: Maresin 1 Mitigates Sepsis-Associated Acute Kidney Injury in Mice via Inhibition of the NF-κB/STAT3/MAPK Pathways

doi: 10.3389/fphar.2019.01323

Figure Lengend Snippet: MaR1 inhibits p65 nuclear translocation in the kidney of sepsis mice. Representative immunofluorescence staining of kidney tissue at 24 h after CLP in each group of mice, and white arrow indicates p65 into the nucleus, magnification 1,000×. MaR1, Maresin 1; CLP, cecal ligation and puncture.

Article Snippet: The membranes were incubated with the following primary antibody at 4°C overnight: phosphorylation index (p-p65, p-Stat3, p-JNK, p-ERK, p-p38), non-phosphorylation index (p65, Stat3), and β-actin (Cell Signaling Technology, Danvers, MA, USA) at 1:1,000.

Techniques: Translocation Assay, Immunofluorescence, Staining, Ligation

Journal: Frontiers in Pharmacology

Article Title: Maresin 1 Mitigates Sepsis-Associated Acute Kidney Injury in Mice via Inhibition of the NF-κB/STAT3/MAPK Pathways

doi: 10.3389/fphar.2019.01323

Figure Lengend Snippet: MaR1 inhibits MAPK pathway activation and p65 nuclear translocation in the kidney of sepsis mice. The expressions of p-JNK, p-ERK, and p-p38 in renal tissues of mice in each group were analyzed by Western blot (A) . The p-JNK (B) , p-ERK (C) , and p-p38 (D) protein levels normalized by β-actin. (E) Nuclear translocation of p65 count/field. Data are shown as mean ± SD, n = 9. ** P 0.01 vs. sham group; ## P 0.01 vs. CLP group; && P 0.01 vs. LD-MaR1 group. MaR1, Maresin 1; MAPK, mitogen-activated protein kinase; CLP, cecal ligation and puncture; LD-MaR1, MaR1 low-dose group.

Article Snippet: The membranes were incubated with the following primary antibody at 4°C overnight: phosphorylation index (p-p65, p-Stat3, p-JNK, p-ERK, p-p38), non-phosphorylation index (p65, Stat3), and β-actin (Cell Signaling Technology, Danvers, MA, USA) at 1:1,000.

Techniques: Activation Assay, Translocation Assay, Western Blot, Ligation

Journal: Frontiers in Immunology

Article Title: Naringenin Modifies the Development of Lineage-Specific Effector CD4 + T Cells

doi: 10.3389/fimmu.2018.02267

Figure Lengend Snippet: Naringenin inhibits Th17 differentiation via affecting the corresponding regulation network. Naïve CD4 + T cells from C57BL/6 mice were activated with anti-CD3/CD28 under Th17-polarizing condition with or without 80 μM naringenin. Intracellular level of IL-17 (A), RORγt (B), and p-STAT3 (C) in differentiated CD4 + T cells was evaluated by flow cytometry. Dot scatters and histogram figures is representative results, and bar figures are mean ± SD of three independent experiments. * P < 0.05 and ** P < 0.01 by Student's t- test. NAR, naringenin.

Article Snippet: The membrane was blocked with 5% non-fat milk in Tris-buffered saline before being incubated, respectively with specific primary antibodies for the following proteins: STAT-3(1:1000), Smad2/3 (1:1000), phosphorylated Smad2/3 (p-Smad2/3) (1:1000), Acetyl-STAT3 (Lys685, Ac-STAT3) (1:1000), phosphorylated STAT3 (p-STAT3) (1:1000) (all from Cell Signaling Technologies, Danvers, MA), and β-actin (1:5000, Sigma-Aldrich).

Techniques: Flow Cytometry

Journal: Frontiers in Immunology

Article Title: Naringenin Modifies the Development of Lineage-Specific Effector CD4 + T Cells

doi: 10.3389/fimmu.2018.02267

Figure Lengend Snippet: Naringenin affects Smad2/3 and STAT3 activation under iTreg-polarizing condition with or without IL-6. Naïve CD4 + T cells from C57BL/6 mice were activated with anti-CD3/CD28 under iTreg-polarizing condition with or without IL-6 with or without 80 μM naringenin. Effect of naringenin on phosphorylated Smad2/3 expression under iTreg-polarizing condition was determined by Western blot (A) . Phosphorylation and acetylation of STAT3 expression were determined by Western blot under iTreg-polarizing condition with IL-6 (B) . The gel pictures are representatives of three independent experiments, which had similar results. The values below band images are the ratios of p-Smad2/3, p-STAT3/STAT3, and Ac-STAT3/STAT3. NAR, naringenin.

Article Snippet: The membrane was blocked with 5% non-fat milk in Tris-buffered saline before being incubated, respectively with specific primary antibodies for the following proteins: STAT-3(1:1000), Smad2/3 (1:1000), phosphorylated Smad2/3 (p-Smad2/3) (1:1000), Acetyl-STAT3 (Lys685, Ac-STAT3) (1:1000), phosphorylated STAT3 (p-STAT3) (1:1000) (all from Cell Signaling Technologies, Danvers, MA), and β-actin (1:5000, Sigma-Aldrich).

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

Journal: Frontiers in Immunology

Article Title: Naringenin Modifies the Development of Lineage-Specific Effector CD4 + T Cells

doi: 10.3389/fimmu.2018.02267

Figure Lengend Snippet: Naringenin affects IL-6 receptor expression under iTreg-polarizing condition with IL-6 and inhibits IL-6 downstream signaling. (A–C) , Naïve CD4 + T cells from C57BL/6 mice were activated with anti-CD3/CD28 under iTreg-polarizing condition with IL-6 in the absence or presence of 80 μM naringenin. mIL-6R and mgp130 expression was determined by flow cytometry and sIL-6R levels in the cultured medium were quantified by ELISA. Values are mean ± SD of three independent experiments. Means without a common letter significantly differ at least at P < 0.05. * P < 0.05 compared with the corresponding control (without NAR) by student's t -test. D, After naïve CD4 + T cells were incubated with 80 μM naringenin for 2 h, IL-6 was added to stimulate cells for 15 min. Phosphorylation and acetylation of STAT3 expression were determined by Western blot. The gel pictures are representatives of three independent experiments, which had similar results. The values below band images are the ratios of Ac-STAT3/STAT3. NAR, naringenin.

Article Snippet: The membrane was blocked with 5% non-fat milk in Tris-buffered saline before being incubated, respectively with specific primary antibodies for the following proteins: STAT-3(1:1000), Smad2/3 (1:1000), phosphorylated Smad2/3 (p-Smad2/3) (1:1000), Acetyl-STAT3 (Lys685, Ac-STAT3) (1:1000), phosphorylated STAT3 (p-STAT3) (1:1000) (all from Cell Signaling Technologies, Danvers, MA), and β-actin (1:5000, Sigma-Aldrich).

Techniques: Expressing, Flow Cytometry, Cell Culture, Enzyme-linked Immunosorbent Assay, Control, Incubation, Phospho-proteomics, Western Blot

Journal: Allergology international : official journal of the Japanese Society of Allergology

Article Title: YTHDF1 regulates the proliferation and differentiation of keratinocytes via the PI3K/AKT signaling pathway in atopic dermatitis.

doi: 10.1016/j.alit.2025.04.005

Figure Lengend Snippet: Fig. 7. YTHDF1 regulates the proliferation and differentiation in the DNCB-induced AD mouse model via the PI3K/AKT pathways. (A)The proliferation and differentiation indicators in different groups by WB. (BeC) PCNA and Ki67 protein levels in mice ear tissues by IHC. (DeE) The expression of FLG and LOR by IHC. Original magnification, 200, scale bar ¼ 50 mm. Data were represented as mean ± SEM, n ¼ 6 mice per group. ns, no significance. *P < 0.05, **P < 0.01, and ***P < 0.001. AAV-Ctrl, AAV-GFP control; AAV-YTHDF1; AAV containing YTHDF1.

Article Snippet: Primary antibodies used were as follows: YTHDF1, PCNA, GAPDH, LOR (Proteintech, NJ, USA), FLG (Biorbyt, Cambridge, UK), K1, K10 (Santa Cruz Biotechnology, CA, USA), PI3K, p-PI3K, AKT, p-AKT, STAT3, p-STAT3 (Abmart, Shanghai, China).

Techniques: Expressing, Control

Journal: Journal of Neurochemistry

Article Title: Rifampicin attenuates experimental autoimmune encephalomyelitis by inhibiting pathogenic Th17 cells responses

doi: 10.1111/jnc.13871

Figure Lengend Snippet: Expressions of p‐ STAT 3 and p‐p65 were reduced in rifampicin‐treated experimental autoimmune encephalomyelitis ( EAE ) mice. Spinal cords were isolated from rifampicin‐treated EAE , vehicle (Veh)‐treated EAE , and normal (Nor) mice on day 26 after immunization ( n = 6). (a) Representative bands of STAT 3, p‐ STAT 3, NF ‐κB/p65, and p‐p65 by immunoblotting. GAPDH was used as an internal control. (b–e) The histograms represent STAT 3, p‐ STAT 3, p65, and p‐p65 levels expressed as folds relative to the loading control. Statistical analysis was performed using one‐way anova followed by LSD post hoc test to compare replicate by time. Values were shown as mean ± SEM of the independent experiments. Statistical significance: # p < 0.05, compared with normal mice; ### p < 0.001, compared with normal mice; * p < 0.05, compared with vehicle‐treated mice; ** p < 0.01, compared with vehicle‐treated mice.

Article Snippet: After being blocked with 5% nonfat dry milk (for detection of non‐phosphorylation proteins) or bovine serum albumin solution (for detection of phosphorylation proteins) in Tween 20‐containing Tris‐buffered saline (20 mM Tris, pH 7.4, 0.1% Tween 20, and 150 mM NaCl), membranes were incubated overnight at 4°C with the following antibodies: p‐STAT3, p‐NF‐κB p65 (1 : 1000; Cell Signaling Technology, Beverly, MA, USA), STAT3, NF‐κB p65, and GAPDH (1 : 2000; Cell Signaling Technology).

Techniques: Isolation, Western Blot, Control