Journal: bioRxiv

Article Title: Opioids trigger breast cancer metastasis through E-Cadherin downregulation and STAT3 activation promoting epithelial-mesenchymal transition

doi: 10.1101/443663

Figure Lengend Snippet: (A) MDA-MB-231 and MCF-7 cells were treated with 1 μM opioid for 5 min – 72 h and analyzed for pSTAT3 Y705 , tSTAT3, SNAIL, TWIST and E-CADHERIN levels by immunoblotting. β-ACTIN and HSC-70 were used as loading controls. ( B ) MDA-MB-231 and MCF-7 cells were treated with 1 μM opioid for 1 h and examined for mRNA levels of E-Cadherin , SNAIL , SLUG and TWIST by real-time qRT-PCR ( * p<0.05, ** p<0.01, n=3 experiments, Student’s t-test). (C) FACS histogram plots (blue: ctrl treated, red: opioid-treated 1 μM, 1 h) of MCF-7 and MDA-MB-231 cells stained against E-Cadherin. Dotted curves represent FACS histogram plots for the isotype-matched controls. (D) Representative H&E, Cleaved Caspase-3 and Vimentin stained lung sections of mice engrafted with MDA-MB-231 cells after primary tumor removal and post-surgical opioid treatment for ten days (original magnification: 40x and 100x; scale bars 200 μm and inserts 100 μm).

Article Snippet: To assess E-Cadherin cell surface expression, cells were stimulated for 1 h with 1 μM of the opioid following incubation with anti-E-Cadherin (dilution 1:1000, CST, 3199).

Techniques: Western Blot, Quantitative RT-PCR, Staining

Journal: Journal of Translational Medicine

Article Title: Long non-coding RNA MEG3 mediates the miR-149-3p/FOXP3 axis by reducing p53 ubiquitination to exert a suppressive effect on regulatory T cell differentiation and immune escape in esophageal cancer

doi: 10.1186/s12967-021-02907-1

Figure Lengend Snippet: Silencing of FOXP3 inhibits regulatory T cell differentiation to repress immune escape in EC mice. A FOXP3 expression in cancer and paracancerous tissues detected by RT-qPCR normalized to GAPDH (n = 56). * p < 0.05 vs. paracancerous tissues. B The percentage of CD25 + FOXP3 + T cells in CD4 + T cells of peripheral blood from EC patients and healthy blood donors assessed by flow cytometry (EC patients: n = 28; healthy blood donors: n = 25). * p < 0.05 vs . peripheral blood from healthy blood donors. C FOXP3 expression in AKR cells detected by RT-qPCR normalized to GAPDH. * p < 0.05 vs . control cells. D The silencing efficiency of sh-FOXP3-1 and sh-FOXP3-2 in AKR cells detected by RT-qPCR normalized to GAPDH. * p < 0.05 vs. AKR cells transfected with sh-NC. Normal mice were used as controls, and 4-NQO-induced mice were transfected or not transfected with sh-NC or sh-FOXP3. E The weight of mice (n = 10). F Pathological changes of tissues in mice measured by HE staining (200×) (n = 10). G FOXP3 expression in tissues of mice detected by RT-qPCR normalized to GAPDH (n = 10). H FOXP3, C-myc, N-myc and Ki-67 positive protein expression in tissues of mice determined by immunohistochemistry (n = 10). I The percentage of CD25 + FOXP3 + T cells in CD4 + T cells in spleen of mice assessed by flow cytometry (n = 10). J The percentage of IL-10 + CD4 + T cells in spleen of mice assessed by flow cytometry (n = 10). K IL-10 level in the spleen of mice assessed by ELISA (n = 10). L The percentage of IL-4 + CD4 + T cells in spleen of mice assessed by flow cytometry (n = 10). M IL-4 level in the spleen of mice assessed by ELISA (n = 10). * p < 0.05 vs . normal mice. # p < 0.05 vs. EC mice treated with sh-NC. Measurement data were expressed as mean ± standard deviation. Data between cancer and paracancerous tissues were compared by paired t test, and data between other two groups were compared by independent sample t test. Comparisons among multiple groups were performed using one way analysis of variance (ANOVA), followed by Tukey's post hoc test, and data at different time points among multiple groups were compared by repeated measures ANOVA, followed by Bonferroni post hoc test. The cell experiment was repeated 3 times

Article Snippet: Peripheral blood mononuclear cells (PBMCs) from human blood were purified via density gradient centrifugation, and stimulated with 10 ng/mL PMA and 1 μM Ionomycin over a period of 6 h. The PBMCs were subsequently stained with antibodies against CD4 (ab133616, 1 μg/mL, Abcam Inc.), CD25 (#43212, 10 μg/mL, CST, Framingham, MA, USA) and FOXP3 (ab215206, 0.5 μg/mL, Abcam Inc.) for 30 min.

Techniques: Cell Differentiation, Expressing, Quantitative RT-PCR, Flow Cytometry, Control, Transfection, Staining, Immunohistochemistry, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: Journal of Translational Medicine

Article Title: Long non-coding RNA MEG3 mediates the miR-149-3p/FOXP3 axis by reducing p53 ubiquitination to exert a suppressive effect on regulatory T cell differentiation and immune escape in esophageal cancer

doi: 10.1186/s12967-021-02907-1

Figure Lengend Snippet: miR-149-3p inhibits regulatory T cell differentiation to repress immune escape in EC mice via FOXP3. Normal mice were used as controls, and EC mice were treated or not treated with NC-agomir, miR-149-3p-agomir, miR-149-3p-agomir + oe-NC or miR-149-3p-agomir + oe-FOXP3. A The weight of mice. B Pathological changes of tissues in mice measured by HE staining (200×). C FOXP3 expression in tissues of mice detected by RT-qPCR normalized to GAPDH. D Immunohistochemistry result of detecting FOXP3, C-myc, N-myc and Ki-67 protein expression. E The percentage of CD25 + FOXP3 + T cells in CD4 + T cells in mice spleen assessed by flow cytometry. F The percentage of IL-10 + CD4 + T cells in mice spleen assessed by flow cytometry. G IL-10 level in the spleen of mice assessed by ELISA. H The percentage of IL-4 + CD4 + T cells in mice spleen assessed by flow cytometry. I IL-4 level in the spleen of mice assessed by ELISA. * p < 0.05 vs . EC mice treated with NC-agomir. # p < 0.05 vs. EC mice treated with miR-149-3p-agomir + oe-NC. Measurement data were expressed as mean ± standard deviation. Data between two groups were compared by independent sample t test. Comparisons among multiple groups were performed using one way analysis of variance (ANOVA), followed by Tukey's post hoc test, and data at different time points among multiple groups were compared by repeated measures ANOVA, followed by Bonferroni post hoc test. n = 10

Article Snippet: Peripheral blood mononuclear cells (PBMCs) from human blood were purified via density gradient centrifugation, and stimulated with 10 ng/mL PMA and 1 μM Ionomycin over a period of 6 h. The PBMCs were subsequently stained with antibodies against CD4 (ab133616, 1 μg/mL, Abcam Inc.), CD25 (#43212, 10 μg/mL, CST, Framingham, MA, USA) and FOXP3 (ab215206, 0.5 μg/mL, Abcam Inc.) for 30 min.

Techniques: Cell Differentiation, Staining, Expressing, Quantitative RT-PCR, Immunohistochemistry, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: Journal of Translational Medicine

Article Title: Long non-coding RNA MEG3 mediates the miR-149-3p/FOXP3 axis by reducing p53 ubiquitination to exert a suppressive effect on regulatory T cell differentiation and immune escape in esophageal cancer

doi: 10.1186/s12967-021-02907-1

Figure Lengend Snippet: p53-activated miR-149-3p results in suppression of regulatory T cell differentiation and immune escape in EC mice via FOXP3. A p53 expression in cancer and paracancerous tissues detected by RT-qPCR normalized to GAPDH (n = 56). * p < 0.05 vs. paracancerous tissues. B p53 expression in AKR cells detected by RT-qPCR normalized to GAPDH. * p < 0.05 vs. control cells. C p53 and miR-149-3p expression in AKR cells after overexpression of p53 detected by RT-qPCR normalized to GAPDH and U6. * p < 0.05 vs. AKR cells treated with oe-NC. D The enrichment of p53 in miR-149-3p promoter measured by ChIP. * p < 0.05 vs. IgG. Normal mice were used as controls, and EC mice were treated or not treated with oe-NC, oe-p53, oe-p53 + NC-inhibitor or oe-p53 + miR-149-3p-inhibitor. E The weight of mice (n = 10). F FOXP3 expression in mice tissues detected by RT-qPCR normalized to GAPDH (n = 10). G Pathological changes of mice tissues measured by HE staining (200 ×) (n = 10). H Immunohistochemistry result of detecting FOXP3, C-myc, N-myc and Ki-67 protein expression (n = 10). I The percentage of CD25 + FOXP3 + T cells in CD4 + T cells in mice spleen assessed by flow cytometry (n = 10). J The percentage of IL-10 + CD4 + T cells in mice spleen assessed by flow cytometry (n = 10). K IL-10 level in mice spleen assessed by ELISA (n = 10). L The percentage of IL-4 + CD4 + T cells in mice spleen assessed by flow cytometry (n = 10). M IL-4 level in mice spleen assessed by ELISA (n = 10). * p < 0.05 vs. EC mice treated with oe-NC. # p < 0.05 vs. EC mice treated with oe-p53 + NC-inhibitor. Measurement data were expressed as mean ± standard deviation. Data between cancer and paracancerous tissues were compared by paired t test, and data between other two groups were compared by independent sample t test. Comparisons among multiple groups were performed using one way analysis of variance (ANOVA), followed by Tukey's post hoc test, and data at different time points among multiple groups were compared by repeated measures ANOVA, followed by Bonferroni post hoc test. The cell experiment was repeated 3 times

Article Snippet: Peripheral blood mononuclear cells (PBMCs) from human blood were purified via density gradient centrifugation, and stimulated with 10 ng/mL PMA and 1 μM Ionomycin over a period of 6 h. The PBMCs were subsequently stained with antibodies against CD4 (ab133616, 1 μg/mL, Abcam Inc.), CD25 (#43212, 10 μg/mL, CST, Framingham, MA, USA) and FOXP3 (ab215206, 0.5 μg/mL, Abcam Inc.) for 30 min.

Techniques: Cell Differentiation, Expressing, Quantitative RT-PCR, Control, Over Expression, Staining, Immunohistochemistry, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: Journal of Translational Medicine

Article Title: Long non-coding RNA MEG3 mediates the miR-149-3p/FOXP3 axis by reducing p53 ubiquitination to exert a suppressive effect on regulatory T cell differentiation and immune escape in esophageal cancer

doi: 10.1186/s12967-021-02907-1

Figure Lengend Snippet: MEG3 suppresses immune escape by decreasing regulatory T cell differentiation through p53/miR-149-3p/FOXP3 axis. Normal mice were used as controls, and EC mice were treated or not treated with oe-NC, oe-MEG3, oe-MEG3 + sh-NC, oe-MEG3 + sh-p53, oe-MEG3 + oe-NC or oe-MEG3 + oe-FOXP3. A The weight of mice. B Pathological changes of tissues in mice measured by HE staining (200×). C Immunohistochemistry result of detecting FOXP3, C-myc, N-myc and Ki-67 protein expression. D MEG3, p53, miR-149-3p and FOXP3 expression in mice tissues detected by RT-qPCR normalized to GAPDH and U6. E The percentage of CD25 + FOXP3 + T cells in CD4 + T cells in mice spleen assessed by flow cytometry. F The percentage of IL-10 + CD4 + T cells in mice spleen assessed by flow cytometry. G The percentage of IL-4 + CD4 + T cells in mice spleen assessed by flow cytometry. H IL-10 level in the spleen of mice assessed by ELISA. I IL-4 level in the spleen of mice assessed by ELISA. * p < 0.05 vs. EC mice treated with oe-NC. # p < 0.05 vs. EC mice treated with oe-MEG3 + sh-NC. $ vs. EC mice treated with oe-MEG3 + oe-NC. Measurement data were expressed as mean ± standard deviation. Data between two groups were compared by independent sample t test. Comparisons among multiple groups were performed using one way analysis of variance (ANOVA), followed by Tukey's post hoc test, and data at different time points among multiple groups were compared by repeated measures ANOVA, followed by Bonferroni post hoc test. n = 10

Article Snippet: Peripheral blood mononuclear cells (PBMCs) from human blood were purified via density gradient centrifugation, and stimulated with 10 ng/mL PMA and 1 μM Ionomycin over a period of 6 h. The PBMCs were subsequently stained with antibodies against CD4 (ab133616, 1 μg/mL, Abcam Inc.), CD25 (#43212, 10 μg/mL, CST, Framingham, MA, USA) and FOXP3 (ab215206, 0.5 μg/mL, Abcam Inc.) for 30 min.

Techniques: Cell Differentiation, Staining, Immunohistochemistry, Expressing, Quantitative RT-PCR, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: The Journal of Experimental Medicine

Article Title: RIP Links TLR4 to Akt and Is Essential for Cell Survival in Response to LPS Stimulation

doi: 10.1084/jem.20040446

Figure Lengend Snippet: Apoptosis and expression of Akt after LPS stimulation of splenocytes from RIP −/− /TNFR1 −/− pups and RIP +/+ /TNFR1 −/− littermates. (A) Annexin V–FITC and B220-PE staining at 0, 16, and 96 h of splenocytes cultured with medium or LPS. Analysis was performed by gating on B220 + cells. Similar results were obtained in two separate experiments. ( B) Akt, IκB, and p38 phosphorylation after stimulation of RIP −/− /TNFR1 −/− and RIP −/+ /TNFR1 −/− splenocytes with LPS ( n = 4) or CpG ODN 1826 ( n = 2). Lysates were probed with Akt as loading control. (C) Fold induction of Akt, IκB, and p38 phosphorylation after LPS stimulation ( n = 4) *P < 0.05.

Article Snippet: Cell lysates were obtained from splenocytes (0.5 × 10 6 cells/condition) suspended in medium containing 1% FCS and stimulated with LPS (10 μg/ml) or CpG ODN1826 (3 μM) for 15 min. Blots were incubated with antibodies specific for phospho-Akt, Akt (Becton Dickinson), phospho-p38, or phospho-IκB (Cell Signaling) followed by horseradish peroxidase–conjugated goat anti–rabbit or goat anti–mouse antibodies (Becton Dickinson).

Techniques: Expressing, Staining, Cell Culture, Phospho-proteomics, Control