anti pstat3 y705  (Cell Signaling Technology Inc)

Journal: Journal of Nanobiotechnology

Article Title: NGO ameliorates psoriasis by modulating mitochondrial function and suppressing pSTAT3–IL-17–expressing CD8 + TRM cells

doi: 10.1186/s12951-025-04020-7

Figure Lengend Snippet: Nano-sized graphene oxide impaired mitochondrial function and potently suppressed pSTAT3 signaling to regulate pathogenic tissue-resident memory cell differentiation and function in vitro (A) Mitochondrial ROS production in splenic immune cells and CD8 + T cells (stimulated with anti CD3 2 µg/ml, aCD28 2 µg/ml, IL-6 10ng/ml) was assessed after nano-sized graphene oxide (NGO) treatment using MitoSOX staining. (B) The mitochondrial oxygen consumption rate in splenic immune cells, under conditions mentioned above, was measured over time following NGO exposure. (C) Immunofluorescence imaging (200x) was performed to examine the distribution of splenic CD8 + T cells in the presence or absence of biotinylated NGO 10 µg/ml (CD8: green; NGO: white; DAPI: blue). (D) Western blotting analysis was used to evaluate phosphorylation of STAT3 at Ser727 and Tyr705, and STAT3 protein expression in NGO-treated splenic immune cells, stimulated under conditions mentioned above. (E – G) Flow cytometry was performed in splenic immune cells at same stimulation conditions to determine the frequencies of IFN-γ + and IL-17 + CD8 + CD103 + TRM-like T cells, as well as CD69 + CD103 + TRM cell subsets, in the presence or absence of NGO and regulatory T (Treg) cells. (H) Cytokine concentrations associated with psoriasis in splenic immune cell culture supernatants were measured by ELISA following treatment with 1 µg/ml NGO. (I) Multiplex immunofluorescence staining in splenic immune cells at same stimulation conditions was used to detect CD8 + CD103 + , CD69 + , pSTAT3 + , and IL-17 + cells, followed by quantitative analysis (CD8: green; CD103: red; CD69, pSTAT3, IL-17: white; DAPI: blue). n = number. All values are shown as the mean ± SD ( n = 6 for aCD3 + aCD28 + IL-6 condition, n = 6 for NGO 1 µg/ml, n = 6 for NGO 10 µg/ml). Each experiment was repeated three times. Statistical significance was determined by non-parametric Mann–Whitney U test for pairwise comparisons and one-way ANOVA using Kruskal-Wallis test for multiple group comparisons. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: After blocking, the membranes were incubated at room temperature for 35 min with primary antibodies against pSTAT3 (Ser727) (#9134; Cell Signaling Technology, Danvers, MA, USA), pSTAT3 (Tyr705) (#9131; Cell Signaling Technology), STAT3 (#9139; Cell Signaling Technology), and GAPDH (ab181602; Abcam), all diluted in 0.4% skim milk in 1× Tris-buffered saline with 0.1% Tween-20 (TBS-T).

Techniques: Cell Differentiation, In Vitro, Staining, Immunofluorescence, Imaging, Western Blot, Phospho-proteomics, Expressing, Flow Cytometry, Cell Culture, Enzyme-linked Immunosorbent Assay, Multiplex Assay, MANN-WHITNEY

Journal: Journal of Nanobiotechnology

Article Title: NGO ameliorates psoriasis by modulating mitochondrial function and suppressing pSTAT3–IL-17–expressing CD8 + TRM cells

doi: 10.1186/s12951-025-04020-7

Figure Lengend Snippet: Nano-sized graphene oxide (NGO) attenuated psoriatic skin inflammation by suppressing pSTAT3–IL-17 immune responses in vivo (A) Schematic overview of the experimental protocol. Psoriasis-like skin inflammation was induced in mice using imiquimod, followed by repeated NGO administration. (B) Measurement of spleen size and weight in control and NGO-treated mice. (C) Clinical scoring of skin scaling, erythema, thickness, and the Psoriasis Area and Severity Index (PASI) in control and NGO-treated mice. (D , G) Histological and immunohistochemical staining to determine epidermal thickness and Ki-67 + , CD8 + , IL-17 + , and pSTAT3 + cells in skin lesions. (E) Representative immunofluorescence images of spleen sections from each group show CD8 + T cells, biotin-labeled NGO, and nuclei in the spleen at magnifications of 200× and 400× (CD8: green; NGO: white; DAPI: blue). (F) Flow cytometric analysis of IFN-γ + and IL-17 + CD4 + and CD8 + T cells in the spleen. n = number. All data are shown as the mean ± SD ( n = 6 for SKG + IMQ, n = 8 for with NGO). Each experiment was repeated three times. Statistical significance was determined by two-way ANOVA with Bonferroni test in multiple groups and non-parametric Mann–Whitney U test for pairwise comparisons. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: After blocking, the membranes were incubated at room temperature for 35 min with primary antibodies against pSTAT3 (Ser727) (#9134; Cell Signaling Technology, Danvers, MA, USA), pSTAT3 (Tyr705) (#9131; Cell Signaling Technology), STAT3 (#9139; Cell Signaling Technology), and GAPDH (ab181602; Abcam), all diluted in 0.4% skim milk in 1× Tris-buffered saline with 0.1% Tween-20 (TBS-T).

Techniques: In Vivo, Control, Immunohistochemical staining, Staining, Immunofluorescence, Labeling, MANN-WHITNEY

Journal: Journal of Nanobiotechnology

Article Title: NGO ameliorates psoriasis by modulating mitochondrial function and suppressing pSTAT3–IL-17–expressing CD8 + TRM cells

doi: 10.1186/s12951-025-04020-7

Figure Lengend Snippet: Nano-sized graphene oxide (NGO) attenuated psoriatic inflammation by inhibiting STAT3 activation and suppressing proinflammatory CD8 + tissue-resident memory (TRM) T cell accumulation in tissue (A) Western blotting analysis of splenic immune cells from psoriatic mice revealed that NGO treatment markedly inhibited phosphorylation of STAT3 at both Ser727 and Tyr705, quantified relative to total STAT3 and GAPDH ( n = 6 for SKG + IMQ, n = 6 for with NGO in western blot). (B) Flow cytometry showed that the frequency of activated CD69 + CD103 + CD8 + T cells, as well as IFN-γ + and IL-17 + CD103 + CD8 + T cell subsets, indicating suppression of key proinflammatory TRM cell populations in splenic immune cells from NGO treated psoriatic mice. (C) Multiplex immunofluorescence imaging and quantification demonstrated significant decreases in CD8 + CD103 + CD69 + TRM cells, CD8 + CD103 + pSTAT3 + , and CD8 + CD103 + IL-17 + cells in spleen tissues after NGO treatment (CD8: green; CD103: red; CD69, pSTAT3, IL-17: white; DAPI: blue). (D) Similar immunofluorescence analyses of skin lesions supported reduced presence of TRM cell markers and effector cytokines with NGO treatment, confirmed by quantitative positive cells counting (CD8: green; CD103: red; CD69, pSTAT3, IL-17: white; DAPI: blue). n = number. All data are shown as the mean ± SD ( n = 6 for SKG + IMQ, n = 8 for with NGO in flow cytometry and tissue data). Each experiment was repeated three times. Statistical significance was determined by non-parametric Mann–Whitney U test for pairwise comparisons. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: After blocking, the membranes were incubated at room temperature for 35 min with primary antibodies against pSTAT3 (Ser727) (#9134; Cell Signaling Technology, Danvers, MA, USA), pSTAT3 (Tyr705) (#9131; Cell Signaling Technology), STAT3 (#9139; Cell Signaling Technology), and GAPDH (ab181602; Abcam), all diluted in 0.4% skim milk in 1× Tris-buffered saline with 0.1% Tween-20 (TBS-T).

Techniques: Activation Assay, Western Blot, Phospho-proteomics, Flow Cytometry, Multiplex Assay, Immunofluorescence, Imaging, MANN-WHITNEY

Journal: Journal of Nanobiotechnology

Article Title: NGO ameliorates psoriasis by modulating mitochondrial function and suppressing pSTAT3–IL-17–expressing CD8 + TRM cells

doi: 10.1186/s12951-025-04020-7

Figure Lengend Snippet: Nano-sized graphene oxide (NGO) restored mitochondrial function and suppressed proinflammatory tissue-resident memory CD8 + T cells in the peripheral blood of patients with psoriasis by regulating the pSTAT3–IL-17 axis. (A, B) Mitochondrial ROS (MitoSOX staining) was measured in whole peripheral blood mononuclear cells (PBMCs) and CD8 + CD103 + T cells from patients with psoriasis following NGO treatment, highlighting altered mitochondrial function. (C) The mitochondrial oxygen consumption rate was analyzed in healthy control PBMCs under conditions for psoriatic IL-17–expressing T cell differentiation (stimulated with anti CD3 2μg/ml, aCD28 2μg/ml, IL-23 10ng/ml). (D) Immunofluorescence imaging showed the co-localization and frequency of CD8 + T cells in the presence or absence of NGO 10μg/ml (CD8: green; NGO: white; DAPI: blue), with marked reduction upon NGO treatment in psoriasis patients’ PBMCs. (E) Western blotting and quantification demonstrated significant inhibition of STAT3 phosphorylation (Ser727 and Tyr705) in psoriasis patients’ PBMCs treated with NGO 10μg/ml. (F, G) Flow cytometric analysis showed that the population of IFN-γ + or IL-17 + CD4 + /CD8 + T cells and CD8 + CD103 + tissue-resident memory (TRM)-like T cells, as well as their cytokine production in psoriasis patients’ PBMCs. (H) The frequency of CD8 + CD69 + CD103 + and IFN-γ + or IL-17 + TRM cell subsets, as determined by flow cytometry in psoriasis patients’ PBMCs. (I) Regulatory T cell (Treg, Foxp3 + CD25 + CD4 + ) frequency assessed by flow cytometry in psoriasis patients’ PBMCs. (J) Multiplex immunofluorescence (200ⅹ) of patient-derived immune cells further confirmed decreased TRM cell marker CD8 + CD103 + , CD69 + , pSTAT3 + , and IL-17 + expression after NGO treatment, and quantitative analysis supported significant reductions in psoriasis patients’ PBMCs (CD8: green; CD103: red; CD69, pSTAT3, IL-17: white; DAPI: blue). Ps: psoriatic, HC: healthy control. n = number. All data are shown as the mean ± SD (n=6 for Psoriasis PBMC, n=6 for with NGO 10 μg/ml). Detailed patient information is described in the supplementary materials. Each experiment was repeated two times. Statistical significance was determined by non-parametric Mann–Whitney U test for pairwise comparisons. * P < 0.05,** P < 0.01, *** P < 0.001,**** P < 0.0001.

Article Snippet: After blocking, the membranes were incubated at room temperature for 35 min with primary antibodies against pSTAT3 (Ser727) (#9134; Cell Signaling Technology, Danvers, MA, USA), pSTAT3 (Tyr705) (#9131; Cell Signaling Technology), STAT3 (#9139; Cell Signaling Technology), and GAPDH (ab181602; Abcam), all diluted in 0.4% skim milk in 1× Tris-buffered saline with 0.1% Tween-20 (TBS-T).

Techniques: Staining, Control, Expressing, Cell Differentiation, Immunofluorescence, Imaging, Western Blot, Inhibition, Phospho-proteomics, Flow Cytometry, Multiplex Assay, Derivative Assay, Marker, MANN-WHITNEY

Journal: Life Science Alliance

Article Title: TRPM7 and magnesium orchestrate human CD4 T-cell activation and differentiation

doi: 10.26508/lsa.202503357

Figure Lengend Snippet: (A) Representative Western blot of total AKT1 signals in total CD4 T cells in presence of 30 μM NS8593 (NS) or DMSO (Ctrl) in resting state and after 30 min α-CD3/α-CD28 stimulation. (B) Respective quantification of total AKT1 signals from blots shown in (A), n = 5. (C) Representative Western blot of total SMAD2/3 signals in CD4 T cells in presence of 30 μM NS8593 (NS) or DMSO control (Ctrl) in resting state and after 10 min TGF-β stimulation. (D) Respective quantification of total SMAD2/3 signals from blots shown in (D), n = 4. (E) Representative Western blot of total SMAD2/3 signals in CD4 T cells in presence of 6 mM MgCl 2 (MgCl 2 ) or H 2 O control (Ctrl) in resting state and after 10 min TGF-β stimulation. (F) Respective quantification of total SMAD2/3 signals from blots shown in E, n = 4. (G) Representative Western blot of pSTAT3 Tyr705 signals in CD4 T cells, in presence of 30 μM NS8593 (NS) or DMSO control (Ctrl), shown in resting state and after 10 min stimulation with IL-6. (H) Respective quantification of pSTAT3 Tyr705 signals of blots shown in (G). Cells treated with 30 μM NS8593 (NS, red) or DMSO control (Ctrl, black), n = 3. (I) Representative FACS plots of pSTAT3 Tyr705 signal in CD4 T cells upon 10 min stimulation with IL-6. Cells treated with 30 μM NS8593 (NS, red) or DMSO control (Ctrl, black), unstimulated Ctrl shown in light gray. (J) Respective quantification of pSTAT3 Tyr705 signal in CD4 T cells of data shown in (I), n = 4. (K) Representative FACS plots of pSTAT3 Tyr705 signal in CD4 T cells upon 10 min stimulation with a-CD3/a-CD28 for cells treated with 30 μM NS8593 (NS, red) or DMSO control (Ctrl, black), unstimulated control in light gray. (L) Respective quantification of pSTAT3 Tyr705 signal of data shown in (K), n = 4. (M) Representative Western blot of pSTAT3 Ser727 signals in CD4 T cells in presence of 30 μM NS8593 (NS) or DMSO control (Ctrl) in resting state and after 10 min stimulation with IL-6. (N) Respective quantification of pSTAT3 Ser727 signals of blots shown in (M). Cells treated with 30 μM NS8593 (NS, red) or DMSO control (Ctrl, black), n = 3. (O) Representative FACS plots of pSTAT3 Ser727 signal in CD4 T cells upon 10 min stimulation with IL-6 of cells treated with 30 μM NS8593 (NS, red) or DMSO control (Ctrl, black), unstimulated control in light gray. (P) Respective quantification of pSTAT3 Ser727 signal of data shown in (O), n = 4. (Q) Representative Western blot of pSTAT3 Ser727 signals in CD4 T cells, in presence of 30 μM NS8593 (NS) or DMSO control (Ctrl) in resting state and after 10 min stimulation with a-CD3/a-CD28. (R) Respective quantification of pSTAT3 Ser727 signals of blots shown in (Q). (S) Representative FACS plots of pSTAT3 Ser727 signal in CD4 T cells upon 10 min stimulation with a-CD3/a-CD28 of cells treated with 30 μM NS8593 (NS, red) or DMSO control (Ctrl, black), unstimulated control in light gray. (T) Respective quantification of pSTAT3 Ser727 signal of data shown in (S), n = 4. (U) Representative Western blot of pSTAT5 Tyr694 signals in CD4 T cells of cells treated with 30 μM NS8593 (NS) or DMSO control (Ctrl) in resting state and after 15 min a-CD3/a-CD28 stimulation. (V) Representative FACS plot of pSTAT5 Tyr694 signal in CD4 T cells upon 15 min stimulation with IL-2 of cells treated with 30 μM NS8593 (NS, red) or DMSO control (Ctrl, black), unstimulated control in light gray. (W) Respective quantification of pSTAT5 Tyr694 signal of data shown in (V), n = 4. (A, D, F, H, J, L, N, P, R, T, W) Statistics: One-way ANOVA (A, D, F, H, N, R) and t test (J, L, P, T, W). *** P < 0.0005, **** P < 0.0001 and n.s., not significant. Data are mean ± SD.

Article Snippet: The following antibodies were used: α-pSMAD2 Ser465/Ser467 (138D4; Cell Signaling), α-SMAD2/3 (D7G7; Cell Signaling), α-AKT1 (D9-9-C9; Thermo Fisher Scientific), α-pSTAT3 Tyr705 (D3A7; Cell Signaling), α-pSTAT3 Ser727 (D8C2Z; Cell Signaling), α-pSTAT5 Tyr 694 (D47E7; Cell Signaling), α-GAPDH (G-9; Santa Cruz).

Techniques: Western Blot, Control