Journal: Science advances

Article Title: The anti-fibrotic drug pirfenidone inhibits liver fibrosis by targeting the small oxidoreductase glutaredoxin-1.

doi: 10.1126/sciadv.abg9241

Figure Lengend Snippet: Fig. 1. PFD inhibits HSC activation and liver fibrosis and induces the expression of Glrx in a Stat5-dependent manner. (A) Immunofluorescence staining of -SMA in primary mouse HSCs treated with PFD (1 mM) for 4 days. Scale bar, 50 m. (B) GSEA of RNA-seq data from PFD-treated primary mouse HSCs. (C to F) Eight-week-old male WT mice were subjected to the CCl4 model and treated with vehicle (n = 4) or PFD (250 mg/kg) (n = 4) by daily gavage (C). -SMA immunostaining and Sirius Red staining are shown. Scale bar, 200 m. (D) mRNA expression of fibrogenic genes (E) and protein expression of -SMA with the quantifications labeled (F). (G) Heat map of HSC gene expression. (H) Protein expression of Glrx. (I) Gene expression in HSCs infected with Ad-shCtrl or Ad-shGlrx and treated with PFD. (J to L) Primary mouse HSCs were treated with PFD (1 mM) for 4 days. GSEA of gene and protein expression by JAK-STAT signaling (J) and mRNA expression of Stat5a, Stat5b (K), and Stat5 downstream genes (L) (n = 3) are shown. (M) ChIP sequencing (ChIP-seq) identified a Stat5-binding peak in mouse Glrx gene promoter. (N) Gene expression in HSCs treated with PFD and STAT5-IN-1 (100 M) for 4 days (n = 3). (O) Glrx promoter luciferase reporter activity in 293T cells cotransfected with Stat5a or Stat5b plasmid (n = 3). (P) Primary mouse HSCs transfected with siCon or siStat5b were treated with PFD (1 mM) for 4 days (n = 3). (Q and R) WT or deletion mutant Glrx promoter reporter activity in 293T cells cotransfected with Stat5b plasmid (n = 3). Data are means ± SD. *P < 0.05 and **P < 0.01. NS, statistically not significant.

Article Snippet: After sonication, sheared chromatin supernatants were added with normal rabbit immunoglobulin G (IgG) (2729, Cell Signaling Technology), anti-Stat5 antibody (94205, Cell Signaling Technology), or anti-Smad3 antibody (ab28379, Abcam) followed by precipitation with Protein A Magnetic Beads (S1425S, New England Biolabs).

Techniques: Activation Assay, Expressing, Immunofluorescence, Staining, RNA Sequencing, Immunostaining, Labeling, Gene Expression, Infection, ChIP-sequencing, Binding Assay, Luciferase, Activity Assay, Plasmid Preparation, Transfection, Mutagenesis

Journal: Journal of Cancer

Article Title: PFKFB4 promotes angiogenesis via IL-6/STAT5A/P-STAT5 signaling in breast cancer.

doi: 10.7150/jca.66773

Figure Lengend Snippet: Figure 4. IL-6 induces STAT5 phosphorylation in HUVEC cells. A) The Western blot analysis showed that conditioned medium collected from MDA-MB-231/PFKFB4 and T47D/PFKFB4 cells induced IL-6R expression of HUVEC cells. β-actin was used as a loading control. B) The representative immunofluorescent photomicrographs illustrated that conditioned medium collected from MDA-MB-231/PFKFB4 (upper panel) and T47D/PFKFB4 (lower panel) augmented IL-6R immunostaining (green) of HUVEC cells. The nuclei (blue) were stained using DAPI. C) The Western blot analysis showed that conditioned medium collected from MDA-MB-231/PFKFB4 and T47D/PFKFB4 cells induced STAT5 phosphorylation, STAT5A and CD31 expression of HUVEC cells, but not STAT1 or STAT3 phosphorylation. STAT1, STAT3, STAT5B, and β-actin were used as a loading control. D) The Western blot analysis showed that knocking down of STAT5A, but not STAT5B expression in HUVEC, abolished STAT5 phosphorylation promoted by the conditioned medium collected from MDA-MB-231/PFKFB4 and T47D/PFKFB4 cells. β-actin was used as a loading control. E) The Western blot analysis showed that anti-IL-6 treatment of HUVEC blocked STAT5 phosphorylation prompted by the conditioned medium collected from MDA-MB-231/PFKFB4 and T47D/PFKFB4 cells. β-actin was used as a loading control.

Article Snippet: Western blot was performed using the following antibodies: PFKFB4 (1:1000, ab137785, Abcam), IL-6 (1:1000, WL02841, Wanleibio), IκBα (phosphor S36) (1:1000, ab133462, Abcam), IκBα (1:1000, ab76429, Abcam), p-NF-κBp65 ser536 (1:1000, WL02169, Wanleibio), NF-κBp65 (1:1000, sc-398442, Santa Cruz), β-actin (1:2000, sc-47778, Santa Cruz), Lamin A/C (1:1000, 2032, CST), α-Tubulin (1:1000, 2144, CST), CD31 (1:1000, WL03674, Wanleibio), IL-6R (1:1000, WL02737, Wanleibio), p-STAT1 (Y701) (1:1000, 7649, CST), p-STAT3 (Y705) (1:1000, 9145, CST), p-STAT5 (Y694) (1:1000, 9359, CST), STAT3 (1:1000, 9139, CST) , STAT1 (1:1000, sc-592, Santa Cruz), STAT5A (1:1000, sc-1081, Santa Cruz), STAT5B (1:1000, sc-1656, Santa Cruz).

Techniques: Phospho-proteomics, Western Blot, Expressing, Control, Immunostaining, Staining

Journal: Journal of Cancer

Article Title: PFKFB4 promotes angiogenesis via IL-6/STAT5A/P-STAT5 signaling in breast cancer.

doi: 10.7150/jca.66773

Figure Lengend Snippet: Figure 5. 5-MPN treatment inhibits PFKFB4-induced angiogenesis signaling in vitro. A) The representative photomicrographs of 3 independent tube formation assays showed that 5-MPN treatment of MDA-MB-231 and T47D cells inhibited PFKFB4-induced HUVEC tube formation compared to the DMSO/PFKFB4 group. This bar graph shows the quantification of tube formation assay that illustrates 5-MPN treatment of MDA-MB-231 and T47D cells significantly decreased HUVEC tube formation versus the DMSO/PFKFB4 group (16.67±0.88 versus 6.00±1.16; 17.00±1.53 versus 5.00±1.16, respectively; ** p<0.01, *** p<0.001). B) The Western blot analysis showed that 5-MPN treatment of MDA-MB-231 and T47D cells diminished PFKFB4-induced NF-κB phosphorylation and IL-6 expression. β-actin was used as a loading control. C) The Western blot analysis showed that 5-MPN treatment of MDA-MB-231 and T47D cells diminished PFKFB4-induced STAT5 phosphorylation and STAT5A, IL-6R, CD31 expression in HUVEC cells. β-actin was used as a loading control.

Article Snippet: Western blot was performed using the following antibodies: PFKFB4 (1:1000, ab137785, Abcam), IL-6 (1:1000, WL02841, Wanleibio), IκBα (phosphor S36) (1:1000, ab133462, Abcam), IκBα (1:1000, ab76429, Abcam), p-NF-κBp65 ser536 (1:1000, WL02169, Wanleibio), NF-κBp65 (1:1000, sc-398442, Santa Cruz), β-actin (1:2000, sc-47778, Santa Cruz), Lamin A/C (1:1000, 2032, CST), α-Tubulin (1:1000, 2144, CST), CD31 (1:1000, WL03674, Wanleibio), IL-6R (1:1000, WL02737, Wanleibio), p-STAT1 (Y701) (1:1000, 7649, CST), p-STAT3 (Y705) (1:1000, 9145, CST), p-STAT5 (Y694) (1:1000, 9359, CST), STAT3 (1:1000, 9139, CST) , STAT1 (1:1000, sc-592, Santa Cruz), STAT5A (1:1000, sc-1081, Santa Cruz), STAT5B (1:1000, sc-1656, Santa Cruz).

Techniques: In Vitro, Tube Formation Assay, Western Blot, Phospho-proteomics, Expressing, Control

Journal: Journal of Cancer

Article Title: PFKFB4 promotes angiogenesis via IL-6/STAT5A/P-STAT5 signaling in breast cancer.

doi: 10.7150/jca.66773

Figure Lengend Snippet: Figure 6. 5-MPN treatment inhibits PFKFB4-induced angiogenesis in vivo. A) Scheme for the in vivo experiment with results shown in panels B, C, D and E. B) Photographs of xenograft MDA-MB-231 tumors formed in NOD/SCID mice harvested on Day 30. The photographs qualitatively indicate that ectopic expression of PFKFB4 increased tumor size versus the MCS group, whereas, 5-MPN treatment inhibited PFKFB4-induced growth of tumor size. C) Growth curve of xenograft MDA-MB-231 tumors formed in NOD/SCID mice. This graph demonstrates that ectopic expression of PFKFB4 significantly increased tumor size versus the MCS group (1364±36.05 versus 1897+44.31, respectively; *** p<0.001), whereas, 5-MPN treatment significantly inhibited PFKFB4-induced growth of tumor size (1897±44.31 versus 746.6±27.59, respectively; *** p<0.001). Tumor volume = length x width x width/2. D) The tumor weight of xenograft MDA-MB-231 tumors formed in NOD/SCID mice. The dot plot demonstrates that ectopic expression of PFKFB4 significantly increased tumor weight versus the MCS group (0.71±0.06 versus 1.22±0.11, respectively; **p<0.01), whereas, 5-MPN treatment significantly inhibited PFKFB4-induced growth of tumor size (1.22±0.11 versus 0.55±0.08, respectively; ***p<0.001). E) The representative micrographs of IL-6, P-NF-κB, IL-6R, CD31, STAT5A, and P-STAT5 immunocytochemical staining in xenograft MDA-MB-231 tumors. Ectopic expression of PFKFB4 increased immunocytochemical staining of above-mentioned molecules versus the MCS group, whereas, 5-MPN treatment inhibited PFKFB4-induced immunocytochemical staining of above-mentioned molecules. Scale bar= 50 µm.

Article Snippet: Western blot was performed using the following antibodies: PFKFB4 (1:1000, ab137785, Abcam), IL-6 (1:1000, WL02841, Wanleibio), IκBα (phosphor S36) (1:1000, ab133462, Abcam), IκBα (1:1000, ab76429, Abcam), p-NF-κBp65 ser536 (1:1000, WL02169, Wanleibio), NF-κBp65 (1:1000, sc-398442, Santa Cruz), β-actin (1:2000, sc-47778, Santa Cruz), Lamin A/C (1:1000, 2032, CST), α-Tubulin (1:1000, 2144, CST), CD31 (1:1000, WL03674, Wanleibio), IL-6R (1:1000, WL02737, Wanleibio), p-STAT1 (Y701) (1:1000, 7649, CST), p-STAT3 (Y705) (1:1000, 9145, CST), p-STAT5 (Y694) (1:1000, 9359, CST), STAT3 (1:1000, 9139, CST) , STAT1 (1:1000, sc-592, Santa Cruz), STAT5A (1:1000, sc-1081, Santa Cruz), STAT5B (1:1000, sc-1656, Santa Cruz).

Techniques: In Vivo, Expressing, Staining

Journal: Journal of Cancer

Article Title: PFKFB4 promotes angiogenesis via IL-6/STAT5A/P-STAT5 signaling in breast cancer.

doi: 10.7150/jca.66773

Figure Lengend Snippet: Figure 7. A proposed model for PFKFB4-induced angiogenesis. PFKFB4 promotes angiogenesis via IL-6/STAT5A/P-STAT5 signaling in breast cancer. PFKFB4 ectopic expression in breast cancer cells elevates lactate levels in the culture medium which initiates NF-κB activation and nuclear translocation. NF-κB within the nucleus binds to the IL-6 promoter region and then enhances IL-6 expression. The resultant IL-6 expression boosts IL-6R and CD31 (a vascular differentiation marker) expression in endothelial cells. Consequently, it appears that STAT5A/P-STAT5 (but not STAT3) is the pivotal signaling molecules involved in the angiogenic process.

Article Snippet: Western blot was performed using the following antibodies: PFKFB4 (1:1000, ab137785, Abcam), IL-6 (1:1000, WL02841, Wanleibio), IκBα (phosphor S36) (1:1000, ab133462, Abcam), IκBα (1:1000, ab76429, Abcam), p-NF-κBp65 ser536 (1:1000, WL02169, Wanleibio), NF-κBp65 (1:1000, sc-398442, Santa Cruz), β-actin (1:2000, sc-47778, Santa Cruz), Lamin A/C (1:1000, 2032, CST), α-Tubulin (1:1000, 2144, CST), CD31 (1:1000, WL03674, Wanleibio), IL-6R (1:1000, WL02737, Wanleibio), p-STAT1 (Y701) (1:1000, 7649, CST), p-STAT3 (Y705) (1:1000, 9145, CST), p-STAT5 (Y694) (1:1000, 9359, CST), STAT3 (1:1000, 9139, CST) , STAT1 (1:1000, sc-592, Santa Cruz), STAT5A (1:1000, sc-1081, Santa Cruz), STAT5B (1:1000, sc-1656, Santa Cruz).

Techniques: Expressing, Activation Assay, Translocation Assay, Marker

Journal: Pharmacological research

Article Title: Ectopic expression of NKG7 enhances CAR-T function and improves the therapeutic efficacy in liquid and solid tumors.

doi: 10.1016/j.phrs.2024.107506

Figure Lengend Snippet: Fig. 4. NKG7 promotes the proliferation of CAR-T cells by enhancing the IL-2 signaling pathway. (A and B) GSEA of DNA replication and cell cycle between B7H3- CAR-TNKG7 and B7H3-CAR-T cells following B7H3 protein stimulation for 6 h. (C) Volcano plot of T cell proliferation-related genes between B7H3-CAR-TNKG7 and B7H3-CAR-T cells. (D) Heatmaps of significant T cell proliferation-related genes. (E and F) IL-2 expression of CAR-T cells was measured by FCM analysis following target cell stimulation for 6 h. (G) Statistics for (E and F). (H) The proliferation of CAR-T cells was detected by a CFSE assay. (I and J) GSEA of the Jak-STAT and PI3K- Akt pathways between B7H3-CAR-TNKG7 and B7H3-CAR-T cells following B7H3 protein stimulation for 6 h. (K) The IL-2 signaling pathway (p-AKT (Ser473), AKT, p- ERK (Thr202/Tyr204), ERK, p-STAT5 (Thr694) and STAT5) in CAR-T cells were explored by WB assays. ***P < 0.001.

Article Snippet: After blocking with 5 % nonfat milk at room temperature for 1 h, the membranes were incubated with primary antibodies against NKG7 (Cat No. 84835, 1:1000, CST), AKT (Cat No. A11016, 1:1000, ABclonal), p-AKT (Ser473) (Cat No. AP0140, 1:1000, ABclonal), ERK (Cat No. 4695, 1:1000, CST), p-ERK (Thr202/Tyr204) (Cat No. 4370, 1:1000, CST), STAT5 (Cat No. 25656, 1:1000, CST), pSTAT5 (Thr694) (Cat No. 4322, 1:1000, CST) and β-actin (Cat No. 66009–1-Ig, 1:5000, Proteintech) overnight at 4◦C.

Techniques: Expressing, Cell Stimulation, CFSE Assay

Journal: European journal of immunology

Article Title: TNF-α and IL-1β sensitize human MSC for IFN-γ signaling and enhance neutrophil recruitment.

doi: 10.1002/eji.201948336

Figure Lengend Snippet: FIGURE 3. Inhibition of NF-kB, p38-MAPK or STAT5 blocks IL-8 release by MSC.

Article Snippet: For immunodetection the following antibodies were used: anti phospho-STAT1(Tyr701) (clone D4A7), STAT5 (total protein) (clone D3N2B), phosphoSTAT5 (Tyr694) (clone D47E7), p38-MAPK (p38α, β, or γ MAPK protein) (clone D13E1), phospho-p38-MAPK (Thr180/Tyr182) (clone D3F9), β-Actin (clone 13E5), Vinculin (clone E1E9V), NF-κB p65 (clone D14E12) and goat anti rabbit IgG HRP (all Cell Signalling Technology, Danvers, MA, USA).

Techniques: Inhibition