Journal: Molecular Oncology

Article Title: Targeted inhibition of STAT3 induces immunogenic cell death of hepatocellular carcinoma cells via glycolysis

doi: 10.1002/1878-0261.13263

Figure Lengend Snippet: Knockdown of STAT3 expression induces membrane translocation of ICD‐related molecules in HCC cells and promotes DC activation. (A,B) Analysis of calreticulin + and ERp57 + cells in Huh7 and HepG2.2.15 cells infected with indicated lentiviral vectors by flow cytometry. (C,D) Flow cytometry analysis of HSP70 + and HSP90 + cells in Huh7 and HepG2.2.15 cells. (E) Assessment of ATP released into culture supernatants of Huh7 and HepG2.2.15 cells. (F,G) Flow cytometry analysis of CD80 + and CD86 + cells in hDCs after coculture with Huh7 or HepG2.2.15 cells for 48 h. Data are shown as mean ± SD from three independent experiments and were analysed with unpaired Student's t ‐test or one‐way ANOVA (* P < 0.05, ** P < 0.01, and *** P < 0.001).

Article Snippet: Plasmid pGFP‐V‐RS encoding mouse STAT3 shRNAs were purchased from Origen (Locus ID 20848, Origene, Rockville, MD, USA).

Techniques: Expressing, Translocation Assay, Activation Assay, Infection, Flow Cytometry

Journal: Molecular Oncology

Article Title: Targeted inhibition of STAT3 induces immunogenic cell death of hepatocellular carcinoma cells via glycolysis

doi: 10.1002/1878-0261.13263

Figure Lengend Snippet: Targeting STAT3 induces immunogenic cell death of HCC in vivo . (A) Immunoblot showing STAT3 and p‐STAT3 Tyr705 levels in Huh7 and Hepa1‐6 cells treated with different concentrations of napabucasin for 12 h ( n = 3). Shown is one representative of at least three independent experiments. (B) After treatment with napabucasin or DMSO at the indicated concentrations and time, Huh7 and Hepa1‐6 cells were labelled with anti‐calreticulin antibody and PI and analysed by flow cytometry. (C) Flow cytometry analysis of calreticulin + , ERp57 + , HSP70 + , and HSP90 + cells in Huh7 and Hepa1‐6 cells treated with napabucasin or DMSO for 12 h. (D) Following coculture with Huh7 or Hepa1‐6 cells treated with napabucasin or DMSO for 48 h, CD80 + , CD40 + , CD86 + , and MHC II + cells in hDCs or mDCs were analysed by flow cytometry. Data are shown as mean ± SD from three independent experiments and were analysed with unpaired Student's t ‐test or one‐way ANOVA (* P < 0.05, ** P < 0.01, and *** P < 0.001). (E) The therapeutic schedule. C57BL/6J mice were inoculated with 5 × 10 6 Hepa1‐6 cells in the left axilla. On day 5 postinoculation, mice were intraperitoneally injected with napabucasin (20 mg·kg –1 ) every 2 days for a total of eight injections. On day 2 after the last injection, the tumours were resected. On day 7 posttumour resection, these mice were inoculated subcutaneously with 5 × 10 6 Hepa1‐6 cells in the right axilla. Solvent was used as vehicle. (F) The levels of calreticulin and ERp57 in tumour tissues were analysed by immunofluorescence. Scale bar = 10 μm. The tumour‐free rate (G) and tumour growth curve (H) of tumour‐rechallenged C57BL/6J mice are shown. Data represent the mean ± SD of n = 6 and tumour‐free analysis were analysed with the Chi‐squared test (* P < 0.05, ** P < 0.01, and *** P < 0.001).

Article Snippet: Plasmid pGFP‐V‐RS encoding mouse STAT3 shRNAs were purchased from Origen (Locus ID 20848, Origene, Rockville, MD, USA).

Techniques: In Vivo, Western Blot, Flow Cytometry, Injection, Immunofluorescence

Journal: Molecular Oncology

Article Title: Targeted inhibition of STAT3 induces immunogenic cell death of hepatocellular carcinoma cells via glycolysis

doi: 10.1002/1878-0261.13263

Figure Lengend Snippet: STAT3 regulates ICD marker eIF2α phosphorylation through interaction with PKR. (A) Western blot analysis of STAT3, p‐STAT3 Tyr705 , PKR, p‐PKR Thr446 , eIF2α, and p‐eIF2α Ser51 expression in Huh7 and HepG2.2.15 cells infected with lentiviral STAT3‐shRNA vector or lentiviral vector. (B) Western blot analysis of STAT3, p‐STAT3 Tyr705 , PKR, p‐PKR Thr446 , eIF2α, and p‐eIF2α Ser51 expression in Huh7 and HepG2.2.15 cells stimulated with IL‐6 at the indicated concentrations for 2 h. (C) Western blot analysis of STAT3, p‐STAT3 Tyr705 , PKR, p‐PKR Thr446 , eIF2α, and p‐eIF2α Ser51 expression in Huh7 and HepG2.2.15 cells infected with indicated lentiviral vectors after stimulation with IL‐6. (D) Coimmunoprecipitation with STAT3 antibody. Huh7 and HepG2.2.15 cell extracts were incubated with anti‐STAT3 antibody and Protein A/G Magnetic Beads. Following incubation, PKR was detected by western blot to verify the protein binding. (E) GST pull‐down assay of the interaction between STAT3 and PKR. Western blotting analysis of PKR binding to purified GST or GST‐STAT3 fusion protein using PKR antibody (top). GST or GST‐STAT3 fusion protein was visualised by Coomassie blue staining (bottom). N = 3. Shown is one representative of at least three independent experiments.

Article Snippet: Plasmid pGFP‐V‐RS encoding mouse STAT3 shRNAs were purchased from Origen (Locus ID 20848, Origene, Rockville, MD, USA).

Techniques: Marker, Western Blot, Expressing, Infection, shRNA, Plasmid Preparation, Incubation, Magnetic Beads, Protein Binding, Pull Down Assay, Binding Assay, Purification, Staining

Journal: Molecular Oncology

Article Title: Targeted inhibition of STAT3 induces immunogenic cell death of hepatocellular carcinoma cells via glycolysis

doi: 10.1002/1878-0261.13263

Figure Lengend Snippet: STAT3 directly regulates “don't eat me” signal CD47 in HCC cells. (A) TCGA database analysis of CD47 expression in HCC patients based on sample types through the UALCAN database. (B) Validation of gene expression correlation between STAT3 and CD47 in HCC patients through the AIPuFu database. Data from the TCGA database. Correlations were determined by Pearson analysis. (C) Huh7 and HepG2.2.15 cells were infected with lentiviral STAT3‐shRNA or control vectors, and CD47 expressions were analysed by flow cytometry. (D) Flow cytometry analysis of CD47 expression in Huh7 and HepG2.2.15 cells stimulated with 50 μ m IL‐6 for 4 h. (E) CM‐Dil‐labelled macrophages derived from THP‐1 cells were cocultured with CFSE‐labelled Huh7 or HepG2.2.15 cells for 2 h, then the phagocytosis was assessed by flow cytometry. (F) The statistical results of phagocytosis. Data are shown as mean ± SD from three independent experiments and were analysed with unpaired Student's t ‐test (* P < 0.05, ** P < 0.01, and *** P < 0.001). (G) Flow cytometry analysis of CD47 + cells in Huh7 cells. (H) The statistical results of phagocytosis. Antihuman CD47 antibody (B6H12) was used to block CD47 on the surface of HCC cells at the 2 μg·mL –1 for 2 h coincubation at 37 °C. Data are shown as mean ± SD from three independent experiments and were analysed with one‐way ANOVA (* P < 0.05, ** P < 0.01, and *** P < 0.001). (I) Schematic representation shows two candidate STAT3 binding sites on human CD47 gene promoter. (J) ChIP assays were performed to identify the binding of STAT3 to the CD47 promoter in HCC cells. Human anti‐p‐STAT3 antibody or control IgG were used for immunoprecipitation with DNA from Huh7 and HepG2.2.15 cells. The immunoprecipitates were then amplified by PCR using primers targeting CD47, n = 3. Shown is one representative of at least three independent experiments.

Article Snippet: Plasmid pGFP‐V‐RS encoding mouse STAT3 shRNAs were purchased from Origen (Locus ID 20848, Origene, Rockville, MD, USA).

Techniques: Expressing, Infection, shRNA, Flow Cytometry, Derivative Assay, Blocking Assay, Binding Assay, Immunoprecipitation, Amplification

Journal: Molecular Oncology

Article Title: Targeted inhibition of STAT3 induces immunogenic cell death of hepatocellular carcinoma cells via glycolysis

doi: 10.1002/1878-0261.13263

Figure Lengend Snippet: STAT3 regulates the glycolysis of HCC cells. (A) CCK‐8 assay of Huh7 and HepG2.2.15 cells treated with saline or the indicated concentrations of 2‐DG for 24, 48, and 72 h. (B) HepG2.2.15 cells were cultured in glucose‐free medium for 6 h to implement glucose starvation, then replaced with fresh medium containing glucose or/and 2‐DG for another 12 h. These cells were harvested for analysis of calreticulin and CD47 levels by flow cytometry. (C,D) Huh7 and HepG2.2.15 cells were infected with lentiviral STAT3‐shRNA vector or control vector for 48 h. The ECAR value was automatically recorded and calculated by the Seahorse XF‐24 analyser in Huh7 and HepG2.2.15 cells. (E) The mRNA levels of HIF1α, GLUT1, HK2, and LDHA in Huh7 and HepG2.2.15 cells infected with lentiviral STAT3‐shRNA vector or control vector were analysed by qRT‐PCR. Data were normalised to β‐actin and are shown as mean ± SD from three independent experiments. Data are shown as mean ± SD from three independent experiments and were analysed with unpaired Student's t ‐test or one‐way ANOVA (* P < 0.05, ** P < 0.01, and *** P < 0.001). (F) AIPuFu database analysis of the correlation between GLUT1 and CD47 in HCC from the TCGA database. Correlations were determined by Pearson analysis. (G) Flow cytometry analysis of GLUT1 + cells in HepG2.2.15 cells treated as indicated. (H) Western blot analysis of PKR, p‐PKR Thr446 , eIF2α, and p‐eIF2α Ser51 expression in HepG2.2.15 cells treated with STF‐31 (0.25 μ m ) for 12 h. The grey scale of protein bands was quantified by imagej (National Institutes of Health, Bethesda, MD, USA). Data are shown as mean ± SD from three independent experiments and were analysed with unpaired Student's t ‐test (* P < 0.05). (I) The ECAR value of HepG2.2.15 cells treated as indicated. The statistical results of glycolysis and glycolysis capacity are shown. Data are shown as mean ± SD from three independent experiments and were analysed with one‐way ANOVA (* P < 0.05, ** P < 0.01, and *** P < 0.001). (J) Analysis of calreticulin + and CD47 + cells in HepG2.2.15 cells treated as indicated by flow cytometry. Data are shown as mean ± SD from three independent experiments and were analysed with one‐way ANOVA (* P < 0.05 and ** P < 0.01). (K) Schematic representation shows two candidate STAT3 binding sites on human GLUT1 gene promoter. (L) ChIP assays were performed to evaluate binding of STAT3 to the GLUT1 promoter in HCC cells. Human anti‐STAT3 antibody or control human IgG was used for immunoprecipitation with DNA from Huh7 and HepG2.2.15 cells. The immunoprecipitates were then amplified by PCR using primers targeting GLUT1. Shown is one representative of at least three independent experiments.

Article Snippet: Plasmid pGFP‐V‐RS encoding mouse STAT3 shRNAs were purchased from Origen (Locus ID 20848, Origene, Rockville, MD, USA).

Techniques: CCK-8 Assay, Cell Culture, Flow Cytometry, Infection, shRNA, Plasmid Preparation, Quantitative RT-PCR, Western Blot, Expressing, Binding Assay, Immunoprecipitation, Amplification

Journal: Molecular Oncology

Article Title: Targeted inhibition of STAT3 induces immunogenic cell death of hepatocellular carcinoma cells via glycolysis

doi: 10.1002/1878-0261.13263

Figure Lengend Snippet: STAT3 inhibition induces ICD and improves the tumour immune‐environment in HCC. Targeting inhibition of STAT3 could induce ICD of HCC cells, which manifested as “eat me” molecules such as calreticulin, ERp57, and HSPs translocated to the HCC cell surface while the “don't eat me” molecule CD47 significantly decreased. This enhanced the recognition and phagocytosis of HCC cells by DCs and macrophages. In parallel, the important antitumour effector CD8 + T cells, with low expression of checkpoint molecules such as LAG‐3 and PD‐1, accumulated in tumour tissues. The underlying mechanism involves the disruption of STAT3‐mediated direct regulation of CD47 and glycolysis via GLUT1 in HCC cells.

Article Snippet: Plasmid pGFP‐V‐RS encoding mouse STAT3 shRNAs were purchased from Origen (Locus ID 20848, Origene, Rockville, MD, USA).

Techniques: Inhibition, Expressing

Journal: Cell death & disease

Article Title: Galectin-3 promotes secretion of proteases that decrease epithelium integrity in human colon cancer cells.

doi: 10.1038/s41419-023-05789-x

Figure Lengend Snippet: Fig. 7 Galectin-3 expression induces activation of PYK2, STAT1 and GSK3α/β signalling. Expression of 37 protein kinases in SW620 cells in response to 10 µg/ml galectin-3 or BSA for 0.5 h was assessed by Proteome Profiler Human Phospho-Kinase Array (A, Percentage changes of the kinases in cell response to galectin-3 in comparison to control are shown at the bottom panel). The presence of galectin-3 increases the phosphorylation of PYK2, GSK3α/β, and STAT1 and decreases phosphorylation of STAT3. SW620 cells treated with 10 µg/ml galectin-3 for different times were assessed by immunoblotting using antibodies against p-PYK2, p-STAT-1, p-GSK3α/β or p-STAT-3 (B). The blots were striped and reprobed with antibodies against PYK2, STAT-1, GSK3α/β or STAT-3. The band density was quantified and expressed as percentages of phospho-/non-phosphorylated proteins (C). In D and E, SW620 cells were treated with 10 µg/ml galectin-3 or BSA followed by introduction of GSK3α/β inhibitor SB 216763 (SB) or PKY2 inhibitor PF-431396 (PF) for 15 min and the levels of phosphorylated PYK2, STAT-1, GSK3α/β or STAT-3 were analysed by immunoblotting. The blots were striped and reprobed with antibodies against PYK2, STAT-1, GSK3α/β or STAT-3. The densities of the blots from three independent experiments were quantified and are expressed as the percentage of phosphorylated/non-phosphorylated levels of each protein. ***P < 0.001, **P < 0.01, *P < 0.05 (ANOVA).

Article Snippet: Biotinylated-anti-galectin-3 (BAF1154) antibody and antibodies against galectin-3 (MAB1154), STAT1 (MAB1490), Phospho-STAT1(MAB2894), STAT3 (MAB1799), Phospho-STAT3 (MAB4934), PYK2 (AF4589), phosphorPYK2 (MAB6210), GSK3α/β (AF2157), Phospho-GSK3α/β (AF1590); Proteome Profiler Human Protease Array (ARY021B), Proteome Profiler Human Phospho-Kinase Array (ARY003C); Human MMP-13 DuoSet ELISA (DY511), Human Cathepsin-B DuoSet ELISA (DY2176) PYK2 inhibitor PF-431396 (4278) and GSK3α/β inhibitor SB 216763 (1616/1) were all purchased from R&D Systems (Abingdon, UK).

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

Journal: bioRxiv

Article Title: In Troyer syndrome Spartin loss induces Complex I impairments and alters pyruvate metabolism

doi: 10.1101/488239

Figure Lengend Snippet: (A) NADH autofluorescence measurement showing an increased level in mutant cells compared to controls. **P < 0.01, means ± SEM. (B) Extracellular lactate content determination by HPLC in SPART wt and SPART 892dupA cells. The extracellular lactate content in culture cell medium was quantified after 24 hours of cell growth by HPLC analysis. The peak area corresponding to lactate was normalized on cell number. (C) Extracellular pyruvate production SPART wt and SPART 892dupA cells measured by HPLC analysis after 72 hours of cell growth. The peak area corresponding to pyruvate was normalized on protein content by Bradford assay. *P < 0.05, means ± SEM. (D) Representative HPLC chromatograms of extracellular media from SPART wt and SPART 892dupA cells. Red line indicates medium from SPART 892dupA cells, green line indicates medium only (DMEM high glucose) and blue line indicates medium from SPART wt cells. Vertical red line indicates pyruvate. (E) Representative western blot analysis showing the expression of phosphorylated and total STAT3 in the two cell lines. Gamma tubulin was used as endogenous control. SPART 892dupA showed an increase of P-STAT3 compared to SPART wt cells, indicating an over-activation of STAT3 in mutant cells. Graph showed the relative quantification of western blot.

Article Snippet: Primary antibodies used were: GAPDH (mouse, 1:10,000; Abcam, Cambridge, UK), γ-tubulin (mouse, 1:10,000; Sigma-Aldrich), T-STAT3 (mouse, 1:500; OriGene, Rockville, MD USA), P-STAT3 (rabbit, 1:500; Cell Signaling, Leiden, Netherlands), Spartin (rabbit, 1:1,000; ProteinTech, Rosemont, IL USA) and GRP75 (goat, 1:500; Santa Cruz, CA USA).

Techniques: Mutagenesis, Bradford Assay, Western Blot, Expressing, Activation Assay

Journal: Nature communications

Article Title: Hypothalamic leptin action is mediated by histone deacetylase 5.

doi: 10.1038/ncomms10782

Figure Lengend Snippet: Figure 1 | Hypothalamic histone deacetylase 5 (HDAC5) expression is regulated by nutrient availability and leptin sensitivity. Hypothalamic Hdac5 mRNA levels were assessed by real-time PCR in (a) lean versus diet-induced obese (DIO) male mice (n ¼ 7) and (b) male Lepob mice treated subcutaneously for 6 days with either saline or 1 mg kg 1 leptin (n ¼ 5–6). Saline-treated mice were fed ad libitum (ad lib) or pair-fed to the lower food consumption of the Leptin ad lib group. (c) Hypothalamic Hdac5 expression was assessed in male C57BL/6J mice that were subjected to 12, 24 or 36 h of fasting as well as refeeding for 6 h with fat-free diet (FFD) or high-fat diet (HFD; n ¼ 6–8). HDAC5, total STAT3, p-STAT3 and ACTIN immunoblots and densitometric analyses of HDAC5 /ACTIN ratios in CLU177 cells (d,e) and primary neurons (f,g) treated for 24 h with leptin. Values are means±s.e.m. Statistical analyses were done by two-tailed unpaired Student’s t-test (a,e,g) or by One-Way ANOVA followed by Bonferroni (b) or Dunnet (c) post hoc tests. *Po0.05, **Po0.01 and ***Po0.001 versus Control; #Po0.05 versus 36-h fasting.

Article Snippet: Adenoviral delivery of HDAC5 was conducted in CLU177 cells that were first transfected with overexpresser mouse STAT3 plasmid (OriGene, Rockville, MD, USA) using lipofectamine.

Techniques: Histone Deacetylase Assay, Expressing, Real-time Polymerase Chain Reaction, Saline, Western Blot, Two Tailed Test, Control

Journal: Nature communications

Article Title: Hypothalamic leptin action is mediated by histone deacetylase 5.

doi: 10.1038/ncomms10782

Figure Lengend Snippet: Figure 4 | Hypothalamic disruption of HDAC5 activity impairs POMC expression and STAT3 signalling: (a) Hypothalamic slices of male POMC–GFP mice were subjected to immunostaining with anti-rabbit HDAC5 antibody, and revealed widespread HDAC5 expression and colocalization to B68.62% of all POMC neurons. (n ¼ 4 slices, Scale bar, 50 mm) (b) Hypothalamic mRNA levels of Pomc and Agrp in rats 2 h after 3rd ventricular injection of 4-phenylbutyric acid (4-PB) or vehicle (n ¼ 8). (c,d) Western blot and densitometric analysis of POMC and ACTIN protein levels in CLU177 cells 24 h after treatment with the selective class IIa HDAC inhibitor MC1568 (n ¼ 3). (e) The number of stained cells in hypothalamic slices after immunohistochemical detection of POMC was counted in male HDAC5 WT and KO mice subjected to 10 weeks of HFD feeding (n ¼ 3). (f,g) Representative western blot and densitometric analysis of reference protein ACTIN, HDAC5 and total STAT3, Ac-STAT3K685 and p-STAT3T705 in primary hypothalamic neurons isolated from KO and WT mice that were subjected to saline or leptin (100 ng ml 1) treatment for 30 min (n ¼ 3). (h) Western blot for HDAC5 STAT3 and pSTAT3T705 following immunoprecipitation with HDAC5 antibody from control CLU177 cells. (i) Representative confocal images of Ac-STAT3K685

Article Snippet: Adenoviral delivery of HDAC5 was conducted in CLU177 cells that were first transfected with overexpresser mouse STAT3 plasmid (OriGene, Rockville, MD, USA) using lipofectamine.

Techniques: Disruption, Activity Assay, Expressing, Immunostaining, Injection, Western Blot, Staining, Immunohistochemical staining, Isolation, Saline, Immunoprecipitation, Control

Journal: Frontiers in Oncology

Article Title: RelA Is an Essential Target for Enhancing Cellular Responses to the DNA Repair/Ref-1 Redox Signaling Protein and Restoring Perturbated Cellular Redox Homeostasis in Mouse PDAC Cells

doi: 10.3389/fonc.2022.826617

Figure Lengend Snippet: STAT3 is not a primary target altering cellular sensitivity to Ref-1 inhibitors in murine PDAC Kras G12D cells. (A) STAT3 was knocked down in KC3590 cells that are RelA deficient (Vector) and clone with functional RelA add back (C13) and knockdown efficiency was assessed by Western bot. Vinculin was used as loading control. The cells were challenged for 48 hours with (B) APX3330, (C) APX2009, (D) APX2014, and (E) RN7-58. Cytotoxicity was measured by alamarBlue. At least three independent experiments were performed (N = 3).

Article Snippet: Cells were transfected by lipofectamine RNAiMax (Invitrogen, CA, USA) with PRDX1 (SR405074, OriGene Technologies, MD, USA), RelA (SR417160, OriGene Technologies, MD, USA), STAT3 (SR427487, OriGene Technologies, MD, USA), and universal scrambled control (SCR) (SR30004, OriGene Technologies, MD, USA) siRNAs.

Techniques: Plasmid Preparation, Functional Assay, Knockdown, Western Blot, Control