Journal: iScience

Article Title: C9ORF72 suppresses JAK-STAT mediated inflammation

doi: 10.1016/j.isci.2023.106579

Figure Lengend Snippet:

Article Snippet: rabbit anti-pSTAT1 , R and D Systems , Cat# AF2894, RRID: AB_2198137.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Reverse Transcription, Bradford Protein Assay, CRISPR, Plasmid Preparation, Software

Journal: Molecular cancer research : MCR

Article Title: Phosphoinositide 3-kinase signaling can modulate MHC class I and II expression

doi: 10.1158/1541-7786.MCR-19-0545

Figure Lengend Snippet: A., SqCC/Y1 cells were treated with vehicle (DMSO) alone, vehicle plus IFN-γ (1 ng/ml) or pictilisib (0.5 mM) plus IFN-γ. Whole cell lysates were prepared 24 hours after the addition of IFN-γ and total-STAT1, phospho-STAT1-Y701 and phospho-STAT1-S727 protein expression analyzed by western blot. Total protein loading on a representative blot is shown in the bottom panel. B., Averaged total-STAT1 (top panel), phospho-STAT1-Y701 (middle panel) and phospho-STAT1-S727 signal intensities (bottom panel) from four experiments are shown normalized to total protein. (*, p<0.05, ***, p<0.001, **** p<0.0001, adjusted P values using 1way ANOVA).

Article Snippet: Antibodies recognizing total STAT1, phospho-STAT1-Y701, phospho-STAT1-S727, total AKT, phospho-AKT-S473, phospho-AKT-T308, and PTEN were all purchased from Cell Signaling (Danvers, MA).

Techniques: Expressing, Western Blot

Journal: Molecular cancer research : MCR

Article Title: Phosphoinositide 3-kinase signaling can modulate MHC class I and II expression

doi: 10.1158/1541-7786.MCR-19-0545

Figure Lengend Snippet: SqCC/Y1 cells were treated with vehicle (DMSO) alone, vehicle plus IFN-γ (100 ng/ml), pictilisib alone (0.5 mM) or pictilisib plus IFN-γ. Forty-eight hours after IFN-γ treatment, chromatin accessibility was assessed using ATAC-seq. A., Principle component analysis of differentially accessible regions (DAR) between the four treatment groups. Circles represent 95% confidence intervals for each group. B. Histogram of chromatin accessibility in each group at STAT1 consensus motifs located in DAR. 100bp surrounding each motif is shown. RPM, reads per million. C., Box plots summarizing accessibility at STAT1 motifs from B. Significance determined by two-tailed Student’s T-test.

Article Snippet: Antibodies recognizing total STAT1, phospho-STAT1-Y701, phospho-STAT1-S727, total AKT, phospho-AKT-S473, phospho-AKT-T308, and PTEN were all purchased from Cell Signaling (Danvers, MA).

Techniques: Two Tailed Test

Journal: Molecular cancer research : MCR

Article Title: Phosphoinositide 3-kinase signaling can modulate MHC class I and II expression

doi: 10.1158/1541-7786.MCR-19-0545

Figure Lengend Snippet: A., HCT116 cells and HCT116 cells lacking PTEN (HCTPTENKD) were left untreated or treated with IFN-γ (IFNG, 1ng/mL) and whole cell lysates prepared 24 hours later. The y-axis represents averaged, HLA-A, HLA-B and HLA-C values from 3 experiments and are normalized to total protein and are expressed relative to untreated parental HCT116 cells. (**, p<0.01, ****, p<0.0001, adjusted p values from 2way ANOVA). B., Representative western blots are shown for HLA-A, HLA-B and HLA-C protein expression and total protein loading for HLA-B western blot. C., STAT1 phosphorylation was assessed 90 minutes after IFN-γ treatment (5 ng/ml) of HCT116 and HCTPTENKD cells and was quantified by western blot. Values are normalized to total STAT1-a and represent the average of three replicate experiments. Representative western blots of STAT1-Y701 and total STAT1 are shown. D., Quantification of STAT1-a and STAT1-b protein levels in HCT116 cells (black bars) or HCTPTENKD cells (gray bars) treated with IFN-γ at the concentrations indicated along the x-axis for 48 hours. STAT1-a and STAT1-b expression are normalized to total protein. The y-axis represents averages from three experiments. Right panel, representative STAT1 western blots and protein loading. Error bars represent the standard deviation. (***, <0.001 adjusted P values using 2way ANOVA). E., Analysis of steady state mRNA levels in HCT116 and HCTPTENKD cells treated with IFN-γ (4 ng/ml). Expression of the genes indicated was measured using quantitative real-time RT-PCR. Values are normalized to beta glucuronidase and are expressed relative to untreated parental HCT116 cells. Values shown represent averages from three experiments. Error bars represent the standard deviation. (*, p<0.05, **, p < 0.01, ****, p<0.0001 using students t-test). F., TCGA RNA-seq data from squamous cell carcinoma of the head and neck showing correlation of expression between HLA-A, HLA-B and HLA-C with PTEN or PIK3CA. G., TCGA RNA-seq data from lung squamous cell carcinoma and pancreatic adenocarcinoma showing violin plots of HLA-A, HLA-B and HLA-C mRNA in patients with mutations or copy number changes in PTEN or PIK3CA (blue) compared to patients without those genetic alterations (yellow). H., TCGA colorectal carcinoma RNA-seq data showing correlation between PTEN mRNA expression and HLA-DRA expression.

Article Snippet: Antibodies recognizing total STAT1, phospho-STAT1-Y701, phospho-STAT1-S727, total AKT, phospho-AKT-S473, phospho-AKT-T308, and PTEN were all purchased from Cell Signaling (Danvers, MA).

Techniques: Western Blot, Expressing, Standard Deviation, Quantitative RT-PCR, RNA Sequencing Assay

Journal: Molecular cancer research : MCR

Article Title: Phosphoinositide 3-kinase signaling can modulate MHC class I and II expression

doi: 10.1158/1541-7786.MCR-19-0545

Figure Lengend Snippet: Top panel, PI3K-activated signaling can occur via upstream activating events, activating mutations in PIK3CA, or via low glucose/oxygen levels as reported by Marijt et al. In addition, PTEN loss or inhibition as well as activation of AKT can activate PI3K signaling. In some settings, these events can have a repressive effect on the expression of MHC molecules and/or their induction by IFN-γ possibly by attenuating the ability of IFN-γ to increase STAT1 protein levels and/or phosphorylation. Decreases in MHC levels may hinder CD8+ and/or CD4+ T-cell activation, tumor recognition, and anti-tumor immunity. Bottom panel, in the setting of PI3K inhibition using PI3K inhibitors such as dactolisib or pictilisib, or the loss of PIK3CA as recently reported by Sivaram et al., the repressive effects of PI3K signaling on MHC expression are diminished. This allows for increased MHC expression and/or induction by IFN-γ possibly mediated by increases in STAT1 protein levels and/or phosphorylation. The increases in MHC expression may promote CD4+ and/or CD8+ activation and anti-tumor immunity. mTOR inhibition by dactolisib, rapamycin or AZD8055 does not alter the impact of PI3K inhibition on MHCI levels and by itself can enhance MHCI levels induced by IFN-γ though mTOR inhibition attenuates the ability of PI3K inhibitors to enhance MHCII induction.

Article Snippet: Antibodies recognizing total STAT1, phospho-STAT1-Y701, phospho-STAT1-S727, total AKT, phospho-AKT-S473, phospho-AKT-T308, and PTEN were all purchased from Cell Signaling (Danvers, MA).

Techniques: Inhibition, Activation Assay, Expressing

Journal: Oncotarget

Article Title: Targeting growth hormone receptor in human melanoma cells attenuates tumor progression and epithelial mesenchymal transition via suppression of multiple oncogenic pathways

doi: 10.18632/oncotarget.15375

Figure Lengend Snippet: a . Representative images of western blot (WB) analyses of phosphorylation levels b . JAK2, c . STAT5, d . STAT1, e . STAT3, f . SRC, g . AKT, h . mTOR and i . ERK1/2, in excess human-GH treated or GHRKD human melanoma cell lysates. SK-MEL-28 cells, 24 hr post-transfection with either scramble (scr)-siRNA or GHR-siRNA were treated for ten mins with GH and lysed as described. WB was performed using appropriate antibodies. Densitometry analyses of individual blots was performed using ImageJ software and the ratio of phosphorylated vs. total protein levels against untreated scr-siRNA transfected controls. Overall, excess GH increased while GHRKD decreased phosphorylation states. Similar results for MALME-3M, MDA-MB-435 and SK-MEL-5 human melanoma cells are presented in . Blots from individual experiments were quantified and the mean of three blots per antibody was taken. Protein levels were normalized against expression of β-actin. [*, p < 0.05, Students t test, n = 3].

Article Snippet: Primary antibodies at specific dilutions were used to detect the following human proteins: GH (Rabbit, 1:100, Abcam #ab155276), GHR (Mouse, 1:300, SCBT #137185; Goat, 1:100, R&D Systems #AF1210; Rabbit, 1:200, Abcam #ab134078), STAT5 (Rabbit, 1:100, CST #9358S), P(Y694/Y699)-STAT5 (Rabbit, 1:100, ActiveMotif #39617, 39618), P(Y701)-STAT1 (Rabbit, 1:100, CST #7649), P(Y705)-STAT3 (Rabbit, 1:100, CST #9145), STAT3 (Rabbit, 1:200, CST #4904), STAT1 (Rabbit, 1:200, CST #9175), p44/42 MAPK (Erk1/2) (Rabbit, 1:2000, CST #9102S), P-p44/42 MAPK (Erk1/2) (Rabbit, 1:3000, CST #4370P), Akt (Rabbit, 1:2000, CST #4685S), P-Akt (Rabbit, 1:1000, CST #4058S), P-Jak2 (Rabbit, 1:200, GeneTex#61122; Rabbit, 1:100, CST #8082), JAK2 (Mouse, 1:200, Sigma Aldrich # SAB4200483), mTOR (Rabbit, 1:1000, CST #2983), P-mTOR (Ser2448) (Rabbit, 1:2000, CST #5536), P-mTOR (Ser2481) (Rabbit, 1:2000, CST #2974), Raptor (Rabbit, 1:500, CST #2280), Rictor (Rabbit, 1:500, CST#2114), GbL (Rabbit, 1:1000, CST #3274), β-Actin (Goat, 1:3000, SCBT #sc1616), GAPDH (Goat, 1:3000, SCBT #sc20357), P(S1524)-BRCA1 (Rabbit, 1:500, CST#9009), P(S139)-histone H2A.X (Rabbit, 1:1000, CST #9718), histone H2A.X (Rabbit, 1:1000, CST #2595), Caspase-3(Rabbit, 1:1000, CST#9665), cleaved (Asp175)-Caspase-3 (Rabbit, 1:1000, CST #9664), P(Y416)-SFK (Rabbit, 1:200, CST #2101), P(Y416)-SRC (Rabbit, 1:200, CST #6943), SRC (Rabbit, 1:500, AbcaM #47405).

Techniques: Western Blot, Transfection, Software, Expressing

Journal: Oncotarget

Article Title: Targeting growth hormone receptor in human melanoma cells attenuates tumor progression and epithelial mesenchymal transition via suppression of multiple oncogenic pathways

doi: 10.18632/oncotarget.15375

Figure Lengend Snippet: Endocrine/paracrine/autocrine GH binds to GHR expressed at high levels in human malignant melanoma cells and activates JAK2 and SRC kinases. This leads to downstream activation of STAT1, STAT3, STAT5, ERK1/2, AKT and mTOR. Subsequent transcription of target genes lead to aggressive phenotypes of tumor cell migration, invasion and proliferation and upregulate autocrine HGF-MET loop, ERBB3 and also drives epithelial-mesenchymal-transition. In our study excess GH upregulated (in green) while siRNA mediated GHR knock-down (GHRKD) downregulated (in red) these effects.

Article Snippet: Primary antibodies at specific dilutions were used to detect the following human proteins: GH (Rabbit, 1:100, Abcam #ab155276), GHR (Mouse, 1:300, SCBT #137185; Goat, 1:100, R&D Systems #AF1210; Rabbit, 1:200, Abcam #ab134078), STAT5 (Rabbit, 1:100, CST #9358S), P(Y694/Y699)-STAT5 (Rabbit, 1:100, ActiveMotif #39617, 39618), P(Y701)-STAT1 (Rabbit, 1:100, CST #7649), P(Y705)-STAT3 (Rabbit, 1:100, CST #9145), STAT3 (Rabbit, 1:200, CST #4904), STAT1 (Rabbit, 1:200, CST #9175), p44/42 MAPK (Erk1/2) (Rabbit, 1:2000, CST #9102S), P-p44/42 MAPK (Erk1/2) (Rabbit, 1:3000, CST #4370P), Akt (Rabbit, 1:2000, CST #4685S), P-Akt (Rabbit, 1:1000, CST #4058S), P-Jak2 (Rabbit, 1:200, GeneTex#61122; Rabbit, 1:100, CST #8082), JAK2 (Mouse, 1:200, Sigma Aldrich # SAB4200483), mTOR (Rabbit, 1:1000, CST #2983), P-mTOR (Ser2448) (Rabbit, 1:2000, CST #5536), P-mTOR (Ser2481) (Rabbit, 1:2000, CST #2974), Raptor (Rabbit, 1:500, CST #2280), Rictor (Rabbit, 1:500, CST#2114), GbL (Rabbit, 1:1000, CST #3274), β-Actin (Goat, 1:3000, SCBT #sc1616), GAPDH (Goat, 1:3000, SCBT #sc20357), P(S1524)-BRCA1 (Rabbit, 1:500, CST#9009), P(S139)-histone H2A.X (Rabbit, 1:1000, CST #9718), histone H2A.X (Rabbit, 1:1000, CST #2595), Caspase-3(Rabbit, 1:1000, CST#9665), cleaved (Asp175)-Caspase-3 (Rabbit, 1:1000, CST #9664), P(Y416)-SFK (Rabbit, 1:200, CST #2101), P(Y416)-SRC (Rabbit, 1:200, CST #6943), SRC (Rabbit, 1:500, AbcaM #47405).

Techniques: Activation Assay, Migration

Journal: Viruses

Article Title: IFNα and β Mediated JCPyV Suppression through C/EBPβ-LIP Isoform

doi: 10.3390/v13101937

Figure Lengend Snippet: STAT1 e16 and e15 single guide RNAs sequence and primer designed for excision assay.

Article Snippet: The following antibodies were used to perform Western blots: mouse anti-α-tubulin (B7) antibody; mouse anti-GAPDH (6C5) antibody; rabbit anti-STAT1 (E-23) p84/p91 antibody and mouse anti-CEBPβ (H7,sc-7962) antibody were all ordered from(Santa Cruz Biotechnology INC., Santa cruz, CA, USA) and were each stored in 4 °C; anti-VP1 antibody (AB597) was kindly provided by W. Atwood (Brown University, Providence, RI, USA) and was stored in −20 °C; rabbit anti-phospho (Y701)-STAT1 (58D6) antibody and rabbit anti-lamin A/C antibody(clone 4C11) were all ordered from (Cell Signaling Technology, Danvers, MA, USA) and were stored at minus 20 °C; mouse anti-FLAG M2 antibody (Sigma-Aldrich St. Luis, MO, USA) was used to detect FLAG-tag SpCas9 in Western blot assay and it was stored at minus 20 °C.

Techniques: Sequencing, Cloning

Journal: Viruses

Article Title: IFNα and β Mediated JCPyV Suppression through C/EBPβ-LIP Isoform

doi: 10.3390/v13101937

Figure Lengend Snippet: Endogenous expression of c/EBPβ-LIP (LIP) after treatment with IFNα or IFNβ . Western blot on protein lysis from SVGA cells collected 4, 8, 12, 24, 36 and 48 h after a single treatment with IFNα ( A – C ) or IFNβ ( D – F ), respectively, in order to evaluate the endogenous expression of LIP ( A , D ). ⍺-tubulin was used as loading control and endogenous expression of STAT1 ( B , E ) and its phosphorylated form ( C , F ) (phosphoSTAT1 or pSTAT1) were also analyzed. For each panel, a densitometry assessment of LIP and STAT1 was also included, using untreated SVGA cells as negative control. At 36 and 24 h there was a significant increase in endogenous expression of LIP after treatment with IFNα ( p < 0.01) and IFNβ ( p < 0.0001), respectively, whereas an important increasing of STAT1 phosphorylation was observed at 24 and 36 h after treatment with IFNα and IFNβ, respectively. Standard deviation bars are depicted on the graph. p values < 0.05 were considered statistically significant. Note: * p < 0.05; ** p < 0.01; *** p < 0.0001.

Article Snippet: The following antibodies were used to perform Western blots: mouse anti-α-tubulin (B7) antibody; mouse anti-GAPDH (6C5) antibody; rabbit anti-STAT1 (E-23) p84/p91 antibody and mouse anti-CEBPβ (H7,sc-7962) antibody were all ordered from(Santa Cruz Biotechnology INC., Santa cruz, CA, USA) and were each stored in 4 °C; anti-VP1 antibody (AB597) was kindly provided by W. Atwood (Brown University, Providence, RI, USA) and was stored in −20 °C; rabbit anti-phospho (Y701)-STAT1 (58D6) antibody and rabbit anti-lamin A/C antibody(clone 4C11) were all ordered from (Cell Signaling Technology, Danvers, MA, USA) and were stored at minus 20 °C; mouse anti-FLAG M2 antibody (Sigma-Aldrich St. Luis, MO, USA) was used to detect FLAG-tag SpCas9 in Western blot assay and it was stored at minus 20 °C.

Techniques: Expressing, Western Blot, Lysis, Control, Negative Control, Phospho-proteomics, Standard Deviation

Journal: Viruses

Article Title: IFNα and β Mediated JCPyV Suppression through C/EBPβ-LIP Isoform

doi: 10.3390/v13101937

Figure Lengend Snippet: Effect of CRISPR/Cas9 editing of STAT1 on JCPyV replication. SVGA cell line stable for the protein SpCas9 by itself or in combination with two specific single-guide RNAs (gRNAs), STAT1e15 and STAT1e16, targeting the exon 15 and 16 of STAT1, respectively, were produced by the plasmid px333 (see ), infected with the Mad-1 strain of JCPyV at MOI 1 and treated with INF-α (50 ng/mL) every 24 h. The cells were harvested and analyzed at 7 days post-infection (p.i.). ( A ) Fifty micrograms of total cell extract were run on a 10% polyacrylamide SDS gel and analyzed by Western blot for VP1, SpCas9, STAT1 and LIP. Alpha tubulin (α-tubulin) and GAPDH were run as loading controls. ( B ) The STAT1 gene sequence, specifically targeted by the endonuclease SpCas9 in presence of the gRNAs STAT1e15 and STAT1e16, was amplified by PCR, electrophoresed on an agarose gel and visualized with ethidium bromide. ( C ) The JC viral load was also assessed by Q-PCR in the culture supernatant. ( D ) As the expression of the protein SpCas9 was confirmed by Western blot ( A ), the gRNAs expression was assessed by reverse transcription (see ). ( E ) Diagram of the STAT1 gene indicating the positions of PCR primers and gRNAs STAT1e15 and STAT1e16. The PCR primers amplified a sequence of 2109 bp. The endonuclease activity of SpCas9 in the presence of the two gRNAs determined an excision of 1309 bp, generating a new sequence of 800 bp. ( F ) Sequence analysis of PCR products for the STAT1 excision (800 bp) from SVGA cell line stable for the protein SpCas9 and the specific gRNAs STAT1e15 and STAT1e16.

Article Snippet: The following antibodies were used to perform Western blots: mouse anti-α-tubulin (B7) antibody; mouse anti-GAPDH (6C5) antibody; rabbit anti-STAT1 (E-23) p84/p91 antibody and mouse anti-CEBPβ (H7,sc-7962) antibody were all ordered from(Santa Cruz Biotechnology INC., Santa cruz, CA, USA) and were each stored in 4 °C; anti-VP1 antibody (AB597) was kindly provided by W. Atwood (Brown University, Providence, RI, USA) and was stored in −20 °C; rabbit anti-phospho (Y701)-STAT1 (58D6) antibody and rabbit anti-lamin A/C antibody(clone 4C11) were all ordered from (Cell Signaling Technology, Danvers, MA, USA) and were stored at minus 20 °C; mouse anti-FLAG M2 antibody (Sigma-Aldrich St. Luis, MO, USA) was used to detect FLAG-tag SpCas9 in Western blot assay and it was stored at minus 20 °C.

Techniques: CRISPR, Produced, Plasmid Preparation, Infection, SDS-Gel, Western Blot, Sequencing, Amplification, Agarose Gel Electrophoresis, Expressing, Reverse Transcription, Activity Assay

Journal: Viruses

Article Title: IFNα and β Mediated JCPyV Suppression through C/EBPβ-LIP Isoform

doi: 10.3390/v13101937

Figure Lengend Snippet: Schematic representation of a novel mechanism of JCPyV transcription and replication control by IFNα and IFNβ by producing a dominate negative isoform LIP of C/EBPβ. IFNα and β binding to IFN receptor (IFNR) activates the signaling pathway which leads to the formation of a phosphorylated STAT1 and STAT2 heterodimer complex to produce transcription factors which binds to IFN-stimulated response elements (ISREs) leading to the production of antiviral effectors ( 1. ). Through these signaling pathways IFNs control viral replication, thus, viral spread through the induction of an antiviral state in infected and uninfected cells . In addition to the JAK-STAT pathway, interferons have shown to exert their antiviral activity by modulating the function of some transcriptional factors such as C/EBPβ families [ , ] ( 2. ). In this study, the dominant negative isoform LIP of C/EBPβ has been shown to be stable in the nucleus after treatment with IFNα and β, and its interaction with the JCPyV NCCR inhibits JCPyV life cycle ( 3. and 4. ).

Article Snippet: The following antibodies were used to perform Western blots: mouse anti-α-tubulin (B7) antibody; mouse anti-GAPDH (6C5) antibody; rabbit anti-STAT1 (E-23) p84/p91 antibody and mouse anti-CEBPβ (H7,sc-7962) antibody were all ordered from(Santa Cruz Biotechnology INC., Santa cruz, CA, USA) and were each stored in 4 °C; anti-VP1 antibody (AB597) was kindly provided by W. Atwood (Brown University, Providence, RI, USA) and was stored in −20 °C; rabbit anti-phospho (Y701)-STAT1 (58D6) antibody and rabbit anti-lamin A/C antibody(clone 4C11) were all ordered from (Cell Signaling Technology, Danvers, MA, USA) and were stored at minus 20 °C; mouse anti-FLAG M2 antibody (Sigma-Aldrich St. Luis, MO, USA) was used to detect FLAG-tag SpCas9 in Western blot assay and it was stored at minus 20 °C.

Techniques: Control, Binding Assay, Protein-Protein interactions, Infection, Activity Assay, Dominant Negative Mutation

Journal: Pharmaceuticals (Basel, Switzerland)

Article Title: EGCG Inhibits Tumor Growth in Melanoma by Targeting JAK-STAT Signaling and Its Downstream PD-L1/PD-L2-PD1 Axis in Tumors and Enhancing Cytotoxic T-Cell Responses.

doi: 10.3390/ph14111081

Figure Lengend Snippet: Figure 2. EGCG inhibits IFN-γ-induced JAK-STAT signaling. (A,B) qRT-PCR analysis of STAT1 (A) and IRF1 (B) in 1205Lu, A375 and HS294T cells after treatment with 0.1% DMSO (control), 10 µM EGCG (EGCG), 10 ng/mL IFN-γ (IFN-γ) or a combination of IFN-γ and EGCG (IFN-γ + EGCG). GAPDH served as a control. (C) Immunoblot analysis of p-STAT1, STAT1 and IRF1 in 1205Lu, A375 and HS294T cells treated with 0.1% DMSO (control), 10 µM EGCG (EGCG), 10 ng/mL IFN-γ (IFN-γ), a combination of IFN-γ and EGCG (IFN-γ + EGCG), 10 µM ruxolitinib (Ruxo) or a combination of IFN-γ and ruxolitinib (IFN-γ + Ruxo). GAPDH served as a control. The band densities of proteins were quantified with image J and normalized to GAPDH. p-STAT1 to STAT1 ratio was calculated and normalized to control. Data are representative of 2 independent experiments and expressed as the mean ± S.D., n = 3 ns, p > 0.05; * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Samples for the same experiment were run simultaneously and probed for multiple proteins. p-STAT1 (Y701) (Cat: 7649) (1:1000), STAT-1 (Cat: 14994) (1:1000) and IRF1 (Cat: 84785) (1:1000) antibodies were purchased from Cell Signaling (Danvers, MA, USA).

Techniques: Quantitative RT-PCR, Control, Western Blot

Journal: Pharmaceuticals (Basel, Switzerland)

Article Title: EGCG Inhibits Tumor Growth in Melanoma by Targeting JAK-STAT Signaling and Its Downstream PD-L1/PD-L2-PD1 Axis in Tumors and Enhancing Cytotoxic T-Cell Responses.

doi: 10.3390/ph14111081

Figure Lengend Snippet: Figure 3. EGCG inhibited mouse melanoma growth in vivo. (A) Flow cytometry data depicting the cell surface expression of PD-L1 in B16F10 cells treated with 0.1% DMSO (control), 10 µM EGCG (EGCG), 10 ng/mL IFN-γ (IFN-γ) or a combination of IFN-γ and EGCG (IFN-γ + EGCG). Histogram (left panel) and quantification of mean fluorescent intensity (MFI) (right panel). (B) qRT-PCR analysis of PD-L1 after treatment with 0.1% DMSO (control), 10 µM EGCG (EGCG), 10 ng/mL IFN-γ (IFN-γ) or a combination of IFN-γ and EGCG (IFN-γ + EGCG) in B16F10 cells. GAPDH served as a control. (C) Immunoblot analysis of p-STAT1, STAT1 and IRF1 in B16F10 cells treated with 0.1% DMSO (control), 10 µM EGCG (EGCG), 10 ng/mL IFN-γ (IFN-γ) or a combination of IFN-γ and EGCG (IFN-γ + EGCG). GAPDH served as a control. The band densities of proteins were quantified with image J and normalized to GAPDH. p-STAT1 to STAT1 ratio was calculated and normalized to control. (D) Tumor growth curve of B16F10 cells injected subcutaneously in C57BL/6 mice. Mice were treated from day 7 with saline daily intraperitoneally (control), 50 mg/kg EGCG daily intraperitoneally (EGCG), or 12.5 mg/kg anti-PD-1 antibody intraperitoneally (anti-PD-1) every 3 days. (E) Ki-67 staining of B16F10 tumors (derived from D) treated with saline (control), 50 mg/kg EGCG (EGCG), or 12.5 mg/kg anti-PD-1 antibody (anti-PD-1). Representative images (left panel) and quantification of positive cells (right panel). Ki-67-positive cells were counted in the whole field under a microscope and presented as % positive tumor cells. Bar = 50 µm. Data are representative of 2 independent experiments and expressed as the mean ± S.D., n = 3 (A,B), 10 (D), or 3 (E). * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Samples for the same experiment were run simultaneously and probed for multiple proteins. p-STAT1 (Y701) (Cat: 7649) (1:1000), STAT-1 (Cat: 14994) (1:1000) and IRF1 (Cat: 84785) (1:1000) antibodies were purchased from Cell Signaling (Danvers, MA, USA).

Techniques: In Vivo, Flow Cytometry, Expressing, Control, Quantitative RT-PCR, Western Blot, Injection, Saline, Staining, Derivative Assay, Microscopy

Journal: Pharmaceuticals (Basel, Switzerland)

Article Title: EGCG Inhibits Tumor Growth in Melanoma by Targeting JAK-STAT Signaling and Its Downstream PD-L1/PD-L2-PD1 Axis in Tumors and Enhancing Cytotoxic T-Cell Responses.

doi: 10.3390/ph14111081

Figure Lengend Snippet: Figure 4. EGCG inhibited JAK-STAT signaling and increased granzyme B expression in CD8+ cells in the B16F10 tumor microenvironment. (A,B) qRT-PCR analysis of STAT1 (A) and IRF1 (B) in B16F10 tumors derived from Figure 3D, after the treatment with saline (control), 50 mg/kg EGCG (EGCG) or 12.5 mg/kg anti-PD-1 antibody (anti-PD-1) (n = 3). GAPDH served

Article Snippet: Samples for the same experiment were run simultaneously and probed for multiple proteins. p-STAT1 (Y701) (Cat: 7649) (1:1000), STAT-1 (Cat: 14994) (1:1000) and IRF1 (Cat: 84785) (1:1000) antibodies were purchased from Cell Signaling (Danvers, MA, USA).

Techniques: Expressing, Quantitative RT-PCR, Derivative Assay, Saline, Control

Journal: American Journal of Respiratory Cell and Molecular Biology

Article Title: Respiratory Syncytial Virus Impairs Macrophage IFN-?/?- and IFN-?-Stimulated Transcription by Distinct Mechanisms

doi: 10.1165/rcmb.2008-0229OC

Figure Lengend Snippet: RSV impairs IFN-mediated transcriptional activation in primary mouse alveolar macrophages. Western immunoblot analysis was performed to determine whether RSV infection (MOI = 1; 24 h) inhibited phosphorylation of STAT1 at Y701 in (A) IFN-β– (100 U/ml) or (C) IFN-γ–stimulated (10 ng/ml, 30-min treatment) primary mouse alveolar macrophages. RSV infection did not inhibit IFN-γ–stimulated STAT1 (Y701) phosphorylation; however, STAT1 phosphorylation in response to IFN-β was significantly impaired in RSV-infected alveolar macrophages. RSV infection increased the level of STAT1. The levels of β-actin were similar in all samples. Multiplex real-time quantitative PCR analysis was performed to determine the mRNA levels of IFN-responsive genes compared with the expression of GAPDH on mRNA harvested from alveolar macrophages stimulated with either IFN-β (100 U/ml) or IFN-γ (10 ng/ml) for 6 hours. RSV infection significantly inhibited the IFN-β–induced expression of Nod1 (B), and the IFN-γ–induced expression of C2ta (D). The data represent three independent experiments run in triplicate, and are presented as means (±SEM); *P < 0.05 compared with IFN-β– or IFN-γ–stimulated samples in B and D.

Article Snippet: The membrane was blocked with 5% powdered milk in Tris-buffered saline (TBS) with 0.1% Tween 20 (TBST) and incubated overnight at 4°C with: rabbit anti-mouse STAT1 (Cell Signaling, Danvers, MA) diluted 1:1,000; rabbit anti-mouse STAT1α (Cell Signaling) diluted 1:1,000; rabbit anti-mouse STAT2 (Cell Signaling) diluted 1:1,000; rabbit anti-human Tyk2 (Abcam, Cambridge, MA) diluted 1:5,000; rabbit anti-mouse phospho-specific STAT1 (Y701) (Cell Signaling) diluted 1:1,000; rabbit anti-mouse phospho-specific STAT1 (S727) (Cell Signaling) diluted 1:1,000; rabbit anti-mouse phospho-specific STAT2 (Cell Signaling) diluted 1:1,000; rabbit anti-mouse phospho-specific Tyk2 (Cell Signaling) diluted 1:1,000; rabbit anti-human CBP antibody (R&D Systems, Minneapolis, MN) diluted 1:3,000; goat anti-mouse lamin B1 (Santa Cruz Biotechnology, Santa Cruz, CA) diluted 1:1,000; rabbit anti-mouse inducible nitric oxide synthase (iNOS) (Upstate, Lake Placid, NY) diluted 1:1,000; or mouse anti-human β-actin (Sigma, St. Louis, MO) diluted 1:20,000 in TBST containing 5% powdered milk.

Techniques: Activation Assay, Western Blot, Infection, Phospho-proteomics, Multiplex Assay, Real-time Polymerase Chain Reaction, Expressing

Journal: American Journal of Respiratory Cell and Molecular Biology

Article Title: Respiratory Syncytial Virus Impairs Macrophage IFN-?/?- and IFN-?-Stimulated Transcription by Distinct Mechanisms

doi: 10.1165/rcmb.2008-0229OC

Figure Lengend Snippet: RSV impairs IFN-α/β– but not IFN-γ–mediated signal transducer and activator of transcription (STAT)-1 phosphorylation. Western immunoblot analysis was performed to determine whether RSV infection (MOI = 1; 24 h) inhibited phosphorylation of STAT1 at Y701 in (A) IFN-α– (100 U/ml), IFN-β– (100 U/ml), or (B) IFN-γ–stimulated (10 ng/ml, 30-min treatment) RAW macrophages. RSV infection did not inhibit IFN-γ–stimulated STAT1 (Y701) phosphorylation; however, STAT1 phosphorylation in response to either IFN-α or IFN-β was significantly impaired in RSV-infected macrophages. RSV infection increased the level of STAT1. The levels of β-actin were similar in all samples. Each lane of the immunoblot was loaded with 20 μg of protein, and the immunoblots shown are representative of three independent experiments.

Article Snippet: The membrane was blocked with 5% powdered milk in Tris-buffered saline (TBS) with 0.1% Tween 20 (TBST) and incubated overnight at 4°C with: rabbit anti-mouse STAT1 (Cell Signaling, Danvers, MA) diluted 1:1,000; rabbit anti-mouse STAT1α (Cell Signaling) diluted 1:1,000; rabbit anti-mouse STAT2 (Cell Signaling) diluted 1:1,000; rabbit anti-human Tyk2 (Abcam, Cambridge, MA) diluted 1:5,000; rabbit anti-mouse phospho-specific STAT1 (Y701) (Cell Signaling) diluted 1:1,000; rabbit anti-mouse phospho-specific STAT1 (S727) (Cell Signaling) diluted 1:1,000; rabbit anti-mouse phospho-specific STAT2 (Cell Signaling) diluted 1:1,000; rabbit anti-mouse phospho-specific Tyk2 (Cell Signaling) diluted 1:1,000; rabbit anti-human CBP antibody (R&D Systems, Minneapolis, MN) diluted 1:3,000; goat anti-mouse lamin B1 (Santa Cruz Biotechnology, Santa Cruz, CA) diluted 1:1,000; rabbit anti-mouse inducible nitric oxide synthase (iNOS) (Upstate, Lake Placid, NY) diluted 1:1,000; or mouse anti-human β-actin (Sigma, St. Louis, MO) diluted 1:20,000 in TBST containing 5% powdered milk.

Techniques: Phospho-proteomics, Western Blot, Infection

Journal: American Journal of Respiratory Cell and Molecular Biology

Article Title: Respiratory Syncytial Virus Impairs Macrophage IFN-?/?- and IFN-?-Stimulated Transcription by Distinct Mechanisms

doi: 10.1165/rcmb.2008-0229OC

Figure Lengend Snippet: RSV infection impairs nuclear pSTAT1-CBP protein–protein interactions. (A) Western immunoblot analysis of cytoplasmic and nuclear fractions for pSTAT1 and STAT1 was performed to determine if RSV infection (MOI = 1; 24 h) inhibited the nuclear translocation of pSTAT1 after IFN-γ treatment (10 ng/ml, 30 min). Nuclear and cytoplasmic fraction enrichment was assessed by Western blot analysis for lamin B and iNOS, respectively. Because interaction of pSTAT1 with CBP/p300 is required for transactivation of the IFN-γ response, the level of CBP/p300-STAT1 interaction in RSV-infected RAW macrophages was assessed. (B) Nuclear extracts from IFN-γ–stimulated (10 ng/ml, 30 min) mock- or RSV-infected (MOI = 1; 24 h) RAW macrophages were subjected to immunoprecipitation (IP) for STAT1 and then Western immunoblot analysis for CBP to determine whether CBP–STAT1 interactions were disrupted in RSV-infected RAW macrophages. (C) Western blot analysis for CBP was performed on nuclear lysates to determine whether diminution of nuclear STAT1–CBP interactions was due to an RSV-induced reduction in nuclear CBP levels. The immunoblots shown are representative of three independent experiments.

Article Snippet: The membrane was blocked with 5% powdered milk in Tris-buffered saline (TBS) with 0.1% Tween 20 (TBST) and incubated overnight at 4°C with: rabbit anti-mouse STAT1 (Cell Signaling, Danvers, MA) diluted 1:1,000; rabbit anti-mouse STAT1α (Cell Signaling) diluted 1:1,000; rabbit anti-mouse STAT2 (Cell Signaling) diluted 1:1,000; rabbit anti-human Tyk2 (Abcam, Cambridge, MA) diluted 1:5,000; rabbit anti-mouse phospho-specific STAT1 (Y701) (Cell Signaling) diluted 1:1,000; rabbit anti-mouse phospho-specific STAT1 (S727) (Cell Signaling) diluted 1:1,000; rabbit anti-mouse phospho-specific STAT2 (Cell Signaling) diluted 1:1,000; rabbit anti-mouse phospho-specific Tyk2 (Cell Signaling) diluted 1:1,000; rabbit anti-human CBP antibody (R&D Systems, Minneapolis, MN) diluted 1:3,000; goat anti-mouse lamin B1 (Santa Cruz Biotechnology, Santa Cruz, CA) diluted 1:1,000; rabbit anti-mouse inducible nitric oxide synthase (iNOS) (Upstate, Lake Placid, NY) diluted 1:1,000; or mouse anti-human β-actin (Sigma, St. Louis, MO) diluted 1:20,000 in TBST containing 5% powdered milk.

Techniques: Infection, Protein-Protein interactions, Western Blot, Translocation Assay, Immunoprecipitation

Journal: American Journal of Respiratory Cell and Molecular Biology

Article Title: Respiratory Syncytial Virus Impairs Macrophage IFN-?/?- and IFN-?-Stimulated Transcription by Distinct Mechanisms

doi: 10.1165/rcmb.2008-0229OC

Figure Lengend Snippet: RSV infection does not inhibit IFN-γ–mediated STAT1 phosphorylation at serine 727 (S727), but does increase IFN-γ–stimulated pSTAT1β. (A) Western immunoblot analysis was performed to determine whether RSV infection (MOI = 1; 24 h) inhibited phosphorylation of STAT1 at S727 in response to IFN-γ (10 ng/ml, 30-min treatment). (B) RAW macrophages infected with RSV were stimulated with IFN-γ (10 ng/ml, 30 min) and cell lysates were subjected to Western immunoblot analysis to determine the levels of pSTAT1α (91 kD) and pSTAT1β (84 kD). (C) Densitometry was used to quantify the levels of pSTAT1β and the ratio of pSTAT1α to pSTAT1β detected by Western blot analysis. Each lane of the immunoblot was loaded with 20 μg of protein, and the immunoblots shown are representative of three independent experiments. (D) RAW macrophages were transfected with 2 μg of the STAT1β-FLAG expression vector or empty vector. After overnight incubation, cells were stimulated with IFN-γ (10 ng/ml) for 6 hours, and mRNA was harvested for quantitative real-time PCR analysis. Multiplex real-time quantitative PCR analysis was performed to determine the mRNA levels of C2ta compared with those of GAPDH. RAW macrophages transfected with STAT1β-FLAG expression vector or empty vector were stimulated with IFN-γ (10 ng/ml, 30 min), and cell lysates were subjected to Western immunoblot analysis to determine the levels of pSTAT1α (91 kD) and pSTAT1β (84 kD) (inset). The data represent three independent experiments run in triplicate. Data are presented as means (±SEM); *P < 0.05 compared with RAW macrophages transfected with empty vector.

Article Snippet: The membrane was blocked with 5% powdered milk in Tris-buffered saline (TBS) with 0.1% Tween 20 (TBST) and incubated overnight at 4°C with: rabbit anti-mouse STAT1 (Cell Signaling, Danvers, MA) diluted 1:1,000; rabbit anti-mouse STAT1α (Cell Signaling) diluted 1:1,000; rabbit anti-mouse STAT2 (Cell Signaling) diluted 1:1,000; rabbit anti-human Tyk2 (Abcam, Cambridge, MA) diluted 1:5,000; rabbit anti-mouse phospho-specific STAT1 (Y701) (Cell Signaling) diluted 1:1,000; rabbit anti-mouse phospho-specific STAT1 (S727) (Cell Signaling) diluted 1:1,000; rabbit anti-mouse phospho-specific STAT2 (Cell Signaling) diluted 1:1,000; rabbit anti-mouse phospho-specific Tyk2 (Cell Signaling) diluted 1:1,000; rabbit anti-human CBP antibody (R&D Systems, Minneapolis, MN) diluted 1:3,000; goat anti-mouse lamin B1 (Santa Cruz Biotechnology, Santa Cruz, CA) diluted 1:1,000; rabbit anti-mouse inducible nitric oxide synthase (iNOS) (Upstate, Lake Placid, NY) diluted 1:1,000; or mouse anti-human β-actin (Sigma, St. Louis, MO) diluted 1:20,000 in TBST containing 5% powdered milk.

Techniques: Infection, Phospho-proteomics, Western Blot, Transfection, Expressing, Plasmid Preparation, Incubation, Real-time Polymerase Chain Reaction, Multiplex Assay