Journal: Molecular Therapy Oncolytics

Article Title: Acquired chemoresistance can lead to increased resistance of pancreatic cancer cells to oncolytic vesicular stomatitis virus

doi: 10.1016/j.omto.2021.11.019

Figure Lengend Snippet: VSV and antiviral protein expression (A and B) C and GR cells were either mock treated or infected with VSV-ΔM51 at MOIs of 1.0, 0.1, and 0.01. Protein sample were analyzed at 24 h p.i. (A) and 48 h p.i. (B) by western blotting for expression of phospho-STAT1 (p-STAT1), phospho-STAT2 (p-STAT2), STAT1, STAT2, and VSV proteins (G, N/P, and M). Cell line and treatment conditions are indicated above blots. Equal protein loading is indicated by Coomassie blue. (C and D) The effect of ruxolitinib on VSV-ΔM51 replication kinetics based on GFP fluorescence. C and GR cells were either mock treated, treated only with ruxolitinib, treated only with VSV-ΔM51, or treated with both VSV-ΔM51 and ruxolitinib. Cells were infected at MOI of 0.01. Ruxolitinib was added to cells after 1 h VSV incubation period. GFP fluorescence was measured from 1 to 80 h p.i. The data points and error bars shown represent the means and SEM of the means, respectively. Control and GR cell lines 1–3 are combined for each treatment.

Article Snippet: Membranes were then incubated in TBS-T with 5% BSA or milk with 0.02% sodium azide and a 1:5,000 dilution of rabbit polyclonal anti-VSV antibodies (raised against VSV virions), a 1:1,000 dilution of rabbit anti-phospho-STAT1 (catalog number 9177S, clone p-S727, Cell Signaling), a 1:1,000 dilution of rabbit anti-STAT1 (catalog number 14994T, clone D1K9Y, Cell Signaling), a 1:1,000 dilution of rabbit anti-phospho-STAT2 (catalog number 600-401-A93S, clone p-Y689, Rockland), a 1:1,000 dilution of rabbit anti-STAT2 (catalog number 4594, Cell Signaling), a 1:1,000 dilution of rabbit anti-phospho-STAT3 (catalog number 9134P, clone Y705, Cell Signaling), a 1:1,000 dilution of mouse anti-STAT3 (catalog number 9139P, clone 124H6, Cell Signaling), a 1:1,000 dilution of rabbit anti-MX1 (catalog number 13750-1-AP, Proteintech), a 1:1,000 dilution of rabbit anti-MX2 (catalog number 43924S, clone E7Y8H, Cell Signaling), a 1:1,000 dilution of rabbit anti-IFI16 (catalog number 14970S, clone D8B5T, Cell Signaling), a 1:1,000 dilution of rabbit anti-APOBEC3B (catalog number 41494S, clone E9A2G, Cell Signaling), a 1:1,000 dilution of rabbit anti-ISG15 (catalog number 2758S, clone 22D2, Cell Signaling), a 1:1,000 dilution of rabbit anti-CDK14-PFTK1 (catalog number 21612-1-AP, Proteintech), a 1:1,000 dilution of rabbit anti-LARGE2/GYLTL1B (catalog number PA5-63331, Invitrogen), a 1:1,000 dilution of rabbit anti-STING (catalog number 13647S, clone D2P2F, Cell Signaling), a 1:1,000 dilution of rabbit anti-phsopho-TBK1/NAK (catalog number 5483P, clone S172, Cell Signaling), a 1:1,000 dilution of rabbit anti-cGAS (catalog number 79978, clone E5V3W, Cell Signaling), or a 1:1,000 dilution of rabbit anti-cyclin B1 (catalog number 12231T, clone D5C10, Cell Signaling).

Techniques: Expressing, Infection, Western Blot, Fluorescence, Incubation

Journal: Stem Cells International

Article Title: Long Noncoding RNA H19 Participates in the Regulation of Adipose-Derived Stem Cells Cartilage Differentiation

doi: 10.1155/2019/2139814

Figure Lengend Snippet: Validation of relative expression levels of representative DEGs in the osteoclast differentiation pathway. (a) Representative 9 miRNAs detected by RNA-Seq were confirmed using qRT-PCR. (b) The mRNA levels of STAT2 and IRF9 detected by RNA-Seq were confirmed using qRT-PCR. (c) Protein levels of STAT2 were confirmed using western blot. (d) Protein levels of IRF9 were confirmed using western blot ( ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001).

Article Snippet: The members were then blocked in 5% fat-free milk for 1 h at room temperature and incubated overnight at 4°C with rabbit anti-collagen II (1 : 1000; ab188570; Abcam), rabbit anti-Aggrecan (1 : 6000; 13880-1-AP; ProteinTech Group, Inc.), rabbit anti-Stat2 polyclonal antibody (1 : 400; GTX59884; GeneTex, Irvine, CA, USA) or rabbit anti-IRF9 polyclonal antibody (1 : 1000; PA5-40357, Thermo Fisher Scientific, Inc. Rockford, IL, USA), or β -actin (1 : 10,000; 051M4892; Sigma-Aldrich, Merck KGaA, Darmstadt, Germany).

Techniques: Biomarker Discovery, Expressing, RNA Sequencing, Quantitative RT-PCR, Western Blot

Journal: Stem Cells International

Article Title: Long Noncoding RNA H19 Participates in the Regulation of Adipose-Derived Stem Cells Cartilage Differentiation

doi: 10.1155/2019/2139814

Figure Lengend Snippet: H19-miRNA-STAT2/IRF9 interactive network in ADSC cartilage differentiation.

Article Snippet: The members were then blocked in 5% fat-free milk for 1 h at room temperature and incubated overnight at 4°C with rabbit anti-collagen II (1 : 1000; ab188570; Abcam), rabbit anti-Aggrecan (1 : 6000; 13880-1-AP; ProteinTech Group, Inc.), rabbit anti-Stat2 polyclonal antibody (1 : 400; GTX59884; GeneTex, Irvine, CA, USA) or rabbit anti-IRF9 polyclonal antibody (1 : 1000; PA5-40357, Thermo Fisher Scientific, Inc. Rockford, IL, USA), or β -actin (1 : 10,000; 051M4892; Sigma-Aldrich, Merck KGaA, Darmstadt, Germany).

Techniques:

Journal: Frontiers in Pharmacology

Article Title: Downregulation of nuclear STAT2 protein in the spinal dorsal horn is involved in neuropathic pain following chronic constriction injury of the rat sciatic nerve

doi: 10.3389/fphar.2023.1069331

Figure Lengend Snippet: The central proteins in the PPI network screened using CentiScaPe analysis.

Article Snippet: The membranes were blocked in NcmBlot blocking buffer (NCM Biotech) for 15 min and subsequently incubated with primary antibodies as follows (4°C, overnight): rabbit anti-STAT2 (1:500, HuaBio), mouse anti-GAPDH (1:5000, ZENBIO), and mouse anti-H3 (1:1000, Cell Signaling Technology).

Techniques:

Journal: Frontiers in Pharmacology

Article Title: Downregulation of nuclear STAT2 protein in the spinal dorsal horn is involved in neuropathic pain following chronic constriction injury of the rat sciatic nerve

doi: 10.3389/fphar.2023.1069331

Figure Lengend Snippet: Chronic constriction injury (CCI) downregulated STAT2 in spinal dorsal horn (SDH) nuclei. (A) The mRNA expression of Stat2 in the SDH was not altered after CCI surgery ( n = 8, two-tailed unpaired Student’s t-test). (B) STAT2 protein expression in the SDH of the CCI group was not different from that in the sham group (protein expression was normalized to the GAPDH level) ( n = 6, two-tailed unpaired Student’s t-test). (C) STAT2 protein expression in the SDH of the CCI group was decreased in the nuclear fraction (protein expression was normalized to the H3 level) but not different from in the cytoplasmic fraction (protein expression was normalized to the GAPDH level) compared to the sham group ( n = 6, ** p < .01, two-tailed unpaired Student’s t-test). (D) Immunofluorescence staining of STAT2 (green) and DAPI (blue) in the injured ipsilateral SDH on day 7 after sham or CCI surgery (scale bar = 50 µm). (E) The proportion of STAT2 co-localized with DAPI (nuclear marker) was decreased in the ipsilateral SDH after CCI surgery (compared with sham-operated rats, ** p < .01, n = 3, two-tailed unpaired Student’s t-test).

Article Snippet: The membranes were blocked in NcmBlot blocking buffer (NCM Biotech) for 15 min and subsequently incubated with primary antibodies as follows (4°C, overnight): rabbit anti-STAT2 (1:500, HuaBio), mouse anti-GAPDH (1:5000, ZENBIO), and mouse anti-H3 (1:1000, Cell Signaling Technology).

Techniques: Expressing, Two Tailed Test, Immunofluorescence, Staining, Marker

Journal: Frontiers in Pharmacology

Article Title: Downregulation of nuclear STAT2 protein in the spinal dorsal horn is involved in neuropathic pain following chronic constriction injury of the rat sciatic nerve

doi: 10.3389/fphar.2023.1069331

Figure Lengend Snippet: Downregulation of STAT2 induced nociceptive hypersensitivity. (A) The specific location of the intraspinal injection of si-STAT2. (B) The time points of si-STAT2 administration in naïve rats. (C,D) Nuclear expression of STAT2 was reduced in the spinal dorsal horn (SDH) after si-STAT2 injection compared with those in rats treated with normal saline (NS) or the corresponding negative control (si-NC) ( n = 4, ** p < .01, * p < .05, one-way ANOVA followed by Bonferroni’s multiple comparisons test). (E,F) Injection of si-STAT2 decreased the paw withdrawal mechanical threshold (PWMT) (E) and paw withdrawal thermal latency (PWTL) (F) in naïve rats from days 3–5 (si-STAT2 vs. si-NC, n = 8, ** p < .01, *** p < .001, two-way ANOVA with repeated measures followed by Bonferroni’s multiple comparisons test).

Article Snippet: The membranes were blocked in NcmBlot blocking buffer (NCM Biotech) for 15 min and subsequently incubated with primary antibodies as follows (4°C, overnight): rabbit anti-STAT2 (1:500, HuaBio), mouse anti-GAPDH (1:5000, ZENBIO), and mouse anti-H3 (1:1000, Cell Signaling Technology).

Techniques: Injection, Expressing, Negative Control

Journal: Frontiers in Pharmacology

Article Title: Downregulation of nuclear STAT2 protein in the spinal dorsal horn is involved in neuropathic pain following chronic constriction injury of the rat sciatic nerve

doi: 10.3389/fphar.2023.1069331

Figure Lengend Snippet: Downregulation of STAT2 increased expression of microglial activation markers. (A) Representative images showing immunoreactivity for STAT2 (green) double labeled with NeuN-labeled neurons (red), IBA1-labeled microglia (red) or GFAP-labeled astrocytes (red) (scale bar = 50 µm). (B) The percentage of marker-positive (NeuN, IBA1, and GFAP) (green) cells relative to STAT2-positive (red) cells ( n = 3). (C) An RNA sequencing analysis heat map shows that major histocompatibility complex class II-associated genes were significantly upregulated in the SDH of chronic constriction injury (CCI) rats compared with those in the sham group. (D,E) Expression of RT1-Ba and Ciita mRNA was increased after CCI surgery and si-STAT2 injection ( n = 6/5 rats/group, ** p < .01, one-way ANOVA followed by Bonferroni’s multiple comparisons test).

Article Snippet: The membranes were blocked in NcmBlot blocking buffer (NCM Biotech) for 15 min and subsequently incubated with primary antibodies as follows (4°C, overnight): rabbit anti-STAT2 (1:500, HuaBio), mouse anti-GAPDH (1:5000, ZENBIO), and mouse anti-H3 (1:1000, Cell Signaling Technology).

Techniques: Expressing, Activation Assay, Labeling, Marker, RNA Sequencing Assay, Injection

Journal: Frontiers in Pharmacology

Article Title: Downregulation of nuclear STAT2 protein in the spinal dorsal horn is involved in neuropathic pain following chronic constriction injury of the rat sciatic nerve

doi: 10.3389/fphar.2023.1069331

Figure Lengend Snippet: Downregulation of STAT2 promoted microglial activation and Tnf and Il1b expression. (A) Negative control (si-NC) rats had resting microglia with a ramified morphology, whereas si-STAT2 rats had activated microglia with an amoeboid morphology. ( n = 3, scale bar = 100 µm). (B) si-STAT2 treatment increased the number of microglia in the spinal dorsal horn (SDH) ( n = 3, * p < .05, Mann-Whitney test). (C,D) Expression of Il1b and Tnf mRNA was significantly increased in the SDH after intraspinal injection of si-STAT2 ( n = 6, ** p < .01, * p < .05, two-tailed unpaired Student’s t-test).

Article Snippet: The membranes were blocked in NcmBlot blocking buffer (NCM Biotech) for 15 min and subsequently incubated with primary antibodies as follows (4°C, overnight): rabbit anti-STAT2 (1:500, HuaBio), mouse anti-GAPDH (1:5000, ZENBIO), and mouse anti-H3 (1:1000, Cell Signaling Technology).

Techniques: Activation Assay, Expressing, Negative Control, MANN-WHITNEY, Injection, Two Tailed Test

Journal: Frontiers in Pharmacology

Article Title: Downregulation of nuclear STAT2 protein in the spinal dorsal horn is involved in neuropathic pain following chronic constriction injury of the rat sciatic nerve

doi: 10.3389/fphar.2023.1069331

Figure Lengend Snippet: STAT2 in the nuclei negatively regulates pro-inflammatory gene expression in microglia. (A) Representative images of HAPI microglia showed that lipopolysaccharides (LPS) activated microglia and minocycline (MN) significantly inhibited microglia activation (Activated microglia had enlarged cytosomes, shortened protrusions, and amoeba-like cell morphology, scale bar = 50 µm). (B) Nuclear expression of STAT2 was reduced in activated HAPI microglia but it returned to basal level after the treatment of MN (HAPI vs. HAPI + LPS, ** p < .01; HAPI + LPS vs. HAPI + LPS + MN, ## p < .01, n = 3, one-way ANOVA followed by Bonferroni’s multiple comparisons test). (C,D) Expression of Il1b and Tnf mRNA was significantly increased in HAPI microglia after transfection of si-STAT2 ( n = 3, * p < .05, ** p < .01, one-way ANOVA followed by Bonferroni’s multiple comparisons test). (E) Nuclear expression of STAT2 was restored after treatment of IFNα (HAPI vs. HAPI + LPS, ** p < .01; HAPI + LPS vs. HAPI + LPS + IFNα, ## p < .01; n = 3, one-way ANOVA followed by Bonferroni’s multiple comparisons test). (F,G) Treatment of IFNα reversed the LPS-induced upregulation of Il1b and Tnf mRNA expression in HAPI microglia (HAPI vs. HAPI + LPS, ** p < .01, *** p < .001; HAPI + LPS vs. HAPI + LPS + IFNα, # p < .05, ### p < .001, n = 3, one-way ANOVA followed by Bonferroni’s multiple comparisons test).

Article Snippet: The membranes were blocked in NcmBlot blocking buffer (NCM Biotech) for 15 min and subsequently incubated with primary antibodies as follows (4°C, overnight): rabbit anti-STAT2 (1:500, HuaBio), mouse anti-GAPDH (1:5000, ZENBIO), and mouse anti-H3 (1:1000, Cell Signaling Technology).

Techniques: Expressing, Activation Assay, Transfection