Journal: Scientific Reports

Article Title: Comparison of anti-inflammatory and anti-angiogenic effects of JAK inhibitors in IL-6 and TNFα-stimulated fibroblast-like synoviocytes derived from patients with RA

doi: 10.1038/s41598-025-94894-2

Figure Lengend Snippet: ( a ) Western blotting was performed to analyze STAT1, pSTAT1, STAT3, pSTAT3, and β-actin. ( b , c ) Comparison of the ratios of pSTAT1 to STAT1 and pSTAT3 to STAT1. Protein expression was determined by semi-quantitative analysis of digitally captured images. All JAK inhibitors significantly suppressed pSTAT1 and pSTAT3 levels compared to those in the control. Compared to TOF, BAR, PEF, UPA, and FIL suppressed pSTAT1 and pSTAT3 levels. STAT, signal transducer and activator of transcription; JAK, Janus kinase; TOF, tofacitinib; BAR, baricitinib; PEF, peficitinib; UPA, upadacitinib; FIL, filgotinib.

Article Snippet: The membrane was blocked with 5% skimmed milk in TBST at 25 °C for 30 min, incubated with antibodies against anti-STAT1, anti-phosphorylated STAT1 (pSTAT1), anti-STAT3, and anti-pSTAT3 (Cell Signaling Technology, Danvers, MA, US) at 4 °C for 12 h, and further incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody at 25 °C for 1 h. The proteins were subsequently visualized using ECL Plus reagent (GE Healthcare Life Sciences, Little Chalfont, UK) on a chemiluminescence analyzer (LAS-3000 mini; Fujifilm, Tokyo, Japan).

Techniques: Western Blot, Comparison, Expressing, Control

Journal: PLOS Pathogens

Article Title: Human cytomegalovirus UL138 interaction with USP1 activates STAT1 in infection

doi: 10.1371/journal.ppat.1011185

Figure Lengend Snippet: Summary of UL138 Interactors ^a .

Article Snippet: pSTAT1 (Y701) , Rabbit , Cell Signaling , WB 1:1000 IF 1:50.

Techniques: Activity Assay, Inhibition, De-Phosphorylation Assay

Journal: PLOS Pathogens

Article Title: Human cytomegalovirus UL138 interaction with USP1 activates STAT1 in infection

doi: 10.1371/journal.ppat.1011185

Figure Lengend Snippet: (A) Fibroblasts were infected (MOI = 1) with a WT or Δ UL138 STOP virus for 24–72 hpi. At each time point, lysates were lysed and immunoblotted. pSTAT1, STAT1, IE1/2, and Tubulin were detected using antibodies. IE proteins serve as a control for infection and tubulin serves as a control for loading. (B) pSTAT1 and STAT1 were normalized to WT 24 hpi and graphed to calculate statistics. Statistical significance was calculated by Two-Way Anova and represented by asterisks; **p-value <0.01, ***p-value <0.001. Graphs represent the mean of three replicates and error bars represent SEM.

Article Snippet: pSTAT1 (Y701) , Rabbit , Cell Signaling , WB 1:1000 IF 1:50.

Techniques: Infection, Virus, Control

Journal: PLOS Pathogens

Article Title: Human cytomegalovirus UL138 interaction with USP1 activates STAT1 in infection

doi: 10.1371/journal.ppat.1011185

Figure Lengend Snippet: (A) Fibroblasts were treated with either DMSO (vehicle control) or 0.88 μ M C527 24 hours prior to being infected (MOI = 1) with a WT or Δ UL138 STOP virus. Lysates were collected and each timepoint and immunoblotted. pSTAT1, STAT1, IE1/2, and Tubulin were detected using antibodies. (B) pSTAT1 was normalized to WT DMSO 24 hpi and graphed for statistical analysis. Statistical significance was calculated by Two-Way Anova and represented by asterisks; *p-value <0.05, **p-value <0.01, ***p-value <0.001. (C) Fibroblasts were reverse transfected with 3 combined siRNA for a non-targeting control (NTC) or USP1. 24 hours post reverse transfection, the media was changed. 48 hours post reverse transfection, fibroblasts were infected (MOI = 1) with either a WT or Δ UL138 STOP virus and lysates were immunoblotted. USP1, pSTAT1, STAT1, IE1/2, and Tubulin were detected using antibodies. (D). USP1 levels in the NTC and USP1 siRNA knockdown were normalized to NTC and graphed to calculate statistics. Statistical significance for USP1 was calculated using an unpaired t test and represented by asterisks; ****p <0.0001. (E) pSTAT1 was normalized to WT NTC 24 hpi and graphed to calculate statistics. Statistical significance was calculated by Two-Way Anova and represented by asterisks; **p-value <0.01. Graphs represent the mean of three replicates and error bars represent SEM.

Article Snippet: pSTAT1 (Y701) , Rabbit , Cell Signaling , WB 1:1000 IF 1:50.

Techniques: Control, Infection, Virus, Transfection, Knockdown

Journal: PLOS Pathogens

Article Title: Human cytomegalovirus UL138 interaction with USP1 activates STAT1 in infection

doi: 10.1371/journal.ppat.1011185

Figure Lengend Snippet: (A) Fibroblasts were reverse transfected with a siRNA against TBK1 for 48 hours prior to being infected (MOI = 1) with a WT or Δ UL138 STOP virus. Lysates were collected and immunoblotted for pSTAT1, STAT1, TBK1, IE1/2, and Tubulin. (B) Quantification of TBK1 levels normalized to NTC and graphed for statistical analysis with an unpaired t test and represented by asterisks; ***p-value <0.001. (C) Fibroblasts were infected (MOI = 1) with either WT or Δ UL138 STOP virus and lysates were immunoblotted. pTBK1, TBK1, pSTING, STING, IE1/2, and Tubulin were detected using antibodies. (D) Fibroblasts were treated with 0.88 μ M C527 for 24 hours prior to being infected (MOI = 1) with a WT or Δ UL138 STOP virus. Lysates were collected and immunoblotted for TBK1 and tubulin. (E) Fibroblasts were infected (MOI = 1) with a WT or Δ UL138 STOP virus and lysates were collected and immunoblotted for pTYK2, TYK2, pJAK1, JAK1, pSTAT1, STAT1, IE1/2, and Tubulin. (F) Fibroblasts were infected (MOI = 1) with a WT or Δ UL138 STOP virus and lysates were collected and immunoblotted for pSTAT1, STAT1, pSHP-2, SHP-2, PTPN2, IE1/2, and Tubulin. (G) Fibroblasts were infected with a WT or Δ UL138 STOP virus (MOI = 1) for 48 hours before treatment with DMSO (vehicle control) or 10 μ M of MG132 for 6 hours before lysates were collected. Lysates were immunoblotted for pSTAT1, STAT1, IE1/2, and Tubulin using antibodies. (H) The fold increase in rescue in the MG132 treated cells over the DMSO treated for mock, WT, and Δ UL138 STOP infected cells were graphed.

Article Snippet: pSTAT1 (Y701) , Rabbit , Cell Signaling , WB 1:1000 IF 1:50.

Techniques: Transfection, Infection, Virus, Control

Journal: PLOS Pathogens

Article Title: Human cytomegalovirus UL138 interaction with USP1 activates STAT1 in infection

doi: 10.1371/journal.ppat.1011185

Figure Lengend Snippet: (A) Fibroblasts were infected (MOI = 1) with a WT or Δ UL138 STOP virus. Cells were fractionated and immunoblotted using antibodies of pSTAT1, STAT1, Lamin B1, and Tubulin. Fraction purity and loading was determined by immunoblotting for Lamin B1 (Nucleus) and Tubulin (Cytoplasm). (B, C, D) Fibroblasts were infected (MOI = 1) with a WT or Δ UL138 STOP virus. DNA was isolated at 24, 48, and 72 hpi. RT-qPCR for ISG expression was performed with TaqMan Gene Expression Assays (Thermo Fischer Scientific). Relative expression was determined using the ΔCT method and values were normalized to the WT 24 hpi and graphed for statistical analysis. Statistical significance was calculated using Two-Way Anova and represented by asterisks; *p-value <0.05, **p-value <0.01, ***p-value <0.001, ****p-value <0.0001. Graphs represent the mean of three replicates and error bars represent SEM.

Article Snippet: pSTAT1 (Y701) , Rabbit , Cell Signaling , WB 1:1000 IF 1:50.

Techniques: Infection, Virus, Western Blot, Isolation, Quantitative RT-PCR, Expressing, Gene Expression

Journal: PLOS Pathogens

Article Title: Human cytomegalovirus UL138 interaction with USP1 activates STAT1 in infection

doi: 10.1371/journal.ppat.1011185

Figure Lengend Snippet: (A) Fibroblasts were infected at MOI of 0.5, 1, and 3 with a WT or Δ UL138 STOP virus and lysates were immunoblotted. pSTAT1, STAT1, IE1/2, and Tubulin were detected using antibodies. (B) Fibroblasts were plated on coverslips at 10,000 cells per coverslip. Fibroblasts were infected (MOI = 1) with either a WT or Δ UL138 STOP virus for 72 hpi. At 72 hpi, mock cells were treated with DPBS or 1000 U/mL of universal type 1 interferon for 30 minutes then the cells were processed per the antibody manufacturer’s recommendations. Coverslips were imaged using a DeltaVision deconvolution microscope.

Article Snippet: pSTAT1 (Y701) , Rabbit , Cell Signaling , WB 1:1000 IF 1:50.

Techniques: Infection, Virus, Microscopy

Journal: PLOS Pathogens

Article Title: Human cytomegalovirus UL138 interaction with USP1 activates STAT1 in infection

doi: 10.1371/journal.ppat.1011185

Figure Lengend Snippet: (A) The HCMV genome was run through PhysBinder Software to obtain potential pSTAT1 consensus sites on the viral genome. Site 1 and 2 are within UL133 and UL136 respectively and are upstream of UL138. (B) Fibroblasts were infected (MOI = 1) with either a WT or Δ UL138 STOP virus. At 72 hpi, uninfected cells were treated with DPBS (vehicle control) or 1000 U/mL of universal type 1 interferon for 30 minutes. After 30 minutes, all cells entered the CUT&RUN protocol developed by Cell Signaling Technologies. qPCR was performed on the input DNA and immunoprecipitated DNA utilizing primers specific to pSTAT1 binding sites of interest. Percent Immunoprecipitated was calculated to 2% of the input sample and graphed normalized to mock cells with interferon treatment. Statistical significance was calculated using One-Way Anova and represented by asterisks; **p-value <0.01, ***p-value <0.001, ****p-value <0.0001. (C) Fibroblasts were infected (MOI = 1) with a WT virus. At the time of infection, cells were treated with DMSO (vehicle control) or 5 μ M Ruxolitinib every 24 hours. At 72 hpi, DNA and RNA was isolated and transcripts encoding UL138 and IE were quantified by RT-qPCR normalized to H6PD. Viral genomes were analyzed through qPCR using primers for b2.7kb and RNaseP as a loading control. Relative expression was determined using the ΔCT method and values were normalized to the DMSO control and graphed for statistical analysis. Statistical significance was calculated using unpaired student t test and represented by asterisks; **p-value <0.01. Graphs represent the mean of three replicates and error bars represent SEM.

Article Snippet: pSTAT1 (Y701) , Rabbit , Cell Signaling , WB 1:1000 IF 1:50.

Techniques: Software, Infection, Virus, Control, Immunoprecipitation, Binding Assay, Isolation, Quantitative RT-PCR, Expressing

Journal: PLOS Pathogens

Article Title: Human cytomegalovirus UL138 interaction with USP1 activates STAT1 in infection

doi: 10.1371/journal.ppat.1011185

Figure Lengend Snippet: (A) CD34+ HPCs were infected with either WT or Δ UL138 STOP virus (MOI = 2). At 24 hpi, CD34+/GFP+ cells were sorted and seeded into long-term culture in the presence of DMSO or 1 μ M C527. After 10 days in culture, populations were either mechanically lysed (pre-reactivation) or whole cells (reactivation) were plated onto fibroblast monolayers in cytokine-rich media. 14 days later, GFP+ wells were scored and frequency of infectious centers was determined by limited dilution analysis. The frequency of infectious centers was normalized to WT pre-reactivation and the average of 3 independent experiments is shown. Due to donor variability in the Δ UL138 STOP infection, data was Log2 transformed and graphed. Statistical significance was calculated using Two-Way Anova and represented by asterisks; *p-value <0.05, **p-value <0.01, ****p-value >0.0001. (B) CD34+ HPCs were infected and sorted for long term culture as described in without the addition of C527. At day 10, CD34+ HPCs were plated onto fibroblast monolayers in cytokine-rich media in the presence of DMSO or 1 μ M C527. 14 days later, GFP+ wells were scored and frequency of infectious centers was determined by limited dilution analysis. The frequency of infectious centers was normalized to WT pre-reactivation and the average of 3 independent experiments is shown. Statistical significance was calculated using Two-Way Anova and represented by asterisks; **p-value <0.01. Graphs represent the mean of three replicates and error bars represent SEM. (C) CD34+ HPCs were infected with either WT or Δ UL138 STOP virus (MOI = 2) in cytokine-rich media in the presence of DMSO or 500 nM Ruxolitinib. Every 24 hours, 500 nM Ruxolitinib was refreshed in the media. At days 1 and 5 post infection, DNA was isolated for qPCR using primers for b2.7kb and RNaseP. Viral genomes were determined using ΔCT method and the day 5 values were normalized to the corresponding day 1 values. For graphing, day 5 genomes were normalized to WT DMSO and Log2 transformed. Statistical significance was calculated using Two-Way Anova and represented by asterisks; *p-value <0.05, **p-value <0.01. Graphs represent the mean of three replicates and error bars represent SEM. (D) To ensure Ruxolitinib inhibited pSTAT1, CD34+ HPCs were cultured in the presence of 100 nM, 500 nM, or 2000 nM Ruxolitinib or equivalent DMSO for 3 hours before being treated with 1000 U/mL universal type 1 interferon for 30 minutes and immunoblotted for pSTAT1 and Tubulin.

Article Snippet: pSTAT1 (Y701) , Rabbit , Cell Signaling , WB 1:1000 IF 1:50.

Techniques: Infection, Virus, Transformation Assay, Isolation, Cell Culture

Journal: PLOS Pathogens

Article Title: Human cytomegalovirus UL138 interaction with USP1 activates STAT1 in infection

doi: 10.1371/journal.ppat.1011185

Figure Lengend Snippet: Antibody description and sources.

Article Snippet: pSTAT1 (Y701) , Rabbit , Cell Signaling , WB 1:1000 IF 1:50.

Techniques: Concentration Assay

Journal: Journal of biomedical science

Article Title: Proteasome inhibitors restore the STAT1 pathway and enhance the expression of MHC class I on human colon cancer cells.

doi: 10.1186/s12929-021-00769-9

Figure Lengend Snippet: Fig. 2 Western blots (A) and the quantification of Western blots (B) for SW620 and SW480 cells in the STAT1 pathway in response to bortezomib, LLnL, MG132, and IFN-γ

Article Snippet: The antibodies applied for Western blots were STAT1 (Cell Signaling Technology, #9172, Danvers, MA, USA), phosphorylated STAT1 (pSTAT1; Cell Signaling Technology, #9167), phospho-JAK1 (pJAK1; Cell Signaling Technology, #3331), JAK1 (Cell Signaling Technology, #3332), phosphoJAK2 (pJAK2; Cell Signaling Technology, #3771), JAK2 (Cell Signaling Technology, #3230), interferon regulatory factor-1 (IRF-1; Cell Signaling Technology, #8478), pan-MHC class I (Origene, AM33035PU-N, Rockville, MD, USA), ß-actin (Abcam, ab8227, Cambridge, UK), GAPDH (Abcam, ab8245), HRP donkey anti-rabbit IgG (BioLegend, 406401), and HRP goat anti-mouse IgG (BioLegend, 405306).

Techniques: Western Blot

Journal: Journal of biomedical science

Article Title: Proteasome inhibitors restore the STAT1 pathway and enhance the expression of MHC class I on human colon cancer cells.

doi: 10.1186/s12929-021-00769-9

Figure Lengend Snippet: Fig. 3 Western blots (A) and the quantification of western blots (B) for SW620 cells in the STAT1 pathway in response to bortezomib and IFN-γ. We further applied DLD-1 cell line with western blots (C) and the quantification of western blots (D) for confirmation. Schema of the mechanism of proteasome inhibitors on MHC class I expression of colon cancer cell lines was summarized in Fig. 3 (E)

Article Snippet: The antibodies applied for Western blots were STAT1 (Cell Signaling Technology, #9172, Danvers, MA, USA), phosphorylated STAT1 (pSTAT1; Cell Signaling Technology, #9167), phospho-JAK1 (pJAK1; Cell Signaling Technology, #3331), JAK1 (Cell Signaling Technology, #3332), phosphoJAK2 (pJAK2; Cell Signaling Technology, #3771), JAK2 (Cell Signaling Technology, #3230), interferon regulatory factor-1 (IRF-1; Cell Signaling Technology, #8478), pan-MHC class I (Origene, AM33035PU-N, Rockville, MD, USA), ß-actin (Abcam, ab8227, Cambridge, UK), GAPDH (Abcam, ab8245), HRP donkey anti-rabbit IgG (BioLegend, 406401), and HRP goat anti-mouse IgG (BioLegend, 405306).

Techniques: Western Blot, Expressing

Journal: Journal of biomedical science

Article Title: Proteasome inhibitors restore the STAT1 pathway and enhance the expression of MHC class I on human colon cancer cells.

doi: 10.1186/s12929-021-00769-9

Figure Lengend Snippet: Fig. 4 One example of IHC staining by the PerkinElmer Opal multiplex system. The merge of all seven different IHC staining within one picture was demonstrated as (A). The individual computerized scanning of STAT1 IHC staining (B), MHC class I IHC staining (C), PD-L1 IHC staining (D), CD4 IHC staining (E), and CD8 IHC staining (F) were demonstrated. Computerized scanning of superimposing all five IHC staining was demonstrated in (G)

Article Snippet: The antibodies applied for Western blots were STAT1 (Cell Signaling Technology, #9172, Danvers, MA, USA), phosphorylated STAT1 (pSTAT1; Cell Signaling Technology, #9167), phospho-JAK1 (pJAK1; Cell Signaling Technology, #3331), JAK1 (Cell Signaling Technology, #3332), phosphoJAK2 (pJAK2; Cell Signaling Technology, #3771), JAK2 (Cell Signaling Technology, #3230), interferon regulatory factor-1 (IRF-1; Cell Signaling Technology, #8478), pan-MHC class I (Origene, AM33035PU-N, Rockville, MD, USA), ß-actin (Abcam, ab8227, Cambridge, UK), GAPDH (Abcam, ab8245), HRP donkey anti-rabbit IgG (BioLegend, 406401), and HRP goat anti-mouse IgG (BioLegend, 405306).

Techniques: Immunohistochemistry, Multiplex Assay

Journal: Journal of biomedical science

Article Title: Proteasome inhibitors restore the STAT1 pathway and enhance the expression of MHC class I on human colon cancer cells.

doi: 10.1186/s12929-021-00769-9

Figure Lengend Snippet: Fig. 5 Counts for mRNA expression in tumor tissue through NanoString analysis for STAT1(A), HLA-A (B), HLA-B (C), HLA-C (D), HLA-E (E), HLA-G (F), and IFN-γ (G). Blue bar indicates the results of the high-STAT1 group in the Table 1. Red bar indicates the results of the low-STAT1 group in Table 1. *p < 0.05

Article Snippet: The antibodies applied for Western blots were STAT1 (Cell Signaling Technology, #9172, Danvers, MA, USA), phosphorylated STAT1 (pSTAT1; Cell Signaling Technology, #9167), phospho-JAK1 (pJAK1; Cell Signaling Technology, #3331), JAK1 (Cell Signaling Technology, #3332), phosphoJAK2 (pJAK2; Cell Signaling Technology, #3771), JAK2 (Cell Signaling Technology, #3230), interferon regulatory factor-1 (IRF-1; Cell Signaling Technology, #8478), pan-MHC class I (Origene, AM33035PU-N, Rockville, MD, USA), ß-actin (Abcam, ab8227, Cambridge, UK), GAPDH (Abcam, ab8245), HRP donkey anti-rabbit IgG (BioLegend, 406401), and HRP goat anti-mouse IgG (BioLegend, 405306).

Techniques: Expressing