Journal: medRxiv

Article Title: Polymorphism in IFNAR contributes to glucocorticoid response and outcome in ARDS and COVID-19

doi: 10.1101/2022.03.10.22272123

Figure Lengend Snippet: (A ) STAT1 expression in the lung after 4-day culture in the presence of IFN beta with or without hydrocortisone (HC). ( B) pSTAT1 expression in the same specimens as in A. ( C) Example photomicrographs showing higher STAT2 expression in a TT patient than in a CT patient and the effect of HC on its nuclear translocation. Most STAT2 remains in the cytoplasm of the CT patients, whereas nuclear expression is prominent in the TT patient. Indicated insets are shown in the bottom row. Arrows. ( D ) Combined results of all patients noting that two CT samples are excluded in the data as the patients were already under glucocorticoid treatment at the time of sample acquisition. Ns, not significant; *P<0.05; **P<0.01; and ***P<0.001

Article Snippet: The first stage antibodies were anti-alpha chain of the IFN alpha/beta receptor (St John’s Laboratory STJ112765) that was used 1:2000 and 1:5000 and anti-Stat1 (1:400, 9175S), anti-pStat1(1:100, 9167S) and anti-Stat2 (1:200, 72604S) all from Cell Signalling.

Techniques: Expressing, Translocation Assay

Journal: Nature Communications

Article Title: Mesenchymal stem cell-derived interleukin-28 drives the selection of apoptosis resistant bone metastatic prostate cancer

doi: 10.1038/s41467-021-20962-6

Figure Lengend Snippet: a , b pSTAT1 ( a ) and pSTAT3 ( b ) levels at baseline and in response to MSC CM (50%) over a 10 min (min) period in PAIII parental (F0) and MSC selected (F2) cell lines. Molecular weights are shown in kDa. Actin was used as a loading control. For densitometry, pSTAT and STAT levels were each normalized to their respective actin controls and time responses for F0 and F2 are compared to their respective 0’ minute controls that were set at a normalized value of 1. All experiments were repeated on at least three separate occasions with similar results. c , d STAT1 and STAT3 DNA binding activity in the PAIII ( c ) and DU145 ( d ) F0 and F2 cell lines was measured in response to MSC CM for 30 min. Results obtained via absorbance (ABS@450 nm) were normalized to respective controls. e , f STAT3 was silenced (si-STAT3) in PAIII ( e ) and DU145 ( f ) F0 parental and F2 MSC-selected cell lines and the resultant impact on STAT3 activity was measured. Blots show total STAT3. Experiments were independently repeated with similar results. g , h The effect of STAT3 silencing on PAIII ( g ) and DU145 ( h ) cell growth in the presence or absence of MSC CM compared to control treated cells using luminescence assay and relative light unit (RLU) measurement or MTT assay ( n ≥ 3 biologically independent samples). Molecular weights are shown in kDa. Error bars in graphs represent the mean ± SEM and statistical analyses were performed by one-way ANOVA with multiple comparisons at 95% CI. Asterisks denote statistical significance (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001) while NS denotes not significant.

Article Snippet: All primary antibodies were purchased from Cell Signaling Technology (Cleaved Caspase 3 #9661S, pSTAT1 Tyr701 #7649S, pSTAT1 Ser727 #9177, pSTAT3 Tyr705 (D3A7) #9145, STAT3 (D3Z26) #12640S, STAT1 #9172S pJAK2 Y1007/1008 # 3776S), Santa Cruz (IL-10Rβ, Cat# sc271969) or Abcam (IL-28Rα, Cat# ab83865).

Techniques: Binding Assay, Activity Assay, Luminescence Assay, MTT Assay

Journal: Nature Communications

Article Title: Mesenchymal stem cell-derived interleukin-28 drives the selection of apoptosis resistant bone metastatic prostate cancer

doi: 10.1038/s41467-021-20962-6

Figure Lengend Snippet: a , b Parental (F0) and MSC-selected (F2) cell lines treated with vehicle control (Control) or the JAK2 inhibitor ruxolitinib (RUX)/STAT3 inhibitor (S3I-201) for 24 h. c F0 and F2 DU145 control (scr-siRNA) or STAT3 silenced (si-STAT3) cells treated with vehicle or S3I-201 for 24 h. d F0 and F2 DU145 growth over time in the presence or absence of STAT3 inhibitor, S3I-201 ( n = 10/group). Representative images of bioluminescence in each group are shown at day 35-time point. Arrow and dashed line represent time of treatment initiation. Graphs illustrate collected RLUs over time for each group. Error bars represent the mean ± SEM and linear regression was used for statistical analysis. e S3I-201 effect on F0 and F2 DU145 at day 42 normalized to respective controls. Box and whisker plots show min to max values obtained. f , g Ex vivo analyses from study endpoint of proliferative and apoptotic indices using phospohistone H3 (pHH3; red arrows; f ) and cleaved caspase 3 (CC3; red, arrows, g ), respectively. Pan-cytokeratin (green) was used to identify prostate cancer cells. Error bars represent the mean ± SEM. Experiments ( a – d ) were repeated on at least three separate occasions with similar results. Error bars in graphs represent the mean ± SEM. Statistical analyses were performed by one-way ANOVA with multiple comparisons at 95% CI. Asterisks denote statistical significance (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001) while NS denotes not significant.

Article Snippet: All primary antibodies were purchased from Cell Signaling Technology (Cleaved Caspase 3 #9661S, pSTAT1 Tyr701 #7649S, pSTAT1 Ser727 #9177, pSTAT3 Tyr705 (D3A7) #9145, STAT3 (D3Z26) #12640S, STAT1 #9172S pJAK2 Y1007/1008 # 3776S), Santa Cruz (IL-10Rβ, Cat# sc271969) or Abcam (IL-28Rα, Cat# ab83865).

Techniques: Whisker Assay, Ex Vivo

Journal: Toxicological Research

Article Title: The Chloroform Fraction of Carpinus tschonoskii Leaves Inhibits the Production of Inflammatory Mediators in HaCaT Keratinocytes and RAW264.7 Macrophages

doi: 10.5487/tr.2012.28.4.255

Figure Lengend Snippet: Fig. 3. Effect of chloroform fraction from Carpinus tschonoskii on the STAT1 signal in IFN--stimulated HaCaT human keratinocytes. (A) Cells were pretreated with the indicated concentration of chloroform fraction of C. tschonoskii for 2 hr, and then the phosphorylation of STAT1 was determined in cells stimulated by IFN- (10 ng/ml) for 30 min. The levels of STAT1 and -actin were identified using West- ern blotting analysis. (B) Cells were pretreated with the chloroform fraction (50 g/ml) of C. tschonoskii for 2 hr, and then the transloca- tion of STAT1 protein was determined in cells stimulated by IFN- (10 ng/ml) for 60 min. Immunofluorescence stain of STAT1 was stained with DyLight488-conjugated 2nd antibody and the fluorescence was identified using confocal microscopy (FV500, OLYMPUS) and the images were acquired at constant conditions (PMT, gain, offset, magnification (20 × objective with zoom factor of 2.5) and res- olution, etc.). These data are representative of three independent experiments.

Article Snippet: Anti-phospho-STAT1 antibody was purchased from Cell Signaling Technology (Beverly, MA); rabbit or mouse origin anti-STAT1 antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) and Becton Dickinson (San Diego, CA), respectively; -actin was obtained from Sigma (St. Louis, MO); and DyLight488 conjugated Donkey anti-Rabbit antibody was purchased from BioLegend Inc. (San Diego, CA).

Techniques: Concentration Assay, Phospho-proteomics, Immunofluorescence, Staining, Fluorescence, Confocal Microscopy