Journal: Metabolism: clinical and experimental

Article Title: Loss of long-chain acyl-CoA synthetase 1 promotes hepatocyte death in alcohol-induced steatohepatitis

doi: 10.1016/j.metabol.2022.155334

Figure Lengend Snippet: Activation of STAT5-ACSL1 signaling pathway protects against alcohol-induced liver injury. Wild-type Flox mice and Acsl1Δhep mice with or without Stat5b overexpression (Stat5b OE) were subjected to alcohol feeding for 8 weeks. (A) Western blot assay of proteins related to apoptotic and necroptotic cell death (n = 3/group). (B) qPCR analysis of hepatic mRNA levels of ACSL1, ACSL3, ACSL4, and ACSL5 (n = 6/group). (C–D) Serum levels of ALT (C) and AST (D) (n = 5/group). (E) Kaplan–Meier survival analysis of survival probability during 8 weeks of alcohol feeding. (F-G) Flow cytometry analysis of hepatic infiltration of Ly6G+CD1+ neutrophils (f) and quantification (g), gated on singlet 7AAD− CD45+ cells. (n = 4/group). (H) qPCR analysis of hepatic mRNA levels of Lcn2 (n = 6/group). (I) Serum levels of LCN2 (n = 5/group). Data are presented as mean ± SD. *P < 0.05, **P < 0.01, values with different superscripts are significantly different.

Article Snippet: After reaching 60 % confluency, cells were transfected with Stat5b Double Nickase Plasmid or Acsl1 Double Nickase Plasmid following the manufacturer’s guidelines (Santa Cruz Biology).

Techniques: Activation Assay, Over Expression, Western Blot, Flow Cytometry

Journal: Metabolism: clinical and experimental

Article Title: Loss of long-chain acyl-CoA synthetase 1 promotes hepatocyte death in alcohol-induced steatohepatitis

doi: 10.1016/j.metabol.2022.155334

Figure Lengend Snippet: STAT5-ACSL1 signaling mediated liver injury in patients with alcoholic hepatitis. (A) The RNA-seq analysis of Stat5a, Stat5b, Acsl1, Bax, Ripk1, Ripk3, and Mlkl in liver tissues from normal subjects (Normal, n = 7) and patients with alcoholic hepatitis (AH, n = 15). GEO accession number: GSE28619, *P < 0.05, **P < 0.01, ***P < 0.001. (B) Western blot assay of hepatic proteins related to STAT5-ACSL1signaling and cell death markers in the liver of normal subjects (Normal, n = 5) and AH patients (AH, n = 5). (C) IF staining of p-MLKL and SATAT5 (Scale bars = 10 μm.), and IHC staining of BAX (Scale bars = 20 μm.) on liver tissue sections from normal subjects and AH patients. Arrows in red indicate the nuclear localization of STAT5. (D) A heatmap of correlative analysis of mRNA levels of Acsl1 with Stat5, apoptotic gene Bax and necroptotic genes including Mlkl and Ripk3. The abscissa and ordinate represent genes, different colors represent different correlation coefficients (blue represents positive correlation whereas red represents negative correlation), the darker the color, the stronger the relation. *P < 0.05, **P < 0.01, values with different superscripts are significantly different.

Article Snippet: After reaching 60 % confluency, cells were transfected with Stat5b Double Nickase Plasmid or Acsl1 Double Nickase Plasmid following the manufacturer’s guidelines (Santa Cruz Biology).

Techniques: RNA Sequencing, Western Blot, Staining, Immunohistochemistry

Journal: Journal of Biological Chemistry

Article Title: Role of the Cytokine-inducible SH2 Protein CIS in Desensitization of STAT5b Signaling by Continuous Growth Hormone

doi: 10.1074/jbc.m004755200

Figure Lengend Snippet: FIG. 1. CIS inhibition of STAT5b signaling is more complete at lower STAT5b levels. COS-1 cells were transfected with expression plasmids encoding rat GHR (100 ng), reporter plasmid 4x-pT109-Luc (400 ng), CIS (200 ng), or SOCS-3 (200 ng) together with increasing amounts of STAT5b (5, 25, 150, or 500 ng). A Renilla luciferase expres- sion plasmid (pRL-TK) (40 ng) was included to normalize samples for transfection efficiencies. After transfection, cells were stimulated for 5 h with 100 ng/ml rat GH. Cell lysates were then prepared and analyzed for STAT5 DNA binding activity by EMSA using a b-casein DNA probe (panel A) or assayed for normalized luciferase reporter activity (panel B). STAT5b protein levels detected by Western blot were maximal at the 150-ng plasmid level (data not shown). Panel A, EMSA gel of STAT5 DNA binding activity, assayed using b-casein probe, in extracts of cells transfected with the indicated factors and then stimulated with GH for 5 h. CIS inhibits GH-inducible STAT5b transcriptional activity more completely at low STAT5b levels, whereas the inhibition by SOCS-3 is already complete at high STAT5b levels. STAT5b EMSA activity in the presence of CIS or SOCS-3 is graphed at each STAT5b plasmid level relative to the corresponding GH stimulated activity obtained in the absence of SOCS/CIS expression plasmid (i.e. STAT5 activity for sam- ples in lanes 2–9, respectively serve as the 100% values for the samples in lanes 11–18 and 20–27). Panel B, firefly luciferase reporter activity normalized for the level of Renilla luciferase activity is graphed as a percentage of the GH stimulated activity obtained in the absence of CIS or SOCS-3 expression (mean 6 S.D., n 5 4).

Article Snippet: STAT5b Western blotting used anti-STAT5b polyclonal antibody sc-835 from Santa Cruz Biotechnology as described elsewhere (32).

Techniques: Inhibition, Transfection, Expressing, Plasmid Preparation, Luciferase, Binding Assay, Activity Assay, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Role of the Cytokine-inducible SH2 Protein CIS in Desensitization of STAT5b Signaling by Continuous Growth Hormone

doi: 10.1074/jbc.m004755200

Figure Lengend Snippet: FIG. 2. Time course of SOCS/CIS protein inhibition of STAT5b EMSA activity. COS-1 cells were transfected with expression plas- mids encoding rabbit GHR (10 ng), STAT5b (25 ng), and each of the indicated SOCS/CIS proteins: CIS (200 ng), SOCS-1 (200 ng), SOCS-2 (500 ng), or SOCS-3 (200 ng). Thirty h later, cells were treated with 200 ng/ml GH for times ranging from 15 to 300 min, followed by preparation of whole cell extracts used for EMSA assays of STAT5 DNA binding activity. Panel A, EMSA analysis showing that CIS protein exerts a complete inhibition of STAT5b DNA binding activity only after 3 h, whereas the inhibitory effect of SOCS-3 is already maximal at 15 min. Inset, Western blot analysis of CIS protein (detection of c-Myc tag) and STAT5b protein (multiple bands correspond to variously phosphoryl- ated STAT5b forms (46)) in duplicate samples co-transfected with GHR, STAT5b, and CIS expression plasmids. Panel B, EMSA gel and quan- titative EMSA data showing GH-inducible STAT5b DNA binding activ- ity is fully blocked by SOCS-3 and SOCS-1 after 60 min and by CIS only after 300 min. Activity is partially blocked in cells expresing SOCS-2 at both 60 and 300 min.

Article Snippet: STAT5b Western blotting used anti-STAT5b polyclonal antibody sc-835 from Santa Cruz Biotechnology as described elsewhere (32).

Techniques: Inhibition, Activity Assay, Transfection, Expressing, Binding Assay, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Role of the Cytokine-inducible SH2 Protein CIS in Desensitization of STAT5b Signaling by Continuous Growth Hormone

doi: 10.1074/jbc.m004755200

Figure Lengend Snippet: FIG. 3. Proteasome inhibitor MG132 prevents GH-induced de- crease in CIS protein. Shown in panels A and B are c-Myc Western blots showing the time dependence of GH-stimulated CIS protein deg- radation and the protective effect of MG132 in transfected COS-1 cells. Cells were transfected for 30 h with expression plasmids encoding GHR (200 ng), STAT5b (50 ng), and c-Myc-tagged CIS or SOCS-3 (200–500 ng, as indicated). Cells were then treated with 50 mM MG132 for 15 min, as indicated, followed by GH stimulation (50 ng/ml) for 30 min to 8 h. Cell extracts were then prepared and blotted with anti-Myc antibody to visualize each of the transfected CIS or SOCS-3 proteins. Panel A shows substantial GH-induced loss of CIS protein in cells transfected with higher levels of CIS plasmid (lanes 9–12 versus 5–8) which is blocked by MG132 (lanes 13–20 versus 1–12). In the absence of MG132, CIS but not SOCS-3 protein is degraded in a GH-dependent manner (panel B, lanes 3 and 4 versus 11 and 12).

Article Snippet: STAT5b Western blotting used anti-STAT5b polyclonal antibody sc-835 from Santa Cruz Biotechnology as described elsewhere (32).

Techniques: Western Blot, Transfection, Expressing, Plasmid Preparation

Journal: Journal of Biological Chemistry

Article Title: Role of the Cytokine-inducible SH2 Protein CIS in Desensitization of STAT5b Signaling by Continuous Growth Hormone

doi: 10.1074/jbc.m004755200

Figure Lengend Snippet: FIG. 4. Proteasome inhibitor MG132 blocks CIS inhibition of GH-stimulated STAT5b activation. Panel A, COS-1 cells transfected with expression plasmids encoding GHR (200 ng), STAT5b (50 ng), and the reporter gene 4x-pT109-Luc were co-transfected with CIS (200 ng), SOCS-1 (50 ng), SOCS-2 (500 ng), or SOCS-3 (200 ng), as indicated. Cells were stimulated with GH (50 ng/ml) for 8 h, and normalized luciferase reporter activity was assayed. MG132 is shown to block the inhibitory effects of CIS but not that of SOCS-1, SOCS-2, or SOCS-3. The CIS and SOCS-3 Western blot of these samples is shown in Fig. 3B. Panel B, Western blot of STAT5b in samples corresponding to control and CIS-transfected groups shown in panel A. Uppermost band seen in lanes 3, 4, 7, 8, 11, and 12 corresponds to the tyrosine-phosphorylated form of STAT5b (46). This band is decreased in relative abundance in CIS-transfected samples (lanes 7 and 8 versus lanes 3 and 4) but is maintained at the uninhibited control levels in the presence of MG132 (lanes 11 and 12). Panel C, experiment carried out as described under panel A, except that the cells were stimulated with GH for either 30 or

Article Snippet: STAT5b Western blotting used anti-STAT5b polyclonal antibody sc-835 from Santa Cruz Biotechnology as described elsewhere (32).

Techniques: Inhibition, Activation Assay, Transfection, Expressing, Luciferase, Activity Assay, Blocking Assay, Western Blot, Control

Journal: Journal of Biological Chemistry

Article Title: Role of the Cytokine-inducible SH2 Protein CIS in Desensitization of STAT5b Signaling by Continuous Growth Hormone

doi: 10.1074/jbc.m004755200

Figure Lengend Snippet: FIG. 5. Dominant-negative CIS mutant R107K blocks inhibition of GH-inducible STAT5b activity by wild type CIS. COS-1 cells were co-transfected with GHR (200 ng), STAT5b (100 ng), and the reporter plasmid 4x-pT109-Luc in the presence of CIS (200 ng), SOCS-1 (50 ng), SOCS-2 (500 ng), or SOCS-3 (100 ng) expression plasmids. Where indicated, CIS-R107K expression plasmid was co-transfected at 500 ng except in the case of SOCS-2, where 800 ng of CIS-R107K was used. Cells were stimulated with GH (200 ng/ml) for 5 h and relative EMSA (panel A) and luciferase reporter activities (panel B) were assayed. CIS-R107K blocks the inhibitory effect of wild-type CIS but not that of SOCS-1, SOCS-2, or SOCS-3. Panel C, Western blot of CIS (c-Myc epitope tag) and CIS-R107K protein (Flag tag) in transfections such as those shown in panels A and B. Panel D, bacterial extract (10 ml) containing equivalent amounts of GST-GHR fusion proteins (c.f. anti-GST probing, lower panel), which encompass amino acids 291–620 or 540–620 of the human GHR, were prepared in the presence or absence of the Elk tyrosine kinase, as indicated. To assay the binding of CIS or CIS-R107K to the GST-GHR fusion proteins, bacterial extracts bound to glutathione-agarose beads were incubated for 2.5 h at 4 °C with total extracts of COS-1 cells transfected with CIS (200 ng), CIS-R107K (500 ng), or CIS in combination with CIS-R107K (200 and 800 ng, respectively) (75 mg of COS-1 cell extract in 0.1 ml of fusion lysis buffer (32)). The beads were washed, eluted in SDS gel buffer, and then analyzed on a Western blot probed sequentially with anti-Myc, anti-Flag, and anti-GST antibodies, as indicated. Recovery of GST-GHR fusion protein was low in lane 1. Lanes 10–12 contain 15 mg of the same SOCS/CIS-transfected COS-1 cell extracts used for the samples shown in lanes 1–9, but were loaded onto the SDS gel directly without binding to the GST-GHR fusion protein. Lower intensity of the Flag signal in lane 11 compared with lane 12 reflects the 4-fold increased level of transfected CIS-R107K plasmid in the latter sample. Panel E, experiment carried out as in panels A and B except that the cells were stimulated with GH for 30 min and STAT5 EMSA activity was assayed.

Article Snippet: STAT5b Western blotting used anti-STAT5b polyclonal antibody sc-835 from Santa Cruz Biotechnology as described elsewhere (32).

Techniques: Dominant Negative Mutation, Mutagenesis, Inhibition, Activity Assay, Transfection, Plasmid Preparation, Expressing, Luciferase, Western Blot, FLAG-tag, Binding Assay, Incubation, Lysis, SDS-Gel

Journal: Journal of Biological Chemistry

Article Title: Role of the Cytokine-inducible SH2 Protein CIS in Desensitization of STAT5b Signaling by Continuous Growth Hormone

doi: 10.1074/jbc.m004755200

Figure Lengend Snippet: FIG. 6. CIS mutant R107K abolishes down-regulation of STAT5b signaling. COS-1 cells transfected with STAT5b (100 ng) and GHR (200 ng) with or without CIS-R107K (500 ng) were treated with GH (200 ng/ml) for times up to 5 h. Cells were treated with 50 mM MG132 for 15 min prior to GH stimulation, as indicated. Extracts were analyzed for STAT5b EMSA activity at each of the indicated time points. Shown is an EMSA gel (panel A) with quantitation by Phosphor- Imager analysis (panel B).

Article Snippet: STAT5b Western blotting used anti-STAT5b polyclonal antibody sc-835 from Santa Cruz Biotechnology as described elsewhere (32).

Techniques: Mutagenesis, Transfection, Activity Assay, Quantitation Assay

Journal: Journal of Biological Chemistry

Article Title: Role of the Cytokine-inducible SH2 Protein CIS in Desensitization of STAT5b Signaling by Continuous Growth Hormone

doi: 10.1074/jbc.m004755200

Figure Lengend Snippet: FIG. 7. Impact of CIS and CIS-R107K on STAT5b nuclear translocation in the liver cell line CWSV-1. CWSV-1 cells were transfected with GFP alone (panels A-D) or GFP in combination with CIS (panels E-H), SOCS-3 (panels I-L), or CIS-R107K (panels M-Q). Cells were stimulated with GH for either 45 or 180 min and the subcelluar localization of STAT5b monitored by indirect immunofluorescence. Shown are confocal microscope images of representative fields revealing the effects of the transfected SOCS/CIS proteins on STAT5b nuclear localization (red fluorescence; bottom sets of panels) in response to GH stimulation. Also shown are the corresponding green fluorescent images (top sets of panels), which directly identify cells that are transfected with plasmid DNA. Cells shown in panels A, F, J, N, and P do not efficiently express GFP and are either non-transfected or poorly transfected. Panels A and B, F and G, J and K, N and O, and P and Q correspond to images of untransfected and transfected cells, respectively, growing on the same coverslip. Results shown are representative of at least three independent sets of experiments.

Article Snippet: STAT5b Western blotting used anti-STAT5b polyclonal antibody sc-835 from Santa Cruz Biotechnology as described elsewhere (32).

Techniques: Translocation Assay, Transfection, Immunofluorescence, Microscopy, Fluorescence, Plasmid Preparation

Journal: Journal of Biological Chemistry

Article Title: Role of the Cytokine-inducible SH2 Protein CIS in Desensitization of STAT5b Signaling by Continuous Growth Hormone

doi: 10.1074/jbc.m004755200

Figure Lengend Snippet: SCHEME 1. Model for dual mechanism whereby CIS inhibits GHR-JAK2 signaling to STAT5b. Step 1 is proposed to involve com- petition between CIS and STAT5b for common phosphotyrosine-bind- ing site(s) on GHR cytoplasmic tail. Step 2 is proposed to involve CIS-dependent degradation of a CIS-(GHR-JAK2) complex via the pro- teasome. Step 2 but not step 1 is blocked in cells treated with the proteasome inhibitor MG132 and in cells expressing the CIS SH2 do- main mutant CIS-R107K, which does not bind GHR.

Article Snippet: STAT5b Western blotting used anti-STAT5b polyclonal antibody sc-835 from Santa Cruz Biotechnology as described elsewhere (32).

Techniques: Expressing, Mutagenesis