Journal: Endocrinology

Article Title: Induction of 3beta-hydroxysteroid dehydrogenase/isomerase type 1 expression by interleukin-4 in human normal prostate epithelial cells, immortalized keratinocytes, colon, and cervix cancer cell lines.

doi: 10.1210/endo.140.10.7038

Figure Lengend Snippet: FIG. 8. Stat6 activation by IL-4. Normal human prostate epithelial cells (PrEC) in primary culture, LnCAP and PC-3 prostate cancer cells, ZR-75–1 and BT-20 breast cancer cells, HaCaT human immor- talized keratinocytes, HT-29 and Caco-2 human colon cancer cells, ME-180 human cervix cancer cells or JAR and JEG-3 human cho- riocarcinoma cells were incubated in the presence of absence of IL-4 (10 ng/ml for 30 min). Analysis of Stat6 activation using EMSA was performed as described in Materials and Methods using a well es- tablished Stat6 responsive element derived from the IgE-promoter. A Stat6 antibody was included in the binding reaction where indicated.

Article Snippet: The membrane was probed with a Stat6 antibody (Santa Cruz Biotechnology, Inc., Santa Clara, CA, catalog no. SC-621X).

Techniques: Activation Assay, Incubation, Derivative Assay, Binding Assay

Journal: Endocrinology

Article Title: Induction of 3beta-hydroxysteroid dehydrogenase/isomerase type 1 expression by interleukin-4 in human normal prostate epithelial cells, immortalized keratinocytes, colon, and cervix cancer cell lines.

doi: 10.1210/endo.140.10.7038

Figure Lengend Snippet: FIG. 7. Stat6 expression in cell types derived from peripheral tissues. Ten micrograms of total cell extract from ZR-75–1 and BT-20 breast cancer cells, normal human prostate epithelial cells (PrEC) in pri- mary culture, LnCAP and PC-3 prostate cancer cells, HaCaT human immortalized keratinocytes; HT-29and Caco-2 human colon cancer cells, ME-180 human cervix cancer cells, JAR and JEG-3 human choriocarcinoma and 293 human embryonic kidney cells were sepa- rated on SDS-PAGE. Western blot analysis was performed as de- scribed in Materials and Methods. Equal sample loading and transfer efficiency was confirmed by staining of the membrane with Ponceau Red.

Article Snippet: The membrane was probed with a Stat6 antibody (Santa Cruz Biotechnology, Inc., Santa Clara, CA, catalog no. SC-621X).

Techniques: Expressing, Derivative Assay, SDS Page, Western Blot, Staining, Membrane

Journal: Endocrinology

Article Title: Induction of 3beta-hydroxysteroid dehydrogenase/isomerase type 1 expression by interleukin-4 in human normal prostate epithelial cells, immortalized keratinocytes, colon, and cervix cancer cell lines.

doi: 10.1210/endo.140.10.7038

Figure Lengend Snippet: FIG. 9. IL-4-activated Stat6 binds to consensus Stat6 sequence sites in the 3b-HSD type 1 gene promoter. Normal human prostate epi- thelial cells (PrEC) in primary culture were incubated in the presence of absence of IL-4 (10 ng/ml for 30 min). Analysis of Stat6 activation using EMSA was performed as described in Materials and Methods using the 3b-HSD type 1 Stat6#1 and Stat6#2 probes from the 3b- HSD type gene promoter. A Stat6 antibody was included in the bind- ing reaction where indicated.

Article Snippet: The membrane was probed with a Stat6 antibody (Santa Cruz Biotechnology, Inc., Santa Clara, CA, catalog no. SC-621X).

Techniques: Sequencing, Incubation, Activation Assay

Journal: Cell death & disease

Article Title: STAT6 contributes to renal fibrosis by modulating PPARα-mediated tubular fatty acid oxidation.

doi: 10.1038/s41419-022-04515-3

Figure Lengend Snippet: Fig. 1 STAT6 is activated in the kidneys of UUO and HFD mice. A Representative micrographs for IHC staining of p-STAT6 in kidney section and quantification are performed. B The mRNA and C protein expression of STAT6, p-STAT6, Arg-1, TGF-β, α-SMA, FN in the indicated groups were determined by qRT-PCR or immunoblot analyses with the quantification on the right panel. D Representative micrographs for Sirius red and relative collagen proportion was quantified. E Representative micrographs for H&E and Oil Red O staining in kidney sections from indicated group. F Kidney TG content were measured in the indicated groups. Results are expressed as the mean ± SD (n = 8, *p < 0.05, Ctrl vs. Treatments). G Nuclear proteins were extracted from kidneys from different individuals as indicated, STAT6 and H3 were assayed by western blot analysis.

Article Snippet: The following antibodies were used: STAT6 (sc-374021), p-STAT6 (sc-136019), Arg-1 (sc166920), TGF-β (sc-146), α-SMA (sc-53142), FN (sc-18827), H3 (sc-517576), GAPDH (sc-32233) from Santa Cruz Biotechnology.

Techniques: Immunohistochemistry, Expressing, Quantitative RT-PCR, Western Blot, Staining

Journal: Cell death & disease

Article Title: STAT6 contributes to renal fibrosis by modulating PPARα-mediated tubular fatty acid oxidation.

doi: 10.1038/s41419-022-04515-3

Figure Lengend Snippet: Fig. 2 STAT6 deficiency in tubular cells attenuates UUO-caused renal fibrosis. The tubular-specific-Stat6 KO and control littermates were sacrificed 1w after UUO operation. A Representative IHC staining of p-STAT6, α-SMA, and micrographs for H&E, Sirius red staining in the control and fibrotic kidneys from the indicated group and quantification was performed. BThe mRNA and C protein expression of p-STAT6, STAT6, Arg-1, TGF-β, α-SMA, FN in the indicated treatments were determined by qRT-PCR or immunoblot analyses with the quantification on the right panel. Results are expressed as the mean ± SD (n = 8, *p < 0.05, Stat6 WT vs. Stat6 cKO).

Article Snippet: The following antibodies were used: STAT6 (sc-374021), p-STAT6 (sc-136019), Arg-1 (sc166920), TGF-β (sc-146), α-SMA (sc-53142), FN (sc-18827), H3 (sc-517576), GAPDH (sc-32233) from Santa Cruz Biotechnology.

Techniques: Control, Immunohistochemistry, Staining, Expressing, Quantitative RT-PCR, Western Blot

Journal: Cell death & disease

Article Title: STAT6 contributes to renal fibrosis by modulating PPARα-mediated tubular fatty acid oxidation.

doi: 10.1038/s41419-022-04515-3

Figure Lengend Snippet: Fig. 3 STAT6 deficiency in tubular cells attenuates UUO-caused lipid accumulation. The tubular-specific-Stat6 KO and control littermates were sacrificed 1w after UUO operation. A Representative IHC staining of PPARα and Oil Red O staining from Stat6 WT and cKO kidneys with or without UUO, and quantification for PPARα was performed. B TG content were determined in the kidneys from Stat6 WT and cKO mice with or without UUO. C mRNA levels of genes related to lipid metabolism in Stat6 WT and cKO mice with or without UUO operation. Results are expressed as the mean ± SD (n = 8, *p < 0.05, Stat6 WT vs. Stat6 cKO).

Article Snippet: The following antibodies were used: STAT6 (sc-374021), p-STAT6 (sc-136019), Arg-1 (sc166920), TGF-β (sc-146), α-SMA (sc-53142), FN (sc-18827), H3 (sc-517576), GAPDH (sc-32233) from Santa Cruz Biotechnology.

Techniques: Control, Immunohistochemistry, Staining

Journal: Cell death & disease

Article Title: STAT6 contributes to renal fibrosis by modulating PPARα-mediated tubular fatty acid oxidation.

doi: 10.1038/s41419-022-04515-3

Figure Lengend Snippet: Fig. 4 STAT6 in tubular cells suppresses PPARα-mediated fatty acid oxidation. HK2 cells were transfected with siRNA or plasmid for STAT6 inhibition or overexpression. Followed by 24 h serum-free medium culture, the cells were treated with TGF-β (5 ng/ml) for another 24 h. A Representative traces of three independent experiments for the measurement of oxygen consumption rate (OCR) were shown. Oligomycin, FCCP, and antimycin/rotenone were administrated at the indicated time point. ATP production was determined according to the OCR values. Data are presented as mean ± SD (n = 3, *P < 0.05). B Representative micrographs for Oil Red O staining of HK2 cells treated as indicated (C) TG content was determined enzymatically. D, E mRNA levels of genes related to lipid metabolism in STAT6 inhibited or overexpressed HK2 cells were determined by qRT-PCR. Results are expressed as the mean ± SD (n = 4 *p < 0.05, SiCtrl/Vector vs. SiSTAT6/STAT6 transfection) F mRNA levels of genes related to FAO and fibrotic proteins expression in primary renal tubular epithelial cells isolated from the above Stat6 WT and cKO mice. Results are expressed as the mean ± SD (n = 4, *p < 0.05, Stat6 WT vs. Stat6 cKO). G HK2 cells were transfected with indicated siRNA or plasmid for 24 h in serum-free medium and followed by TGF-β (5 ng/ml) treatment for another 24 h. Cell lysates were harvested and subjected to immunoblot analyses with the indicated antibodies. Quantification of relative protein expression was determined. Results are expressed as the mean ± SD (n = 4, *p < 0.05, Ctrl vs. TGF-β; #p < 0.05, SiCtrl/Vector vs.SiSTAT6/STAT6 transfection).

Article Snippet: The following antibodies were used: STAT6 (sc-374021), p-STAT6 (sc-136019), Arg-1 (sc166920), TGF-β (sc-146), α-SMA (sc-53142), FN (sc-18827), H3 (sc-517576), GAPDH (sc-32233) from Santa Cruz Biotechnology.

Techniques: Transfection, Plasmid Preparation, Inhibition, Over Expression, Staining, Quantitative RT-PCR, Expressing, Isolation, Western Blot

Journal: Cell death & disease

Article Title: STAT6 contributes to renal fibrosis by modulating PPARα-mediated tubular fatty acid oxidation.

doi: 10.1038/s41419-022-04515-3

Figure Lengend Snippet: Fig. 5 STAT6 regulates FAO and fibrotic related protein expression by targeting PPARα in tubular cells. HK2 cells were transfected with siRNA or plasmid for STAT6 or PPARα. After 24 h incubation, cells were treated with TGF-β (5 ng/ml) for another 24 h. A Cell lysates were harvested and subjected to immunoblot analyses with the indicated antibodies. Quantification of relative protein expression was determined. Results are expressed as the mean ± SD (n = 4 *p < 0.05, SiCtrl/Vector vs. siSTAT6/STAT6 transfection; #p < 0.05, SiCtrl/Vector vs. SiPPARα/PPARα transfection). B Representative micrographs showing the Oil Red O staining of indicated treatments in HK2 cells. C Identification of four SIEs in the promoter of PPARα. The potential site of STAT6 binding to PPARα promoter with detected by ChIP assay in HK2 cells with or without TGF-β (5 ng/ml) 24 h treatment. Results are expressed as the mean ± SD (n = 4 *p < 0.05, IgG vs. STAT6 immunoprecipitation). D The different human PPARα promoter constructs were cloned upstream of a luciferase reporter gene. HK2 cells were either transfected with empty vector or these constructs along with Renilla luciferase reporter for 24 h and followed by another 24 h TGF-β treatment, dual-luciferase activities were measured. The experiment was repeated three times, each with triplicate samples. Data are expressed as mean ± SD (n = 3, *p < 0.05, Vector vs. PPARα promoter constructs). E, F HK2 cells were transfected with indicated siRNA and plasmid for 24 h in serum-free medium, followed by TGF-β (5 ng/ml) 24 h and harvested for qRT-PCR analysis of the indicated genes. Results are expressed as the mean ± SD (n = 4 *p < 0.05, SiCtrl/ Vector vs. siSTAT6/STAT6 transfection; #p < 0.05, SiCtrl/Vector vs. SiPPARα/PPARα transfection).

Article Snippet: The following antibodies were used: STAT6 (sc-374021), p-STAT6 (sc-136019), Arg-1 (sc166920), TGF-β (sc-146), α-SMA (sc-53142), FN (sc-18827), H3 (sc-517576), GAPDH (sc-32233) from Santa Cruz Biotechnology.

Techniques: Expressing, Transfection, Plasmid Preparation, Incubation, Western Blot, Staining, Binding Assay, Immunoprecipitation, Construct, Clone Assay, Luciferase, Quantitative RT-PCR

Journal: Cell death & disease

Article Title: STAT6 contributes to renal fibrosis by modulating PPARα-mediated tubular fatty acid oxidation.

doi: 10.1038/s41419-022-04515-3

Figure Lengend Snippet: Fig. 6 AS1517499 attenuates aberrant lipid metabolism and tubulointerstitial fibrosis in UUO mice. A Representative micrographs for p-STAT6 staining in the kidney from the indicated groups, and quantification of relative protein expression in the kidneys from the indicated groups. B Representative Sirius red staining and H&E staining in the kidney from the indicated groups, and quantification of relative collagen proportion in the kidneys from the indicated groups. C Representative Oil O Red staining in the kidney from the indicated groups. D TG content was determined in the kidneys from the indicated groups. E Kidney tissue lysates from each group were subjected to immunoblot analyses with the indicated antibodies. Representative blots of three independent samples in each group were shown and quantification of relative protein expression was determined. F The mRNA levels of genes related to lipid metabolism and renal fibrosis in the kidneys from the indicated groups. Results are expressed as the mean ± SD (n = 6-8, *p < 0.05, UUO vs. Ctrl, #p < 0.05, UUO vs. UUO + AS1517499).

Article Snippet: The following antibodies were used: STAT6 (sc-374021), p-STAT6 (sc-136019), Arg-1 (sc166920), TGF-β (sc-146), α-SMA (sc-53142), FN (sc-18827), H3 (sc-517576), GAPDH (sc-32233) from Santa Cruz Biotechnology.

Techniques: Staining, Expressing, Western Blot