Journal: International Journal of Oncology

Article Title: Combined inhibition of IL-6 and IL-8 pathways suppresses ovarian cancer cell viability and migration and tumor growth

doi: 10.3892/ijo.2022.5340

Figure Lengend Snippet: Baze and SCH inhibit the expression of target genes downstream of the IL-6 and IL-8 pathways in human ovarian cancer cells. SKOV3 and CAOV3 cells were treated with DMSO, a single drug or their combination. The levels of p-STAT3, p-AKT, p-S6 and survivin were determined by western blot analysis. (A) CAOV3. (B) SKOV3. * P<0.05, ** P<0.01, *** P<0.001 and **** P<0.0001 (DMSO vs. Baze, SCH or Baze + SCH). B+S, Baze + SCH; B10, BAZE 10 µ M; B5, BAZE 5 µ M; BAZE, Baze; S50, SCH 50 µ M; p-phosphorylated; SCH, SCH.

Article Snippet: The following primary antibodies were purchased from Cell Signaling Technology, Inc. and diluted at 1:1,000 in 5% milk: Phosphorylated (p)-STAT3 (Y705; rabbit mAb; cat. no. 9131S), STAT3 (rabbit mAb; cat. no. 4904S), p-S6 (rabbit mAb; cat. no. 4858S), S6 (rabbit mAb; cat. no. 2217S), p-AKT (rabbit mAb; cat. no. 4060S), AKT (rabbit mAb; cat. no. 4691S), survivin (rabbit mAb; cat. no. 2808S) and GAPDH (rabbit mAb; cat. no. 2118S).

Techniques: Expressing, Western Blot

Journal: BMJ Open Diabetes Research & Care

Article Title: Anti-inflammatory, antioxidant and renoprotective effects of SOCS1 mimetic peptide in the BTBR ob/ob mouse model of type 2 diabetes

doi: 10.1136/bmjdrc-2020-001242

Figure Lengend Snippet: JAK/STAT, inflammatory and oxidative stress pathways in kidney tissue of BTBR ob/ob model. (A) Representative images of phosphorylated (p-)STAT3, p-p65 NF-κB and p-NRF2, and quantification of positive cells in glomerular and tubular fields of BTBR WT and ob/ob mice. Magnification ×400 and ×630. Arrows indicate positive stained cells. (B) Representative images of SOCS1/SOCS3 proteins and quantification of positive stained area per tubular field. Magnification ×200. (C) Real-time PCR analysis of JAK/STAT, inflammatory and oxidative stress genes. Values normalized by endogenous control gene 18s are expressed as n-fold of the average value from BTBR WT. Data are shown as scatter dot plots and mean±SD of each group (n=5–7 mice/group); *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 versus BTBR WT control. (D) Schematic protein–protein interaction prediction of JAK/STAT, inflammatory and oxidative stress markers in Mus musculus according to STRING software. More information can be found at https://string-db.org/ . a.u., arbitrary units; BTBR, black and tan brachyuric; GCS, glomerular cross section; JAK/STAT, Janus kinase/signal transducers and activators of transcription; NF-κB, nuclear factor-κB; NRF2, nuclear factor erythroid 2-related factor 2; ob/ob, obese/obese; SOCS, suppressor of cytokine signaling; WT, wild type.

Article Snippet: The primary antibodies for immunodetection were sourced as follows: phosphorylated (p-) STAT3 serine 727 (Cell Signaling Technology Cat# 9134, RRID:AB_331589, dilution 1:100), p-STAT1 tyrosine 701 (Cell Signaling Technology Cat# 7649, RRID:AB_10950970, dilution 1:50), p-p65 subunit of nuclear factor-κB (NF-κB) serine 536 (Santa Cruz Biotechnology Cat# sc-33020, RRID:AB_2179018, dilution 1:100), p-nuclear factor erythroid 2-related factor 2 (NRF2) serine 40 (Abcam Cat# ab76026, RRID:AB_1524049, dilution 1:2000), SOCS1 (Abcam Cat# ab62584, RRID:AB_956316, dilution 1:1000), SOCS3 (Abcam Cat# ab16030, RRID:AB_443287, dilution 1:200), F4/80 monocytes/macrophages (Bio-Rad Cat# MCA497, RRID:AB_2098196, dilution 1:70), CD3 T lymphocytes (Agilent Cat# M7254, RRID:AB_2631163, dilution 1:100), Wilms tumor protein-1 (WT-1; Agilent Cat# M3561, RRID:AB_2304486, dilution 1:100), perilipin-1 (sc-390169, dilution 1:50, Santa Cruz Biotechnology, USA) and 4-hydroxy-2-nonenal (4-HNE; Abcam Cat# ab46545, RRID:AB_722490, dilution 1:200).

Techniques: Staining, Real-time Polymerase Chain Reaction, Control, Software

Journal: BMJ Open Diabetes Research & Care

Article Title: Anti-inflammatory, antioxidant and renoprotective effects of SOCS1 mimetic peptide in the BTBR ob/ob mouse model of type 2 diabetes

doi: 10.1136/bmjdrc-2020-001242

Figure Lengend Snippet: MiS1 treatment inhibits kidney JAK/STAT activation and renal microinflammatory milieu in the BTBR ob/ob model. (A) Graphs and images represent the changes observed in JAK/STAT activation (p-STATs) in diabetic mice treated with active MiS1 (2 µg and 4 µg) and inactive mutated peptide (Mut 4 µg) compared with vehicle controls (Veh), quantified per number of positive cells p-STAT1+ and p-STAT3+, both at the glomerular and tubular fields. Magnification ×630. (B) Representative images of immunohistochemistry against F4/80 and CD3. Magnification ×200 and ×630. Graphs represent the quantification of average number of monocytes/macrophages F4/80+ and CD3+ T lymphocytes, both at the glomerular and interstitial fields. Arrows indicate positively stained cells. (C) Gene expression analysis of mRNA related with JAK/STAT pathway ( Stat1 , Stat3 , Socs1 and Socs3 ), inflammatory cytokines ( Tnfα and Il-12 ) and chemokines ( Mcp-1 and Rantes ), and kidney damage markers ( Kim-1 and Ngal ) were evaluated by real-time PCR, being normalized in each sample by endogenous control gene 18s and expressed as n-fold the average value obtained in the vehicle group (Veh). Data are shown as scatter dot plots and mean±SD of each group (n=6–7 mice/group); *p<0.05, **p<0.01, ****p<0.0001 versus diabetic vehicle control. BTBR, black and tan brachyuric; GCS, glomerular cross section; JAK/STAT, Janus kinase/signal transducers and activators of transcription; ob/ob, obese/obese.

Article Snippet: The primary antibodies for immunodetection were sourced as follows: phosphorylated (p-) STAT3 serine 727 (Cell Signaling Technology Cat# 9134, RRID:AB_331589, dilution 1:100), p-STAT1 tyrosine 701 (Cell Signaling Technology Cat# 7649, RRID:AB_10950970, dilution 1:50), p-p65 subunit of nuclear factor-κB (NF-κB) serine 536 (Santa Cruz Biotechnology Cat# sc-33020, RRID:AB_2179018, dilution 1:100), p-nuclear factor erythroid 2-related factor 2 (NRF2) serine 40 (Abcam Cat# ab76026, RRID:AB_1524049, dilution 1:2000), SOCS1 (Abcam Cat# ab62584, RRID:AB_956316, dilution 1:1000), SOCS3 (Abcam Cat# ab16030, RRID:AB_443287, dilution 1:200), F4/80 monocytes/macrophages (Bio-Rad Cat# MCA497, RRID:AB_2098196, dilution 1:70), CD3 T lymphocytes (Agilent Cat# M7254, RRID:AB_2631163, dilution 1:100), Wilms tumor protein-1 (WT-1; Agilent Cat# M3561, RRID:AB_2304486, dilution 1:100), perilipin-1 (sc-390169, dilution 1:50, Santa Cruz Biotechnology, USA) and 4-hydroxy-2-nonenal (4-HNE; Abcam Cat# ab46545, RRID:AB_722490, dilution 1:200).

Techniques: Activation Assay, Immunohistochemistry, Staining, Gene Expression, Real-time Polymerase Chain Reaction, Control

Journal: International Journal of Molecular Medicine

Article Title: SPHK1 contributes to cisplatin resistance in bladder cancer cells via the NONO/STAT3 axis

doi: 10.3892/ijmm.2021.5037

Figure Lengend Snippet: SPHK1 binds to the novel protein NONO and thus affects STAT3 expression in bladder cancer cells. (A) Co-immunoprecipitation confirmed that SPHK1 bound to certain proteins in bladder cancer cells. (B) Mass spectrogram of the target protein NONO. (C) NONO and its downstream target STAT3 were validated via western blotting ( ** P<0.01 and *** P<0.001). SPHK1, sphingosine kinase 1; NONO, non-POU domain containing octamer binding; IP, immunoprecipitation; Vector, negative control; OE, overexpression.

Article Snippet: After blocking with 5% skimmed milk for 2 h at room temperature, the membranes were incubated overnight at 4°C with the following primary antibodies: SPHK1 (cat. no. 10670-1-AP; ProteinTech Group, Inc.; 1:1,000), GAPDH (cat. no. AB0037; Shanghai Abways Biotechnology Co., Ltd.; 1:5,000;), NONO (cat. no. 385171; ZenBio, Inc.; 1:1,000), phosphorylated (p)-STAT3 (cat. no. 380906; ZenBio, Inc.; 1:1,000), Bax (cat. no. 50599-2-Ig; Cell Signaling Technology, Inc.; 1:1,000), Bcl2 (cat. no. 12789-1-AP; Cell Signaling Technology, Inc.; 1:1,000) and cleaved caspase-3 (cat. no. 9661; Cell Signaling Technology, Inc.; 1:1,000).

Techniques: Expressing, Immunoprecipitation, Western Blot, Binding Assay, Plasmid Preparation, Negative Control, Over Expression

Journal: International Journal of Molecular Medicine

Article Title: SPHK1 contributes to cisplatin resistance in bladder cancer cells via the NONO/STAT3 axis

doi: 10.3892/ijmm.2021.5037

Figure Lengend Snippet: SPHK1 inhibition reverses the antiapoptotic effect in bladder cancer cells. (A) Inhibition efficiency of SPHK1 inhibitor FTY-720 as determined via western blotting ( *** P<0.001). (B) Inhibition of SPHK1 decreased the expression of NONO and p-STAT3 ( *** P<0.001). (C) Inhibition of SPHK1 decreased apoptosis in bladder cancer cells ( *** P<0.001). SPHK1, sphingosine kinase 1; NONO, non-POU domain containing octamer binding; p-, phosphorylated; OE, overexpression.

Article Snippet: After blocking with 5% skimmed milk for 2 h at room temperature, the membranes were incubated overnight at 4°C with the following primary antibodies: SPHK1 (cat. no. 10670-1-AP; ProteinTech Group, Inc.; 1:1,000), GAPDH (cat. no. AB0037; Shanghai Abways Biotechnology Co., Ltd.; 1:5,000;), NONO (cat. no. 385171; ZenBio, Inc.; 1:1,000), phosphorylated (p)-STAT3 (cat. no. 380906; ZenBio, Inc.; 1:1,000), Bax (cat. no. 50599-2-Ig; Cell Signaling Technology, Inc.; 1:1,000), Bcl2 (cat. no. 12789-1-AP; Cell Signaling Technology, Inc.; 1:1,000) and cleaved caspase-3 (cat. no. 9661; Cell Signaling Technology, Inc.; 1:1,000).

Techniques: Inhibition, Western Blot, Expressing, Binding Assay, Over Expression

Journal: International Journal of Molecular Medicine

Article Title: SPHK1 contributes to cisplatin resistance in bladder cancer cells via the NONO/STAT3 axis

doi: 10.3892/ijmm.2021.5037

Figure Lengend Snippet: Validation of SPHK1 and its corresponding proteins in the T24/DDP cell line. (A) DPP-resistant T24 bladder cancer cells were established and drug resistance was detected by performing the CCK-8 assay (1 µ g/ml: *** P<0.001 T24/DDP vs. T24; 2 µ g/ml: *** P<0.001 T24/DDP vs. T24). (B) Apoptosis was analyzed in T24 vs. T24/DDP (1 µ g/ml: *** P<0.001 T24/DDP vs. T24; 2 µ g/ml: *** P<0.001 T24/DDP vs. T24). (C and D) SPHK1, NONO and p-STAT3 were overexpressed in the T24/DDP cell line ( *** P<0.001). Following SPHK1 knockdown in the T24/DDP cell line, the results of (E and F) western blotting, (G) flow cytometry and (H) CCK-8 assay were similar to those reported in previous studies ( * P<0.05, ** P<0.01 and *** P<0.001). SPHK1, sphingosine kinase 1; DPP, cisplatin; CCK-8, Cell Counting Kit-8; NONO, non-POU domain containing octamer binding; p-, phosphorylated; OE, overexpression.

Article Snippet: After blocking with 5% skimmed milk for 2 h at room temperature, the membranes were incubated overnight at 4°C with the following primary antibodies: SPHK1 (cat. no. 10670-1-AP; ProteinTech Group, Inc.; 1:1,000), GAPDH (cat. no. AB0037; Shanghai Abways Biotechnology Co., Ltd.; 1:5,000;), NONO (cat. no. 385171; ZenBio, Inc.; 1:1,000), phosphorylated (p)-STAT3 (cat. no. 380906; ZenBio, Inc.; 1:1,000), Bax (cat. no. 50599-2-Ig; Cell Signaling Technology, Inc.; 1:1,000), Bcl2 (cat. no. 12789-1-AP; Cell Signaling Technology, Inc.; 1:1,000) and cleaved caspase-3 (cat. no. 9661; Cell Signaling Technology, Inc.; 1:1,000).

Techniques: Biomarker Discovery, CCK-8 Assay, Knockdown, Western Blot, Flow Cytometry, Cell Counting, Binding Assay, Over Expression