Journal: Medicina

Article Title: Meta-Analysis of Survival Effects of Receptor Tyrosine Kinase-like Orphan Receptor 1 (ROR1)

doi: 10.3390/medicina58121867

Figure Lengend Snippet: Characteristics included for the study of ROR1.

Article Snippet: H. Chang (2015) [ ] , Republic of Korea , Gastric cancer , 424 , IHC , Rabbit polyclonal antibody (1:25; Abcam) , Staining > 50% , - , 0.8 (0.53–1.21) p = 0.189.

Techniques: Staining, Expressing, Fluorescence

Journal: Cell Death & Disease

Article Title: m 6 A methylation controls pluripotency of porcine induced pluripotent stem cells by targeting SOCS3/JAK2/STAT3 pathway in a YTHDF1/YTHDF2-orchestrated manner

doi: 10.1038/s41419-019-1417-4

Figure Lengend Snippet: a qPCR analysis of JAK2 and SOCS3 in control and METTL3 knockdown piPSCs. GAPDH was used as an internal control. b Western blot analysis of JAK2 and SOCS3 in piPSCs with or without METTL3 knockdown. β-Actin was used as loading control. c Western blot analysis of JAK2 and SOCS3 in piPSCs with or without METTL3 overexpression. d Western blot analysis of JAK2, pSTAT3, STAT3, KLF4, and SOX2 in piPSCs with or without METTL3 overexpression and transfected with negative control or JAK2 siRNA. e Western blot of nuclear and cytoplasmic distribution of pSTAT3, KLF4, and SOX2 in piPSCs with or without METTL3 overexpression and transfected with negative control or JAK2 siRNA. f qPCR analysis of SOX2 and KLF4 expression in piPSCs with or without METTL3 overexpression and transfected with negative control or JAK2 siRNA. g Western blot analysis of SOCS3, pSTAT3, STAT3, KLF4, and SOX2 in piPSCs with or without METTL3 knockdown and transfected with negative control or SOCS3 siRNA. h Western blot of nuclear and cytoplasmic distribution of pSTAT3, KLF4, and SOX2 in piPSCs with or without METTL3 knockdown and transfected with negative control or SOCS3 siRNA. Histone H3 and Tubulin serve as nuclear and cytoplasmic markers, respectively. i qPCR analysis of SOX2 and KLF4 expression in piPSCs with or without METTL3 knockdown and transfected with negative control or SOCS3 siRNA. Data were presented as mean ± SD of three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001 compared with the control group

Article Snippet: The primary antibodies used for western blot were as follows: METTL3 (1:2000, 15073-1-AP, Proteintech, IL, USA), SOCS3 (1:1000, 14025-1-AP, Proteintech, IL, USA), JAK2 (1:2000, M1501-8, Huabio, Hangzhou, China), pSTAT3 (1:2000, ab76315, Abcam, MA, USA), STAT3 (1:2000, ab68153, Abcam, MA, USA), SOX2 (1:500, sc-365964, Santa Cruz, CA, USA), KLF4 (1:2000, R1308-1, Huabio, Hangzhou, China), FLAG (1:1000, 20543-1-AP, Proteintech, IL, USA), YTHDF1 (1:1000, 17479-1-AP, Proteintech, IL, USA), YTHDF2 (1:1000, ABE542, Millipore, Darmstadt, Germany), Histone H3 (1:1000, 17168-1-AP, Proteintech, IL, USA), Tubulin (1:1000, 66031-1-Ig, Proteintech, IL, USA), β-Actin (1:1000, ab8227, Abcam, MA, USA). β-Actin was used as a loading control.

Techniques: Western Blot, Over Expression, Transfection, Negative Control, Expressing

Journal: Cell Death & Disease

Article Title: m 6 A methylation controls pluripotency of porcine induced pluripotent stem cells by targeting SOCS3/JAK2/STAT3 pathway in a YTHDF1/YTHDF2-orchestrated manner

doi: 10.1038/s41419-019-1417-4

Figure Lengend Snippet: a m 6 A dot blot analysis in piPSCs transfected with control, wild-type (WT), and mutant (MUT) METTL3 plasmid. Equal loading of mRNA was verified by methylene blue staining (lower panel). b Western blot analysis of METTL3, JAK2, and SOCS3 in piPSCs transfected with control, WT, and MUT METTL3 plasmid. β-Actin was used as loading control. c AP staining of piPSCs transfected with control, WT, and MUT METTL3 plasmid. d Quantification of AP-positive colonies of piPSCs transfected with control, WT, and MUT METTL3 plasmid. e qPCR analysis of piPSCs transfected with control, WT, and MUT METTL3 plasmid. GAPDH was used as an internal control. f Western blot analysis of KLF4 and SOX2 in piPSCs transfected with control, WT, and MUT METTL3 plasmid. g Top consensus motif identified by HOMER with m 6 A-seq peaks in piPSCs. h Distribution of m 6 A peaks across the length of mRNA transcripts. Each region of 5′UTRs, CDSs, and 3′UTRs were binned into 100 segments, and the percentage of m 6 A peaks that fall within each bin was determined. i The m 6 A abundances in JAK2 and SOCS3 mRNA transcripts in piPSCs as detected by m 6 A-seq. The m 6 A peaks were shown in the black rectangles. j Methylated RNA immunoprecipitation (MeRIP)-qPCR analysis of m 6 A levels of JAK2 and SOCS3 in piPSCs with or without METTL3 knockdown. k MeRIP-qPCR analysis of m 6 A levels of JAK2 and SOCS3 in piPSCs transfected with control or METTL3 plasmid. l Relative luciferase activity of WT or MUT (A-to-T mutation) SOCS3-3′UTR (or JAK2-3′UTR) luciferase reporter in piPSCs transfected with control, WT, or MUT METTL3 plasmid. Firefly luciferase activity was measured and normalized to Renilla luciferase activity. Data were presented as mean ± SD of three independent experiments. ** P < 0.01, *** P < 0.001 compared with the control group

Article Snippet: The primary antibodies used for western blot were as follows: METTL3 (1:2000, 15073-1-AP, Proteintech, IL, USA), SOCS3 (1:1000, 14025-1-AP, Proteintech, IL, USA), JAK2 (1:2000, M1501-8, Huabio, Hangzhou, China), pSTAT3 (1:2000, ab76315, Abcam, MA, USA), STAT3 (1:2000, ab68153, Abcam, MA, USA), SOX2 (1:500, sc-365964, Santa Cruz, CA, USA), KLF4 (1:2000, R1308-1, Huabio, Hangzhou, China), FLAG (1:1000, 20543-1-AP, Proteintech, IL, USA), YTHDF1 (1:1000, 17479-1-AP, Proteintech, IL, USA), YTHDF2 (1:1000, ABE542, Millipore, Darmstadt, Germany), Histone H3 (1:1000, 17168-1-AP, Proteintech, IL, USA), Tubulin (1:1000, 66031-1-Ig, Proteintech, IL, USA), β-Actin (1:1000, ab8227, Abcam, MA, USA). β-Actin was used as a loading control.

Techniques: Dot Blot, Transfection, Mutagenesis, Plasmid Preparation, Staining, Western Blot, Methylation, Immunoprecipitation, Luciferase, Activity Assay

Journal: Cell Death & Disease

Article Title: m 6 A methylation controls pluripotency of porcine induced pluripotent stem cells by targeting SOCS3/JAK2/STAT3 pathway in a YTHDF1/YTHDF2-orchestrated manner

doi: 10.1038/s41419-019-1417-4

Figure Lengend Snippet: a Western blot analysis of FLAG, YTHDF1, and JAK2 in piPSCs transfected with control and YTHDF1-FLAG plasmid. β-Actin was used as loading control. b RIP analysis of the interaction of JAK2 with FLAG in piPSCs transfected with YTHDF2-FLAG plasmid. Enrichment of JAK2 with FLAG was measured by qPCR and normalized to input. c Relative luciferase activity of WT or MUT JAK2-3′UTR luciferase reporter in piPSCs transfected with control or YTHDF2 plasmid. Firefly luciferase activity was measured and normalized to Renilla luciferase activity. d Western blot analysis of JAK2 in piPSCs transfected with control or YTHDF1 plasmid and treated with or without 20 n m rapamycin (Rap). e Western blot analysis of JAK2, KLF4, and JAK2 in piPSCs with or without METTL3 knockdown and transfected with control or YTHDF1 plasmid. f AP staining of piPSCs with or without METTL3 knockdown and transfected with control or YTHDF1 plasmid. g Quantification of AP-positive colonies of piPSCs with or without METTL3 knockdown and transfected with control or YTHDF1 plasmid. h qPCR analysis of SOX2 and KLF4 expression in piPSCs with or without METTL3 knockdown and transfected with control or YTHDF1 plasmid. Data were presented as mean ± SD of three independent experiments. ** P < 0.01, *** P < 0.001 compared with the control group

Article Snippet: The primary antibodies used for western blot were as follows: METTL3 (1:2000, 15073-1-AP, Proteintech, IL, USA), SOCS3 (1:1000, 14025-1-AP, Proteintech, IL, USA), JAK2 (1:2000, M1501-8, Huabio, Hangzhou, China), pSTAT3 (1:2000, ab76315, Abcam, MA, USA), STAT3 (1:2000, ab68153, Abcam, MA, USA), SOX2 (1:500, sc-365964, Santa Cruz, CA, USA), KLF4 (1:2000, R1308-1, Huabio, Hangzhou, China), FLAG (1:1000, 20543-1-AP, Proteintech, IL, USA), YTHDF1 (1:1000, 17479-1-AP, Proteintech, IL, USA), YTHDF2 (1:1000, ABE542, Millipore, Darmstadt, Germany), Histone H3 (1:1000, 17168-1-AP, Proteintech, IL, USA), Tubulin (1:1000, 66031-1-Ig, Proteintech, IL, USA), β-Actin (1:1000, ab8227, Abcam, MA, USA). β-Actin was used as a loading control.

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

Journal: Cell Death & Disease

Article Title: m 6 A methylation controls pluripotency of porcine induced pluripotent stem cells by targeting SOCS3/JAK2/STAT3 pathway in a YTHDF1/YTHDF2-orchestrated manner

doi: 10.1038/s41419-019-1417-4

Figure Lengend Snippet: m 6 A methyltransferase METTL3 increases the m 6 A levels of JAK2 and SOCS3 mRNA, leading to enhancing YTHDF1-mediated translation of JAK2 and attenuating YTHDF2-dependent mRNA stability of SOCS3, resulting in increased protein expression of JAK2 and decreased protein expression of SOCS3, thereby activating STAT3 phosphorylation and enhancing expression of core pluripotency genes KLF4 and SOX2 to facilitates piPSCs pluripotency

Article Snippet: The primary antibodies used for western blot were as follows: METTL3 (1:2000, 15073-1-AP, Proteintech, IL, USA), SOCS3 (1:1000, 14025-1-AP, Proteintech, IL, USA), JAK2 (1:2000, M1501-8, Huabio, Hangzhou, China), pSTAT3 (1:2000, ab76315, Abcam, MA, USA), STAT3 (1:2000, ab68153, Abcam, MA, USA), SOX2 (1:500, sc-365964, Santa Cruz, CA, USA), KLF4 (1:2000, R1308-1, Huabio, Hangzhou, China), FLAG (1:1000, 20543-1-AP, Proteintech, IL, USA), YTHDF1 (1:1000, 17479-1-AP, Proteintech, IL, USA), YTHDF2 (1:1000, ABE542, Millipore, Darmstadt, Germany), Histone H3 (1:1000, 17168-1-AP, Proteintech, IL, USA), Tubulin (1:1000, 66031-1-Ig, Proteintech, IL, USA), β-Actin (1:1000, ab8227, Abcam, MA, USA). β-Actin was used as a loading control.

Techniques: Expressing

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Salvianolic acid B combined with bone marrow mesenchymal stem cells piggybacked on HAMA hydrogel re-transplantation improves intervertebral disc degeneration

doi: 10.3389/fbioe.2022.950625

Figure Lengend Snippet: (A) Western-blotting assays show that SalB activates the JAK2-STAT3 signaling pathway, (B–E) : Western-blotting grayscale values show that SalB activates this pathway by promoting JAK2 and STAT3 phosphorylation, while WP1066 can inhibit this activation effect. n = 3, *: compared with control group, * p < 0.05, ** p < 0.001; #: compared with H2 O 2 group, # p < 0.05, ## p < 0.001; &: compared with Sal B + H 2 O 2 group, & p < 0.05, && p < 0.001.

Article Snippet: JAK2, p-JAK2, STAT3, p-STAT3, BAX, and Bcl2 antibodies were purchased from Affinity Biosciences (China), DAPI and FITC-labeled ghost cyclic peptides were purchased from Sloarbio (China), and the Calcein-AM/PI Double Stain Kit was purchased from Yeasen Biotechnology (China), Sprague-Dawley (SD) rat bone marrow MSC osteogenesis, adipogenesis, and chondrogenesis differentiation kits were purchased from Cyagen Biotech (China).

Techniques: Western Blot, Phospho-proteomics, Activation Assay, Control

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Salvianolic acid B combined with bone marrow mesenchymal stem cells piggybacked on HAMA hydrogel re-transplantation improves intervertebral disc degeneration

doi: 10.3389/fbioe.2022.950625

Figure Lengend Snippet: (A) Western-blotting detects that SalB can regulate the expression of BAX and Bcl2 proteins through JAK2-STAT3 signaling pathway, (B,C) : By analyzing the grayscale values of Western-blotting, it shows that SalB can reduce the expression of BAX and elevate Bcl2 at the same time, inhibit H 2 O 2 -induced apoptosis, and WP1066 could inhibit this effect . n = 3,*:compared with control group,* p < 0.05,** p < 0.001;#:compared with H 2 O 2 group, # p < 0.05,## p < &:compared with Sal B + H 2 O 2 group, & p < 0.05,&& p < 0.001.

Article Snippet: JAK2, p-JAK2, STAT3, p-STAT3, BAX, and Bcl2 antibodies were purchased from Affinity Biosciences (China), DAPI and FITC-labeled ghost cyclic peptides were purchased from Sloarbio (China), and the Calcein-AM/PI Double Stain Kit was purchased from Yeasen Biotechnology (China), Sprague-Dawley (SD) rat bone marrow MSC osteogenesis, adipogenesis, and chondrogenesis differentiation kits were purchased from Cyagen Biotech (China).

Techniques: Western Blot, Expressing, Control

Journal: The Journal of Biological Chemistry

Article Title: A thiazole-derived oridonin analogue exhibits antitumor activity by directly and allosterically inhibiting STAT3

doi: 10.1074/jbc.RA119.009801

Figure Lengend Snippet: CYD0618 disturbs the function of STAT3–SH2 domain via covalent modification of Cys-542. A and F, recombinant His–WT–STAT3 or His–C542S–STAT3 was treated with DMSO or CYD0618 for 1 h at room temperature. The mixtures were then incubated with the indicated purified FLAG kinases for 1 h at room temperature. Immunoblotting (IB) using the pTyr-705 STAT3 antibody reflects the effects of CYD0618 on kinase-induced phosphorylation of WT–STAT3 (A) or C542S–STAT (F) in vitro. B and C, CYD0618 inhibits the association of upstream kinases and STAT3. A2780 cells were pre-treated with CYD0618 (3 μm) for 2 h before stimulation with IL-6 (10 ng/ml) or EGF (20 ng/ml) for 30 min. Afterward, the whole-cell extracts were subjected to immunoprecipitation (IP) and immunoblotted with the indicated antibodies. D, C542S mutation regains the association of EGFR and STAT3. HeLa cells transfected with WT–STAT3 or C542S–STAT3 vector were pre-treated with CYD0618 for 2 h, followed by incubation of EGF (20 ng/ml) for 30 min. The whole-cell lysates were subjected to immunoprecipitation and immunoblotted with the indicated antibodies. E, effects of CYD0618 on the binding of Ac-pYLPQTV-NH2 to WT–STAT3 or C542S–STAT3 using the pulldown assay. Following incubation of CYD0618 for 1 h at room temperature, the recombinant His–WT–STAT3 or His–C542S–STAT3 was incubated with biotinylated Ac-pYLPQTV-NH2 (PPB) or biotinylated Ac-YLPQTV-NH2 (PB) and streptavidin beads for 1 h at room temperature. The mixtures were then subjected to immunoblotted with His antibody. G, representative global view of the GSH-mediated allosteric effect of STAT3 protein (PDB code 4E68). STAT3 protein alone is shown by the gray cartoon model; GSH–STAT3 complex is shown by the orange model. The disulfide bond between GSH and Cys-542 is identified and indicated by yellow line. H, representative view of the conformation change of the SH2 domain in STAT3 protein. The STAT3 protein alone is shown in gray, and GSH–STAT3 complex is shown in orange. The critical residues Lys-591, Arg-609, Ser-611, and Ser-613 are identified and indicated. The interaction between these two residues and pTyr-705 (stick model) of other STAT3 protein is also indicated.

Article Snippet: Antibodies Phospho-STAT1 (Tyr-701), phospho-STAT3 (Tyr-705), phospho-STAT3 (Ser-723), phospho-STAT5 (Tyr-694), phospho-JAK1 (Tyr-1034), phospho-JAK2 (Tyr-1007), phospho-EGFR (Tyr-1068), phospho-Src (Tyr-418), STAT1, STAT3, STAT5, JAK1, JAK2, EGFR, Src, Bcl-2, cyclin D1, c-Myc, survivin, MMP2, and MMP9 antibodies were from Signalway Antibody (Baltimore, MD).

Techniques: Modification, Recombinant, Incubation, Purification, Western Blot, Phospho-proteomics, In Vitro, Immunoprecipitation, Mutagenesis, Transfection, Plasmid Preparation, Binding Assay