stat5b sirna  (OriGene)

Journal: EMBO Reports

Article Title: The Derlin-1-Stat5b axis maintains homeostasis of adult hippocampal neurogenesis

doi: 10.1038/s44319-024-00205-7

Figure Lengend Snippet: ( A ) Experimental scheme to induce the transition of control and Derl1 knockdown mouse hippocampal NSCs from active to quiescent states. ( B ) Representative images of EdU (red) and Hoechst (blue) staining in siControl and siDerl1 mouse NSCs with or without induction of quiescence with diazepam (100 μM) or BMP4 (50 ng/mL) for 2 days. NSCs were fixed 30 min after the addition of EdU. Scale bars: 50 μm. ( C ) Quantification of the percentage of EdU + proliferating NSCs among total Hoechst + cells in siControl and siDerl1 mouse NSCs with proliferative conditions or induction of quiescence with diazepam or BMP4 for 2 days ( n = 3 biological replicates). 2WA two-way ANOVA, k knockdown, c condition. ( D ) Experimental scheme for investigating the Stat5b expression of control and Derl1 knockdown mouse NSCs from active to quiescent states. ( E ) Expression of Stat5b in siControl and siDerl1 mouse NSCs under proliferation and quiescent conditions. Gene expression levels were estimated by qPCR and normalized to that of S18 ( n = 4 biological replicates; Proliferation, n = 3 biological replicates; Quiescence). ( F ) Experimental scheme to investigate NSC proliferation with conditioned medium derived from control and Derl1 knockdown NSCs over 2 days. ( G ) Quantification of the percentage of EdU + proliferating NSCs among total Hoechst + cells cultured for 2 days in siControl and siDerl1 NSC–derived conditioned quiescence medium ( n = 4 biological replicates). ( H ) Experimental scheme to investigate NSC proliferation of control and Derl1 knockdown NSCs with conditioned medium derived from wild-type (WT) NSCs over 2 days. ( I ) Quantification of the percentage of EdU + proliferating NSCs among total Hoechst + cells cultured for 2 days in siControl and siDerl1 NSCs with WT NSC–derived conditioned quiescence medium ( n = 3 biological replicates). Bar graphs are presented as the mean ± SEM. Significance was determined using Student’s t -test ( E , G , I ) or two-way ANOVA ( C ). * P < 0.05 and ** P < 0.01 by Student’s t -test ( E , I ). n.s. not significant.

Article Snippet: The following siRNAs were used for the knockdown of adult rat- and mouse-derived hippocampal NSCs: Stealth RNAi™ siRNA Derl1-MSS289837 (Invitrogen), Stealth RNAi™ siRNA Stat5b-RSS332572 (Invitrogen).

Techniques: Control, Knockdown, Staining, Expressing, Derivative Assay, Cell Culture

Journal: EMBO Reports

Article Title: The Derlin-1-Stat5b axis maintains homeostasis of adult hippocampal neurogenesis

doi: 10.1038/s44319-024-00205-7

Figure Lengend Snippet: ( A ) Experimental scheme for investigating the molecular mechanism underlying the impairment of NSC transition to quiescence by Derl1 knockdown. ( B ) GSEA showing differential expression of 92 genes in the NSCs categorized by the GO term “Response to ER stress.” GSEA shows gene expression changes in siDerl1 NSCs relative to siControl NSCs. The enrichment plot shows the distribution of genes in each set that are positively (red) or negatively (blue) correlated with Derl1 knockdown. ( C ) Venn diagrams showing the overlap between transcription factor (TF) genes and upregulated (left) or downregulated (right) genes in siDerl1 NSCs. ( D ) Quantification of the efficiency of each viral infection in siControl and siDerl1 NSCs ( n = 4 biological replicates). ( E ) Quantification of the percentage of EdU + proliferating NSCs among total GFP + cells in siControl and siDerl1 NSCs with or without exogenous expression of mutant Stat5b (Y699F) [ n = 5 biological replicates; + Control, n = 4 biological replicates; + Stat5b (Y699F)]. ( F ) GSEA showing differential expression of 80 candidate Stat5b target genes. GSEA shows gene expression changes in siDerl1 NSCs relative to siControl NSCs. The enrichment plot shows the distribution of genes in each set that are positively (red) or negatively (blue) correlated with Derl1 knockdown. Bar graphs are presented as the mean ± SEM. Significance was determined using the nominal P value of GSEA software ( B , F ) or one-way ANOVA ( E ). * P < 0.05 and ** P < 0.01 by one-way ANOVA followed by Bonferroni’s post hoc test ( E ). n.s. not significant.

Article Snippet: The following siRNAs were used for the knockdown of adult rat- and mouse-derived hippocampal NSCs: Stealth RNAi™ siRNA Derl1-MSS289837 (Invitrogen), Stealth RNAi™ siRNA Stat5b-RSS332572 (Invitrogen).

Techniques: Knockdown, Expressing, Infection, Mutagenesis, Control, Software

Journal: EMBO Reports

Article Title: The Derlin-1-Stat5b axis maintains homeostasis of adult hippocampal neurogenesis

doi: 10.1038/s44319-024-00205-7

Figure Lengend Snippet: ( A ) Experimental scheme for evaluating the relevance of Stat5b underlying the impairment of NSC transition to quiescence by Derl1 knockdown. ( B ) Expression of Stat5b in siControl and siDerl1 NSCs under proliferation and quiescent conditions. Gene expression levels were estimated by qPCR and normalized to that of β-actin ( n = 3 biological replicates). ( C ) Representative immunoblots (IB) of siControl, siDerl1, and siStat5b NSCs after induction of quiescence. Whole-cell lysates were analyzed by IB with Stat5b and actin antibodies. ( D ) Representative images of EdU (red), Ki67 (green), and Hoechst (blue) staining in siControl and siStat5b NSCs after induction of quiescence with BMP4 for 2 days. NSCs were fixed 30 min after the addition of EdU. Scale bars: 50 m. ( E , F ) Quantification of the percentages of EdU + ( E ) and Ki67 + ( F ) proliferating NSCs among total Hoechst + cells in siControl and siStat5b NSCs after induction of quiescence with BMP4 for 2 days ( n = 3 biological replicates). ( G ) Experimental scheme for investigating the requirement of Stat5b for impairment of NSC transition to quiescence by Derl1 knockdown. ( H ) Representative images of GFP (green), EdU (red), and Hoechst (gray; insets) staining in siControl and siDerl1 quiescence-conditioned NSCs with or without exogenous Stat5b expression. NSCs were fixed 30 min after the addition of EdU. Scale bars: 50 μm. ( I ) Quantification of the percentage of EdU + proliferating NSCs among total GFP + cells in siControl and siDerl1 NSCs with or without exogenous Stat5b expression ( n = 4 biological replicates). ( J ) Experimental scheme for assessing the effect of Stat5b expression in the DG on NSC proliferation in Derl1 NesCre mice. ( K ) Representative immunofluorescence images of the DG with HA (red), Sox2 (cyan), Ki67 (green), and Hoechst staining (gray; insets). Scale bars: 25 μm. ( L ) Quantification of the number of Ki67 + proliferating cells in the SGZ of Derl1 NesCre mice with or without exogenous Stat5b expression ( n = 3 mice). ( M ) Quantification of the percentage of Ki67 + Sox2 + HA + proliferating NS/PCs among total Sox2 + HA + cells in the DG of Derl1 NesCre mice with or without exogenous Stat5b expression ( n = 3 mice). Bar graphs are presented as the mean ± SEM. * P < 0.05, ** P < 0.01, and *** P < 0.001 by Student’s t -test ( B , E , F , L , M ) or one-way ANOVA followed by Bonferroni’s post hoc test ( I ). .

Article Snippet: The following siRNAs were used for the knockdown of adult rat- and mouse-derived hippocampal NSCs: Stealth RNAi™ siRNA Derl1-MSS289837 (Invitrogen), Stealth RNAi™ siRNA Stat5b-RSS332572 (Invitrogen).

Techniques: Knockdown, Expressing, Western Blot, Staining, Immunofluorescence

Journal: EMBO Reports

Article Title: The Derlin-1-Stat5b axis maintains homeostasis of adult hippocampal neurogenesis

doi: 10.1038/s44319-024-00205-7

Figure Lengend Snippet: ( A ) Experimental scheme for evaluating the effect of 4-PBA on the impairment of NSC transition to quiescence by Derl1 knockdown. ( B ) Representative images of Hoechst (gray) and EdU (red) staining in siControl and siDerl1 NSCs treated with or without 4-PBA (1 mM). NSCs were fixed 30 min after the addition of EdU. Scale bars: 50 μm. ( C ) Quantification of the percentage of EdU + proliferating NSCs among total Hoechst + cells in 4-PBA-treated siControl and siDerl1 NSCs induced to enter the quiescent state by the administration of BMP4 for 2 days ( n = 3 biological replicates; Vehicle, n = 4 biological replicates; 4-PBA). ( D ) Experimental scheme for assessing the expression of Stat5b in siControl and siDerl1 NSCs treated with or without 4-PBA. ( E ) Expression of Stat5b in siControl and siDerl1 NSCs with or without 4-PBA (1 mM) treatment. Gene expression levels were estimated by qPCR and normalized to that of β-actin ( n = 5 biological replicates; Vehicle, n = 4 biological replicates; 4-PBA). ( F ) Representative images of Hoechst (gray) and EdU (red) staining in siDerl1, siControl, and siDerl1, siStat5b NSCs treated with or without 4-PBA (1 mM). NSCs were fixed 30 min after the addition of EdU. Scale bars: 50 μm. ( G ) Quantification of the percentage of EdU + proliferating NSCs among total Hoechst + cells in 4-PBA-treated siDerl1, siControl, and siDerl1, siStat5b NSCs induced to enter the quiescent state by the administration of BMP4 for 2 days ( n = 3 biological replicates). Bar graphs are presented as the mean ± SEM. * P < 0.05, ** P < 0.01, and *** P < 0.001 by one-way ANOVA followed by Bonferroni’s post hoc test. n.s. not significant. .

Article Snippet: The following siRNAs were used for the knockdown of adult rat- and mouse-derived hippocampal NSCs: Stealth RNAi™ siRNA Derl1-MSS289837 (Invitrogen), Stealth RNAi™ siRNA Stat5b-RSS332572 (Invitrogen).

Techniques: Knockdown, Staining, Expressing

Journal: EMBO Reports

Article Title: The Derlin-1-Stat5b axis maintains homeostasis of adult hippocampal neurogenesis

doi: 10.1038/s44319-024-00205-7

Figure Lengend Snippet: ( A ) Experimental scheme for assessing the expression of Stat5b in siControl and siDerl1 NSCs treated with or without TUDCA (50 μM), trehalose (10 mM), VPA (1 mM), or VPM (1 mM). ( B – E ) Expression of Stat5b in siControl and siDerl1 NSCs with or without TUDCA ( B ) ( n = 4 biological replicates; Vehicle, n = 3 biological replicates; TUDCA), trehalose ( C ) ( n = 4 biological replicates), VPA ( D ) ( n = 3 biological replicates), or VPM ( E ) treatment ( n = 3 biological replicates). Gene expression levels were estimated by qPCR and normalized to that of β-actin . ( F ) Experimental scheme for evaluating the effect of VPA (1 mM) or TUDCA (50 μM) on the impairment of the transition of NSCs to quiescence by Derl1 knockdown. ( G , H ) Quantification of the percentage of EdU + proliferating NSCs among total Hoechst + cells in VPA-treated ( G ) ( n = 3 biological replicates) or TUDCA-treated ( H ) ( n = 4 biological replicates; Vehicle, n = 5 biological replicates; TUDCA) siControl and siDerl1 NSCs induced to enter the quiescent state by the administration of BMP4 for 2 days. ( I ) Experimental scheme for investigating the proliferation of NS/PCs in Derl1 NesCre mice with or without VPA treatment. Derl1 NesCre mice treated with vehicle or VPA daily for 2 weeks were simultaneously injected with BrdU daily for 7 days during the latter and fixed 1 day after the last BrdU injection. ( J ) Representative immunofluorescence images of the DG stained for BrdU (red) and Hoechst (blue) in Derl1 NesCre mice treated with or without VPA. Scale bars: 100 μm. ( K ) Quantification of the number of BrdU + proliferating cells in the SGZ of Derl1 NesCre mice treated with or without VPA ( n = 3 mice). ( L ) Representative immunofluorescence images of the DG stained for DCX (cyan) and Hoechst (gray; insets) in Derl1 NesCre mice treated with or without VPA. The areas outlined by a white rectangle are enlarged to the right. The yellow arrowhead indicates DCX + ectopic immature neurons in the hilus, and dashed white lines indicate the boundaries between the GCL and hilus. Scale bars, 100 μm (left images) and 20 μm (right images). ( M ) Quantification of the number of DCX + cells in the hilus in Derl1 NesCre mice treated with or without VPA ( n = 3 mice). ( N ) Expression of Chop in the DG of 2-month-old Derl1 f/f and Derl1 NesCre mice. Gene expression levels were estimated by qPCR and normalized to that of S18 ( n = 7; Derl1 f/f mice, n = 6; Derl1 NesCre mice). Bar graphs are presented as the mean ± SEM. * P < 0.05, ** P < 0.01, and *** P < 0.001 by one-way ANOVA followed by Bonferroni’s post hoc test ( B – E , G , H ) or Student’s t -test ( K , M , N ). n.s. not significant.

Article Snippet: The following siRNAs were used for the knockdown of adult rat- and mouse-derived hippocampal NSCs: Stealth RNAi™ siRNA Derl1-MSS289837 (Invitrogen), Stealth RNAi™ siRNA Stat5b-RSS332572 (Invitrogen).

Techniques: Expressing, Knockdown, Injection, Immunofluorescence, Staining

Journal: Journal of Cancer

Article Title: The Role of MiR-5094 as a Proliferation Suppressor during Cellular Radiation Response via Downregulating STAT5b

doi: 10.7150/jca.39679

Figure Lengend Snippet: MiR-5094 directly targets STAT5b. (A) Alignment of wild-type seed sequence of the 3'-UTR of STAT5b mRNA (WT STAT5b 3'-UTR) and a mutated seed sequence of the miR-5094-binding site (Mut STAT5b 3'-UTR). The seed region is shown in bold. (B) Luciferase reporter assays. Luciferase reporter containing wild-type or mutant STAT5b 3'UTR was co-transfected with exogenous miR-5094 mimics (miR-5094) or negative mock control (NC) into HeLa cells. Luciferase activity was measured 24 h after transfection. Renilla luciferase activity was used to normalize the firefly luciferase activity. (C) MiR-5094 suppresses STAT5b mRNA expression in different cells at 24 h after transfection. The relative expression levels were normalized to same cells transient transfected with NC at same time point. (D) MiR-5094 suppresses STAT5b protein expression in different cells at 24 h after transfection. Mcs: miR-5094 mimics; inhibitor: miR-5094 inhibitor; si-1, si-2 and si-3: STAT5b siRNA. *P < 0.05 and **P < 0.01 represent the comparison with NC.

Article Snippet: For HeLa and Beas-2B cells, 2×10 5 cells were seeded in 35mm dishes and cultured for 24 h prior to be transfected with small RNAs including miR-5094 mimics (GeneCopoeia, HmiR1458, China), negative control (GeneCopoeia, CmiR0001-MR04), miR-5094 inhibitor (GeneCopoeia, HmiR-AN2605), and the inhibitor negative control (GeneCopoeia, CmiR-AN0001-AM01) or STAT5b siRNA (RioNeer, 1145682, 1145685, 1145687, CA, USA) and its negative control (BioNeer, Daejeon, Korea) at 40-60% confluence using Lipofectamine TM 2000 (Invitrogen, 11668-019, USA).

Techniques: Sequencing, Binding Assay, Luciferase, Mutagenesis, Transfection, Control, Activity Assay, Expressing, Comparison

Journal: Journal of Cancer

Article Title: The Role of MiR-5094 as a Proliferation Suppressor during Cellular Radiation Response via Downregulating STAT5b

doi: 10.7150/jca.39679

Figure Lengend Snippet: Radiation induces increase expression of miR-5094 and decrease expression of STAT5b. (A) STAT5b and miR-5094 expression in HeLa cells at different time points after radiation. U6 was used as control of miR-5094 expression, and GAPDH mRNA was used as control of STAT5b mRNA. (B) Expression of miR-5094 and STAT5b mRNA in HeLa cells after different dosages of irradiation treatment. U6 and GAPDH were used as controls. (C) Expression of miR-34a, miR-134, miR-150-5p and miR-200a after radiation in HeLa cells. The qRT-PCR was conducted to quantify the expression levels of miR-34a, miR-134, miR-150-5p and miR-200a at 12 h and 24 h after 2 Gy X-rays. U6 was used as controls. *P < 0.05 or **P < 0.01 represents statistical significance of comparison against non-irradiated control.

Article Snippet: For HeLa and Beas-2B cells, 2×10 5 cells were seeded in 35mm dishes and cultured for 24 h prior to be transfected with small RNAs including miR-5094 mimics (GeneCopoeia, HmiR1458, China), negative control (GeneCopoeia, CmiR0001-MR04), miR-5094 inhibitor (GeneCopoeia, HmiR-AN2605), and the inhibitor negative control (GeneCopoeia, CmiR-AN0001-AM01) or STAT5b siRNA (RioNeer, 1145682, 1145685, 1145687, CA, USA) and its negative control (BioNeer, Daejeon, Korea) at 40-60% confluence using Lipofectamine TM 2000 (Invitrogen, 11668-019, USA).

Techniques: Expressing, Control, Irradiation, Quantitative RT-PCR, Comparison

Journal: Journal of Cancer

Article Title: The Role of MiR-5094 as a Proliferation Suppressor during Cellular Radiation Response via Downregulating STAT5b

doi: 10.7150/jca.39679

Figure Lengend Snippet: Influence of miR-5094 on expression of STAT5b and its down-stream genes. (A) Expression of miR-5094 and mRNA levels of STAT5b, Bcl-2, Cyclin D1, Igf-1 and p21 in HeLa cells at 24 h after radiation. U6 and GADPH were used as controls. (B) Protein expression of STAT5b, Bcl-2, Cyclin D1, Igf-1 and p21 in HeLa cells at 24 h after radiation. NC: Negative mock control; 5094 mcs: miR-5094 mimics. *P < 0.05 and **P < 0.01 represent the comparison with NC, while #P < 0.05 represent the comparison with NC plus 2 Gy X-rays (NC-2Gy).

Article Snippet: For HeLa and Beas-2B cells, 2×10 5 cells were seeded in 35mm dishes and cultured for 24 h prior to be transfected with small RNAs including miR-5094 mimics (GeneCopoeia, HmiR1458, China), negative control (GeneCopoeia, CmiR0001-MR04), miR-5094 inhibitor (GeneCopoeia, HmiR-AN2605), and the inhibitor negative control (GeneCopoeia, CmiR-AN0001-AM01) or STAT5b siRNA (RioNeer, 1145682, 1145685, 1145687, CA, USA) and its negative control (BioNeer, Daejeon, Korea) at 40-60% confluence using Lipofectamine TM 2000 (Invitrogen, 11668-019, USA).

Techniques: Expressing, Control, Comparison

Journal: Journal of Cancer

Article Title: The Role of MiR-5094 as a Proliferation Suppressor during Cellular Radiation Response via Downregulating STAT5b

doi: 10.7150/jca.39679

Figure Lengend Snippet: miR-5094 regulates cellular radiation response by targeting STAT5b. (A) Survival fraction of HeLa cells. Left graph shows quantitative bar of colony formation assay; right graph is plates of colony formation assay . (B) Cell viability assay of HeLa cells at 48 h after 2 Gy X-rays. (C) The apoptotic rate of HeLa cells at 24 h after 2 Gy X-rays. (D) Cell cycle distribution of HeLa cells treated with 2 Gy of X-rays at 24 h post-irradiation. (E) Proliferation curves of Jurkat cells. (F) The expression of STAT5b in HeLa cells and Jurkat cells at 24 h after radiation. None: non-transfected control; NC: negative mock control; mcs: miR-5094 mimics; siRNA: STAT5b siRNA; inhibitor: miR-5094 inhibitor. *P < 0.05 and **P < 0.01 represent comparison with NC; #P < 0.05 represent comparison with NC plus 2 Gy X-rays (NC-R).

Article Snippet: For HeLa and Beas-2B cells, 2×10 5 cells were seeded in 35mm dishes and cultured for 24 h prior to be transfected with small RNAs including miR-5094 mimics (GeneCopoeia, HmiR1458, China), negative control (GeneCopoeia, CmiR0001-MR04), miR-5094 inhibitor (GeneCopoeia, HmiR-AN2605), and the inhibitor negative control (GeneCopoeia, CmiR-AN0001-AM01) or STAT5b siRNA (RioNeer, 1145682, 1145685, 1145687, CA, USA) and its negative control (BioNeer, Daejeon, Korea) at 40-60% confluence using Lipofectamine TM 2000 (Invitrogen, 11668-019, USA).

Techniques: Colony Assay, Viability Assay, Irradiation, Expressing, Transfection, Control, Comparison

Journal: Journal of Cancer

Article Title: The Role of MiR-5094 as a Proliferation Suppressor during Cellular Radiation Response via Downregulating STAT5b

doi: 10.7150/jca.39679

Figure Lengend Snippet: miR-5094 regulates T cells proliferation by targeting STAT5b. (A) Proliferation curves of CD4 + CD25 + T cells from human Peripheral blood. (B) Proliferation curves of CD4 + T cells from human Peripheral blood. (C) Percentage of CD4 + CD25 + T cells in CD4 + T cells at 72 h after radiation. (D) Expression of miR-5094 and STAT5b mRNA in CD4 + T cells at 24 h after radiation. U6 and GADPH mRNAs were used as controls. (E) Expression and activation of STAT5b in CD4 + T cells at 24 h after radiation. Cells were cultured with anti-CD3/CD28-coated beads (1:4) and subjected to IL-2 (20 U/mL) stimulation as indicated. NC: negative mock control; mcs: miR-5094 mimics; inhib: miR-5094 inhibitor; R: 2 Gy X-rays. *P < 0.05 and **P < 0.01 represent the comparison with NC.

Article Snippet: For HeLa and Beas-2B cells, 2×10 5 cells were seeded in 35mm dishes and cultured for 24 h prior to be transfected with small RNAs including miR-5094 mimics (GeneCopoeia, HmiR1458, China), negative control (GeneCopoeia, CmiR0001-MR04), miR-5094 inhibitor (GeneCopoeia, HmiR-AN2605), and the inhibitor negative control (GeneCopoeia, CmiR-AN0001-AM01) or STAT5b siRNA (RioNeer, 1145682, 1145685, 1145687, CA, USA) and its negative control (BioNeer, Daejeon, Korea) at 40-60% confluence using Lipofectamine TM 2000 (Invitrogen, 11668-019, USA).

Techniques: Expressing, Activation Assay, Cell Culture, Control, Inhibition, Comparison

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: Transcriptional Regulation of Nos2 via STAT5B Binding to Nos2 Gene Promoter Mediates Nitric Oxide Production: Relevance in β-Cell Maintenance.

doi: 10.33594/000000010

Figure Lengend Snippet: Fig. 1. STAT5B is a transcriptional regulator of Nos2. A) Chromosome ideogram of mouse Stat5b and Nos2 on chromosome 11(http://genome.ucsc.edu/). B) Cartoon representation of the putative binding site of STAT5B on Nos2 promoter, as identified from ChampionChiP Transcription Factor Search Portal (SABiosciences). C) Chromatin immunoprecipitation in MIN6 cells identified Nos2 as a direct target of STAT5B. Lane 1 - Input DNA, lane 2 - mouse control IgG, lane 3 - anti-RNA polymerase II antibody, lane 4 - anti-STAT5B antibody, lane 5 - DNA ladder. The image is a representation of experiment done thrice independently. D) Bar graph representation of 2-ΔΔCt of Stat5b and Nos2 transcripts showing downregulation in prolactin-induced Stat5b siRNA treated versus control siRNA treated MIN6 cells, calculated with respect to untreated cells and 18SrRNA as the endogenous control. (n = 3). E) Western blots and F) densitometric analysis of STAT5B and NOS2, showing a downregulation in PRL-induced siRNA treated samples, with actin as the loading control (n = 3). P-value was calculated using Student’s t-test.

Article Snippet: Control siRNA (sc-37007) and Stat5b siRNA (sc-37011) were from Santa Cruz Biotechnology (SCBT), Dallas, TX, USA.

Techniques: Binding Assay, Chromatin Immunoprecipitation, Control, Western Blot

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: Transcriptional Regulation of Nos2 via STAT5B Binding to Nos2 Gene Promoter Mediates Nitric Oxide Production: Relevance in β-Cell Maintenance.

doi: 10.33594/000000010

Figure Lengend Snippet: Fig. 4. Impact of Stat5b silencing on SOD2 and H2O2. A) Bar graph representation of increased relative transcript levels (2-

Article Snippet: Control siRNA (sc-37007) and Stat5b siRNA (sc-37011) were from Santa Cruz Biotechnology (SCBT), Dallas, TX, USA.

Techniques:

Journal: Cell Death & Disease

Article Title: Inhibition of STAT5A promotes osteogenesis by DLX5 regulation

doi: 10.1038/s41419-018-1184-7

Figure Lengend Snippet: a Protein levels of STAT5A, STAT5B, DLX5, and RUNX2 during osteogenesis of hBMSCs. b Alizarin Red S staining demonstrating the effect of STAT5 overexpression on the osteogenesis at day 14. Scale bar, 60 μm. c Quantification of Alizarin Red S staining. d Western blot analysis of protein expression of osteogenic master genes, DLX5 and Runx2 after STAT5 overexpression. GAPDH was used as a control. e Quantification of DLX5 and RUNX2 protein levels. f Gene expression of DLX5 and expression of downstream genes of DLX5 after 4 days with STAT5 overexpression in hBMSCs using real-time PCR. All mRNA and protein levels were normalized with GAPDH. Each experiment was performed in triplicate ( n = 3). All error bars indicate ± SEM. * P < 0.01; ** P < 0.01; *** P < 0.001

Article Snippet: Synthetic siRNAs for STAT5A, STAT5B, and DLX5 mRNA were purchased from Bioneer (Bioneer, Daejeon, South Korea).

Techniques: Staining, Over Expression, Western Blot, Expressing, Real-time Polymerase Chain Reaction

Journal: Cell Death & Disease

Article Title: Inhibition of STAT5A promotes osteogenesis by DLX5 regulation

doi: 10.1038/s41419-018-1184-7

Figure Lengend Snippet: a Alizarin Red S staining to detect suppression of STAT5A and STAT5B on osteogenesis using targeted siRNA. Staining was performed on day 14 of osteogenesis. Scale bar, 30 μm. b Quantification of Alizarin Red S staining. c Western blot analysis of DLX5 protein expression after silencing of STAT5A in hBMSCs. d Quantification of DLX5 protein level compared with GAPDH expression level. e Alizarin Red S staining for the effect of STAT5 inhibitor during osteogenesis of hBMSCs. STAT5 inhibitor (sc-355797) was used at concentrations of 0, 2, 6, and 10 μM. Staining was performed at day 14 of osteogenesis. Scale bar, 30 μm. f Quantification of Alizarin Red S staining. g Analysis of DLX5 mRNA level after 10 μM STAT5 inhibitor treatment. mRNA level was checked on day 5 of osteogenesis. h Western blot analysis of DLX5 protein expression after STAT5 inhibitor treatment. Protein level was detected on day 5 of osteogenesis. Each experiment was performed in triplicate ( n = 3). All error bars indicate ±SEM. * P < 0.01; ** P < 0.01; *** P < 0.001

Article Snippet: Synthetic siRNAs for STAT5A, STAT5B, and DLX5 mRNA were purchased from Bioneer (Bioneer, Daejeon, South Korea).

Techniques: Staining, Western Blot, Expressing

Journal: Cell Death & Disease

Article Title: Inhibition of STAT5A promotes osteogenesis by DLX5 regulation

doi: 10.1038/s41419-018-1184-7

Figure Lengend Snippet: a ALP staining of 10-week-old wild-type and Stat5a -/- mBMSCs at day 0, as indicated. Scale bar, 60 μm. b Relative ALP activity assay results at days 0 in wild-type and Stat5a -/- mBMSCs. c and d Alizarin Red S staining ( c ) and quantitative analysis ( d ) of at day 8 after induction of osteogenesis in 10-week-old, 20-week-old, and 30-week-old wild-type and Stat5a -/- mBMSCs. e Protein levels of DLX5 in 10-week-old, 20-week-old, and 30-week-old male wild-type and Stat5a -/- mBMSCs using western blotting at 5 days after osteogenesis. f mRNA levels of Dlx5 in wild-type and Stat5a -/- mBMSCs at day 3 after the induction of osteogenesis. g mRNA levels of osteoblast-related genes Alp , Bsp, and Opn at day 3 and mRNA level of Ocn at day 7 after the induction of osteogenesis. h – j Relative mRNA ( h ) and protein levels ( I and j ) of DLX5 depending on exogenously increased Stat5a expression in wild-type and Stat5a -/- mBMSCs. pcDNA-mStat5a was transfected in Stat5a -/- mBMSCs. mRNA and protein levels of Dlx5 were checked at day 3 and 5 after osteogenesis, respectively. Osteogenesis was induced after overexpression of Stat5a by transfection. k The osteoblast differentiation of Stat5a -/- mBMSCs upon overexpression of STAT5A using Alizarin Red S and Von Kossa staining at day 8 after induction of osteogenesis. l Quantification of Alizarin Red S staining in Stat5a -/- mBMSCs. Each experiment was performed in triplicate ( n = 3). All error bars indicate ±SEM. * P < 0.01; ** P < 0.01; *** P < 0.001

Article Snippet: Synthetic siRNAs for STAT5A, STAT5B, and DLX5 mRNA were purchased from Bioneer (Bioneer, Daejeon, South Korea).

Techniques: Staining, ALP Activity Assay, Western Blot, Expressing, Transfection, Over Expression

Journal: Cell Death & Disease

Article Title: Inhibition of STAT5A promotes osteogenesis by DLX5 regulation

doi: 10.1038/s41419-018-1184-7

Figure Lengend Snippet: a Representative longitudinal sections of fractured femurs from wild-type and Stat5a -/- mice at 2 and 4 weeks post-fracture. 6-week-old male mice were used for the fracture model. Scale bar, 4 mm ( n = 16 per each group). b Quantitative analysis of newly formed callus volume at 2 and 4 weeks post-fracture. c – f Representative histological analysis of paraffin sections of calluses from wild-type and Stat5a -/- mice at 2 and 4 weeks post-fracture stained with ( c ) Safranin O/Fast Green staining for cartilaginous bone callus. d Quantitative analysis of remaining cartilaginous callus area at 2 and 4 weeks post-fracture ( n = 8 per each group). Scale bar, 0.5 mm. e Masson’s Trichrome staining for total callus. f Quantitative analysis of total callus area at 2 and 4 weeks post-fracture ( n = 8 per each group). Scale bar, 0.5 mm. g Immunohistochemistry against DLX5 at 2 weeks post-fracture of wild-type and Stat5a -/- mice in the fractured femoral section. Scale bars, 100 μm ( n = 5 for each group). All error bars indicate ±SEM. * P <0.01; ** P <0.01; *** P <0.001

Article Snippet: Synthetic siRNAs for STAT5A, STAT5B, and DLX5 mRNA were purchased from Bioneer (Bioneer, Daejeon, South Korea).

Techniques: Staining, Immunohistochemistry

Journal: Oncotarget

Article Title: STAT5 induces miR-21 expression in cutaneous T cell lymphoma

doi: 10.18632/oncotarget.10160

Figure Lengend Snippet: ( A ) Expression of pri-miR-21 and pri-miR-155 in MyLa2059 after treatment with JAK3 inhibitor (50 μmol/L) Tofacitinib, or DMSO control for 24 hours, measured by qRT-PCR. N = 3. ( B ) pri-miR-21 expression after siRNA mediated knock-down of STAT3 and STAT5A and STAT5B in MyLa2059 cells as measured by qRT-PCR. ( N = 3). ( C ) ChIP-seq reads from the miR21HG promoter region in malignant MyLa2059 cells. Reads obtained from immunoprecipitation with STAT5, STAT3, RelB, RelA and a negative control (Rabbit IgG antibody, bottom). Forward reads are indicated in green and reverse reads in red.

Article Snippet: 2 × 10 6 cells per sample were transfected with small interfering RNA (siRNA) against STAT3, STAT5A, STAT5B or non-targeting control (ON-TARGETplus SMARTpool, Thermo Scientific).

Techniques: Expressing, Control, Quantitative RT-PCR, Knockdown, ChIP-sequencing, Immunoprecipitation, Negative Control

Journal: Oncotarget

Article Title: STAT5 induces miR-21 expression in cutaneous T cell lymphoma

doi: 10.18632/oncotarget.10160

Figure Lengend Snippet: ( A ) pri-miR-21 expression in SeAx cells cultured with IL-2 and treated with JAK3 inhibitor Tofacitinib (50 μmol/L). N = 5, p < 0.05 (Wilcoxon signed rank test). ( B ) pri-miR-21 expression after siRNA mediated knock-down of STAT3, STAT5A and STAT5B in SeAx cells cultured with IL-2. N = 5, p < 0.05 (Wilcoxon signed rank test). ( C ) Expression of miR-21 and miR-155 in SeAx cells cultured with the IL-2Rg cytokines IL-2, IL-15 and IL-21 for 24 hours. Data are shown from one experiment.

Article Snippet: 2 × 10 6 cells per sample were transfected with small interfering RNA (siRNA) against STAT3, STAT5A, STAT5B or non-targeting control (ON-TARGETplus SMARTpool, Thermo Scientific).

Techniques: Expressing, Cell Culture, Knockdown