Fig 1: Transient DUSP5 siRNA+T3 therapy stimulates cardiomyocyte proliferation and number expansion. (A) Schematic shows the Confetti construct (left) and potential for expression of green (GFP), yellow (YFP), red (RFP) and cyan fluorescent protein (CFP) in cardiomyocytes (right) following Cre-mediated recombination. (B), Schematic (left) shows the experimental protocol for 4-hydroxytamoxifen treatment to initiate Cre-mediated recombination for expression of fluorescent proteins in cardiomyocytes and timing of DUSP5 siRNA and T3 therapy. Bar graph (right) shows quantification of monochromatic singlet or clustered cardiomyocytes in DUSP5 scrambled siRNA+T3 and DUSP5 siRNA+T3 treated mice (n = 4-5 mice per group); A pie chart analysis (below left) of either red (RFP) or yellow (YFP) colored monochromatic singlets, doublets and triplets distribution in DUSP5 siRNA+T3 or scrambled siRNA+T3 treated mice shows that Triplets (3-cell clusters) only occur in DUSP5 siRNA+T3 treated mouse hearts. Representative images (below right) of monochromatic cardiomyocyte (CM) clusters with cell outlines delineated using wheat germ agglutinin (WGA) staining. (C) Schematic (left) shows the experimental protocol. Bar graph (right) shows ventricular cardiomyocyte numbers analysis after in vivo DUSP5 scrambled siRNA, DUSP5 scrambled siRNA+T3, DUSP5 siRNA or DUSP5 siRNA+T3 therapy (n = 5-7 mice per group). **P < 0.01, ***P < 0.001. Individual data points and mean ± s.e.m are shown. Comparisons were made using a 2-tailed t-test (B) or ANOVA with Sidak multiple comparison test (C).
Fig 2: The expression of DUSP5 in SD rat chondrocytes. (A, B) The protein expression and quantitation of DUSP5 in SD rat chondrocytes incubated with IL-1ß at 0, 5, 10, and 20 ng/mL for 24 h. (C, D) The protein expression and quantitation of DUSP5 in SD rat chondrocytes incubated with IL-1ß at 10 ng/mL for 0, 6, 12, and 24 h. (E, F) The protein expression and quantitation of DUSP5 in SD rat chondrocytes incubated with IL-1ß at 10 ng/mL for 0, 6, 12, 24, 48, 72, and 96 h. The medium and IL- 1ß were added at time Zero. Then every 24 hours changed the medium and re-added IL- 1ß. GAPDH was used as the control. All data are expressed as mean±S.D. (n = 3). *p<0.05, **p<0.01.
Fig 3: IHC staining for DUSP5 in tissues. (a) DUSP5 expression in TMAs of FTC and PTC samples. Representative images of DUSP5 immunohistochemistry on FTC tissues and PTC tissues. Magnification, x50 (left panels) and x400 (right panels). (b) DUSP5 expression in clinical samples of FTC and PTC. Representative images of DUSP5 immunohistochemistry on FTC tissues and PTC tissues. Magnification, x100 (left panels) and x200 (right panels). (c) Quantification of positive‐staining for DUSP5 in TMAs of FTC and PTC groups. DUSP5 was significantly less in FTC than PTC samples. The values denote the positive area/ total area ± SEM. *, statistically significant difference (P < 0.05). (d) Quantification of positive‐staining for DUSP5 in clinical samples of FTC and PTC groups. DUSP5 was dramatically less in FTC than PTC samples. The values denote the positive area/total area ± SEM. *, statistically significant difference (P < 0.05).
Fig 4: DUSP5 expression is essential for the regulation of TNFa-induced ERK 1/2 phosphorylation. (A) RNA was isolated from 3T3-L1 pre-adipocytes and differentiated adipocytes and (A) DUSP5 mRNA and (B) protein expression was assessed via qPCR and immunoblotting, respectively. Pre-adipocytes and adipocytes were stimulated in parallel with TNFa (100 pM) and RNA isolated over time post-treatment prior to examination of DUSP5 mRNA expression via qPCR in (C) preadipocytes and (D) differentiated adipocytes. (E) 3T3-L1 adipocytes were stimulated without TNFa (100pM) and protein harvested over time. Whole cell lysate was subjected to immunoblotting for the detection of DUSP5, phospho-ERK and JNK as well as total ERK and JNK. Adipocytes were transiently transfected with small, non-targeting control (100 nM) siRNAs or siRNAs (100 nM) targeting independent regions of the DUSP5 gene for 48 hrs prior to stimulation with TNFa. (F) RNA was isolated 30 min post-treatment and qPCR used to examine DUSP5 expression. (G) Whole cell lysate was collected over time post-TNFa stimulation and immunoblotting used to assess phospho-ERK and JNK as well as total ERK and JNK. (H) Adipocytes (n = 3/group) were transiently transfected with small, non-targeting control (100 nM) siRNAs or siRNAs (100 nM) targeting independent regions of the DUSP5 gene for 48 hrs prior to stimulation with TNFa. Protein was collected 15 minutes post-TNFa stimulation and immunoblotted for phospho- and total-ERK. Image J software was used to quantify ERK phosphorylation to total ERK expression and one-way ANOVA with Tukey’s post-hoc analysis used to determine significance p < 0.05.
Fig 5: Effect of QNZ and GDC on IL-1β-induced inflammation in DUSP5 knockdown chondrocytes. (A, B) The protein expression and quantitation of DUSP5, iNOS, COX2, and MMP9. (C, D) The protein expression and quantitation of DUSP5, iNOS, COX2, and MMP9. GAPDH was used as the control. DUSP5 knockdown chondrocytes were treated with 10 μM QNZ or 6.1 nM GDC for 2 h before incubation with IL-1β (10 ng/mL) for 24 h. All data are expressed as mean±S.D. (n = 3). *p<0.05, **p<0.01.
Supplier Page from Abcam for Anti-DUSP5 antibody [EPR19684]