Fig 1: SNORD3A, SNORA13 and SNORA28 up-regulate GADD45A and MYC, downregulate TOP2A. U-2OS cells were transfected with empty vector (U-2OS) or expression vectors for SNORD3A, SNORA13 and SNORA28. U-2OS/DX30, U-2OS/DX100 and U-2OS/DX580 cells were used as control of Dox-resistant cells. (A) Heatmaps of changes in the expression of genes relevant for osteosarcoma pathogenesis and progression, measured by PCR arrays (n = 4). Yellow circles: GADD45A hits; orange circles: MYC hits; blue circles: TOP2A hits. (B) Immunoblot of the indicated proteins. The figure is representative of one out of three experiments. Tubulin was used as control of equal protein loading. (C) Immunoblot quantitation. The quantitation of the band density was performed using the ImageJ software. Data are means + SD (n = 3). * p < 0.05, *** p < 0.001: U-2OS/DX30, U-2OS/DX100 or snoRNA-over-expressing U-2OS cells vs. parental U-2OS cells.
Fig 2: Absence of Gadd45a induction in skeletal muscle myocytes results in increased atrophy of multiple fiber types and loss of fiber type identity.Baseline minimum Feret diameters of type I, type IIa, and non–I/IIa myofibers did not differ among WT, heterozygous, Gadd45afl, HSA-Cre:Gadd45afl (muscle-specific Gadd45a deletion), and CMV-Cre:Gadd45afl (ubiquitous Gadd45a deletion) genotypes (A–E, scale bar: 100 µm; K, multiple linear regression, with pairwise comparisons determined by linear combination). Type I, type IIa, and non–I/IIa myofibers from denervated soleus of HSA-Cre:Gadd45afl and CMV-Cre:Gadd45afl mice all showed significantly reduced minimum Feret diameters by 14 days postdenervation compared with all other genotypes (F–J, and L, multiple linear regression, with pairwise comparisons determined by linear combination). The box plots depict the minimum and maximum values (whiskers), the upper and lower quartiles, and the median. The length of the box represents the interquartile range. In addition, denervated soleus from HSA-Cre:Gadd45afl and CMV-Cre:Gadd45afl genotypes showed a significant increase (P < 0.001) in the proportion of non–I/IIa myofibers, with prominent loss of Myh2-expressing fibers (type IIa), suggesting accelerated loss of myofiber identity in the absence of GADD45A expression (M, Pearson’s ?2 test).
Fig 3: Gadd45a induction is an acute and sustained response to denervation injury.Gadd45a is the most significantly differentially expressed transcript in acutely denervated mouse gastrocnemius and shows sustained induction up to 3 months postdenervation (DN) (A–D; **P < 0.01, ***P < 0.001, paired t test with Bonferroni’s correction, n = 4 for each time point). GADD45A is similarly induced in a diverse range of denervated human skeletal muscle from individuals with acute denervation injury or acute flaccid myelitis and remains elevated up to 4 years after denervation (F–H, Welch’s t test). Error bars in F show mean ± SEM. All the normalized GADD45A threshold detection cycles (?Ct) from denervated skeletal muscle are substantially lower than controls, indicating higher mean expression levels (H).
Fig 4: Knockdown of STIM1 disruption multiple critical pathways in human tongue squamous carcinoma cells(A) Heatmap representation of 1841 genes (833 genes up-regulated and 1008 genes down-regulated) with differential expressions in Tca-8113 cells infected with lentivirus expressing either shCtrl or shSTIM1 (criteria: P<0.05, fold change >1.5). (B) Functional pathway enrichment of differential genes was analyzed based on KEGG and BIOCARTA databases. (C) Interactional network was constructed between STIM1 and genes involved in KEGG pathway cell cycle. Green circles represent down-regulated genes, red circles represent up-regulated genes and genes of gray circles represent no expression changing. (D) STIM1 regulates the expression of MDM2, CDK6, and GADD45A. Tca-8113 cells were infected with shCtrl or shSTIM1 for 48 h and then Western blot was performed to analyze the expression of MDM2, CDK6, and GADD45A.
Fig 5: Activation of FOXO signaling by T3-EV.A Heatmap of clustering dysregulated mRNA expression profiles with microarray in T3-EV-treated BMSCs compared to un-EV treated control. B Volcano plot of mRNA expression profiles in T3-EV-treated BMSC recipient. C Dysregulated typical chondrogenic markers derived from the microarray results with T3-EV treatment. D All differentially expressed genes were subjected to gene ontology (GO) analysis (DEG with fold change >2 or <0.5, p value <0.01). BP biological processes, MF molecular function, CC cellular component. E Significantly enriched pathways for dysregulated genes enriched with T3-EV treatment in KEGG pathways. F, G FOXO1 expression(red) assayed with western blotting and fluorescent immunostaining (red for FOXO1; green for cytoskeleton) in T3-EV treated BMSC recipient. Cells were counterstained with DAPI for the nucleus (blue). Treatment with saline served as control. H Quantification of gene expression with the same BMSCs and the same EVs (n = 3 for each) with qRT-PCR for Chondrogenic genes (SOX9, ACAN, COL2A1, and MMP13) and FOXO signaling-related genes (FOXO1, Gadd45a, p27, and Cathepsin L). *P < 0.05, **P < 0.01, ***P < 0.001, NS not significant.
Supplier Page from Abcam for Anti-GADD45A antibody