Fig 2: Tumor cell-mediated induction of mRNA and proteins in MC3T3-E1 cells with potential involvement in the up-regulation of MMP-13. A, Affymetrix microarray analysis of mRNAs showing increased expression (>2-fold) in MC3T3-E1 grown in the presence of MDA-1833 conditioned medium (CM) compared with base-level expression (Base). The heat map indicates the range of mRNA expression levels (A-values) and the fold changes in mRNA expression are indicated. All fold changes are significant with BH-adjusted p values < 0.05. MC3T3-E1 cells were treated with either serum-free or MDA-1833-conditioned media: B, quantitative real time PCR of MMP-13, MMP-2, OSMR, and gp130 mRNA levels. C, Western blotting of OSMR and gp130 protein with actin levels used as a comparative loading control.

Fig 3: EcoHIV neither induces OSM secretion, nor the expression of gp130 or GLAST mRNA, but stimulates the expression of GLT-1 and OSMR-ß in primary cortical astrocytes. a Shows real-time PCR analyses of HIV LTR mRNA in control and EcoHIV-infected (35,000 pg of p24, for 24 h) cortical astrocyte cultures. ****p < 0.0001, n = 3. b Shows ELISA analysis of secreted OSM proteins in culture supernatants collected from control and EcoHIV-infected primary astrocyte cultures. n = 6. c, d Shows real-time PCR analyses of GLAST and GLT-1 (c) and gp130 and OSMR-ß (d) mRNA in control and EcoHIV-infected cortical astrocyte cultures. *p < 0.05, **p < 0.01, n = 3. e Shows Western blot analyses for OSMR-ß proteins in control and EcoHIV-infected cortical astrocyte culture lysates. The left panel shows representative blot of three independent experiments. The right panel shows densitometric analysis of OSMR-ß proteins. Data are presented as percentage of each respective ratio between optical density value of OSMR-ß band (110 kDa) intensity and optical density value of the matched ß-actin (42 kDa; loading control) band intensity. *p = 0.027, n = 3; two-tailed Student’s t test

Fig 4: MMP-13 induction by cytokines that induce STAT-3-dependent signaling. A, QRT-PCR analysis of MMP-13, OSMR, gp130, and MMP-2 mRNA levels in MC3T3-E1 cells treated with the indicated dose of recombinant OSM or MDA-1833 cell-conditioned medium. B, Western blot analysis of OSM, LIF, and IL-6 expression in MDA-1833 cell-conditioned medium. MC3T3-E1 cells were incubated with 10 ng/ml OSM or MDA-1833 cell-conditioned medium and analyzed by Western blot for MMP-13 induction, with the zymogram of MMP-2 included as a control (C) and phospho-STAT-3 signaling in the absence or presence of a JAK-1 inhibitor with total STAT-3 as a loading control and analysis of MMP-13 mRNA induction by QRT-PCR in the presence of the inhibitor (D). MC3T3-E1 cells treated with MDA-1833 cell-conditioned medium in the absence or presence of a function-blocking antibody against the common gp130 subunit of the OSM, LIF, and IL-6 receptors. Phospho-STAT-3 signaling was assessed by Western blotting (D) and MMP-13 mRNA induction by QRT-PCR (D) and Western blotting (E), with the zymogram of MMP-2 included to show selectivity of the response to MMP-13. CM, cell-conditioned medium; Std, pro and active MMP-13 as standards.

Fig 5: Loss of miR-1/133a facilitates cell cycle reentry of cardiomyocytes.(A and B) GSEA of microarray data from adult mouse hearts showing enrichment of genes related to cell cycle phase transition (A) and cell division (B) after inactivation of miR-1/133a. UO126 treatment or additional knockout of Osmr (tKO OR) or Fgfr1 (tKO FR1) abolished enrichment. Heatmaps of the 10 most up-regulated genes in dKO hearts derived from the respective gene sets confirm normalization of gene expression [ctrl: n = 4; dKO, dKO + UO, tKO (OR), tKO (FR1): n = 3]. (C and D) EdU incorporation in an isolated cTNT+ adult miR-1/133a mutant cardiomyocyte and quantification of EdU incorporation in cardiomyocytes of dKO, tKO, and control mice after EdU administration for 7 days before isolation [ctrl, n = 13; dKO, n = 10; tKO (OR), n = 14; tKO (FR1), n = 11 animals; one-way analysis of variance (ANOVA)]. Scale bars, 20 µm. (E and F) Electron microscope images of adult cardiomyocytes (ctrl, n = 3; dKO, n = 2). Scale bars, 1 µm. Sarcomeres of dKO cardiomyocytes show fuzzy Z-discs (arrowhead in inlet) and signs of disorganization. (G and H) qRT-PCR analysis of cell cycle markers in isolated adult dKO (n = 7 to 9) and control cardiomyocytes (n = 8 to 12) and noncardiomyocytes (ctrl non-CM, n = 6 to 7; dKO, n = 6 to 7; one-tailed unpaired t test). *P < 0.05 and **P < 0.01 compared to dKO; ##P < 0.01 and ###P < 0.001 compared to WT.