Fig 2: Hepatic fibrosis is alleviated in mice given CCl4 with T-MSC-derived sEVs(A) qRT-PCR analysis of Tgf-ß, a-Sma, Vimentin, and Ctgf in the livers from CON+PBS, CCl4, CCl4+PBS, and CCl4+sEV groups (n = 3 per group). Data are presented as mean ± SEM (unpaired two-sample Student’s t test; *p < 0.05, **p < 0.005). (B and C) Western blot (B) and cumulative densitometric (C) analyses of Tgf-ß (25 kDa), a-Sma (42 kDa), Vimentin (57 kDa), Col1a1 (140 kDa), and Gapdh (36 kDa) in the livers from each group (n = 4 per group). Immunoblots shown represent one of three independent experiments with similar results. Band densities were normalized to the expression level of GAPDH, which was used as an internal control. Data are presented as mean ± SEM (unpaired two-sample Student’s t test; *p < 0.05, **p < 0.005). (D) Representative images of Sirius-red-stained (top) and a-Sma-stained (bottom) liver sections from each group (original magnification, ×10; scale bars, 100 µm). (E) Relative levels of hydroxyproline contents in the livers from each group (CON+PBS, n = 4; CCl4, n = 4; CCl4+PBS, n = 5; and CCl4+sEV, n = 6). Data are presented as mean ± SEM (unpaired two-sample Student’s t test; *p < 0.05).

Fig 3: Effect of CBP and P300 knockdown on KLF9 expression and enhancer activity from KLF9 GBSs.A–B, A549 cells were incubated with pools of four siRNAs for 48 h. The pools were either nontargeting siRNA (siControl; black), or siRNAs targeted to CBP (siCBP; orange), P300 (siP300; light blue), or combined CBP/P300 siRNAs (dark red). A, cells were harvested for western blot analysis of CBP, P300, and GAPDH. Representative blots are shown (upper panel). Following densitometric analysis, normalized data (N = 4), expressed as log2 (gene/GAPDH), are plotted as box-and-whiskers plots. B, cells were either not stimulated (NS) or treated with 300 nM budesonide (Bud) for 6 h prior to qPCR analysis of: mature KLF9 mRNA, KLF9 usRNA, and the four eRNAs, 1, 2.1, 2.2, and 4. For normalization, GAPDH and U6 were also assayed. Normalized data (N = 4), expressed as log2 (mRNA/GAPDH, usRNA/U6, or eRNA/U6), are plotted as means ± SE. C, A549 cells stably transfected with empty vector or reporter constructs for the (?) orientation of P1+2, P1, P2, P3, and P4 GBSs were treated with siRNAs and Bud as in (B) prior to luciferase assay. The RLU measurements were normalized to that of the empty vector for each condition (siControl, siCBP, siP300, or siCBP + siP300). Normalized data (N = 4), expressed as log2 (RLU/RLUempty), are plotted as means ± SE. Significance, using normalized data relative to siControl group for each treatment condition was tested using ANOVA with Tukey’s post hoc test. *p = 0.05, **p = 0.01, ***p = 0.001.

Fig 4: Proinflammatory cytokines and glucocorticoids synergistically induce TLR2 mRNA and transcription.A, A549 cells, or pHBECs grown in submersion culture, or B, pHBECs cultured at air-liquid interface (ALI) were either not stimulated (NS) or treated with IL1B (1 ng/ml) and/or dexamethasone (Dex; 1 µM) or budesonide (Bud; 300 nM) for the times indicated. C, A549 cells were treated with IL1B and Dex, as indicated, for 6 h. A–C, cells were harvested for qPCR analysis. TLR2 data from N independent experiments were normalized to GAPDH and plotted as fold of NS (of the earliest time point/graph). D, A549 cells and E, pHBECs grown in submersion culture were treated as in A and harvested after 2 h for qPCR analysis of unRNA of TLR2 and U6. Data (upper panels) from N independent experiments were normalized to U6 and are plotted as fold relative to untreated control. Significance was tested using ANOVA with a Sidak post-hoc test. In A, ‘$’ and ‘#’ represent significance relative to IL1B and Dex, respectively. In A, B, D, and E (lower panels), the sum effect (i.e., fold - 1) of both treatments (sum) and the effect (fold - 1) for the combination treatment (comb) is plotted. Significance at each time was tested by paired t test. *p = 0.05, **p = 0.01, ***p = 0.001. pHBEC, primary human bronchial epithelial cell; qPCR, quantitative PCR; TLR, toll-like receptor; unRNA, unspliced nuclear RNA.

Fig 5: TR;HSALR bi-transgenic mice as a therapy reporter model for DM1. In the HSALR transgenic mouse model of DM1, exclusion of Atp2a1 exon 22 is upregulated. To validate the TR-ATP2A1 splice switch as a reporter for DM1, we crossed the TR transgenic with the HSALR transgenic model to create TR;HSALR bi-transgenic mice. a In vivo imaging of DsRed and GFP fluorescence in TR transgenic and TR;HSALR bi-transgenic mice (IVIS Spectrum) under general anesthesia in the prone position. The scale shows radiant efficiency (photons/second/cm2). Bars = 5 mm. b DsRed and GFP fluorescence measured in regions of interest in bilateral gastrocnemius (Gast) and paraspinal (P-sp) muscles of TR (N = 4) and TR;HSALR mice (N = 3). Error bars indicate mean ± s.e.m. ****P < 0.0001 TR vs. TR;HSALR (one-way ANOVA). c Splicing analysis in gastrocnemius muscle by RT-PCR of the TR transgene (Tg) exon 22 (ex 22) and endogenous Atp2a1 ex 22. N = 3 each group. WT FVB wild-type. d Quantitation of splicing results in c, displayed as the percentage of exon 22 inclusion. Error bars indicate mean ± s.e.m. ****P < 0.0001 (two-way ANOVA). e Western blot of GFP protein expression in gastrocnemius and paraspinal muscles of TR and TR;HSALR mice (N = 3 each group), with GAPDH as loading control. kD kilodaltons. f Representative DsRed and GFP expression in TR (upper row) and TR;HSALR (lower row) gastrocnemius muscle cryosections. Fluorescence images are the extended focus of deconvolved Z-series. Fluorescence intensity range = 0–5080 grayscale units. Laminin (yellow) and DAPI (blue) highlight muscle fibers and nuclei, respectively. Merge DsRed (red) + GFP (green) + laminin + DAPI, H&E hemotoxylin and eosin. Bars = 50 µm