Additional file 2: Figure S2. of FGF19/FGFR4 signaling contributes to the resistance of hepatocellular carcinoma to sorafenib

The structure diagram of electrochemical biosensors with a three-electrode system. (TIF 355 kb

Additional file 1: Figure S1. of FGF19/FGFR4 signaling contributes to the resistance of hepatocellular carcinoma to sorafenib

Representative images of sorafenib-induced ROS-associated cell apoptosis. HCC cell lines were treated with Sora (4 μM for MHCC97L, MHCC97H and SMCC-7721, and 6 μM for HepG2) over a series of time points. Apoptosis was determined by DAPI staining (A), and ROS generation was determined by DCFH-DA staining (B). (TIF 4418 kb

Additional file 4: Figure S4. of FGF19/FGFR4 signaling contributes to the resistance of hepatocellular carcinoma to sorafenib

Sorafenib-resistant MHCC97H cells highly resistant to sorafenib-induced apoptosis and ROS generation. (A–F) The effect of Sora-resistant cells on Sora-induced HCC cell apoptosis and ROS generation. Sora-naive (WT) and Sora-resistant MHCC97H (MHCC97H Sora-R) cells were exposed to 20 μM of Sora over a series of time points before analysis. Morphological changes of cells were observed under microscope (A); cell viability was determined by MTS assays (B); apoptosis was determined by DAPI staining (C) and Western blot of c-PARP (D); ROS generation was determined by DCFH-DA staining (E); and O2 •− generation was determined by electrochemical biosensor (F). In C, expression levels were normalized against actin and reported relative to controls (fold changes shown below each lane).* p < 0.05; ** p < 0.01

Additional file 5: Figure S5. of FGF19/FGFR4 signaling contributes to the resistance of hepatocellular carcinoma to sorafenib

FGF19 knockdown in sorafenib-resistant HepG2 cells enhances ROS-associated apoptosis by sorafenib. (A) The knockdown effect of FGF19 in Sora-resistant HepG2 (HepG2 Sora-R) cells. (B–E) The effect of FGF19 knockdown on Sora-induced apoptosis in HepG2 Sora-R cells. FGF19 was knocked down in HepG2 Sora-R cells by lentiviral shRNA. FGF19 knockdown cells (shFGF19) and the control cells (shNC) were treated with different doses of Sora for 24 h. Cell viability was determined by MTS assays (B); apoptosis was determined by DAPI staining (C); ROS generation was determined by DCFH-DA staining (D), and O2 •− generation was determined by electrochemical biosensor (E). In A, expression levels were normalized against actin and reported relative to controls (fold changes shown below each lane). * p < 0.05; ** p < 0.01

Additional file 3: Figure S3. of Inhibition of Oct 3/4 mitigates the cardiac progenitor-derived myocardial repair in infarcted myocardium

Showing immunostaining signals for CD31 and α-SMA from sham and MI myocardium. Hearts from sham and MI + PBS groups were stained with CD31 (red) and GFP (green) to determine the development of c-kit+ CSC-derived capillaries. c-kit+ CSC-derived vascular smooth muscle cells were stained with α-SMA (green) and GFP (red). (Positive staining of c-kit+ CSCs from other groups are presented in Fig. 4a, b) Scale bar: 100 μm. The detailed procedure is described in Materials and Methods. (TIF 295 kb

Hsp20 stimulates HUVEC proliferation, migration and capillary-like tube formation.

<p>(A) Recombinant human Hsp20 protein was added to HUVECs at various doses (80–2000 ng/ml) for 24 h. BSA was used as a control. Cell proliferation was determined by MTS. (B) Time-course effects of the Hsp20 protein(1000 ng/ml) on the HUVEC proliferation. (C) Representative photographs indicated the effects of recombinant human Hsp20 protein on the trans-well and tube formation of HUVECs. (D) Migration was quantified by counting cells that were moved through the membrane (Trans-well assay). (E) Tube formation was evaluated by the measurement of relative tube length. Similar results were observed in three additional, independent experiments (*, p<0.05 <i>vs.</i> Control).</p

Hsp20 is secreted from cardiomyocytes <i>in vivo</i>.

<p>(A) The serum Hsp20 level was increased in response to <i>in vivo</i> 30 min-LAD occlusion followed by 24 h-reperfusion. Cardiac-specific overexpression of Hsp20 increased the Hsp20 concentration in the serum under basal and myocardial ischemia/reperfusion conditions. (n = 6; *, p<0.05 <i>vs.</i> WTs; #, p<0.05 <i>vs.</i> Sham groups). (B) The levels of Hsp20 in hearts from Hsp20-transgenic mice were determined by Western blot. α-Actin was used as an internal control (n = 4). (C) Myocardial ischemia/reperfusion stimulated the translocation of Hsp20 to the cardiomyocyte membrane, which was detected by fluorescence microscopy. Images are representative sections from four mice per group (green, Hsp20; red, α-Actin; Scale bar, 100 µm). (D) Quantitative data for expression of Hsp20 was evaluated using IPP 5.1 (n = 4; *, p<0.05 <i>vs.</i> WTs; #, p<0.05 <i>vs.</i> Sham groups).</p

Proposed mechanism of the Hsp20 release and its regulation of myocardial angiogenesis.

<p>Intracellular Hsp20 is released outside cardiomyocytes via exosomes, and then interacts with VEGFR2. Consequently, its downstream signaling pathways (i.e. Akt and ERK) are activated, which promote myocardial angiogenesis.</p

Hsp20 is secreted from adult rat cardiomyocytes <i>via</i> exosomes, independent of the ER-Golgi protein export pathway.

<p>(A) Brefeldin A (BFA), which inhibits the classical protein transport pathway, did not block Hsp20 release into the media under either basal or hypoxia conditions (20 µM H₂O₂). However, the release of Hsp20 from cardiomyocytes was reduced by both dimethyl amiloride (DMA), an exosome inhibitor, and Methyl-β-cyclodextrin (MBC), an inhibitor of lipid raft formation via depletion of membrane cholesterol. (B) The activity of acetylcholine esterase was used to quantify the amount of exosomes present in the media after various treatments. Similar results were observed in three additional, independent experiments (*, p<0.05 vs. Basal-Control; #, p<0.05 <i>vs.</i> H₂O₂-Control).</p

Cardiac-specific overexpression of Hsp20 promotes myocardial angiogenesis.

<p>(A) Blood vessels were stained for CD31 (capillary density) in heart sections of WT and Hsp20 TG mice, and (B) their quantitative analysis. For quantification of positively stained vessels, five sections of each heart (n = 4 hearts per group) were analyzed by an investigator who was blinded with respect to samples. Blood vessels were detected at low magnification (×200). Images are representative sections from four mice per group (green, α-Actin; red, CD31). Scale bar, 50 µm.</p