Experimental protocols of thioacetamide (TAA)-induced hepatic fibrosis in rats pretreated with Sho-Saiko-To (SST).

<p>Eight-week-old male rats were intraperitoneally injected with TAA (200 mg/kg) twice weekly at weeks 1 and 2 and then once at weeks 5 and 6. A cohort of rats receiving vehicle without treatment of SST (TAA only) was assigned as Group 1. Group 2 (TAA +0.25 g/kg SST) was administered moderate dose (0.25 g/kg) of SST two days before the first TAA exposure and sustained for 6 weeks. Group 3 (TAA +1 g/kg SST) was administered high dose (1 g/kg) of SST.</p

Comparison of biochemical markers in TAA-induced liver fibrosis in rats.

<p>Group 1 (TAA only); Group 2 (TAA+0.25 g/kg SST); Group 3 (TAA+1 g/kg SST).</p><p>TAA: Thioacetamide; STT: Sho-saiko-to; AST: Alanine aminotranferease; ALT: Aspartate aminotransferase; GGT: Gamma-glutamyl transpeptidase; ALP:Alkaline phosphatase; LDH: Lactic acid dehydrogenase. Data represent as mean±SE.</p><p>Comparison of biochemical markers in TAA-induced liver fibrosis in rats.</p

Gross appearance and histological assessments of liver fibrosis induced by thioacetamide (TAA) in rats.

<p>(A) Histological observation of liver fibrosis by hematoxylin and eosin (H&E) staining under light-field microscope with 200×magnification. The biopsies and liver tissues were sectioned in 5-µm thickness at weeks 2 (a–d) and 6 (e–f) and sections were stained with H&E. (a, e) Normal controls showed normal liver parenchyma at week 2 and week 6. (b, f) Group 1 (TAA only) showed spot necrosis of liver parenchyma at week 2 and bridging necrosis at week 6. (c, g) Group 2 (TAA +0.25 g/kg SST) showed lymphocyte infiltration at week 2 and mild fibrosis at week 6. (d, h) Group 3 (TAA+1 g/kg SST) showed relatively normal liver parenchyma at weeks 2 and 6. (B) Representative photographs at week 6 were shown. Gross examination of liver taken from Group 1 (TAA only, left panel), Group 2 (TAA +0.25 g/kg SST, middle panel) and Group 3 (TAA +1 g/kg SST, right panel) showed smooth surface.</p

Detection of Mrp2, Oatp1 and α-Sma expression in liver fibrosis induced by thioacetamide (TAA) in rats by immunohistochemistry and Western blots.

<p>(A) Immunohistochemistry of Mrp2 (upper panel), Oatp1 (middle panel) and α-Sma (lower panel) in rat liver sections of normal controls, Group 1 (TAA only), 2 (TAA+0.25 g/kg SST) and 3 (TAA+1 g/kg SST) was shown under light-field microscope with 200×magnifications. (B) Graph showed quantification of percentage of positive Mrp2 in Groups 1, 2 and 3. Bar, SE; **p<0.01; ***p<0.001. (C) Western blots of liver tissues from Mrp2, Oatp1 and α-Sma in rat liver tissues of normal controls, Groups 1, 2 and 3. α-tubulin serves as internal control. (D) Graph showed quantification of percentage of positive Oatp1 in Groups 1, 2 and 3. Bar, SE. (E) Graph showed quantification of percentage of positive α-Sma in Groups 1, 2 and 3. Bar, SE.</p

Collagen deposition in liver fibrosis induced by thioacetamide (TAA) in rats using Sirius red staining.

<p>(A) Histological observation of collagen deposition by Sirius red staining under light-field microscope with 40×(upper panel) and 200×(lower panel) magnifications. The biopsies and livers were sectioned 5-µm thickness at weeks 2 (a–d) and 6 (e–h) and sections were stained with Sirius red. (a, e) Normal controls showed no collagen deposition at weeks 2 and 6. (b, f) Group 1 (TAA only) showed extended collagen deposition and large septa of hepatic lobules, (c, g) Group 2 (TAA +0.25 g/kg SST) reduced the severity of hepatic fibrosis at weeks 2 and 6. (d, h) Group 3 (TAA +1 g/kg SST) markedly reduced the severity of hepatic fibrosis at weeks 2 and 6. (B) Quantitative analysis of Sirius red staining of each group at week 6. In comparison with Group 1, Groups 2 and 3 show decreased percentages of immune intensity. Bar, SE; *p<0.05.</p

Examination of thioacetamide (TAA)-induced liver fibrosis in rats by gadoxetic acid-enhanced MRI.

<p>(A) T1-weighted MRI images of Group 1 (TAA only, upper panel), 2 (TAA +0.25 g/kg SST) and 3 (TAA +1 g/kg SST) were shown at different time points (10, 20, 30, 40 50 and 60 min). (B) Quantification of ratios of relative enhancement of each group at time point of 10 min. Significant decreases in ratios were observed among Groups 1, 2 and 3. Bar, SE; *p<0.05. (C) Quantification of the signal drop slope in each group was determined by dividing the ratio difference at different points by the ratio at 10 min. A significant drop of the slope was observed between Groups 1 and 2. *p<0.05.</p

Examination of thioacetamide (TAA)-induced liver fibrosis in rats by sonoelastography.

<p>(A) Sonoelastographic images before and after SST administration (weeks 0 and 6) in Groups 1 (TAA only, upper panel), 2 (TAA +0.25 g/kg SST, middle panel) and 3 (TAA +1 g/kg SST, lower panel). In the elastography frame, blue color stands for hard issues and red stands for soft issues. (B) Quantification of difference in liver stiffness of each group at weeks 0 and 6 by sonoelastography. Significant decrease in liver stiffness was observed in Groups 1 and 3. Bar, SE; *p<0.1.</p

Evaluation of TAA-induced liver injury in rat models using Ishak and Metavir scores.

<p>Group 1 (TAA only); Group 2 (TAA+0.25 g/kg SST); Group 3 (TAA+1 g/kg SST).</p><p>TAA: Thioacetamide; STT: Sho-saiko-to; Fibrosis score: Ishak: 0–6; Metavir: F0 = 0, F1 = 1, F2 = 2, F3 = 3. Data represent as median.</p><p>Evaluation of TAA-induced liver injury in rat models using Ishak and Metavir scores.</p

Additional file 7: of Isocitrate dehydrogenase 1–snail axis dysfunction significantly correlates with breast cancer prognosis and regulates cell invasion ability

Figure S3. IDH1 stable knockdown significantly promoted MDA-MB-231 cell motility. (a) The expression levels of IDH1 were examined in two IDH1 stable knockdown MDA-MB-231 cells (shIDH1#1 and shIDH1#2) through western blotting. (b) Invasion ability was assessed using the Transwell assay in MDA-MB-231 cells with IDH1 stable knockdown and a scrambled control. The cell images of the representative experiment are provided. (c) Values were quantified using Ascent software, as detailed. Data are reported as the number of invading cells relative to the control (means Âą standard deviation (SD)). (d) Expression levels of IDH1, snail, slug, twist, and actin were examined in shIDH1#1, shIDH1#2, and the scrambled control through western blotting. (TIFF 3572 kb

Additional file 9: of Isocitrate dehydrogenase 1–snail axis dysfunction significantly correlates with breast cancer prognosis and regulates cell invasion ability

Figure S5. IDH1 knockdown accelerated MCF7 cell proliferation and migration ability. (a) the expression levels of IDH1 were examined in MCF7 cells with siIDH1#1, siIDH1#2, and control transfection through western blotting. (b) A wound healing assay was employed to examine MCF7 cells transfected with siIDH1#1, siIDH1#2, and the scrambled control. (c) The expression levels of IDH1, snail, slug, twist and actin were examined in siIDH1#1, siIDH1#2, and the scrambled control through western blotting. (d) The proliferation assay was performed in MCF-7 cells transfected with the scrambled control, siIDH1#1 and siIDH1#2, respectively. (TIFF 4527 kb