Journal: Communications Biology

Article Title: Selectivity matters: selective ROCK2 inhibitor ameliorates established liver fibrosis via targeting inflammation, fibrosis, and metabolism

doi: 10.1038/s42003-023-05552-0

Figure Lengend Snippet: a C57BL/6 female mice were treated with vehicle, GV101 (30, 100, and 150 mg/kg) or KD025 (150 mg/kg) by oral gavage on Week 6 after starting TAA in drinking water and continued for 6 weeks. b Hydroxyproline was assessed in the median lobe of liver collected on week 6 (normal W6 and TAA W6 with 5 mice/group) or harvest day on Week 12 (normal W9, TAA W9 and treatments with 10 mice/group). c KD025 dose was lowered to 100 mg/kg after 4 weeks of treatment due to toxicities. Representative images of Picrosirius Red (PSR) staining are shown for each group. d The levels of leptin, insulin, IL-1β and IL-17 in serum collected on Week 12 were measured by ELISA. e Liver tissue lysates were prepared in RIPA buffer and levels of pAMPK, pAkt, pCofilin, and pSTAT3 were determined by Western Blot. Graphs represent mean +/− s.e.m. Unpaired t-test statistical analysis was performed: * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001.

Article Snippet: On day 1, cells were seeded in 24-well plates at density of 50,000 cell/well in 1 ml culture medium; On day 2, cells was switched to starve EMEM containing 0.5% FBS; On day3, cells were treated with indicated concentration of ROCK inhibitors, incubated for 1 h, and then TGF-β1 (2.5 ng/mL) was applied to the cells; On day 5 (48 h after treatment), cultured medium was collected for Col1α1 ELISA (human Pro-Collagen 1α1 DuoSet ELISA kit, R&D systems), cells were collected for RNA extraction or for western analysis.

Techniques: Staining, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Communications Biology

Article Title: Selectivity matters: selective ROCK2 inhibitor ameliorates established liver fibrosis via targeting inflammation, fibrosis, and metabolism

doi: 10.1038/s42003-023-05552-0

Figure Lengend Snippet: Human subcutaneous preadipocytes were induced adipogenesis with a medium containing differentiation cocktail (DM) for 7 days in the presence of indicated inhibitors. Differentiated cells were stained at day 8 by Oil Red O ( a ), quantification of lipid accumulation was measured by absorbance at 492 nM of extracted Oil Red O, all the absorbance was normalized to vehicle (DMSO/DM) treated differentiated cells ( a ), total RNA was extracted at day 8 and Glut4 gene expression was accessed by real-time RT-PCR ( b ). Human subcutaneous preadipocytes were treated with indicated inhibitors for 2 h before whole cell extracts were collected for Western blot analysis of phosphorylated AMPK and β-Actin ( c ). MRC-5 cells were pre-treated with various concentration of indicated inhibitors for 1 h before TGF-β1 stimulation for 48 h. Whole cell extracts were subjected to Western blot for the expression of α-SMA, pCofilin and β-actin ( d ). Western blots were quantified and normalized to the β-actin, and values are indicated under the corresponding immunoblots. Col1α levels in supernatants were measured by ELISA. All the Col1α levels were normalized with that of vehicle (DMSO/TGF-β1) treated cells ( d ), gene expression of α-SMA, CTGF, Col3α levels were measured by real-time RT-PCR. All the levels were first normalized with internal 18s control and then were normalized with the level of vehicle (DMSO/TGF-β1) treated cells ( e ). The data ( a , d ) are representative of three repeated experiments, ( b , e ) are average of three repeated experiments.

Article Snippet: On day 1, cells were seeded in 24-well plates at density of 50,000 cell/well in 1 ml culture medium; On day 2, cells was switched to starve EMEM containing 0.5% FBS; On day3, cells were treated with indicated concentration of ROCK inhibitors, incubated for 1 h, and then TGF-β1 (2.5 ng/mL) was applied to the cells; On day 5 (48 h after treatment), cultured medium was collected for Col1α1 ELISA (human Pro-Collagen 1α1 DuoSet ELISA kit, R&D systems), cells were collected for RNA extraction or for western analysis.

Techniques: Staining, Gene Expression, Quantitative RT-PCR, Western Blot, Concentration Assay, Expressing, Enzyme-linked Immunosorbent Assay, Control

Journal: Communications Biology

Article Title: Selectivity matters: selective ROCK2 inhibitor ameliorates established liver fibrosis via targeting inflammation, fibrosis, and metabolism

doi: 10.1038/s42003-023-05552-0

Figure Lengend Snippet: PBMCs were treated with indicated doses of GV101 or KD025 1.5 h before stimulation with LPS. After 24 h, TNF, IL-23, and IL-10 secretion in supernatants was analyzed by ELISA and normalized ( n = 12 except GV101 0.3 and 0.6 μM: n = 4) ( a ) and Western blots for the indicated proteins were performed on whole cell extracts ( b ). Human ( c , e ) or murine primary Kupffer cells ( d ) were treated with indicated doses of GV101 or KD025 1.5 h before stimulation with LPS and 24 h before collection of Kupffer cells supernatants for analysis of IL-6 and TNF secretion by ELISA followed by normalization ( c : n = 8 except GV101/KD025 2.5 μM, n = 5 ; d : n = 4), and preparation of human Kupffer cell extracts for Western blots analysis of the indicated proteins ( e ). All experiments represent at least 4 independent repeats. Graphs represent mean +/− s.e.m. In ( a , c , d ), one way ANOVA was performed followed by multiple comparisons Sidak tests to allow two-by-two comparisons. ns: not significant, * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001. In ( b , e ), Western blots were quantified and normalized to the LPS-stimulated conditions, and values are indicated under the corresponding immunoblots.

Article Snippet: On day 1, cells were seeded in 24-well plates at density of 50,000 cell/well in 1 ml culture medium; On day 2, cells was switched to starve EMEM containing 0.5% FBS; On day3, cells were treated with indicated concentration of ROCK inhibitors, incubated for 1 h, and then TGF-β1 (2.5 ng/mL) was applied to the cells; On day 5 (48 h after treatment), cultured medium was collected for Col1α1 ELISA (human Pro-Collagen 1α1 DuoSet ELISA kit, R&D systems), cells were collected for RNA extraction or for western analysis.

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Circulating Monocyte Chemoattractant Protein-1 (MCP-1) in Patients with Primary Biliary Cholangitis

doi: 10.3390/ijms25021333

Figure Lengend Snippet: Correlation between serum MCP-1 concentration and level of ( a ) Pro-Collagen type III, ( b ) Hyaluronic acid—HA, ( c ) FIB-4 index, ( d ) Collagen type IV, ( e ) APRI in patients with primary biliary cholangitis.

Article Snippet: Human procollagen type III concentration was evaluated by using a commercial ELISA kit (Causabio Technology LLC., Houston, TX, USA), Hyaluronic acid and collagen type IV concentration was evaluated by using a commercial ELISA kit (Aviva Systems Biology, San Diego, CA, USA).

Techniques: Concentration Assay