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: Breast Cancer Research : BCR

Article Title: Membrane associated collagen XIII promotes cancer metastasis and enhances anoikis resistance

doi: 10.1186/s13058-018-1030-y

Figure Lengend Snippet: Collagen XIII expression is increased during breast cancer development. a Western blot analysis of Collagen XIII (Col13) in the malignant and non-malignant mammary epithelial cell lines in vitro. Red stands for triple negative breast cancer cell lines, blue stands for luminal type breast cancer cell lines, black stands for non-malignant mammary epithelial cell lines. b Col13 mRNA expression in human breast cancer and normal mammary tissues in the TCGA dataset. n = 593 ** p < 0.01. c Col13 mRNA expression in human breast cancer and normal mammary tissues in the Finak dataset. n = 59 ** p < 0.01. d Col13 mRNA levels in ER negative ( n = 91) and ER positive ( n = 270) subgroups of breast cancer patients; ** p < 0.01. e Col13 mRNA expression in triple negative breast cancer tissues ( n = 48) and other subtypes ( n = 473); results are presented as the mean ± s.e.m.; ** p < 0.01. f Kaplan-Meier analysis of recurrence free survival of breast cancer patients grouped into high and low expression levels of Col13. The patients were equally divided into two groups based on the mRNA levels of Col13 in breast cancer tissues, n = 3554; *** p < 0.001. g Kaplan-Meier analysis of recurrence free survival of estrogen receptor negative patients grouped into high and low expression levels of Col13. n = 671; *** p < 0.001

Article Snippet: Anti-human Collagen XIII α1 (R&D systems, AF6346, 1:200 for WB).

Techniques: Expressing, Western Blot, In Vitro

Journal: bioRxiv

Article Title: Single cell landscape of hypertrophic scars identifies serine proteases as key regulators of myofibroblast differentiation

doi: 10.1101/2020.06.17.157073

Figure Lengend Snippet: A,B) Western blot of primary FBs stimulated with active TGFβ1 for 24h to differentiate FBs into alpha-smooth muscle actin-expressing (αSMA) myofibroblasts. Myofibroblast differentiation inhibited with A) DPP4-inhibitor Sitagliptin or B) urokinase-inhibitor BC-11. C, D) Collagen I or E, F) fibronectin in supernatants of stimulated primary skin FBs, detected by Enzyme-linked Immunosorbent Assay (ELISA). Whiskers represent range maximum and minimum values with < 1.5 interquartile range, boxes represent 25 th −75 th quartiles, line represents mean. Statistical significance was tested using one-way ANOVA with Tukey post-test. NS p>0.05, *p<0.05, **p<0.01, ***p<0.001. Experiments were performed in duplicates of five donors each.

Article Snippet: Human pro-collagen Ia1 ELISA (R&D Systems) and human fibronectin ELISA (R&D Systems) were performed with supernatants of TGFβ 1-stimulated FBs according to the manufacturer’s manual.

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

Journal:

Article Title: Collagen XXIII--a potential biomarker for the detection of primary and recurrent non-small cell lung cancer

doi: 10.1158/1055-9965.EPI-09-1095

Figure Lengend Snippet: Collagen XXIII immunohistochemical analysis of multiple cancer tissues

Article Snippet: Antibody Validation A new human collagen XXIII mouse monoclonal antibody was made against the cleaved portion of human collagen XXIII by R&D Systems.

Techniques: Immunohistochemical staining, Staining

Journal:

Article Title: Collagen XXIII--a potential biomarker for the detection of primary and recurrent non-small cell lung cancer

doi: 10.1158/1055-9965.EPI-09-1095

Figure Lengend Snippet: Representative images showing collagen XXIII staining in the NSCLC subtypes: A) lung adenocarcinoma, B) large cell carcinoma, and C) squamous cell carcinoma. D) Collagen XXIII staining is absent in small cell lung cancer tissues. Staining was performed using the 468642 antibody and images were acquired using a 20X objective lens.

Article Snippet: Antibody Validation A new human collagen XXIII mouse monoclonal antibody was made against the cleaved portion of human collagen XXIII by R&D Systems.

Techniques: Staining

Journal:

Article Title: Collagen XXIII--a potential biomarker for the detection of primary and recurrent non-small cell lung cancer

doi: 10.1158/1055-9965.EPI-09-1095

Figure Lengend Snippet: A) Bar graph showing percentage of patient tumor or normal tissue cores expressing collagen XXIII across all NSCLC TMAs and each individual TMA. Single asterisks indicate unavailable data. Patients lacking detectable collagen XXIII staining in all of their representative tissue cores were considered negative for collagen XXIII protein expression (Supplementary Table 1). Chi-squared analysis of a 3 by 2 contingency table for the combined TMA data was highly significant (p<0.001). A two-tailed p-value was calculated for each comparison between NSCLC subtype and normal lung tissue using Fisher’s Exact Test. Highly significant differences (p<0.001) are indicated (***). For immunohistochemical analysis, the 468642 antibody was used on the US Biomax and M.D. Anderson Cancer Center TMAs, while the 6.1B antibody was used on the Brigham and Women’s Hospital TMA. B) ROC curve plotting the true positive rate against the false positive rate for the different possible collagen XXIII staining intensity thresholds. (Tabulated data in supplementary table 2) Area under the curve = 0.860. C) Representative Kaplan Meier curves demonstrating reduced recurrence-free survival in patients with high (>85) collagen XXIII staining intensity (p= 0.013). Data for all examined threshold values is included in supplementary table 4.

Article Snippet: Antibody Validation A new human collagen XXIII mouse monoclonal antibody was made against the cleaved portion of human collagen XXIII by R&D Systems.

Techniques: Expressing, Staining, Two Tailed Test, Comparison, Immunohistochemical staining

Journal:

Article Title: Collagen XXIII--a potential biomarker for the detection of primary and recurrent non-small cell lung cancer

doi: 10.1158/1055-9965.EPI-09-1095

Figure Lengend Snippet: A) Collagen XXIII is detected in urine from 23 of 29 (79%) NSCLC patients but only in 15 of 54 (28%) control urine samples (Fisher’s Exact test; p<0.0001 ***). B) Representative Western blot demonstrating collagen XXIII positivity in a subset of urine samples using the 468642 antibody. Recombinant collagen XXIII corresponding to the cleaved ectodomain was used as a positive control (+ con) and is the predominant form of urinary collagen XXIII. Arrow indicates potential non-reduced protein complexes.

Article Snippet: Antibody Validation A new human collagen XXIII mouse monoclonal antibody was made against the cleaved portion of human collagen XXIII by R&D Systems.

Techniques: Control, Western Blot, Recombinant, Positive Control