Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Magnesium isoglycyrrhizinate ameliorates radiation-induced pulmonary fibrosis by inhibiting fibroblast differentiation via the p38MAPK/Akt/Nox4 pathway.

doi: 10.1016/j.biopha.2019.108955

Figure Lengend Snippet: Fig. 4. MgIG regulated the expression of TGF-β1 and Nox4, and the phosphorylation of p38MAPK and Akt in vivo. The protein level of TGF-β1 in lung tissues at 12 weeks post-irradiation was determined using (A) im- munohistochemical staining (×200, n = 6) and (B) western blotting (n = 3), and quanti- tative analyses were performed. (C) TGF-β1 content in serum at 12 weeks post-irradiation was measured using an ELISA kit (n = 6). The protein levels of Nox4, and the phosphoryla- tion of p38MAPK and Akt in lung tissues at 12 weeks post-irradiation were observed using (D) immunohistochemical staining (×200, n = 6) and (E) western blotting (n = 3), and quanti- tative analyses were performed. All data were expressed as the mean ± SEM. #P < 0.05 vs. the control group; *P < 0.05 vs. the IR group; &P < 0.05 vs. the IR + MgIG group; Bar =50 μm.

Article Snippet: The serum was used to measure the TGF-β1 concentration using an ELISA kit (EK0515, Boster Bioengineering Institute, Huhan, China), according to the manufacturer's instructions.

Techniques: Expressing, Phospho-proteomics, In Vivo, Irradiation, Staining, Western Blot, Enzyme-linked Immunosorbent Assay, Immunohistochemical staining, Control

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: MicroRNA-150 relieves vascular remodeling and fibrosis in hypoxia-induced pulmonary hypertension.

doi: 10.1016/j.biopha.2018.11.058

Figure Lengend Snippet: Fig. 4. Effect of miR-150 on pulmonary fibrosis of pulmonary hypertension rats. (A) Pulmonary fibrosis was detected by Masson’s staining (200× magnification). Scale bars, 100 μm. (B) The area of pulmonary fibrosis was calculated and shown. The mRNA expressions of TGF-β1 (C) and collagen I (D) in lung tissues were evaluated by qPCR. (E) The protein levels of TGF-β1 and collagen I in lung tissues were measured by western blot assay. (F)&(G) Relative grey values of the protein bands were shown. (H) The expressions of TGF-β1 and collagen I in lung tissues were detected by immunohistochemical staining (400× magnification). Scale bars, 50 μm. Data were presented as mean ± SD. **P < 0.01, ***P < 0.001 versus the indicated group.

Article Snippet: The membranes were blocked in 5% nonfat milk for 1 h at room temperature, then incubated with primary antibodies against TGF-β1 (1:400, BOSTER, China), Collagen I (1:300, BOSTER, China), p-AKTser473 (1:500, KeyGen, China), AKT (1:500, KeyGen, China), p-mTORser2481 (1:500, Sangon Biotech, China), mTOR (1:1000, Cell signaling Technology, USA), and β-actin (1:500, Bioss, China) at 4°C overnight, followed by incubation with HRP-labeled Goat AntiRabbit or Goat Anti-Mouse IgG (1:5000, Beyotime, China) at 37°C for 45min.

Techniques: Staining, Western Blot, Immunohistochemical staining

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: MicroRNA-150 relieves vascular remodeling and fibrosis in hypoxia-induced pulmonary hypertension.

doi: 10.1016/j.biopha.2018.11.058

Figure Lengend Snippet: Fig. 6. Effect of miR-150 on the expressions of fibrosis-related molecules. The mRNA expressions of TGF-β1 (A) and collagen I (B) in PASMCs were measured by qPCR. (C) The protein levels of TGF-β1 and collagen I in PASMCs were detected by western blot assay. (D)&(E) Relative grey values of the protein bands were shown. Data were presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 versus the indicated group.

Article Snippet: The membranes were blocked in 5% nonfat milk for 1 h at room temperature, then incubated with primary antibodies against TGF-β1 (1:400, BOSTER, China), Collagen I (1:300, BOSTER, China), p-AKTser473 (1:500, KeyGen, China), AKT (1:500, KeyGen, China), p-mTORser2481 (1:500, Sangon Biotech, China), mTOR (1:1000, Cell signaling Technology, USA), and β-actin (1:500, Bioss, China) at 4°C overnight, followed by incubation with HRP-labeled Goat AntiRabbit or Goat Anti-Mouse IgG (1:5000, Beyotime, China) at 37°C for 45min.

Techniques: Western Blot

Journal: iScience

Article Title: Kidney fibrosis molecular mechanisms Spp1 influences fibroblast activity through transforming growth factor beta smad signaling

doi: 10.1016/j.isci.2024.109839

Figure Lengend Snippet:

Article Snippet: TGF-β1 , Cell Signaling, USA , 5231LF.

Techniques: Recombinant, Staining, CCK-8 Assay, Enzyme-linked Immunosorbent Assay

Journal: Communications Medicine

Article Title: Suppression of NRAS -mutant melanoma growth with NRAS-targeting Antisense Oligonucleotide treatment reveals therapeutically relevant kinase co-dependencies

doi: 10.1038/s43856-025-00932-5

Figure Lengend Snippet: a Analysis of response of cell lines from the Dependency Map portal (DepMap) database to CRISPR-knockout (blue curve) or RNAi-mediated inhibition of NRAS -mRNA (green curve) shows that the vast majority of cell lines presented no dependency on NRAS -mRNA expression (dependency score 0, black dotted line). b Filtering for melanoma cell lines showed that specifically NRAS -mutant melanoma cells presented a strong vulnerability on NRAS -mRNA expression (dependency score ≤ -1, red dotted line). Dot plots represent all analyzed cell lines (black: non-melanoma, yellow: NRAS wild type melanoma, red: NRAS -mutant melanoma), highlighting that the dependent melanoma cell lines harbor NRAS mutations. c Subcellular mRNA enrichment analysis was done using qRT-PCR to compare the ratio of nuclear versus cytoplasmic mRNA levels of NRAS , GAPDH and B-ACTIN in D04 and MM415 cells. The data are presented as fold-change of nuclear to cytoplasmic ratio normalized to GAPDH ( n = 3) and show higher nuclear enrichment of NRAS-mRNA , when compared to reference genes. The error bars represent Standard Error (s.e.m.). d , e Representative images of RNA in situ hybridization (RNA-ISH) derived from d D04 and e MM415 cell pellets. Fluorescent signals are either produced by DAPI DNA staining to mark the nuclear regions (blue) or probes that stain the NRAS -mRNA (red). f Quantification of punctua per nucleus from fluorescent signals produced by probes that stain NRAS -mRNA in D04 and MM415 cells. The calculations included > 1000 cells per cell line. g Intronic (small bars) and exonic (large bars) regions of the NRAS gene (ENSG00000213281.5) as annotated in the Genecode database (V44). NRAS ASO target regions are highlighted in black and the codons Q61 and G12 are highlighted in red. h NRAS -mRNA (Genecode ID: ENST00000369535.5) secondary structure as predicted by the Minimum Free Energy (MFE) model. NRAS ASO target regions are highlighted in black, provided in additional cutout and zoom. Codon Q61 is highlighted in red. The ASO target regions represent stable and accessible structures.

Article Snippet: Fluorescent signals were produced by DAPI DNA staining to mark the nuclear regions (blue), probes that stain the NRAS -mRNA (red), and two different antibodies that stain for NRAS protein (ProteinTech 10724-1-AP – green, LsBio LS-C174539 – orange).

Techniques: CRISPR, Knock-Out, Inhibition, Expressing, Mutagenesis, Quantitative RT-PCR, RNA In Situ Hybridization, Derivative Assay, Produced, Staining

Journal: Communications Medicine

Article Title: Suppression of NRAS -mutant melanoma growth with NRAS-targeting Antisense Oligonucleotide treatment reveals therapeutically relevant kinase co-dependencies

doi: 10.1038/s43856-025-00932-5

Figure Lengend Snippet: a Using qRT-PCR to compare RNA levels in D04 and MM415 cells that were either treated with NRAS ASO-1 or NRAS ASO-2, showed a robust reduction of NRAS -mRNA levels after 6, 24, 48, and 72 hours, when compared to treatment with non-targeting Control ASO. Final oligonucleotide concentration was 100 nM; error bars represent s.e.m. ( n = 3). b , c Representative images of RNA in situ hybridization (RNA-ISH) derived from pellets of b D04 or c MM415 cells, either treated with NRAS ASO-1, or Control ASO. Fluorescent signals were produced by DAPI DNA staining to mark the nuclear regions (blue), probes that stain the NRAS -mRNA (red), and two different antibodies that stain for NRAS protein (ProteinTech 10724-1-AP – green, LsBio LS-C174539 – orange). NRAS ASO-1 treatment strongly reduced NRAS -mRNA levels in the cytoplasm and nucleus of the cells and NRAS protein expression. Final oligonucleotide concentration was 100 nM and treatment period lasted for 24 h. d Immunoblotting showing a strong decrease in NRAS protein levels 1 day after NRAS ASO-1 treatment compared to Control ASO treatment in D04 (−66%) and MM415 (−87%) cell lysates. B-ACTIN served as loading control and normalization parameter. e Immunoblotting showing a decrease in p-ERK1/2 protein levels 2 days after NRAS ASO treatment compared to Control ASO treatment in D04 (−50%) and MM415 (−50%) cell lysates, while total ERK1/2 levels were not altered significantly. GAPDH served as loading control and normalization parameter. f Immunoblotting showing a decrease in p-S6 protein levels 2 days after NRAS ASO-1 treatment compared to Control ASO treatment in D04 (−70%) and MM415 (−71%) cell lysates, while total S6 levels were not altered significantly. g Immunoblotting showing a small increase in p-AKT protein levels 2 days after NRAS ASO-1 treatment compared to Control ASO treatment in D04 (+18%) and MM415 (+12%) cell lysates. Total AKT levels were not altered significantly. Final oligonucleotide concentration was 100 nM. h A simplified illustration depicting key signaling pathways in NRAS -mutant melanoma, emphasizing the activation of crucial proteins contributing to cellular survival. Through transcription, the mutations in the NRAS gene are carried over to the NRAS -mRNA, which is translated into the constitutively active mutant NRAS protein, initiating downstream signaling cascades. This activation prompts the RAF kinase (not shown) to activate MEK, which, in turn, activates ERK. ERK signaling influences the activation of S6 ribosomal protein and translocates to the nucleus, regulating transcription and supporting cellular proliferation. S6 plays a pivotal role in translation, facilitating protein synthesis. The activation of this signaling pathways enhances cellular survival in NRAS -mutant melanoma. Phosphorylation-dependent activation steps are denoted by (P). i A simplified illustration highlighting the impact of NRAS ASO treatment: NRAS ASOs reduce NRAS -mRNA levels in both the cytoplasm and nucleus. This reduction is followed by decreased NRAS protein levels and the inhibition of MAPK-pathway signaling activity, as evidenced by diminished p-ERK and p-S6 protein levels. The pathway is essential for the NRAS-mutant cancer cells’ ability to proliferate and survive.

Article Snippet: Fluorescent signals were produced by DAPI DNA staining to mark the nuclear regions (blue), probes that stain the NRAS -mRNA (red), and two different antibodies that stain for NRAS protein (ProteinTech 10724-1-AP – green, LsBio LS-C174539 – orange).

Techniques: Quantitative RT-PCR, Control, Concentration Assay, RNA In Situ Hybridization, Derivative Assay, Produced, Staining, Expressing, Western Blot, Protein-Protein interactions, Mutagenesis, Activation Assay, Phospho-proteomics, Inhibition, Activity Assay

Journal: Communications Medicine

Article Title: Suppression of NRAS -mutant melanoma growth with NRAS-targeting Antisense Oligonucleotide treatment reveals therapeutically relevant kinase co-dependencies

doi: 10.1038/s43856-025-00932-5

Figure Lengend Snippet: a Treatment with NRAS ASO-1 caused significant inhibition of cell growth in the NRAS -mutant melanoma cell lines D04 ( p = 0.000002), MM415 ( p = 0.00002), WM1366 ( p = 0.0005), Sk-Mel-2 ( p = 0.00001), VMM39 ( p = 0.00004), WM3060 ( p = 0.003), NZM40 ( p = 0.0006), WM3629 ( p = 0.0008), and the primary derived cell line Hs852T ( p = 0.000006). b Treatment with NRAS ASO-2 caused significant inhibition of cell growth in the NRAS -mutant melanoma cell lines D04 ( p = 0.000004) and MM415 ( p = 0.0001). The antiproliferative outcomes are similar when compared to treatment with NRAS ASO-1. c NRAS ASO treatment did not cause significant antiproliferative effects in primary human melanocytes (PHM, p = 0.33), primary human liver cells (Hs775li, p = 0.29), human colon cells (FHC, p = 0.29), and BRAF-mutant melanoma cells (Sk-Mel-28, p = 0.13). d NRAS ASO treatment significantly inhibited colony formation in the D04 ( p = 0.0017) and MM415 ( p = 0.008) cell lines compared to treatment with non-targeting Control ASOs. Treatment period was 7 days (50 nM final oligonucleotide concentration, n = 3). e Representative images of D04 colonies in 6 cm dishes after ASO treatment. f Dot plot graph of flow cytometric analysis of PI and Annexin V staining after 1 day of ASO-treatment (100 nM) shows increased apoptotic cell death in D04-cells treated with NRAS ASO (15,780 total events) compared to Control ASO treatment (44,285 total events). g Distribution of overall cell populations from panel f ) in regards of their apoptotic state. Bars represent the percentage of vital (Q2), early apoptotic (Q3), late apoptotic (Q4) and dead (Q1) cells. h NRAS ASO-mediated induction of apoptosis was confirmed by measurement of significantly increased activity levels of the apoptosis markers Caspase-3 & -7 after 1 day of treatment with either NRAS or Control ASOs (100 nM) in the D04 ( p = 0.002) and MM415 ( p = 0.0002) cell lines ( n = 4). i Treatment with NRAS ASO−1 caused significant inhibition of cell growth in the NRAS -mutant multiple myeloma (MM) cell line H929 ( p = 0.0005), and small cell lung cancer (SCLC) cell line SW1271 ( p = 0.0001). j Significant tumor growth reduction was observed when comparing treatment groups for subcutaneous systemic treatment with either NRAS ASO (X) or Control ASO (O) in mouse models carrying xenografts of the D04 melanoma cell line (3 × 200 µg ASO/week, n = 6, days of measurement and p -values: −3 –0.38, 1 –0.27, 3 –0.02, 5 –0.04, 8 –0.05, 10 – 0.02, 12 –0.06, 15 –0.02, 17 – 0.02, 19 – 0.03). At the endpoint of the experiment (day 19), the average tumor size in the NRAS ASO treatment group was 48% smaller compared to control. k NRAS -mRNA levels were significantly reduced (0.68-fold, s.e.m = 0.03, p = 0.0003) in tumors of the NRAS ASO treatment group compared to the Control ASO treatment group at the end of study period. Tumors were harvested at end of treatment period; gene expression was normalized to Β-ACTIN expression and NRAS -mRNA expression in NRAS ASO treated tumors was normalized to expression in Control ASO treated tumors ( n of each group = 5). l No significant weight changes were observed between the NRAS ASO (X) and Control ASO (O) groups at any time-point (days of measurement and p -values: -3 – 0.3, 1 – 0.36, 3 – 0.33, 5 – 0.46, 8 – 0.43, 10 – 0.5, 12 – 0.47, 15 – 0.49, 17 – 0.48, 19 – 0.49). m Blood of mice that either received a dose of NRAS ASO-1 (200 µg/injection), or ASO-free PBS was drawn 24 hours after injection and analyzed for parameters of liver function (Serum transaminases – ALT, AST, bilirubin - TBIL, direct (conjugated) bilirubin - DBIL, total protein - TP, albumin - ALB, and alkaline phosphatase – ALKP). All growth and weight curves are presented as polynomial trend lines (order: 2). Data in ( a – c , i ) were normalized to treatment with non-targeting Control ASO, final oligonucleotide concentration was 50 nM, treatment period was 5 days ( n = 3). The error bars in a – d ), h , i , m ) represent s.d., in j − l they represent s.e.m. Significance is shown as p -values calculated by Student’s t-test. * = p < 0.05, ** = p < 0.01, *** = p < 0.001.

Article Snippet: Fluorescent signals were produced by DAPI DNA staining to mark the nuclear regions (blue), probes that stain the NRAS -mRNA (red), and two different antibodies that stain for NRAS protein (ProteinTech 10724-1-AP – green, LsBio LS-C174539 – orange).

Techniques: Inhibition, Mutagenesis, Derivative Assay, Control, Concentration Assay, Staining, Activity Assay, Gene Expression, Expressing, Injection

Journal: Communications Medicine

Article Title: Suppression of NRAS -mutant melanoma growth with NRAS-targeting Antisense Oligonucleotide treatment reveals therapeutically relevant kinase co-dependencies

doi: 10.1038/s43856-025-00932-5

Figure Lengend Snippet: a Schematic illustration of HT-KAM analysis of the phosphor-catalytic activity of kinases. D04 and MM415 cells were either treated with NRAS or Control ASOs (50 nM, 1 day). Cells were lysed, and protein lysate was investigated for peptide-associated phosphorylation activity of kinases. b Comparison of kinase activity in treatment groups (NRAS ASO VS. Control ASO) showed that kinase activity of several kinases was significantly upregulated in the D04 and MM415 cell lines upon NRAS ASO treatment. Kinases are ranked by their relative increase of activity (from bottom to top). The top 3 kinases with strongest shift in activity increase are MAP2K1 (MEK1), FGFR2, and CDK4. The RET kinase activity shift is shown as a representative example for kinases that were downregulated in activity. c QRT-PCR analysis showing elevated NRAS -mRNA levels in D04 and MM415 cells after three days of drug-induced Inhibition of MEK (MEKi), using the small molecule inhibitor Trametinib (20 nM or 40 nM), when compared to control, treated with DMSO ( n = 3). d QRT-PCR analysis showing elevated NRAS -mRNA levels in the MEKi resistant cell lines D04RM and MM415RM, which were chronically exposed to Trametinib, when compared to their paternal treatment naïve cell lines D04 and MM415 ( n = 3). Error bars in panel ( c , d ) represent s.e.m. e Treatment with NRAS ASO-1 caused significant inhibition of cell growth in the MEKi resistant NRAS mutant melanoma cell lines D04RM (p = 0.011), MM415RM ( p = 0.001), WM3629RM ( p = 0.0002), and Sk-Mel-2RM ( p = 0.015). Data were normalized to treatment with non-targeting Control ASO; treatment period was 5 days, final oligonucleotide concentration was 50 nM, and error bars represent s.d. ( n = 3). f – i Dual treatment with 20 nM of NRAS ASO and Trametinib (Tram, 0.5 nM −25 nM) caused robust synergistic effects in D04 ( f , g ) and MM415 ( h , i ) cells after 3 ( f , h ) and 5 ( g , i ) days of treatment ( n = 2). Dose response curves show NRAS ASO treatment (blue), trametinib treatment (yellow) and dual treatment (red). Synergism of dual cell growth inhibition is shown as bar graphs and determined by the HSA synergy score.

Article Snippet: Fluorescent signals were produced by DAPI DNA staining to mark the nuclear regions (blue), probes that stain the NRAS -mRNA (red), and two different antibodies that stain for NRAS protein (ProteinTech 10724-1-AP – green, LsBio LS-C174539 – orange).

Techniques: Activity Assay, Control, Phospho-proteomics, Comparison, Quantitative RT-PCR, Inhibition, Mutagenesis, Concentration Assay