Journal: Journal of Biological Chemistry

Article Title: Platelet-derived Growth Factor and Reactive Oxygen Species (ROS) Regulate Ras Protein Levels in Primary Human Fibroblasts via ERK1/2

doi: 10.1074/jbc.m502851200

Figure Lengend Snippet: FIGURE 1. Induction of Ras proteins by PDGF in primary fibroblasts. Primary fibroblasts derived from healthy donors (2–5 passages) were incubated in 0.2% of FCS for 48 h and then stimulated with PDGF 15 ng/ml for the indicated time. A, total proteins were extracted, immunoprecipitated with pan-Ras antibodies, and immunoblotted with specific antibodiesagainstHa-RasandKi-Ras.Theimmunoblotshownisrepresentativeofthreeexperimentsperformedinduplicate. DUontheordinatesindicatesanarbitrarydensitometric unit, normalized to the band of Ha-Ras without treatment with PDGF set to 1. B, fluorescence microscopy of fibroblasts preincubated in 0.2% FCS for 48 h and stimulated with PDGF (15 ng/ml) for 15 min. Cells were stained by indirect immunofluorescence with specific antibodies to Ha-Ras and Ki-Ras. Representative results from 1 of 5 experiments are shown. Neg indicates immunofluorescence with non-immune sera. 40 magnification. C, FACS analysis with anti-Ha-Ras antibodies of cells treated as indicated in B. The histograms of a representative experiment are shown. Values are mean 1 S.D. of three independent experiments performed in duplicate. Both the monoclonal (F235) and polyclonal (SC520) antibodies to Ha-Ras were used with identical results (see “Materials and Methods”). D, Ha-Ras expression in normal fibroblasts stimulated with PDGF (15 ng/ml) in the presence or absence of cycloheximide (10 mg/ml for 6 h). A representative of three experiments is shown. E, semiquantitative RT-PCR of RNA extracted from cells stimulated with 15 ng of PDGF for 60 min. The reactions were carried out as described under “Materials and Methods.” Here a representative of three reactions (20 cycles) is shown. Under these conditions the intensity of the specific bands was linearly dependent on the concentration of cDNA. Ki long and short represent the two major Ki4B mRNAs differing in the length of 3-untranslated region.

Article Snippet: Ras Immunoprecipitation—Ras proteins were immunoprecipitated from cultured fibroblasts with polyclonal anti-pan Ras antibody (Santa Cruz Biotechnology) following recommended procedures from the manufacturer.

Techniques: Derivative Assay, Incubation, Immunoprecipitation, Fluorescence, Microscopy, Staining, Immunofluorescence, Expressing, Reverse Transcription Polymerase Chain Reaction, Concentration Assay

Journal: Journal of Biological Chemistry

Article Title: Platelet-derived Growth Factor and Reactive Oxygen Species (ROS) Regulate Ras Protein Levels in Primary Human Fibroblasts via ERK1/2

doi: 10.1074/jbc.m502851200

Figure Lengend Snippet: FIGURE 5. Accelerated senescence of scleroderma fibroblasts. A, activation of DNA damage checkpoint in scleroderma fibroblasts. Immunoblot with a specific antibody that recognize phosphorylated histone H2AX or p21WAF. Representative results from 1 of 3 experiments are shown. A polyclonal antibody against ERK1/2 was used to normalize the amount of protein loaded. B, chromosomal damage in scleroderma fibroblasts. Karyotype analysis of three lines of primary fibroblasts derived from scleroderma lesions. These lines were cultured for 2 passages and treated 48 h with FTI-277 (10 g/ml). At least 50 metaphases were scored for each line, included three normal primary fibroblasts. The frequency of altered metaphases in normal lines was 4% 2 in the presence or absence of FTI (48 h, 10 g/ml) (data not shown). The aberrations found were not artifact-derived from in vitro growth of the cells, since they were present in the first passages. Some alterations were more frequent, depending on the time elapsed from the initial passages in culture to the time of analysis. The right panel shows representative chromosomal alterations found in SSc cells. C, oxidative stress-induced apoptosis in scleroderma cells. Normal (N) and SSc fibroblasts were stimulated for 2 h with increasing concentrations of H2O2 in the presence or absence of MEK inhibitor PD 98059 (40 M). Apoptosis was then detected by FACS analysis of annexin V-C-stained cells. Diagram represents the percentage of annexin V-positive cells. The data shown are the mean 1 S.D. of three independent experiments. D, activation of ROS-inducible genes in scleroderma cells. Activation of collagen promoter by ROS in SSc cells. N or SSc cells were transfected with the empty vector (pGL3) or with 2(I) collagen promoter driving the expression of luciferase gene (Col1A2) with or without a vector expressing the human catalase gene (pCat, 10). The ratio of Renilla to firefly luciferase values was used to normalize co-transfection experiments. Data are expressed as mean 1 S.D. (n 3). E, 1(I) and 2(I) collagen mRNA after incubation of SSc fibroblasts with PD98059 (40 M for 24 h), FTI-277 (20 M for 24 h) or transfection with an expression vector carrying the human catalase gene. Collagen mRNAs in normal cells stimulated with H2O2. (for 24 h). Representative results from 1 of 3 experiments are shown.

Article Snippet: Ras Immunoprecipitation—Ras proteins were immunoprecipitated from cultured fibroblasts with polyclonal anti-pan Ras antibody (Santa Cruz Biotechnology) following recommended procedures from the manufacturer.

Techniques: Activation Assay, Western Blot, Derivative Assay, Cell Culture, In Vitro, Staining, Transfection, Plasmid Preparation, Expressing, Luciferase, Cotransfection, Incubation

Journal: Journal of Hematology & Oncology

Article Title: RAS signaling in carcinogenesis, cancer therapy and resistance mechanisms

doi: 10.1186/s13045-024-01631-9

Figure Lengend Snippet: Other inhibitors in clinical trials

Article Snippet: Pan-RAS/ RAS wild-type inhibitor , 1 , RMC-6236 (Revolution Medicines) (ON state; tri-complex inhibitor inhibitor) , NCT05379985 , NSCLC ( n = 40); PDAC ( n = 46) (12) , NSCLC ORR: 38% PDAC ORR: 20%.

Techniques: Adjuvant, Biomarker Assay