Journal: Journal of Biological Chemistry

Article Title: The Leukocyte Chemotactic Receptor FPR1 Is Functionally Expressed on Human Lens Epithelial Cells

doi: 10.1074/jbc.m112.411181

Figure Lengend Snippet: FIGURE 1. Lens epithelial cells constitutively express FPR1; RNA and immunophenotypic evidence. A, RNA is shown. FHL 124 cells nucleofected with the shRNA indicated on the x axis were analyzed for FPR1 mRNA expression by PCR. B, immunophenotype is shown. FHL 124 cells nucleofected with the shRNAs indicated on the x axis were analyzed by FACS with a PE-labeled anti-FPR1 mAb. C, ligand binding; FHL 124 cells nucleofected with the shRNAs indicated on the x axis were analyzed by FACS with the fluorescent agonist ligand fNLFNYK-Fl (10 nM). D, Ca2 assay; FHL 124 cells nucleofected with the shRNAs indicated in the legend were stimulated with 1 M fMLF, and the Ca2 response was followed for 150 s. The base line (first 20 s) of each individual curve and a background curve recorded with only buffer (no fMLF) were subtracted from each signal. Then all experiments were pooled and transformed to % of the peak signal intensity of the siRNA-free (only H2O) control. All data are the means S.E.

Article Snippet: 100 pmol of FPR1 siRNA (three 20–25-nucleotide siRNAs, catalog no. sc-40121, Santa Cruz Biotechnology, Santa Cruz, CA), 100 pmol of scrambled fluorescein-labeled siRNA (Santa Cruz Biotechnology, catalog no. sc-36869), or water was added to 100 l of cell suspension and nucleofected with program X-005 (FHL 124) or Q-001 (HEK 293-FPR1 ) of the Nucleofector II Device (Lonza).

Techniques: shRNA, Expressing, Labeling, Ligand Binding Assay, Transformation Assay, Control

Journal: Oncotarget

Article Title: Epigenomic study identifies a novel mesenchyme homeobox2-GLI1 transcription axis involved in cancer drug resistance, overall survival and therapy prognosis in lung cancer patients

doi: 10.18632/oncotarget.17715

Figure Lengend Snippet: (A) mRNA expression analysis was performed for the A427 and A549 lung adenocarcinoma cell lines in the presence or absence of cisplatinum-based treatment, and the effects of negative control scramble siRNAs and specific anti-MEOX2 siRNAs were assessed. The results are shown for two representative biological experiments performed in triplicate, with * p ≤0.05, and ** p ≤0.01. (B) A427 and A549 lung adenocarcinoma cells exhibited a cisplatinum-inducible GLI-1 protein expression pattern at IC:12.5 (8 μM), while reduced inducible GLI-1 expression was observed following transfection with anti-MEOX2 siRNA in the presence of 8 μM cisplatinum. Western blot statistical analyses, assessed via quantitative densitometry, were performed to determine * p ≤0.05 and ** p ≤0.005 using Student's t -test as well as one-way ANOVA with Dunnett's and Tukey's multiple comparison tests. Western blot bands were quantified as the pixel total intensity rate and expressed as a Change Index normalized to GAPDH. Images are representative of 3 biological replicates. Quantification analyses were performed using cisplatinum (0 μM) treatment as a negative control reference. Images were obtained using a C-DIGIT device (LICOR). Pixel quantification and data analyses were carried out using Image Studio software; the total pixel intensity for each specific protein product was normalized to GAPDH.

Article Snippet: Two non-human related siRNAs obtained from Santa Cruz Biotechnology (Dallas, TX, USA) were used as negative controls (sc-37007 and sc-44230).

Techniques: Expressing, Negative Control, Transfection, Western Blot, Comparison, Software

Journal: Oncotarget

Article Title: Epigenomic study identifies a novel mesenchyme homeobox2-GLI1 transcription axis involved in cancer drug resistance, overall survival and therapy prognosis in lung cancer patients

doi: 10.18632/oncotarget.17715

Figure Lengend Snippet: (A) A549 and H1975 lung cancer cells demonstrated an inducible GLI-1 protein expression pattern following treatment with 8 μM cisplatinum and reduced GLI-1 inducible expression following the application of specific anti-MEOX2 siRNA and/or anti-MEOX2 siRNA plus anti-GLI1 siRNA in the presence of 8 μM cisplatinum. Western blot statistical analyses, assessed via quantitative densitometry, were performed to determine * p ≤0.05 by one-way ANOVA and Dunnett's test for multiple comparisons to identify significant differences with respect to controls. Student's t -test was performed to identify significant differences between control and cisplatinum treatment. Quantification analyses were normalized to scrambled siRNA as a negative control for gene silencing. Images were obtained using a C-DIGIT device (LICOR), and pixel quantification and data analyses were carried out using Image Studio software. Total pixel intensity for each specific protein product was normalized to GAPDH. (B) Cell culture images and graphs showing the quantitative analysis of cellular migration as a percentage (transwell migration assays) indicated significant MEOX2 and GLI-1 protein-dependent functions following the individual and combined application of anti-MEOX2 and anti-GLI1 siRNAs in A549, NH2347 and H1975 lung adenocarcinoma cells; ** p ≤0.005 and *** p ≤0.0001 based on one-way ANOVA and Dunnett's multiple comparisons test. (C) Cell culture images and graphs showing the quantitative analysis of cellular proliferation (clonogenic assays) indicated significant MEOX2 and GLI-1 protein-dependent functions following the individual and combined application of anti-MEOX2 and anti-GLI1 siRNAs in A549, NH2347 and H1975 lung adenocarcinoma cells; ** p ≤0.005 and *** p ≤0.0001 based on one-way ANOVA and Dunnett's multiple comparisons test. Transwell migration index and colony growth (clonogenic assays) rates were normalized and quantified using the ImageJ Colony Number plugin tool (see Materials and Methods).

Article Snippet: Two non-human related siRNAs obtained from Santa Cruz Biotechnology (Dallas, TX, USA) were used as negative controls (sc-37007 and sc-44230).

Techniques: Expressing, Western Blot, Control, Negative Control, Software, Cell Culture, Migration

Journal: Cancer Management and Research

Article Title: Chrysin inhibits sphere formation in SMMC-7721 cells via modulation of SHP-1/STAT3 signaling pathway

doi: 10.2147/CMAR.S193647

Figure Lengend Snippet: Knockdown of SHP-1 induced the p-STAT3 and Twist1 expression. Western blot analysis has performed the levels of SHP-1, p-STAT3 (Tyr705) and Twist1 in SMMC-7721 cells transfected with SHP-1 siRNA; β-actin was used as an internal control. ( A , C , and E ) Representative blot from three independent experiments. ( B , D , and F ) relative density or phosphorylation level (Mean ± SD, n=3); * p <0.05 vs SMMC-7721 cell line; # p <0.05 vs SMMC-7721 cell line transfected with control.

Article Snippet: The siRNA components used in experiments, control (sc-37007) and SHP-1 (sc-44101 primers: sense-GCAGGAGUCCGAGGAUACATT, antisense-UGUACCUCGGACUCCUGCTT)) were purchased from Santa Cruz Biotechnology (Santa Cruz, MO, USA).

Techniques: Knockdown, Expressing, Western Blot, Transfection, Control, Phospho-proteomics

Journal: Cancer Management and Research

Article Title: Chrysin inhibits sphere formation in SMMC-7721 cells via modulation of SHP-1/STAT3 signaling pathway

doi: 10.2147/CMAR.S193647

Figure Lengend Snippet: Effect of SHP-1 siRNA transfection on chrysin inhibition of sphere formation, upregulation of SHP-1 expression and decreasing expression of p-STAT3 and Twist1 in SMMC-7721 cells. ( A ) SHP-1 siRNA transfection abrogated chrysin’s inhibition of sphere formation in SMMC-7721 cells. Representative phase contrast microscopy images are shown (×10). ( B ) statistical analysis of sphere formation rate (mean ± SD; n=9). * p <0.05 vs control; # p <0.05 vs SMMC-7721 cells transfected with si-SHP-1. ( C , E , and G ) Representative blot from three independent experiments. ( D , F , and H ) relative density or phosphorylation of target band (mean ± SD; n=3); * p <0.05 vs control; # p <0.05 vs SMMC-7721 cells transfected with si-SHP-1.

Article Snippet: The siRNA components used in experiments, control (sc-37007) and SHP-1 (sc-44101 primers: sense-GCAGGAGUCCGAGGAUACATT, antisense-UGUACCUCGGACUCCUGCTT)) were purchased from Santa Cruz Biotechnology (Santa Cruz, MO, USA).

Techniques: Transfection, Inhibition, Expressing, Microscopy, Control, Phospho-proteomics

Journal: BMC cardiovascular disorders

Article Title: Single-cell RNA sequencing reveals hub genes of myocardial infarction-associated endothelial cells.

doi: 10.1186/s12872-024-03727-z

Figure Lengend Snippet: Fig. 7 Timp1 and Fn1 were upregulated in hypoxia-induecd HUVECs. a The expressions of Timp1 and Fn1 in HUVECs induced by hypoxia detected by Western blot. b The expressions of Timp1 or Fn1 in HUVECs transfected with siRNA of Timp1 or Fn1 detected by Western blot. *P < 0.05 compared with the Control group; **P < 0.01 compared with the Control group

Article Snippet: In line with the manufacturer’s instructions, tissue inhibitor of matrix metalloproteinase 1 (Timp1) siRNA or control siRNA (JTS scientific company, Wuhan, China), and Fibronectin 1 (Fn1) siRNA or control siRNA (Santa Cruz Biotechnology, Dallas, TX, USA, cat#: sc-29,315) were transfected into cells using lipofectamine 3000 (Invitrogen, MD, USA).

Techniques: Western Blot, Transfection, Control

Journal: BMC cardiovascular disorders

Article Title: Single-cell RNA sequencing reveals hub genes of myocardial infarction-associated endothelial cells.

doi: 10.1186/s12872-024-03727-z

Figure Lengend Snippet: Fig. 8 Timp1 and Fn1 inhibited cell migration and tube formation of HUVECs. a Wound healing assays were used to examine the migration of HUVECs transfected with siRNA of Timp1 or Fn1. b Tube formation of HUVECs after transfection with the siRNA of Timp1 or Fn1. *P < 0.05 Timp1 siRNA compared with the NC siRNA group; #P < 0.05 Fn1 siRNA compared with the Control group

Article Snippet: In line with the manufacturer’s instructions, tissue inhibitor of matrix metalloproteinase 1 (Timp1) siRNA or control siRNA (JTS scientific company, Wuhan, China), and Fibronectin 1 (Fn1) siRNA or control siRNA (Santa Cruz Biotechnology, Dallas, TX, USA, cat#: sc-29,315) were transfected into cells using lipofectamine 3000 (Invitrogen, MD, USA).

Techniques: Migration, Transfection, Control

Journal: Cell Death & Disease

Article Title: Rewiring melanoma cell fate: TRPM8 modulators trigger apoptosis and boost NK cell cytotoxicity

doi: 10.1038/s41419-026-08469-8

Figure Lengend Snippet: A – C WM266-4 cells were transfected with control siRNA (siRNA ctrl) or TRPM8-targeting siRNA (siRNA TRPM8). A Western blot analysis was performed on total cell lysates using the indicated antibodies. B Cells were then left untreated or treated with compound 4 or 9 (1 μM, 24 h), and cell death was assessed by PI staining. Total cells were stained in green with acridin orange. C Quantification of PI-positive cells corresponding to ( B ). Representative Western blots showing transient TRPM8 overexpression (TRPM8 OE) in WM266-4 ( D ) and AMM16 ( F ) melanoma cells. E , G Densitometric analysis of TRPM8 and GAPDH protein levels, represented as TRPM8/GAPDH ratios (from three independent experiments). WM266-4 ( H ) and AMM16 ( I ) cells transfected with control plasmid (ctrl plasmid) or TRPM8 plasmid (TRPM8 OE) were left untreated or treated with compounds 4 and 9 (1 or 10 μM) for 6 h. Representative images (contrast phase, PI staining, and overlays) and corresponding quantitative graphs (below panels) are shown. Scale bar, 100 μm. In C , E , G , H , I Data are presented as mean ± SD from n independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001. In H , I Red asterisks indicate statistical significance between ctrl and OE groups at the corresponding treatment conditions.

Article Snippet: A non-targeting siRNA (sc-44236; Santa Cruz Biotechnology) was used as a negative control.

Techniques: Transfection, Control, Western Blot, Staining, Over Expression, Plasmid Preparation

Journal: Cell Death & Disease

Article Title: Rewiring melanoma cell fate: TRPM8 modulators trigger apoptosis and boost NK cell cytotoxicity

doi: 10.1038/s41419-026-08469-8

Figure Lengend Snippet: A Representative images of crystal violet-stained colonies, derived from WM266-4 cells, after 21-day treatment with TRPM8 modulators. B Western blot analysis showing ULBP1 expression in WM266-4 cells after treatment with TRPM8 modulators. The α-tubulin was used as a loading control. C WM266-4 derived spheroids treated for 21 days as indicated, in absence (upper panel; -NK cells) or presence (lower panel; + NK cells) of NK cells. Images are representative of three different experiments. Bar, 100 µm. D The graph represents the dead cells/total cells. Values of dead (red stained cells) and total cells (green stained cells) were analyzed using NIH Image J. They derive from red fluorescence mean/green fluorescence mean intensity and are expressed as mean ± SD of 3 different experiments ( n = 3); ** p < 0.01; *** p < 0.001. E NK cell cytotoxicity assay. WM266-4 cells pre-treated with TRPM8 modulators were co-cultured with primary NK cells at the indicated effector:target (E:T) ratios. Where indicated, neutralizing antibodies against ULBP1 or NKG2D were added 1 h before co-culture to melanoma cells or NK, respectively. Data are presented as percentage of lysis. * p < 0.05; ** p < 0.01. F WM266-4 cells were transfected with control siRNA (siRNA ctrl) or TP53-targeting siRNA (siRNA p53) at two different concentrations (300 pmol and 400 pmol, respectively). After 4 days, cells were collected, lysed, and Western blot analysis was performed on cell lysates using the indicated antibodies. α-Tubulin was used as a loading control. G WM2664 cells transfected with control siRNA (siRNA ctrl) or TP53-targeting siRNA (siRNA p53; 300 pmol) were unstimulated or stimulated with compounds 4 and 9 (at 1 μM) for 4 days and then collected and lysed. Western blot analysis was performed using the indicated antibodies. α-Tubulin was used as a loading control. H Phosphorylated AKT (Ser473) levels in melanoma cells treated with the PI3K agonist, 740 Y-P, in absence or presence of TRPM8 modulators (used at 1 μM) for 4 days, analyzed by Western blot. GAPDH was used as a loading control.

Article Snippet: A non-targeting siRNA (sc-44236; Santa Cruz Biotechnology) was used as a negative control.

Techniques: Staining, Derivative Assay, Western Blot, Expressing, Control, Fluorescence, Cytotoxicity Assay, Cell Culture, Co-Culture Assay, Lysis, Transfection

Journal: Acta Pharmaceutica Sinica. B

Article Title: Evolution-guided design of mini-protein for high-contrast in vivo imaging

doi: 10.1016/j.apsb.2025.07.015

Figure Lengend Snippet: In vitro pharmacodynamic characterization of BindHer. (A) Flow cytometry analysis of HER2 levels in SK-BR-3 cells treated with HER2 siRNA, using Trastuzumab-PE/Cy7 to confirm HER2 expression. (B) ELISA confirming BindHer's binding specificity to HER2 across concentrations from 100 to 0.098 nmol/L. (C) In vivo immunogenicity of BindHer and ABY-025 with hIgG used as a positive control. (D) Western blot of SK-BR-3 cell lysates following BindHer treatment at various concentrations, with EGF as a positive control. Equal protein amounts were loaded in each lane; SDS-PAGE gels were run, and membranes were sectioned by molecular weight for analysis of total HER2, phospho-HER2, and β -actin. Phospho-HER2/HER2 quantification is depicted in graphs, where ∗∗∗∗ P < 0.0001; ns denotes non-significant results.

Article Snippet: SK-BR-3 cells were plated in 6-well plates at a density of 10 6 cells per well and incubated for 24 h. HER2 siRNA I (#6282, Cell Signaling Technology) and control siRNA (#6568, Cell Signaling Technology) were transfected into the cells using LipofectamineTM 3000 (Invitrogen) according to the manufacturer's instructions.

Techniques: In Vitro, Flow Cytometry, Expressing, Enzyme-linked Immunosorbent Assay, Binding Assay, In Vivo, Immunopeptidomics, Positive Control, Western Blot, SDS Page, Molecular Weight