Journal: Molecular Therapy. Nucleic Acids

Article Title: miR-200-mediated inactivation of cancer-associated fibroblasts via targeting of NRP2-VEGFR signaling attenuates lung cancer invasion and metastasis

doi: 10.1016/j.omtn.2024.102194

Figure Lengend Snippet: VEGF-D and PTN derived from CAFs activate cancer cell migration and invasion (A and B) RT-qPCR analysis of Vegfd and Ptn expression in CAF-vec and CAF-200 (A), and CAFs transfected with NTC or Nrp2 siRNA #2 ( siNrp2 #2) (B). The expression levels were normalized to the Rpl32 mRNA level, and the values relative to those of the control (set at 1.0) are presented. Mean ± SD ( n = 3). P , two-tailed Student’s t test. (C, D) ELISA of secreted VEGF-D (C) and PTN (D) in the CAF-vec and CAF-200 culture media. Mean ± SD ( n = 3). P , two-tailed Student’s t test. (E) RT-qPCR analysis of Vegfd expression in murine CAFs transfected with NTC or Vegfd siRNAs (#1, #2, and #3). The expression levels were normalized to the Rpl32 mRNA level, and the values relative to those of NTC (set at 1.0) are presented. Mean ± SD ( n = 3). P, two-tailed Student’s t test. (F) Transwell migration assay of 344SQ cells co-cultured with CAFs transfected with NTC or Vegfd siRNAs (si Vegfd #2 and #3). CAFs were seeded in the bottom wells, and 344SQ cells were seeded in the upper wells. After 24 h, the migrated 344SQ cells were photographed and counted. Mean ± SD ( n = 3). P , two-tailed Student’s t test. (G) Spheroid invasion assay of 344SQ cells co-cultured with CAFs transfected with NTC or si Vegfd . Mean ± SD (+NTC, n = 59; +si Vegfd #2, n = 73; +si Vegfd #3, n = 45). P , two-tailed Student’s t test. (H) RT-qPCR analysis of Ptn expression in murine CAFs transfected with NTC or Ptn siRNAs (#1, #2, and #3). Mean ± SD ( n = 3). P , two-tailed Student’s t test. (I) Transwell migration assay of 344SQ cells co-cultured with CAFs transfected with NTC or Ptn siRNAs (si Ptn #1 and #3). Mean ± SD ( n = 3). P , two-tailed Student’s t test. (J) Spheroid invasion assay in 344SQ cells co-cultured with CAFs transfected with NTC or si Ptn . Mean ± SD (+NTC, n = 37; +si Ptn #1, n = 41; +si Ptn #3, n = 78). P , two-tailed Student’s t test. (K) Transwell migration assay of 344SQ cells treated with VEGF-D and PTN recombinant proteins. 344SQ cells were seeded in the upper inserts and VEGF-D and PTN recombinant proteins (20 ng/mL) were added to the bottom wells. After 24 h, migrated 344SQ cells were photographed and counted. Mean ± SD ( n = 3). P , two-tailed Student’s t test. (L) Spheroid invasion assay of 344SQ cells treated with recombinant VEGF-D and PTN. Mean ± SD (+BSA, n = 67; +VEGF-D, n = 73; +PTN, n = 70; +VEGF-D/PTN, n = 149). P , two-tailed Student’s t test. (M) RT-qPCR analysis of Vegfd and Ptn expression in CAFs treated with VEGF-A plus inhibitors of FAK (PF-562271, 10 μM) and p38 MAPK (SB203580, 10 μM). Mean ± SD ( n = 3). ∗ p < 0.05, ∗∗ p < 0.01; two-tailed Student’s t test. (N) Western blots of phospho-FAK, FAK, phospho-MAPKAPK2, and MAPKAPK2 (a substrate of p38 MAPK) in CAF-vec and CAF-200. Actin was used as a control. Relative quantitation results (phospho-form/total) are shown below. (O) A diagram suggesting that miR-200 suppresses CAF-derived VEGF-D and PTN secretion by targeting NRP2/VEGFR signaling, leading to the inhibition of cancer cell migration and invasion.

Article Snippet: Antibodies against NRP2 (1:1,000 dilution, #sc-13117; Santa Cruz Biotechnology, Dallas, TX, USA), phospho-FAK (1:1,000 dilution, #sc-81493; Santa Cruz Biotechnology), FAK (1:1,000 dilution, #sc-557; Santa Cruz Biotechnology), phospho-MAPKAPK2 (1:1,000 dilution, #3041; Cell Signaling Technology, Danvers, MA, USA), MAPKAPK2 (1:1,000 dilution, #3042; Cell Signaling Technology), and β-actin (1:5,000 dilution, #BS6007M; Bioworld Technology, St. Louis Park, MN, USA) were used.

Techniques: Derivative Assay, Migration, Quantitative RT-PCR, Expressing, Transfection, Control, Two Tailed Test, Enzyme-linked Immunosorbent Assay, Transwell Migration Assay, Cell Culture, Invasion Assay, Recombinant, Western Blot, Quantitation Assay, Inhibition

Journal: Oncogene

Article Title: Hyperactivation of MEK-ERK1/2 signaling and resistance to apoptosis induced by the oncogenic B-RAF inhibitor, PLX4720, in mutant N-RAS melanoma cells

doi: 10.1038/onc.2010.408

Figure Lengend Snippet: PLX4720-induced MEK-ERK1/2 hyperactivation occurs rapidly and regulates FAK serine 910 phosphorylation. (a) Sbcl2, WM1361A and WM1366 cell lines were treated with DMSO (−), 1 μM PLX4720 or 10 μM U0126 for 16 h. Lysates were analyzed by western blotting (Boisvert-Adamo and Aplin, 2006) using antibodies for (p)FAK-S910 (Biosource Int, Camarillo, CA, USA), (p)FAK-Y397 (Bd Biosciences, San Jose, CA, USA), and total FAK (Cell Signaling Technology). (b) Sbcl2 and WM1366 cells were treated with 1 μM PLX4720 for 15 min to 20 h. Lysates were analyzed by western blot analysis for (p)MEK, MEK, (p)ERK1/2 and ERK1/2.

Article Snippet: Together, these data demonstrate that PLX4720 treatment of mutant N-RAS and wild-type N-RAS/B-RAF melanoma cell lines leads to enhanced activation of the MEK-ERK1/2 pathway. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 2 caption a7 PLX4720-induced MEK-ERK1/2 hyperactivation occurs rapidly and regulates FAK serine 910 phosphorylation. (a) Sbcl2, WM1361A and WM1366 cell lines were treated with DMSO (−), 1 μM PLX4720 or 10 μM U0126 for 16 h. Lysates were analyzed by western blotting ( Boisvert-Adamo and Aplin, 2006 ) using antibodies for (p)FAK-S910 (Biosource Int, Camarillo, CA, USA), (p)FAK-Y397 (Bd Biosciences, San Jose, CA, USA), and total FAK (Cell Signaling Technology). (b) Sbcl2 and WM1366 cells were treated with 1 μM PLX4720 for 15 min to 20 h. Lysates were analyzed by western blot analysis for (p)MEK, MEK, (p)ERK1/2 and ERK1/2.

Techniques: Western Blot

Journal: Oncology Letters

Article Title: Diallyl disulfide effect on the invasion and migration ability of HL-60 cells with a high expression of DJ-1 in the nucleus through the suppression of the Src signaling pathway

doi: 10.3892/ol.2018.8139

Figure Lengend Snippet: The expression of p-Src and p-Fak in the HL-60 cells with a high expression of DJ-1 in the nucleus induced by diallyl disulfide for 0, 5, 15, 30 or 60 min. (A) A representative western blot analysis presenting the expression of p-Src and p-Fak. (B) Comparisons between the expression levels of p-Src and p-Fak. *P<0.05 vs. the control. p-Src, phosphorylated Src protein; p-Fak, phosphorylated Fak protein; DJ-1, parkinsonism associated deglycase.

Article Snippet: Phosphorylated Src (p-Src) was purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA; cat. no. 2101; 1:1,000 dilution) and phosphorylated Fak (p-Fak) was purchased from Abgent, Inc. (cat no. AJ1285e; 1:1,000 dilution). β-actin monoclonal antibodies were purchased from Abgent, Inc. (cat. no. AM1021B-ev; 1:2,000 dilution).

Techniques: Expressing, Western Blot

Journal: Oncology Letters

Article Title: Diallyl disulfide effect on the invasion and migration ability of HL-60 cells with a high expression of DJ-1 in the nucleus through the suppression of the Src signaling pathway

doi: 10.3892/ol.2018.8139

Figure Lengend Snippet: DADS affects the Src signaling pathway in a control group, empty vector group and high expression group. (A) Protein expression of p-Src, Src, P-Fak, Fak and integrin was examined using western blot analysis. (B) Quantified protein expression of p-Src and p-Fac across the three groups. *P<0.05 vs. the control. DADS, diallyl disulfide; DJ-1, parkinsonism associated deglycase; p-Src, phosphorylated Src protein; p-Fak, phosphorylated Fak protein.

Article Snippet: Phosphorylated Src (p-Src) was purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA; cat. no. 2101; 1:1,000 dilution) and phosphorylated Fak (p-Fak) was purchased from Abgent, Inc. (cat no. AJ1285e; 1:1,000 dilution). β-actin monoclonal antibodies were purchased from Abgent, Inc. (cat. no. AM1021B-ev; 1:2,000 dilution).

Techniques: Plasmid Preparation, Expressing, Western Blot

Journal: Oncology Letters

Article Title: Diallyl disulfide effect on the invasion and migration ability of HL-60 cells with a high expression of DJ-1 in the nucleus through the suppression of the Src signaling pathway

doi: 10.3892/ol.2018.8139

Figure Lengend Snippet: DADS and Src inhibitor inhibit the Src signaling pathway in the control group, the empty vector group and the high expression group. Protein expression of p-Src, Src, P-Fak, Fak and integrin was examined by (A) western blot analysis and (B) quantitative analysis. *P<0.05 vs. the control. DADS, diallyl disulfide; p-Src, phosphorylated Src protein; p-Fak, phosphorylated Fak protein; DJ-1, parkinsonism associated deglycase.

Article Snippet: Phosphorylated Src (p-Src) was purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA; cat. no. 2101; 1:1,000 dilution) and phosphorylated Fak (p-Fak) was purchased from Abgent, Inc. (cat no. AJ1285e; 1:1,000 dilution). β-actin monoclonal antibodies were purchased from Abgent, Inc. (cat. no. AM1021B-ev; 1:2,000 dilution).

Techniques: Plasmid Preparation, Expressing, Western Blot