Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: MMP28 recruits M2-type tumor-associated macrophages through MAPK/JNK signaling pathway-dependent cytokine secretion to promote the malignant progression of pancreatic cancer

doi: 10.1186/s13046-025-03321-x

Figure Lengend Snippet: MMP28 promotes the secretion of IL-8 and VEGFA from cancer cells by regulating the MAPK/JNK signaling pathway. A - B WB analysis of JNK, p-JNK, ERK, p-ERK, P38, and p-P38 in pancreatic cancer cells with MMP28 knockdown or overexpression compared with the control. C - D GEPIA database analysis of the relationships between JNK expression and the expression of IL-8 and VEGFA in pancreatic cancer. E ELISAs were used to measure IL-8 and VEGFA protein secretion in empty vector-treated pancreatic cancer cells, MMP28-overexpressing pancreatic cancer cells, and pancreatic cancer cells treated with the JNK inhibitor SP600125 (20 μM). **** P < 0.0001. ### P < 0.01, and #### P < 0.0001

Article Snippet: The membranes were blocked with 5% bovine serum albumin (GC305010-5 g, Servicebio) for one hour and incubated overnight at 4°C with primary antibodies against MMP28 (1:500, sc-515010, Santa Cruz Biotechnology), GAPDH (1:1000, GB12002, Servicebio), β-actin (1:1000, GB15003-100, Servicebio), JNK (1:500, BM4329, BOSTER), p-JNK (1:500 BM4380, BOSTER), P38-MAPK (1:500, BM4439, BOSTER), p-P38-MAPK (1:500, M00176T180, BOSTER), ERK (1:500, BM3426, BOSTER), p-ERK (1:500, BM4156, BOSTER), ANXA2 (1:1000, 11256-1-AP, Proteintech), Vimentin (1:500, PB9359, BOSTER), CDH1 (1:500, BM4166, BOSTER), CDH2 (1:500, BA0673, BOSTER), BAX (1:500, BA0315-2, BOSTER), and BCL-2 (1:500, A00040-2, BOSTER).

Techniques: Knockdown, Over Expression, Control, Expressing, Plasmid Preparation

Journal: Translational Neuroscience

Article Title: Euxanthone inhibits traumatic spinal cord injury via anti-oxidative stress and suppression of p38 and PI3K/Akt signaling pathway in a rat model

doi: 10.1515/tnsci-2021-0012

Figure Lengend Snippet: Effect of Euxanthone on p-p38 protein expression in t-SCI-induced rats. (a) Quantification and (b) representative Western blot images of the effect of Euxanthone on p-p38 protein expression. GAPDH served as an internal control. The data are represented as the mean value ± standard error of mean. # p < 0.01 relative to sham group, * p < 0.05 relative to t-SCI model group, and $ p < 0.05 relative to t-SCI model group and Eux30 study group. t-SCI, traumatic spinal cord injury; Eux30 and Eux60, Euxanthone 30 and 60 mg/kg, respectively, in t-SCI-induced rats; p, phosphorylated.

Article Snippet: The membranes were then incubated for 12 h at 4°C with primary antibodies purchased from Bio-rad Laboratories, Shanghai, China, namely – Receptor activator of nuclear factor-κB ligand (RANKL) (MCA5971), toll-like receptor 4(TLR4) (AHP1822), Nuclear factor (NF)-κB (VPA00015), phosphorylated (p)-NF-κB (AHP1342), PI3K (AHP2224), Akt (VMA00253) and GAPDH (HCA272), p38 (AHP2410), and p-p38 (#9211, Cell Signaling Technology, Shanghai, China).

Techniques: Expressing, Western Blot

Journal: PLoS ONE

Article Title: Endogenous Semaphorin-7A Impedes Human Lung Fibroblast Differentiation

doi: 10.1371/journal.pone.0170207

Figure Lengend Snippet: HLF derived from non-fibrotic lungs were treated with control-siRNA (Ctrl.) or semaphorin7A-siRNA (sema7A) for 24 h before starvation in BSA for 24 h. A/ Then cells were activated with 1% or 10% FBS for 15 min. ERK phosphorylation (P-ERK) was analyzed by western-blot. The graph shows ratios of phospho-ERK versus total ERK1, and is an average of 3 experiments. * indicates that the increase of phospho-ERK after semaphorin-7A (Sema7A)-siRNA-treatment is statistically different from phospho-ERK after control-siRNA (Ctrl) treatment. B/ Cells were activated with 1% FBS for 30 min and a representative blot of 2 experiments shows phosphorylation of p38 (P-p38). C/ Cells were treated with the indicated siRNA and cultured for 48 h in 1% FBS along with a phospho-ERK inhibitor (U0126) or its inactive analog (U0124) during the last 24 h. Proliferation was determined by adding BrdU in the last 6 h of the culture. One representative experiment of 2 using 2 different HLF lines is shown. Average ±SEM of 4 wells is shown.

Article Snippet: The rabbit anti-phospho-p38 antibody was from Genetel Laboratories (Madison, WI).

Techniques: Derivative Assay, Control, Phospho-proteomics, Western Blot, Cell Culture

Journal: Oncology Research

Article Title: ROR2 promotes invasion and chemoresistance of triple-negative breast cancer cells by activating PI3K/AKT/mTOR signaling

doi: 10.32604/or.2024.045433

Figure Lengend Snippet: ROR2 is upregulated in TNBC cancer tissues. ROR2 expression was examined by IHC (A and B) and RT-qPCR (C) in primary TNBC and metastatic TNBC tissues (n = 20). Scale bar: 100 μm. ** p < 0.01. TT: TNBC tumor, TM: TNBC metastases. (D) ROR2 expression in TNBC cell lines BT-549, MDA-MB-435, Hs 578T, MDA-MB-468 and HCC1599. (E and F) Relative protein expression of ROR2 in BT-549, MDA-MB-435, Hs 578T, MDA-MB-468 and HCC1599 cells. Data are presented as mean ± SD (n = 3, ** p < 0.01 vs . MDA-MB-435 or MDA-MB-468 cells).

Article Snippet: The membrane was incubated with primary antibodies against ROR2 (EnoGene, Cat. E38PA4208, USA), Akt (Phospho-Ser473) (EnoGene, Cat. E011054-2, USA), Akt (Ab-473) (EnoGene, Cat. E021054-2, USA), mTOR (Abcam, Cat. ab134903, UK), mTOR (phospho S2448) (Abcam, Cat. ab109268, UK), PI3Kinase p85 alpha (phospho Y607) (Abcam, Cat. ab182651, UK), PI3Kinase p85 alpha (Abcam, Cat. ab191606, UK), E-cadherin (Abcam, Cat. ab227639, UK), MMP2 (Abcam, Cat. ab181286, UK), N-cadherin (Abcam, Cat. ab76011, UK), SNAIL (Abcam, Cat. ab216347, UK), Vimentin (Abcam, Cat. ab16700, UK), and GAPDH (EnoGene, Cat. E1C604-2, USA) at 4°C overnight.

Techniques: Expressing, Quantitative RT-PCR

Journal: Oncology Research

Article Title: ROR2 promotes invasion and chemoresistance of triple-negative breast cancer cells by activating PI3K/AKT/mTOR signaling

doi: 10.32604/or.2024.045433

Figure Lengend Snippet: Generating HCC1599 and MDA-MB-435 cells with stable knockdown and overexpression of ROR2. HCC1599 cells were transfected with ROR2 siRNA plasmid or non-targeting control, and selected by 5 μg/ml puromycin. Stable ROR2 expression MDA-MB-435 cells were generated by transfection with pLenti-ROR2 plasmid and selected by 5 μg/ml puromycin. ROR2 expression was analyzed at mRNA (A) and protein (B and C) levels. Data are presented as mean ± SD (n = 3, ** p < 0.01 vs . parent or vector transfected control cells).

Article Snippet: The membrane was incubated with primary antibodies against ROR2 (EnoGene, Cat. E38PA4208, USA), Akt (Phospho-Ser473) (EnoGene, Cat. E011054-2, USA), Akt (Ab-473) (EnoGene, Cat. E021054-2, USA), mTOR (Abcam, Cat. ab134903, UK), mTOR (phospho S2448) (Abcam, Cat. ab109268, UK), PI3Kinase p85 alpha (phospho Y607) (Abcam, Cat. ab182651, UK), PI3Kinase p85 alpha (Abcam, Cat. ab191606, UK), E-cadherin (Abcam, Cat. ab227639, UK), MMP2 (Abcam, Cat. ab181286, UK), N-cadherin (Abcam, Cat. ab76011, UK), SNAIL (Abcam, Cat. ab216347, UK), Vimentin (Abcam, Cat. ab16700, UK), and GAPDH (EnoGene, Cat. E1C604-2, USA) at 4°C overnight.

Techniques: Knockdown, Over Expression, Transfection, Plasmid Preparation, Control, Expressing, Generated

Journal: Oncology Research

Article Title: ROR2 promotes invasion and chemoresistance of triple-negative breast cancer cells by activating PI3K/AKT/mTOR signaling

doi: 10.32604/or.2024.045433

Figure Lengend Snippet: ROR2 regulated Adriamycin induced apoptosis of TNBC cells. MDA-MB-435 (A and B) and HCC1599 (A and C) cells with altered ROR2 expression were exposed to 2 uM adriamycin and apoptosis was examined by flow cytometry. ROR2 altered chemosensitivity of MDA-MB-435 (D and E) and HCC1599 (D and F). CCK-8 assay of cells treated with adriamycin for 72 h, and IC50 values were calculated (D and E). Data are presented as mean ± SD (n = 3, ** p < 0.01 vs . parent or vector control cells).

Article Snippet: The membrane was incubated with primary antibodies against ROR2 (EnoGene, Cat. E38PA4208, USA), Akt (Phospho-Ser473) (EnoGene, Cat. E011054-2, USA), Akt (Ab-473) (EnoGene, Cat. E021054-2, USA), mTOR (Abcam, Cat. ab134903, UK), mTOR (phospho S2448) (Abcam, Cat. ab109268, UK), PI3Kinase p85 alpha (phospho Y607) (Abcam, Cat. ab182651, UK), PI3Kinase p85 alpha (Abcam, Cat. ab191606, UK), E-cadherin (Abcam, Cat. ab227639, UK), MMP2 (Abcam, Cat. ab181286, UK), N-cadherin (Abcam, Cat. ab76011, UK), SNAIL (Abcam, Cat. ab216347, UK), Vimentin (Abcam, Cat. ab16700, UK), and GAPDH (EnoGene, Cat. E1C604-2, USA) at 4°C overnight.

Techniques: Expressing, Flow Cytometry, CCK-8 Assay, Plasmid Preparation, Control

Journal: Oncology Research

Article Title: ROR2 promotes invasion and chemoresistance of triple-negative breast cancer cells by activating PI3K/AKT/mTOR signaling

doi: 10.32604/or.2024.045433

Figure Lengend Snippet: ROR2 activated PI3K/AKT/mTOR pathway in TNBC cells. (A) Western blot analysis of p-PI3K p85 (Tyr458)/p55 (Tyr199), PI3K, p-AKT (Ser 473), AKT, p-mTOR (Ser 2448) and mTOR proteins in TNBC cell lines (mean ± SD, n = 3, ** p < 0.01 vs . parental cells). (B) IHC of p-PI3K, p-AKT, and p-mTOR in primary and metastatic TNBC tissues (n = 20). Scale bar: 100 μm. Data are presented as mean ± SD. ** p < 0.01. vs . TT. TT: TNBC tumor, TM: TNBC metastases.

Article Snippet: The membrane was incubated with primary antibodies against ROR2 (EnoGene, Cat. E38PA4208, USA), Akt (Phospho-Ser473) (EnoGene, Cat. E011054-2, USA), Akt (Ab-473) (EnoGene, Cat. E021054-2, USA), mTOR (Abcam, Cat. ab134903, UK), mTOR (phospho S2448) (Abcam, Cat. ab109268, UK), PI3Kinase p85 alpha (phospho Y607) (Abcam, Cat. ab182651, UK), PI3Kinase p85 alpha (Abcam, Cat. ab191606, UK), E-cadherin (Abcam, Cat. ab227639, UK), MMP2 (Abcam, Cat. ab181286, UK), N-cadherin (Abcam, Cat. ab76011, UK), SNAIL (Abcam, Cat. ab216347, UK), Vimentin (Abcam, Cat. ab16700, UK), and GAPDH (EnoGene, Cat. E1C604-2, USA) at 4°C overnight.

Techniques: Western Blot, Paraffin-embedded Immunohistochemistry

Journal: Oncology Research

Article Title: ROR2 promotes invasion and chemoresistance of triple-negative breast cancer cells by activating PI3K/AKT/mTOR signaling

doi: 10.32604/or.2024.045433

Figure Lengend Snippet: ROR2 promoted migration and invasion potential of TNBC cells. (A) Representative images (Left panel) showing wound closure after 24 h in TNBC cells. magnification 200×. Right panel: The quantification of wound area. Cells that invaded to the lower side of the chamber were counted (B). Transwell invasion assay. (C and D) The quantification of invaded cells. Scale bar: 100 μm. Data are presented as mean ± SD (n = 5, ** p < 0.01 vs . controls).

Article Snippet: The membrane was incubated with primary antibodies against ROR2 (EnoGene, Cat. E38PA4208, USA), Akt (Phospho-Ser473) (EnoGene, Cat. E011054-2, USA), Akt (Ab-473) (EnoGene, Cat. E021054-2, USA), mTOR (Abcam, Cat. ab134903, UK), mTOR (phospho S2448) (Abcam, Cat. ab109268, UK), PI3Kinase p85 alpha (phospho Y607) (Abcam, Cat. ab182651, UK), PI3Kinase p85 alpha (Abcam, Cat. ab191606, UK), E-cadherin (Abcam, Cat. ab227639, UK), MMP2 (Abcam, Cat. ab181286, UK), N-cadherin (Abcam, Cat. ab76011, UK), SNAIL (Abcam, Cat. ab216347, UK), Vimentin (Abcam, Cat. ab16700, UK), and GAPDH (EnoGene, Cat. E1C604-2, USA) at 4°C overnight.

Techniques: Migration, Transwell Invasion Assay

Journal: Oncology Research

Article Title: ROR2 promotes invasion and chemoresistance of triple-negative breast cancer cells by activating PI3K/AKT/mTOR signaling

doi: 10.32604/or.2024.045433

Figure Lengend Snippet: ROR2 expression correlated positively with metastatic phenotype of TNBC cells. Representative blots (Left panel) and histograms (Right panel) of E-cadherin, N-cadherin, vimentin, Snail and MMP-2 proteins in MDA-MB-435/pLenti-ROR2, HCC1599/siRNA-ROR2, and their respective parental cells. Data are presented as mean ± SD (n = 3, ** p < 0.01 vs . parental cells).

Article Snippet: The membrane was incubated with primary antibodies against ROR2 (EnoGene, Cat. E38PA4208, USA), Akt (Phospho-Ser473) (EnoGene, Cat. E011054-2, USA), Akt (Ab-473) (EnoGene, Cat. E021054-2, USA), mTOR (Abcam, Cat. ab134903, UK), mTOR (phospho S2448) (Abcam, Cat. ab109268, UK), PI3Kinase p85 alpha (phospho Y607) (Abcam, Cat. ab182651, UK), PI3Kinase p85 alpha (Abcam, Cat. ab191606, UK), E-cadherin (Abcam, Cat. ab227639, UK), MMP2 (Abcam, Cat. ab181286, UK), N-cadherin (Abcam, Cat. ab76011, UK), SNAIL (Abcam, Cat. ab216347, UK), Vimentin (Abcam, Cat. ab16700, UK), and GAPDH (EnoGene, Cat. E1C604-2, USA) at 4°C overnight.

Techniques: Expressing

Journal: Oncology Research

Article Title: ROR2 promotes invasion and chemoresistance of triple-negative breast cancer cells by activating PI3K/AKT/mTOR signaling

doi: 10.32604/or.2024.045433

Figure Lengend Snippet: ROR2 affects PI3K/AKT/mTOR signaling in MDA-MB-435/Adr cells. (A) Western blot analysis of ROR2, p-PI3K, PI3K, p-AKT, AKT, p-mTOR and mTOR proteins in MDA-MB-435/Adr cells. Data present as mean ± SD. n = 3, ** p < 0.01 vs . parental cells. (B and D) Apoptosis of MDA-MB-435 and MDA-MB-435/Adr cells exposed to 2 uM adriamycin for 24 h. Data present as mean ± SD (n = 3, ** p < 0.01). (C and E) CCK-8 assay of MDA-MB-435 and MDA-MB-435/Adr cells exposed to 2 uM adriamycin for 72 h, and IC50 values were calculated. Data are presented as mean ± SD (n = 3, ** p < 0.01 vs . MDA-MB-435/Adr group. ## p < 0.01 vs . MDA-MB-435 adriamycin treated group).

Article Snippet: The membrane was incubated with primary antibodies against ROR2 (EnoGene, Cat. E38PA4208, USA), Akt (Phospho-Ser473) (EnoGene, Cat. E011054-2, USA), Akt (Ab-473) (EnoGene, Cat. E021054-2, USA), mTOR (Abcam, Cat. ab134903, UK), mTOR (phospho S2448) (Abcam, Cat. ab109268, UK), PI3Kinase p85 alpha (phospho Y607) (Abcam, Cat. ab182651, UK), PI3Kinase p85 alpha (Abcam, Cat. ab191606, UK), E-cadherin (Abcam, Cat. ab227639, UK), MMP2 (Abcam, Cat. ab181286, UK), N-cadherin (Abcam, Cat. ab76011, UK), SNAIL (Abcam, Cat. ab216347, UK), Vimentin (Abcam, Cat. ab16700, UK), and GAPDH (EnoGene, Cat. E1C604-2, USA) at 4°C overnight.

Techniques: Western Blot, CCK-8 Assay

Journal: Oncology Research

Article Title: ROR2 promotes invasion and chemoresistance of triple-negative breast cancer cells by activating PI3K/AKT/mTOR signaling

doi: 10.32604/or.2024.045433

Figure Lengend Snippet: ROR2 promoted migration and invasion of MDA-MB-435/Adr cells. (A) Representative images (Left panel) showing wound closure after 24 h. The quantification of wound area (Right panel). (B) Transwell invasion assay. Scale bar: 100 μm. (C) The analysis of E-cadherin, N-cadherin, vimentin, Snail, and MMP-2 protein in MDA-MB-435, MDA-MB-435/Adr and MDA-MB-435/Adr/siROR2 cells. Data are presented as mean ± SD (n = 3, ** p < 0.01 vs . MDA-MB-435/Adr cells).

Article Snippet: The membrane was incubated with primary antibodies against ROR2 (EnoGene, Cat. E38PA4208, USA), Akt (Phospho-Ser473) (EnoGene, Cat. E011054-2, USA), Akt (Ab-473) (EnoGene, Cat. E021054-2, USA), mTOR (Abcam, Cat. ab134903, UK), mTOR (phospho S2448) (Abcam, Cat. ab109268, UK), PI3Kinase p85 alpha (phospho Y607) (Abcam, Cat. ab182651, UK), PI3Kinase p85 alpha (Abcam, Cat. ab191606, UK), E-cadherin (Abcam, Cat. ab227639, UK), MMP2 (Abcam, Cat. ab181286, UK), N-cadherin (Abcam, Cat. ab76011, UK), SNAIL (Abcam, Cat. ab216347, UK), Vimentin (Abcam, Cat. ab16700, UK), and GAPDH (EnoGene, Cat. E1C604-2, USA) at 4°C overnight.

Techniques: Migration, Transwell Invasion Assay

Journal: Oncotarget

Article Title: Klotho inhibits EGF-induced cell migration in Caki-1 cells through inactivation of EGFR and p38 MAPK signaling pathways

doi: 10.18632/oncotarget.25481

Figure Lengend Snippet: (A) Cells were pretreated with EGF (100ng/ml) and harvested after the indicated times. Lysates were collected, and p38 MAPK phosphorylation levels were analyzed by Western blotting using the antiphospho-p38 antibody. The same membrane was striped and re-probed with a different antibody for measuring total-p38 MAPK protein levels. (B) Caki-1 cells were preincubated with 400pM of the secreted form of the Klotho (KL) protein at the indicated times followed by EGF (100ng/ml) stimulation for additional 5 min. Cell lysates were subjected to Western blot analysis as described in (A). Plots indicate means ± S.E.M of three independent experiments.

Article Snippet: Rabbit anti-human polyclonal active p38 MAPK antibody and SB203580 were purchased from Promega (Madison, WI).

Techniques: Phospho-proteomics, Western Blot, Membrane

Journal: Oncotarget

Article Title: Klotho inhibits EGF-induced cell migration in Caki-1 cells through inactivation of EGFR and p38 MAPK signaling pathways

doi: 10.18632/oncotarget.25481

Figure Lengend Snippet: (A) Classical in vitro wound healing assay was performed using near-confluent serum-starved Caki-1 cells grown on collagen type 1. Cells were either pretreated with 400pM Klotho (KL) or 1μM p38 MAPK-specific inhibitor SB203580 as positive control, for 60 min followed by EGF (100ng/ml) treatment. Cell culture images shown here were taken at 0 and 24 h. (B) Plots of quantification of the resultant cell motility values were computed by gap surface area measurements for four selected microscopic fields in each assay condition. The degree of migration is expressed as % wound closure compared with zero time point. The results represent means ± S.E.M of three independent experiments. (C) Wound healing assays performed under 3D settings. Images are microphotographs of Caki-1 cells showing hole-closure of “tissue openings” generated with magnetic pattering as described in the materials and method. Cells were pretreated the same way with either Klotho (KL) or SB203580 and stimulated with EGF as described for the classical wound healing assay. (D) Plots of rate closure of holes for Caki-1 cells as a function of EGF exposure in the presence or absence of KL or SB203580.

Article Snippet: Rabbit anti-human polyclonal active p38 MAPK antibody and SB203580 were purchased from Promega (Madison, WI).

Techniques: In Vitro, Wound Healing Assay, Positive Control, Cell Culture, Migration, Generated