Journal: PLoS ONE

Article Title: p38α regulates actin cytoskeleton and cytokinesis in hepatocytes during development and aging

doi: 10.1371/journal.pone.0171738

Figure Lengend Snippet: a. Old wild type and p38α knock out livers were Western blotted for phosphorylated MKK3/6 and MKK4, for phosphorylated p38 and total levels of p38α. Densitometric quantification of p-MKK3/6 (S189/207)/α-tubulin, p-MKK4 (S257/T261)/a tubulin were done. b. Old wild type and p38α knock out livers were Western blotted for MNK1 and phosphorylated MNK1 on Thr197/202, for MK2 and phosphorylated MK2 on Thr334 and Thr222, for AKT and phosphorylated AKT on Ser473, for GSK3β and phosphorylated GSK3β on serine 9 and for HSP27 and phosphorylated HSP27 on Ser 82. α-tubulin was used as a loading control. Densitometric quantification of p-MNK-1(T197/202)/MNK1, p-MK2 (T334)/MK2, p-MK2 (T222) /MK2, p-AKT(S473)/AKT, p-GSK3β(S9)/GSK3β and p-HSP27(S82)/HSP27 were determined. Data are shown as mean ± SD. **P < 0.01 WT versus KO.

Article Snippet: Antibodies used were as follows: AKT (Genscript A00301); α tubulin (Sigma Aldrich T6074); cyclin B1 (sc-245), cyclin D1 (sc-20044), GSK3β (sc-9166), HSP27 (sc-59562), MK2 (sc-7871), p21 (sc-6246), p38 alpha (sc-535), p-GSK3β (BS4084) from Santa Cruz Biotechnology; tata binding protein (abcam, 818); MNK1 (Novus biological, H00008569-M14); p-AKT (Ser473) (4058), β catenin (9562), p-cofilin (Ser 3) (3313), cofilin (5175), p-HSP27 (Ser82)(2401), p-H3 (Ser10) (3377), H3 (4499), p-MKK3/6 (Ser189/207) (9231), p-MKK4 (Ser257/Thr261) (9156), p-MK2 (Thr334) (3007), p-MK2 (Thr222) (3316), p-p38 (Thr 180/Tyr188) (4511XP), p-MNK1(Thr197/202) (2111), p27 (2552), from Cell Signaling Technology.

Techniques: Knock-Out, Western Blot, Control

Journal: PLoS ONE

Article Title: p38α regulates actin cytoskeleton and cytokinesis in hepatocytes during development and aging

doi: 10.1371/journal.pone.0171738

Figure Lengend Snippet: The Rho family plays a central role in organizing the actin cytoskeleton and in the regulation of cytokinesis. RhoA activity may be inhibited by p27, and additionally the RhoA downstream pathway may be blocked by p21 or cofilin. MNK1 and MK2 are major downstream targets of the p38α pathway that have been implicated in the regulation of cytokinesis and actin dynamics. HSP27 is another downstream target of p38α than can be also activated by MK2 and regulates the stability of actin filaments.

Article Snippet: Antibodies used were as follows: AKT (Genscript A00301); α tubulin (Sigma Aldrich T6074); cyclin B1 (sc-245), cyclin D1 (sc-20044), GSK3β (sc-9166), HSP27 (sc-59562), MK2 (sc-7871), p21 (sc-6246), p38 alpha (sc-535), p-GSK3β (BS4084) from Santa Cruz Biotechnology; tata binding protein (abcam, 818); MNK1 (Novus biological, H00008569-M14); p-AKT (Ser473) (4058), β catenin (9562), p-cofilin (Ser 3) (3313), cofilin (5175), p-HSP27 (Ser82)(2401), p-H3 (Ser10) (3377), H3 (4499), p-MKK3/6 (Ser189/207) (9231), p-MKK4 (Ser257/Thr261) (9156), p-MK2 (Thr334) (3007), p-MK2 (Thr222) (3316), p-p38 (Thr 180/Tyr188) (4511XP), p-MNK1(Thr197/202) (2111), p27 (2552), from Cell Signaling Technology.

Techniques: Activity Assay

Journal: Molecular Medicine Reports

Article Title: Rsf-1 regulates malignant melanoma cell viability and chemoresistance via NF-κB/Bcl-2 signaling

doi: 10.3892/mmr.2019.10610

Figure Lengend Snippet: Rsf-1 expression in melanoma cell lines and Rsf-1 knockdown efficiency. (A) Western blotting and RT-qPCR analysis revealed the endogenous expression levels of Rsf-1 in three melanoma cell lines (MV3, M14 and A375). (B) Western blotting and RT-qPCR analysis demonstrated that Rsf-1 siRNA transfection significantly decreased Rsf-1 expression levels in MV3 and A375 cells, while Rsf-1 plasmid transfection upregulated the protein and mRNA expression of Rsf-1 in M14 cells. Data were presented as the mean ± standard deviation of at least three experiments. *P<0.05 vs. control. Rsf-1, remodeling and spacing factor 1; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; siRNA, small interfering RNA.

Article Snippet: The targeting sequences were as follows: Rsf-1 siRNA, 5′-GGAAAGACAUCUCUACUAU-3′; and control siRNA, 5′-GCGCGATAGCGCGAATATA-3′. pCMV6-Rsf-1 and control empty plasmids were purchased from OriGene Technologies, Inc. (Rockville, MD, USA), and M14 cells were transfected with 1 µg plasmid using Lipofectamine 3000 according to the manufacturer's protocols.

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Transfection, Plasmid Preparation, Standard Deviation, Real-time Polymerase Chain Reaction, Small Interfering RNA

Journal: Molecular Medicine Reports

Article Title: Rsf-1 regulates malignant melanoma cell viability and chemoresistance via NF-κB/Bcl-2 signaling

doi: 10.3892/mmr.2019.10610

Figure Lengend Snippet: Rsf-1 regulates melanoma cell viability and invasion. (A) An MTT assay (96-well plate) revealed that Rsf-1 depletion decreased the viability of MV3 and A375 cells; conversely, Rsf-1 overexpression increased M14 cell viability. (B) A colony formation assay (culture dish diameter, 6 cm) demonstrated that the colony number was reduced in MV3 and A375 cells transfected with Rsf-1 siRNA, while Rsf-1 overexpression promoted colony formation ability in M14 cells. (C) A Transwell invasion assay (24-well plate) revealed that the number of invading cells decreased following Rsf-1 depletion in MV3 and A375, and increased following Rsf-1 overexpression in M14 cells. Data were presented as the mean ± standard deviation of at least three experiments. *P<0.05 vs. control. Magnification, ×200. Rsf-1, remodeling and spacing factor 1; siRNA, small interfering RNA.

Article Snippet: The targeting sequences were as follows: Rsf-1 siRNA, 5′-GGAAAGACAUCUCUACUAU-3′; and control siRNA, 5′-GCGCGATAGCGCGAATATA-3′. pCMV6-Rsf-1 and control empty plasmids were purchased from OriGene Technologies, Inc. (Rockville, MD, USA), and M14 cells were transfected with 1 µg plasmid using Lipofectamine 3000 according to the manufacturer's protocols.

Techniques: MTT Assay, Over Expression, Colony Assay, Transfection, Transwell Invasion Assay, Standard Deviation, Small Interfering RNA

Journal: Molecular Medicine Reports

Article Title: Rsf-1 regulates malignant melanoma cell viability and chemoresistance via NF-κB/Bcl-2 signaling

doi: 10.3892/mmr.2019.10610

Figure Lengend Snippet: Rsf-1 regulates cell cycle progression of melanoma and expression of MMP2, cyclin E and p-IκB. (A) Cell cycle analysis revealed that Rsf-1 depletion increased the percentage of G1 phase cells and decreased that of S phase cells in MV3 and A375 cell groups; Rsf-1 overexpression in M14 cells exhibited opposing effects. (B) Western blotting demonstrated that Rsf-1 depletion decreased the levels of MMP2, cyclin E and p-IκB in MV3 and A375 cell lines. Rsf-1 overexpression upregulated expression of MMP2, cyclin E and p-IκB in M14 cells. Data were presented as the mean ± standard deviation of at least three experiments. *P<0.05 vs. control. MMP2, matrix metalloproteinase-2; p, phosphorylated; Rsf-1, remodeling and spacing factor 1; siRNA, small interfering RNA.

Article Snippet: The targeting sequences were as follows: Rsf-1 siRNA, 5′-GGAAAGACAUCUCUACUAU-3′; and control siRNA, 5′-GCGCGATAGCGCGAATATA-3′. pCMV6-Rsf-1 and control empty plasmids were purchased from OriGene Technologies, Inc. (Rockville, MD, USA), and M14 cells were transfected with 1 µg plasmid using Lipofectamine 3000 according to the manufacturer's protocols.

Techniques: Expressing, Cell Cycle Assay, Over Expression, Western Blot, Standard Deviation, Small Interfering RNA

Journal: Molecular Medicine Reports

Article Title: Rsf-1 regulates malignant melanoma cell viability and chemoresistance via NF-κB/Bcl-2 signaling

doi: 10.3892/mmr.2019.10610

Figure Lengend Snippet: Rsf-1 regulates chemoresistance and the MMP of melanoma cells. (A) An MTT assay revealed that cell viability was decreased following Rsf-1 depletion in MV3 and A375 cells treated with cisplatin. Rsf-1 overexpression promoted cell viability in M14 cells treated with cisplatin. (B) Annexin V/propidium iodide analysis revealed that the percentage of apoptotic cells was significantly increased in Rsf-1-depleted MV3 and A375 cells compared with controls. Rsf-1 overexpression downregulated cisplatin-induced apoptosis in M14 cells. (C) Rsf-1 overexpression reduced MMP depolarization in M14 cells, while Rsf-1 depletion increased depolarization in MV3 and A375 cells treated with cisplatin. Data were presented as the mean ± standard deviation of at least three experiments. *P<0.05 vs. control. FITC, fluorescein isothiocyanate; MMP, mitochondrial membrane potential, Rsf-1, remodeling and spacing factor 1; siRNA, small interfering RNA.

Article Snippet: The targeting sequences were as follows: Rsf-1 siRNA, 5′-GGAAAGACAUCUCUACUAU-3′; and control siRNA, 5′-GCGCGATAGCGCGAATATA-3′. pCMV6-Rsf-1 and control empty plasmids were purchased from OriGene Technologies, Inc. (Rockville, MD, USA), and M14 cells were transfected with 1 µg plasmid using Lipofectamine 3000 according to the manufacturer's protocols.

Techniques: MTT Assay, Over Expression, Standard Deviation, Small Interfering RNA

Journal: Molecular Medicine Reports

Article Title: Rsf-1 regulates malignant melanoma cell viability and chemoresistance via NF-κB/Bcl-2 signaling

doi: 10.3892/mmr.2019.10610

Figure Lengend Snippet: Rsf-1 regulates Bcl-2 expression via NF-κB signaling. (A) Western blotting revealed that Bax expression levels increased, whereas cIAP1, cIAP2 and Bcl-2 expression decreased significantly following Rsf-1 depletion in MV3 and A375 cells. Rsf-1 overexpression in M14 cells exhibited opposing effects. (B) NF-κB inhibition significantly downregulated p-IκB and NF-κB p65 protein levels in M14 cells. NF-κB inhibition also eradicated the effects of Rsf-1 overexpression on Bcl-2 upregulation. Total IκB expression was markedly altered. Data were presented as the mean ± standard deviation of at least three experiments. *P<0.05 vs. control. Bax, Bcl-2-associated X protein; Bcl-2, B-cell lymphoma 2; cIAP1, cellular inhibitor of apoptosis protein 1; NF-κB, nuclear factor κ-light-chain-enhancer of activated B cells; p, phosphorylated; Rsf-1, remodeling and spacing factor 1; siRNA, small interfering RNA.

Article Snippet: The targeting sequences were as follows: Rsf-1 siRNA, 5′-GGAAAGACAUCUCUACUAU-3′; and control siRNA, 5′-GCGCGATAGCGCGAATATA-3′. pCMV6-Rsf-1 and control empty plasmids were purchased from OriGene Technologies, Inc. (Rockville, MD, USA), and M14 cells were transfected with 1 µg plasmid using Lipofectamine 3000 according to the manufacturer's protocols.

Techniques: Expressing, Western Blot, Over Expression, Inhibition, Standard Deviation, Small Interfering RNA