Journal: Oncology reports

Article Title: Hsp90 inhibitor induces KG-1a cell differentiation and apoptosis via Akt/NF-κB signaling.

doi: 10.3892/or.2017.5797

Figure Lengend Snippet: Figure 4. SNX-2112 inhibits Akt and NF-κB signaling. (A) KG-1a cells were treated with indicated concentrations of SNX-2112 for 48 h. Hsp90, IKKα, IKKβ, Akt and p-Akt were analyzed by western blotting relative to that of GADPH. (B) KG-1a cells were treated with an NF-κB pathway inhibitor (Bar, 0.5 µM) or left untreated (control) for 48 h, and cell proliferation was assessed with CCK-8 assay. (C and D) KG-1a cells were treated with Bar (0.5 µM) or a vehicle for 48 h and Annexin V/PI staining was detected after 48 h by flow cytometry. Data are presented as the mean ± SEM (n=3/group); ****P<0.0001 compared to the corresponding control.

Article Snippet: Antibodies against glyceraldehyde 3-phosphate dehydrogenase (GAPDH), caspase-3, Bcl-xl, Bcl-2, Akt, p-Akt (Thr308), IKKα, IKKβ, p65, p-p65, IκB, and PU.1 were purchased from Cell Signaling Technology (Beverly, MA, USA), and cluster of differentiation (CD)11b was purchased from BD Biosciences (Franklin Lakes, NJ, USA).

Techniques: Western Blot, Control, CCK-8 Assay, Staining, Flow Cytometry

Journal: Oncology reports

Article Title: Hsp90 inhibitor induces KG-1a cell differentiation and apoptosis via Akt/NF-κB signaling.

doi: 10.3892/or.2017.5797

Figure Lengend Snippet: Figure 4. Continued. SNX-2112 inhibits Akt and NF-κB signaling. (E) KG-1a cells were treated with Bar (0.5 µM) or left untreated (control) for 48 h. IKKα, IKKβ and p65 levels were analyzed by western blotting relative to that of GADPH. (F) KG-1a cells were treated with indicated concentrations of SNX-2112 for 48 h. IκB, p65 and p-p65 levels were analyzed by western blotting relative to that of GADPH. (G) KG-1a cells were treated with indicated concentrations of SNX-2112 for 48 h and p65 expression was detected by immunofluorescence labeling. Nuclear cytoplasmic expression of NF-κB p65 was decreased by SNX-2112 treatment.

Article Snippet: Antibodies against glyceraldehyde 3-phosphate dehydrogenase (GAPDH), caspase-3, Bcl-xl, Bcl-2, Akt, p-Akt (Thr308), IKKα, IKKβ, p65, p-p65, IκB, and PU.1 were purchased from Cell Signaling Technology (Beverly, MA, USA), and cluster of differentiation (CD)11b was purchased from BD Biosciences (Franklin Lakes, NJ, USA).

Techniques: Control, Western Blot, Expressing, Immunofluorescence, Labeling

Journal: Cellular signalling

Article Title: Minocycline Inhibits PDGF-BB-induced Human Aortic Smooth Muscle Cell Proliferation and Migration by reversing miR-221- and -222-mediated RECK suppression

doi: 10.1016/j.cellsig.2019.01.014

Figure Lengend Snippet: A, PDGF-BB induces time-dependent NF-κB activation. Quiescent SMC were incubated with PDGF-BB (10 ng/ml). At the indicated time periods, activation of NF-κB was analyzed by immunoblotting using antibodies that specifically detect phosphorylated p65 at Ser536. B, PDGF-BB induces time-dependent AP-1 activation. Quiescent SMC incubated as in A were analyzed for AP-1 activation by immunoblotting using antibodies that specifically detect phosphorylated c-Jun at Ser73. C, Silencing IKKβ or pre-treatment with minocycline inhibit PDGF-BB-induced NF-κB activation. SMC incubated with lentiviral IKKβ shRNA (moi0.5 for 48 h) were made quiescent and treated with PDGF-BB (10 ng/ml for 30 min). In a subset of experiments, quiescent SMC were incubated with minocycline (10 μM for 15 min) and then treated with PDGF-BB (10 ng/ml for 30 min). Activation of NF-κB was analyzed as in A. D, Silencing JNK2 or pre-treatment with minocycline inhibits PDGF-BB-induced AP-1 activation. SMC incubated with lentiviral JNK2 shRNA (moi 0.5 for 48 h) were made quiescent and treated with PDGF-BB (10 ng/ml for 30 min). In a subset of experiments, quiescent SMC were incubated with minocycline (10 mM for 15 min) and then treated with PDGF-BB (10 ng/ml for 30 min). Activation of AP-1 was analyzed as in B. Silencing IKKβ and JNK2 was confirmed by immunoblotting. JNK2 and IKKβ served as off-targets in IKKβ and JNK2 silenced cells, respectively (right hand panels in C and D). Tubulin served as a loading control. Bar graphs at the bottom of panels in A-D represent densitometric analyses from three independent experiments. *P<0.05 control, †P<0.05 versus PDGF-BB (n=3).

Article Snippet: Adeno- and Lentiviral transduction The following lentiviral shRNA vectors against IKKβ (#sc-35645-V), p65 (#sc-29410-V), JNK2 (#sc-39101-V), and c-Jun (#sc-29223-V) were purchased from Santa Cruz Biotechnology, Inc. Lentiviral shRNA against RECK (SHCLNV- {"type":"entrez-nucleotide","attrs":{"text":"NM_021111","term_id":"1519313840","term_text":"NM_021111"}} NM_021111 ; TRCN0000376461) and eGFP (SHC005V) were purchased from Sigma-Aldrich.

Techniques: Activation Assay, Incubation, Western Blot, shRNA

Journal: Cellular signalling

Article Title: Minocycline Inhibits PDGF-BB-induced Human Aortic Smooth Muscle Cell Proliferation and Migration by reversing miR-221- and -222-mediated RECK suppression

doi: 10.1016/j.cellsig.2019.01.014

Figure Lengend Snippet: A, B, PDGF-BB induces miR-221 (A) and miR-222 (B) expression via IKKβ, NF-κB, JNK and AP-1. Quiescent SMC incubated with PDGF-BB (10 ng/ml) for lh were analyzed for miR-221 (A) and miR-222 (B) expression by TaqMan® Advanced miRNA assays. The results were normalized to corresponding U6 expression. In a subset of experiments, SMC were incubated with lentiviral IKKβ, p65, JNK2 or c-Jun shRNA (moi0.5 for 48 h), made quiescent and then treated with PDGF-BB addition. C, D, miR-221 and miR-222 mediate PDGF-induced SMC migration (C) and proliferation (D), without affecting cell viability (E). SMC were transduced with miR-221 or miR-222 inhibitors prior to the addition of PDGF-BB (10 ng/ml). Cell migration was analyzed after 18 h using transwell migration assays (C). Cell proliferation was analyzed after 48h by CyQUANT® Cell Proliferation Assay (D). Cleaved caspase-3 levels, indicative of cells undergoing apoptosis, was analyzed after 8 h by immunoblotting using antibodies that detect both total and cleaved caspase-3 levels (E). Hydrogen peroxide (H2O2, 100 μM) served as a positive control. *P

Article Snippet: Adeno- and Lentiviral transduction The following lentiviral shRNA vectors against IKKβ (#sc-35645-V), p65 (#sc-29410-V), JNK2 (#sc-39101-V), and c-Jun (#sc-29223-V) were purchased from Santa Cruz Biotechnology, Inc. Lentiviral shRNA against RECK (SHCLNV- {"type":"entrez-nucleotide","attrs":{"text":"NM_021111","term_id":"1519313840","term_text":"NM_021111"}} NM_021111 ; TRCN0000376461) and eGFP (SHC005V) were purchased from Sigma-Aldrich.

Techniques: Expressing, Incubation, shRNA, Migration, Transduction, CyQUANT Assay, Proliferation Assay, Western Blot, Positive Control