Journal: Molecular Cancer

Article Title: Prosaposin down-modulation decreases metastatic prostate cancer cell adhesion, migration, and invasion

doi: 10.1186/1476-4598-9-30

Figure Lengend Snippet: Effect of PSAP down-modulation on β 1A -integrin expression and PCa cell adhesion to FN and LN . (A) PSAP down-modulation reduces β 1 -integrin expression. Total RNA was extracted for RT-PCR with primers specific for integrin-β 1A , -β 1B , and -β 1C , and GAPDH. Cell lysates were analyzed by western blotting with antibodies against β 1 -integrin and its isoforms β 1A , β 1B , and β 1C and GAPDH. (B) Transient down modulation of β 1 -integrin expression. Previously established control stable clones of PC-3 and DU-145 cells were transiently transfected with β 1 integrin- or scrambled-siRNA oligos. After 48 h, cell lysates were analyzed for integrin β 1 and β 1A expression by western blotting. (C) Inhibition of PCa cell adhesion by transient transfection with integrin β 1 -siRNA. After siRNA transfection, cells were subjected to adhesion assay on FN- or LN-coated 96-well plates as described in "Materials and Methods". (D) β 1A -integrin stability. The steady-state β 1A protein levels were evaluated by treating cells with cycloheximide (12.5 μg/ml) for up to 72 h and immunoblotting with anti-β 1A antibody. (E) The half-live of β 1A -integrin was evaluated by densitometric analysis of immunoblotting bands using the Quantity One software and the β 1A protein levels were calculated as percentage of non-treatment values after normalization using GAPDH for loading control. (F) Effect of inhibition of lysosomal proteolysis on β 1A -integrin expression. Cells were incubated in the presence or absence of the lysosomal proteolysis inhibitor, NH 4 Cl (50 mM) for up to 24 h. Cell lysates were analyzed for β 1A protein expression by immunoblotting. The β 1A -integrin degradation curve was calculated as described above. Columns , mean of three independent samples run together; bars , ± SEM, p < 0.0001, Two-sample t -tests with Satterthwaite corrections were used to compare β 1 -siRNA versus scrambled siRNA oligos transfected cells following adhesion to FN or LN. ANOVA was used to examine the significance of the data ( p < 0.05) among different cycloheximide treatment periods in PSAP-KD versus control clones. Similar results were obtained from three independent experiments.

Article Snippet: We used the following antibodies for immunoblotting of cell adhesion molecules: Mouse anti-integrin β 1 (Santa Cruz, clone JB1B at 1:200), rabbit anti-integrin β 1A (Millipore, AB1952P at 1:1000), rabbit anti-integrin β 1B and β 1C antibodies provided by Dr. L.R.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Clone Assay, Transfection, Inhibition, Cell Adhesion Assay, Software, Incubation

Journal: Frontiers in Pharmacology

Article Title: Veratrilla baillonii Franch Could Alleviate Lipid Accumulation in LO2 Cells by Regulating Oxidative, Inflammatory, and Lipid Metabolic Signaling Pathways

doi: 10.3389/fphar.2020.575772

Figure Lengend Snippet: Primers used for real time PCR (RT-PCR).

Article Snippet: Total protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (50 μg), and was then transferred to Millipore Corp, Billerica, MA membranes and sealed with 5% skim milk powder at 37°C for 1 h. Subsequently, rabbit anti-mouse monoclonal antibodies against PPARα, SREBP-1c, FASN, Nrf2, and GAPDH (1:1,000; Abcam, Cambridge, MA) were added to the membrane and it was shaken at 4°C overnight.

Techniques: Real-time Polymerase Chain Reaction, Sequencing

Journal: Frontiers in Pharmacology

Article Title: Veratrilla baillonii Franch Could Alleviate Lipid Accumulation in LO2 Cells by Regulating Oxidative, Inflammatory, and Lipid Metabolic Signaling Pathways

doi: 10.3389/fphar.2020.575772

Figure Lengend Snippet: Effect of water extract of Veratrilla baillonii (WVBF) on non-alcoholic fatty liver disease (NAFLD) lipid, oxidative stress, and inflammation of free fatty acids (FFA)-induced cell. (A) Effect of WVBF on the expression of PPARα (a), SREBP-1c (b), FASN (c), ACC (d), Nrf2 (e), NF-ĸB (f), and TNF-α (g) messenger RNA (mRNA). (B) Protein expression of PPARα, SREBP-1c, fatty acid synthase (FAS), acetyl-CoA carboxylase (ACC), and Nrf2 in FFA-induced LO2 cell was detected by western blot. (C) Effect of WVBF on the expression of Nrf2, ACC, FASN, SREBP-1c, and PPARα protein. n =3 ( n , the number of experiment), ( ## P < 0.01, ### P < 0.001, vs . control group; * P < 0.05, ** P < 0.01, and *** P < 0.001 vs . FFA group).

Article Snippet: Total protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (50 μg), and was then transferred to Millipore Corp, Billerica, MA membranes and sealed with 5% skim milk powder at 37°C for 1 h. Subsequently, rabbit anti-mouse monoclonal antibodies against PPARα, SREBP-1c, FASN, Nrf2, and GAPDH (1:1,000; Abcam, Cambridge, MA) were added to the membrane and it was shaken at 4°C overnight.

Techniques: Expressing, Western Blot