Journal:

Article Title: Fas-Mediated Apoptosis Is Regulated by the Extracellular Matrix Protein CCN1 (CYR61) In Vitro and In Vivo ▿

doi: 10.1128/MCB.00064-09

Figure Lengend Snippet: ROS-dependent hyperactivation of p38 MAPK is critical for FasL/CCN1-induced apoptosis. (A, top) Apoptosis of HSFs treated with vehicle (dimethyl sulfoxide), the p38 MAPK inhibitor SB203580 or SB202190 (10 μM each), or the JNK inhibitor SP600125 (12 μM) prior to stimulation with FasL with or without CCN1. *, P < 0.05; **, P < 0.01. (Bottom) Immunoblot analysis of caspase-9 processing in lysates of cells treated as indicated (B, BSA; C, CCN1; F, FasL; CF, CCN1 and FasL), with or without preincubation with 40 μM SB203580. (B) HSFs were treated with various proteins, and total cell lysates, collected after 4 h of incubation, were analyzed for phospho-JNK1/2 (Pi-JNK; Thr183/Tyr185) and total JNK1/2 by immunoblotting. (C) HSFs were pretreated with various concentrations of SP600125 for 1 h and left uninduced or induced with CCN1 and FasL (C+F) for 4 h. Whole-cell lysates were immunoblotted against phospho-c-Jun (Ser63) and β-actin. (D, top) Lysates from cells transfected with control or p38α siRNA were immunoblotted with antibodies against p38 MAPK and β-actin. (Bottom) Transfected cells were stimulated with CCN1 and/or FasL; apoptosis was scored 4 h thereafter. (E) HSFs were treated as indicated in the presence or absence of NAC. After 4 h, total lysates were collected and analyzed for phospho-p38 MAPK (Thr180/Tyr182) and total p38 by immunoblotting. (F) Cells transfected with control or SMPD2 siRNA or treated with the nSMase inhibitor GW4869 (20 μM) were stimulated with CCN1 and/or FasL as described above. Cell lysates were immunoblotted for phosphorylated and total p38.

Article Snippet: Antibodies for caspase-8 and phospho-c-Jun N-terminal protein kinase 1/2 (phospho-JNK1/2; Thr183/Tyr185) were from Assay Designs, those for caspase-9, -3, phospho-p38 MAPK (Thr180/182), phospho-c-Jun (Ser63), and JNK1/2 were from Cell Signaling Technology, and those for p38 MAPK, Fas (C-20), and poly(ADP ribose) polymerase (PARP; H-250) were from Santa Cruz.

Techniques: Western Blot, Incubation, Transfection

Journal: Annals of Translational Medicine

Article Title: Repressing PDCD4 activates JNK/ABCG2 pathway to induce chemoresistance to fluorouracil in colorectal cancer cells

doi: 10.21037/atm-20-4292

Figure Lengend Snippet: MiR-21 increased the phosphorylation of c-Jun (Ser63) and ABCG2 via targeting PDCD4. (A) RT-PCR of miR-21 in RKO and HCT116 cells upon 10 µM of 5-FU treatment for 48 hours. (B) RT-PCR of the baseline expression of miR-21 in HCT116 and HCT116/FUR cells. (C) RT-PCR of the baseline expression of pdcd4 in RKO and RKO-KO (miR-21 knockout) cells. (D) Western blotting of the indicated proteins in RKO-KO and RKO cells. Relative expression of proteins was quantified using Image J, and normalized to GAPDH. (E) Western blotting of the indicated proteins in RKO cells, and RKO-KO cells transfected with mimic-miR-21 normal control (NC) or mimic-miR-21. Relative expression of proteins was quantified using Image J, normalized to GAPDH, and is shown as a ratio relative to that of RKO cells. (F) RT-PCR of pdcd4 in RKO and HCT116 cells transfected with miR-21 inhibitor NC or miR-21 inhibitor. (G) Western blotting of the indicated proteins in RKO or HCT116 cells transfected with miR-21 inhibitor NC or miR-21 inhibitor. Relative expression of proteins was quantified using Image J, normalized to GAPDH, and is shown as a ratio relative to that of miR-21 inhibitor NC. Results are expressed as mean ± SD of three independent experiments. *, P≤0.05; ***, P≤0.001.

Article Snippet: Antibodies specific for c-Jun (#380397), phospho-c-Jun (Ser63) (#382955), phospho-JNK (Thr183/Tyr185) (#619878), and b-actin (#200068-6D7) were obtained from Zen-Bio (Chengdu, China).

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Knock-Out, Western Blot, Transfection