Journal: Cell & Bioscience
Article Title: MST1R-targeted therapy in the battle against gallbladder cancer
doi: 10.1186/s13578-024-01290-w
Figure Lengend Snippet: Synergistic antitumor effect of MST1R inhibitor and JMJD6 inhibitor combination in vitro. ( A ) RT-qPCR validation of transcription levels of the top 30 upregulated genes from transcriptomic sequencing. ( B ) Schematic representation of drug screening and statistical results of drug interaction Q values. ( C ) Effect of MGCD-265 in combination with SKLB325 on in vivo proliferative capacity of gallbladder cancer cells GBC-SD, NOZ, SGC-996. ( D ) Immunohistochemical staining illustrating the expression levels of JMJD6, CDC25A, HSP90AA1, SLC7A11, and SLC1A5 in subcutaneous tumor tissues of mice treated with 10 mg/kg MGCD-265. * p < 0.05, ** p < 0.01, *** p < 0.001
Article Snippet: Anti-p21 (Santa Cruz Biotechnology, #sc-6246, working dilution 1:2000), anti-HSP90AA1 (Abcam, ab303516l, working dilution 1:500), anti-Cyclin D1 (Abcam, ab226977, working dilution 1:2000), anti-Cyclin B1 (Abcam, ab181593, working dilution 1:2000), anti-CDK4 (Abcam, ab137675, working dilution 1:2000), anti-JMJD6 (Abcam, ab307654, working dilution 1:500), anti-CDC25A (Abcam, ab2357, working dilution 1:500), anti-SLC1A5 (Abcam, ab237704, working dilution 1:500), anti-SLC7A11 (Abcam, ab307602, working dilution 1:500), anti-GAPDH (Abcam, ab8227, working dilution 1:5000), anti-Cleaved-caspase3 (Cell Signaling Technology, #9664, working dilution 1:1500), anti-Caspase3 (Cell Signaling Technology, #9662, working dilution 1:1500), anti-Cleaved-PARP (Cell Signaling Technology, #5625, working dilution 1:1500), anti-β-actin (Cell Signaling Technology, #4970, working dilution 1:5000), Goat Anti-Rabbit IgG H&L (HRP) secondary antibody (Abcam, ab6721, working dilution 1:8000), and Goat Anti-Mouse IgG H&L (HRP) secondary antibody (Abcam, ab205719, working dilution 1:8000).
Techniques: In Vitro, Quantitative RT-PCR, Sequencing, In Vivo, Immunohistochemical staining, Staining, Expressing
![Regulation of CDC25 A and Cyclin D1 by miR-449a may prevent CRNA. A and B , rat cortical neurons were transfected with a luciferase reporter plasmid containing <t>CDC25A</t> 3′-UTR ( A ) or cyclin D1 3′-UTR ( B ) along with transduction of miR-449a or pLKO (control) lentivirus and treated with Aβ 42 for 48 h. In addition, a mutant in which miR-449a site in these UTRs was disrupted was also transfected. Cell lysates were prepared, luciferase activity assays were performed, and fold change in activity with respect to pLKO transduced cells was determined. Data represent mean ± SEM, two-tailed paired t test, ∗ p < 0.05, ∗∗∗ p < 0.001, N = 3. A predicted miR-449a–binding site in CDC25A ( A ) and cyclin D1- 3′-UTR ( B ) is indicated in red . C and D , rat cortical neurons were transfected with anti-miR-449a or control antagomiR. Neurons were harvested after 48 h and lysates were subjected to immunoblotting for CDC25A ( C ) or cyclin D1 ( D ). Actin was used as a loading control. Densitometry was performed to determine fold change in these proteins [ Right/bottom panel , mean ± SEM, two tailed paired t test, ∗ p < 0.05, ∗∗ p < 0.01, N = 5 ( C ), N = 3 ( D )]. E and F , cortical neurons from WT/TgAD mice were transduced with miR-449a or control lentivirus. After 48 h, lysates were prepared and Western blotting was performed with indicated antibodies. Bottom panel , densitometry was performed and fold change in cyclin D1 or CDC25A with respect to pLKO transduced WT neurons is provided (mean ± SEM, one-way ANOVA with Sidak multiple comparison, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001, N = 4 ( E ), N = 3 ( F )). G , cortical neurons treated with Aβ 42 for 48 h or left untreated were transduced with miR-449a or control lentivirus 48 h. Subsequently, neurons were also infected with adenovirus expressing CDC25A-HA or GFP (control). Western blotting was performed with indicated antibodies. Bottom panel , densitometry was performed to determine the levels of PCNA and cl_caspase 3 which were normalized with respect to actin and fold change in their expression with respect to pLKO-transduced neurons was determined. Data represent mean ± SEM, two-way ANOVA with Tukey’s multiple comparison, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.001, N = 3. H , rat cortical neurons were transduced with miR-449a or control lentivirus, treated with Aβ 42 for 48 h or left untreated. Subsequently, neurons were also infected with adenovirus expressing cyclin D1-HA or GFP (control). Western blotting was performed with indicated antibodies. Right panel , densitometry was performed to determine the levels of PCNA and cl_caspase 3 which were normalized with respect to actin and fold change in their expression with respect to pLKO-transduced neurons was determined. Data represent mean ± SEM, two-way ANOVA with Tukey’s multiple comparison, ∗ p < 0.05, ∗∗ p < 0.01, N = 3.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_9829/pmc11419829/pmc11419829__gr4.jpg)
