Journal: Journal of Translational Medicine

Article Title: UBC9-mediated regulation of K144 ubiquitination of Lamin A and its implications for hepatocellular carcinoma

doi: 10.1186/s12967-026-07722-0

Figure Lengend Snippet: Impact of UBC9 knockdown on HCC cells using a doxycycline-inducible system. ( A ) schematic of the doxycycline (Dox)-inducible gene repression system used to downregulate UBC9 expression. ( B ) Western blot analysis shows reduced UBC9 protein levels after 72-hour of Dox treatment. ( C ) colorimetric CCK8 proliferation assay reveals significantly decreased cell proliferation in UBC9-depleted cells over 72 hours. ( D ) scratch assay shows reduced cell migration in UBC9 knockdown cells, 48 hours post-Dox induction. ( E ) Matrigel invasion assay demonstrates a marked decrease in the invasive capacity of HCC cells upon UBC9 knockdown. ( F ) Western blot analysis and quantification confirming UBC9 knockdown efficiency in Huh-7 cells. ( G ) CCK-8 assay revealing the effect of UBC9 depletion on Huh-7 cell proliferation. ( H ) wound healing assay showing the impact of UBC9 knockdown on the migration of Huh-7 cells. ( I ) Transwell invasion assay demonstrating the effect of UBC9 knockdown on the invasion of Huh-7 cells. ( J ) in vivo xenograft tumor assay. Images of dissected tumors, tumor weights, and tumor volume growth curves from nude mice injected with control (NC) or UBC9-shRNA Hep3B cells. ns, p > 0.05; **, p < 0.01; ***, p < 0.001

Article Snippet: UBC9 shRNA and scrambled control shRNA sequences were cloned into the pTRIPZ lentiviral vector containing a tetracycline (doxycycline, Dox)-inducible system (Thermo Scientific, Waltham, MA, USA).

Techniques: Knockdown, Expressing, Western Blot, Proliferation Assay, Wound Healing Assay, Migration, Invasion Assay, CCK-8 Assay, Transwell Invasion Assay, In Vivo, Injection, Control, shRNA

Journal: Scientific reports

Article Title: Transcription factor FoxO1 regulates myoepithelial cell diversity and growth.

doi: 10.1038/s41598-024-51619-1

Figure Lengend Snippet: Figure 2. Overexpression of FoxO1 in ME cells. (A) Scatter plot of CD49f (x-axis) and EpCAM (y-axis). The cells were isolated from SMG in TP53 mutant female mice (n = 4) and analyzed by flow cytometry. EpCAMlowCD49fhigh-cells were sorted as ME cells (6.5%). (B) A schematic for integration of PiggyBac transposon vector plasmid. The Tet-On inducible gene expression system was used. FoxO1 expression was induced by doxycycline (Dox). (C) mCherry fluorescence merged with phase contrast in MEPB-FoxO1 cells treated with and without Dox (2 µg/mL) for 48 h. (D) Expression of FoxO1 mRNA in MEPB-FoxO1 cells treated with and without Dox for 24 h. *P < 0.05. n = 3. (E) Immunoblotting for FoxO1, αSMA, Krt14, Krt5, and β-actin in MEPB-FoxO1 cells treated with and without Dox for 72 h. (F) FoxO1 luciferase assay in the presence of FoxO1 inhibitor (Inh.; AS1842856) at the indicated concentrations. pGL4 luciferase reporter vector (upper) was constructed to include three FoxO1-binding elements (daf16:TTGTTTA and mdaf16:TTGCTTA). FoxO1 transcriptional activity was measured. pRL-TK was used as internal control. The Renilla luciferase normalized the firefly luciferase. #P < 0.05 vs. control (Ctrl). *P < 0.05 vs. Dox. n = 5. (G) Expression of αSMA mRNA in ME cells treated with and without FoxO1 inhibitor (Inh.; AS1842856, 1 μM) for 72 h. *P < 0.05. n = 3. (H) Expression of FoxO1 and αSMA mRNA in siRNA-mediated knockdown of FoxO1 (siFoxO1) or control (si Ctrl) in ME cells. *P < 0.05. n = 3. (I) Immunoblotting for NF-κB/p65 and phospho-NF-κB/p65 in MEPB-FoxO1 cells treated with and without Dox at the indicated time-points. The signal intensity of phospho-NF-κB/p65 was normalized to that of NF-κB/p65 (ratio). All data were representative of three independent experiments. See also Supplementary Figs. S2 and S3.

Article Snippet: The PiggyBac plasmid carried a tetracycline response element to drive doxycycline (Dox) induction (2 μg/mL; LKT Labs, St. Paul, MN).

Techniques: Over Expression, Isolation, Mutagenesis, Flow Cytometry, Plasmid Preparation, Gene Expression, Expressing, Fluorescence, Western Blot, Luciferase, Construct, Binding Assay, Activity Assay, Control, Knockdown