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

Journal: Journal of Cell Science

Article Title: ALKBH5 regulates somatic cell reprogramming in a phase-specific manner

doi: 10.1242/jcs.259824

Figure Lengend Snippet: ALKBH5 overexpression in the late phase of reprogramming stabilizes Nanog transcripts, resulting in increased Nanog expression. (A) Immunoblot analysis of ALKBH5 protein levels on day 12 of reprogramming. MEFs and lentivirus-infected MEFs with empty vector (EV), ALKBH5 or ALKBH5–HA were treated with or without Dox (1 µg/ml) on day 10, and cells were harvested on day 12. α-tubulin (A-TUB) was used as a loading control. (B) Endogenous expression of pluripotency factors ( Oct4, Sox2 and Klf4 ) in reprogrammed MEFs on day 12. MEFs and lentivirus-infected MEFs with empty vector, ALKBH5 or ALKBH5–HA were treated with or without Dox (1 µg/ml) on day 10, and cells were harvested on day 12 and analyzed by qPCR. The data were normalized to the housekeeping gene Gapdh . (C) Expression of pluripotency markers in reprogrammed MEFs on day 12. MEFs and lentivirus-infected MEFs with empty vector, ALKBH5 or ALKBH5–HA were treated with or without Dox (1 µg/ml) on day 10, and cells were harvested on day 12 and analyzed by qPCR. The data were normalized to the housekeeping gene Gapdh . (D) m 6 A-IP qPCR data of Nanog , Gapdh and Stat3 from reprogrammed MEFs on day 12. MEFs were infected with empty vector, or were MEFs overexpressing ALKBH5 or ALKBH5–HA, and were analyzed on day 12 of reprogramming. m 6 A qPCR data were normalized to the inputs. (E) Stability of Nanog transcripts in reprogrammed MEFs as a control, or MEFs with ALKBH5–HA overexpression, on day 12 of reprogramming. Actinomycin D (ActD) was added on day 12. Cells were treated with either DMSO or 5 µM ActD at different time points from 0 to 9 h. Gapdh and Stat3 were used as negative controls, and the data of cells treated with 5 µM ActD were normalized to that from DMSO-treated cells. (F) Fraction of Δ-PE-Oct4-GFP-positive cells on day 14 of reprogramming determined by FACS analysis using Dox-inducible overexpression of NANOG. Cells use were WT MEFs or lentivirus-infected MEFs with scrambled shRNA (SCR), or two different shRNAs targeting Alkbh5 (KD1 and KD2). Reprogrammed cells were treated with or without Dox (1 µg/ml) from day 8. (G) Number of Δ-PE-Oct4-GFP-positive colonies on day 14 of reprogramming. Images in A and F are representative of three repeats. Quantitative data are shown as the mean±s.d.; n =3. * P <0.05, ** P <0.01, *** P <0.001 (paired Student's t -test).

Article Snippet: Two shRNAs for targeting mouse Alkbh5 ( Table S1 ) were annealed in annealing buffer by heating for 10 min at 95°C in thermocycler then cooling by gradual decreasing the temperature to 4°C for 30 min. Then the annealed oligonucleotides were ligated using T4 DNA Ligase (5 U/µl) (Thermo Fisher Scientific #EL0011) to either pLKO.1 puro (Addgene plasmid # 8453 ) for constitutive knockdown or Tet-pLKO-puro (Addgene plasmid # 21915 ) for dox inducible knockdown which was linearized with AgeI-HF (NEB #R3552L) and EcoRI-HF (NEB #R3101S) restriction enzymes.

Techniques: Over Expression, Expressing, Western Blot, Infection, Plasmid Preparation, shRNA

Journal: Nature Communications

Article Title: Deep mutational scanning of the human insulin receptor ectodomain to inform precision therapy for insulin resistance

doi: 10.1038/s41467-025-64178-4

Figure Lengend Snippet: A Schematic of saturation mutagenesis of the INSR ectodomain, including barcoding of an entry vector with 30 random nucleotides, PCR-based INSR ectodomain mutagenesis to generate the barcode-variant library, and long read DNA sequencing to phase barcodes and mutations. B Transfection of the barcoded library into Igf1r knockout Mouse Embryo Fibroblasts (MEFs) with doxycycline-inducible INSR knockdown and a Bxb1-targetable landing pad to generate a large library of cells, each with a uniquely barcoded, conditionally expressible INSR variant. C Sorting of the cellular variant library into four bins based on binding assays (left) or signalling assays (right). Bin thresholds are shown above the plots, with WT (blue), variant libraries (red), and control (grey, no transfected MEFs) overlaid. At least 13 million cells were sorted into each bin. D Extraction of gDNA from each bin and amplification of barcodes with primers linked to Illumina p5 and p7 sequences. E Violin plots representing distribution of insulin binding (left panel) and cell surface expression (right panel) barcode scores for WT, synonymous, missense, and three known pathogenic variants. Boxes = interquartile range (IQR), with median line. Whiskers denote 1.5 × IQR. Two-sided Mann–Whitney U tests were used to compare insulin binding score distributions between wild-type (WT) and missense variants ( p < 10⁻³⁰⁸), and between WT and synonymous variants ( p = 0.8). The same testing was undertaken for WT and missense ( p < 10⁻³⁰⁸), and WT and synonymous ( p = 0.052) expression scores. *** indicates p < 0.005; n.s. = not significant. F A detail of a sequence-function map representing variant scores for insulin binding for amino acids 705-751. Cell colour indicates the score for a single amino acid change. Variants are arrayed by position (column) and variant amino acid (row). Positive scores (in red) and negative scores (in blue) indicate better and worse function than wild-type in the assay, respectively. Grey cells are missing data and white cells with a dot in centre indicate the wild type residue at that position. Figure created in BioRender. Luijten, I. ( https://BioRender.com/z6ak0zq ).

Article Snippet: A previously described DOX-inducible BxB1 DNA recombinase landing pad (Tet-coBxb1-2A-BFP_IRES-iCasp9-2A-Blast_rtTA3, Addgene 171588) was introduced into R-MmINSR KD cells using the Lenti-X Packaging Single Shot (VSV-G) (Cat 631275) system.

Techniques: Mutagenesis, Plasmid Preparation, Variant Assay, DNA Sequencing, Transfection, Knock-Out, Knockdown, Binding Assay, Control, Extraction, Amplification, Expressing, MANN-WHITNEY, Sequencing, Residue