Journal: Autophagy

Article Title: Kit-mediated autophagy suppression driven by a viral oncoprotein emerges as a crucial survival mechanism in Merkel cell carcinoma

doi: 10.1080/15548627.2025.2477385

Figure Lengend Snippet: MCPyV truncated LT induces paranuclear retention and stabilization of KIT. (A) Immunofluorescence detection of KIT (green) and MCPyV LT (CM2B4, red) or sT (CM8E6, red) in KIT-HEK293 cells transfected with MCPyV T-antigens or vector control ( n = 6). (B) Top : illustration of the expression constructs of LT339 and the VPS39-interaction defective mutant LT339 W209A . Bottom : Representative images showing the effect of LT339 and LT339 W209A mutant on localization of KIT (green). LT was detected by CM2B4 (red). ( n = 3) (C) Immunoblots showing the effect of the LT339 and LT339 W209A on KIT expression. The quantification of KIT level is shown below the immunoblots ( n = 9). (D) Top : immunoblot analysis of the effect LT339 and LT339 W209A on KIT protein stability in the presence of cycloheximide (CHX) up to 4 h. Bottom : quantification of KIT protein stability after normalization to 0 h time point ( n = 4). (E) Immunoblots showing KIT protein stability in KIT-HEK293 cells transfected with LT339, LT339 W209A or plasmid control (CTR) treated with KIT ligand (KITLG, 100 ng/mL) or solvent control (PBS) in the presence of CHX (100 μg/mL). (F) Quantification of KIT protein stability after normalization to 0 h time point ( n = 5). Solid lines represent PBS control (KITLG − ) and dotted lines represent KITLG treatment (KITLG + ). (A and B) Nuclei were stained by DAPI (blue). Scale bar: 10 μm. Numbers below the images refer to the proportion of cells with KIT paranuclear dot-like staining to the total number of cells analyzed. (C, D and F) Error bars represent mean ± SEM. * p < 0.05, *** p < 0.001, ns = not significant were calculated by one-way ANOVA with post-hoc Tukey’s test (C), two-way ANOVA (D) or two-way ANOVA with post-hoc Bonferroni’s test (F).

Article Snippet: For KITLG induced KIT degradation experiments, KIT-293 cells were transfected with LT339, LT339 W209A or CTR for 48 h, followed by addition of CHX (100 μg/mL) and KITLG (100 ng/mL; Proteintech Group, HZ-1024) or PBS.

Techniques: Immunofluorescence, Transfection, Plasmid Preparation, Control, Expressing, Construct, Mutagenesis, Western Blot, Solvent, Staining

Journal: bioRxiv

Article Title: Distinct Microstructural Heterogeneities Underpin Specific Micromechanical Properties in Human ACL Femoral and Tibial Entheses

doi: 10.1101/2023.08.16.553628

Figure Lengend Snippet: Quantitative proteome analysis of the difference in expression of FE and TE interfaces compared to Ligament and SB. (A) Experimental procedure for the proteomic investigation. Volcano plots of univariate statistical analysis as applied to ligament-bone samples (n = 3 for each tissue). The plots are based on the fold change (log2) and the P-value (-log10) of all proteins identified in (B) proteins from FE and FL; (C) proteins from FE and FB; (D) proteins from TE and TL; (E) proteins from TE and TB. (F) Immunofluorescence staining of SLC24A1 (cyan) and CLEC11A (magenta) in ACL-femur tissues and SRPX (cyan) and MGP (magenta) in ACL-tibia tissues. Scale bar, 20 μm.

Article Snippet: Next, the FE samples were subjected to incubation with various primary antibodies, including SLC25A1 antibody (1:50, 15235-1-AP, Proteintech) and CLEC11A antibody (1:100, 55019-1-AP, Proteintech).

Techniques: Expressing, Immunofluorescence, Staining

Journal: Oncotarget

Article Title: Hypoxia-inducible factors regulate pluripotency factor expression by ZNF217- and ALKBH5-mediated modulation of RNA methylation in breast cancer cells

doi: 10.18632/oncotarget.11743

Figure Lengend Snippet: A-C. Breast cancer cell lines were exposed to 20% or 1% O 2 for 24 h and NANOG (A), KLF4 (B), and SOX2 (C) mRNA levels were determined by RT-qPCR, relative to 18S rRNA, and normalized to the mean value for MDA-MB-231 cells (MDA231) at 20% O 2 (mean ± SEM; n = 3). * P < 0.05, ** P < 0.01, *** P < 0.001 vs . same cell line at 20% O 2 by Student's t test. D and E. HCC-1954 (D) and MCF-7 (E) subclones, which were stably transfected with an expression vector encoding a non-targeting control (NTC) shRNA, or vector encoding shRNA targeting HIF-1α (sh1α) or HIF-2α (sh2α), or vectors encoding shRNAs targeting both HIF-1α and HIF-2α (DKD), were exposed to 20% or 1% O 2 for 24 h and RT-qPCR was performed to determine NANOG (D) or KLF4 (E) mRNA levels relative to 18S rRNA. The results were normalized to NTC at 20% O 2 (mean ± SEM; n = 3). * P < 0.05, ** P < 0.01, *** P < 0.001 vs . NTC at 20% O 2 ; # P < 0.05, ## P < 0.01, ### P < 0.001 vs . NTC at 1% O 2 by ANOVA. F . ZR75.1 cells treated with vehicle or digoxin (200 nM) were exposed to 20% or 1% O 2 for 24 h and SOX2 mRNA was measured (mean ± SEM; n = 3). * P < 0.05, ** P < 0.01 vs . NTC at 20% O2; ### P < 0.001 vs . NTC at 1% O2 by ANOVA. G and H . NTC and DKD subclones of HCC-1954 (G) and MCF-7 (H) were exposed to 20% or 1% O 2 for 48 h, whole cell lysates were prepared, and immunoblot assays were performed to analyze HIF-1α, HIF-2α, NANOG and KLF4 protein expression. Actin was also analyzed as a loading control. I. ZR75.1 cells were treated with vehicle or digoxin (200 nM), exposed to 20% or 1% O 2 for 48 h, and HIF-1α, NANOG and SOX2 immunoblot assays were performed.

Article Snippet: Blots were probed with HIF-1α (#610959, BD Biosciences, San Jose, CA), HIF-2α (#NB100-122, Novus Biologicals, Littleton, CO), ALKBH5 (#NBP1-82188, Novus Biologicals), NANOG (#NB100-588, Novus Biologicals), KLF4 (#NBP1-83940, Novus Biologicals), SOX2 (NBP1-42823, Novus Biologicals), ZNF217 (#NBP1-78189, Novus Biologicals), and Actin (#sc-1616, Santa Cruz Biotechnology, Dallas, TX) antibodies.

Techniques: Quantitative RT-PCR, Stable Transfection, Transfection, Expressing, Plasmid Preparation, Control, shRNA, Western Blot

Journal: Oncotarget

Article Title: Hypoxia-inducible factors regulate pluripotency factor expression by ZNF217- and ALKBH5-mediated modulation of RNA methylation in breast cancer cells

doi: 10.18632/oncotarget.11743

Figure Lengend Snippet: Induction of BCSCs, pluripotency factors and m 6 A-regulating proteins by hypoxia

Article Snippet: Blots were probed with HIF-1α (#610959, BD Biosciences, San Jose, CA), HIF-2α (#NB100-122, Novus Biologicals, Littleton, CO), ALKBH5 (#NBP1-82188, Novus Biologicals), NANOG (#NB100-588, Novus Biologicals), KLF4 (#NBP1-83940, Novus Biologicals), SOX2 (NBP1-42823, Novus Biologicals), ZNF217 (#NBP1-78189, Novus Biologicals), and Actin (#sc-1616, Santa Cruz Biotechnology, Dallas, TX) antibodies.

Techniques: