Journal: Advanced Science

Article Title: The Crosstalk Between CRL5 and APC/C E3 Ligases Regulates Metastasis and Chemosensitivity of Cancer Cells

doi: 10.1002/advs.202512652

Figure Lengend Snippet: ITCH stabilizes APC11 by adding K27, K29, and K33‐linked polyubiquitin chains to APC11 at Lys83 in a CUL5‐dependent manner. a) Immunoblot of APC11 and ITCH in Hep3B and PLC/PRF/5 cells upon ITCH knockdown by two distinct siRNA oligos. b) Immunoblot of APC11 and APC/C substrate securin in HEK293 cells after transfection with wild‐type ITCH or its C830S mutant for 48 h. c) The stability of APC11 in Hep3B and PLC/PRF/5 cells upon ITCH knockdown. Hep3B and PLC/PRF/5 cells were transfected with indicated siRNA oligos for 72 h and then treated with CHX (100 µg mL −1 ) for the indicated time periods, followed by IB analysis. Densitometry quantifications were performed with ImageJ, and the decay curves are shown (right). d) K27/K29/K33‐linked ubiquitylation of APC11 promoted by overexpression of ITCH but not its C830S mutant. HEK293 cells were co‐transfected with indicated plasmids, followed by purification with Ni‐NTA. Pull‐downs (top) and WCE (bottom) were subjected to IB with indicated Abs. e) Co‐IP of exogenous FLAG‐tagged APC11 with endogenous ITCH or CUL5 in HEK293 cells. f) The interaction of exogenous FLAG‐tagged CUL5 with endogenous ITCH or APC11 in HEK293 cells. g) Co‐IP of endogenous ITCH with CUL5 or APC11 in Hep3B and PLC/PRF/5 cells. h) Co‐IP of exogenous FLAG‐tagged APC11 in HeLa cells upon CUL5 knockout mediated by CRISPR‐Cas9. HeLa cells with or without CUL5 were infected with lentivirus expressing FLAG‐APC11 for 72 h, and then harvested for IP analysis. Band intensities were quantified using ImageJ, and the relative ITCH‐APC11 binding affinity in sgCUL5 cells was compared to sgCtrl cells. i) Immunoblot of APC11 in sgCtrl or sgCUL5 HeLa cells after transfection with GFP‐ITCH for 48 h. Band intensities were quantified using ImageJ and expressed as relative gray values (normalized to sgCtrl/Vector cells), shown beneath each band. j,k) ITCH facilitates K27/K29/K33‐linked ubiquitylation of wild‐type APC11 but not its W14A (j) or K83R (k) mutant. HEK293 cells were co‐transfected with indicated plasmids, followed by purification with Ni‐NTA. Pull‐downs (top) and WCE (bottom) were subjected to IB with indicated Abs. Data are presented as mean ± SEM, n = 3 (c, h, i; right). For statistical analysis, significances were determined by Student's t ‐test.* p < 0.05, ** p < 0.01, ns, not significant. l A model illustrating that CUL5 stabilizes APC11 through ITCH‐mediated K27/K29/K33‐linked polyubiquitylation at Lys83.

Article Snippet: HeLa cells with CUL5 knockout by CRISPR‐Cas9 were established by puromycin (Selleck, S7417) selection after transfection with a sequence‐verified CRISPR plasmid.

Techniques: Western Blot, Knockdown, Transfection, Mutagenesis, Over Expression, Purification, Co-Immunoprecipitation Assay, Knock-Out, CRISPR, Infection, Expressing, Binding Assay, Plasmid Preparation

Journal: bioRxiv

Article Title: MEG3 Enhances Survival of Developing Human Neurons with CLCN4 -Linked Autophagy Impairment

doi: 10.1101/2025.07.16.665078

Figure Lengend Snippet: (a) Representative images of day 6 neurons after Lysotracker treatment. Lysotracker is shown in magenta, and DAPI in blue. Scale bar = 20 μm. (b) Quantification of Lysotracker signal in day 6 neurons. The relative Lysotracker intensity of CLCN4 -variant neurons compared to the WT was presented. Each dot represents one biological replicate. (n=3-4, mean ± SEM, one-way ANOVA with Dunnett’s multiple comparisons test * p =0.039 for variant A; *** p =0.0007 for variant B; ** p =0.0092 for variant C) The magenta dashed line indicates the value for WT. (c) Relative acidity in acidic subcellular organelles. Each dot represents one biological replicate. (n=3, mean ± SEM, unpaired Student’s t-test; p =0.2894) (d-g) Dynamics of endo-lysosomal vesicles. RAB5, RAB7, RAB11 and LAMP1 indicates early endosome, late endosome, recycling endosome and lysosome markers, respectively. (RAB5, n=6; RAB7, n=3; RAB11, n=4; and LAMP1, n=3 or 4; mean ± SEM, unpaired Student’s t-test) (d) Representative images of a neuron with GFP-labeled vesicular organelles. TUJ1 and DAPI are shown in red and blue, respectively. Scale bar = 10 μm. (e) Relative GFP intensity expressed in vesicular organelles (* p =0.0265 for RAB5, * p =0.0158 for RAB7, * p =0.0475 for RAB11, and ** p =0.0067 for LAMP1) (f) Number of GFP-expressing vesicles in neurons. (* p =0.0186 for RAB5, * *p =0.0066 for RAB7, and p =0.9031 for LAMP1) (g) Average distance of GFP-expressing vesicles from the center of nucleus, representing the mean linear distance of puncta from the center of the nucleus in each cell. (** p =0.0011 for RAB5, * p =0.0463 for RAB7, and * p =0.0103 for LAMP1) (h) Scheme of autophagic flux analysis using tandem fluorescent protein fused to LC3. (i) Representative images of day 6 neurons expressing tandem fluorescent-tagged LC3. Red: RFP, green: GFP, white: TUJ1. Scale bar = 5 μm. (j) Quantification of the GFP-/RFP+ puncta. (n=5, mean ± SEM, unpaired Student’s t-test; * p =0.0164)

Article Snippet: For specific subcellular vesicle visualization, lentiviral constructs encoding GFP-tagged RAB5 (Addgene #134858), RAB7 (Addgene #133027), RAB11 (Addgene #134860), and LAMP1 (Addgene #134868) were utilized.

Techniques: Variant Assay, Labeling, Expressing