Journal: STAR Protocols

Article Title: Protocol for 3D-guided sectioning and deep cell phenotyping via light sheet imaging and 2D spatial multiplexing

doi: 10.1016/j.xpro.2025.104296

Figure Lengend Snippet: Illustration of MACSima™ Imaging Cyclic Staining (MICS) principle MICS technology was applied (Step 46). (0) Image acquisition of 3D-IF staining in autofluorescence channel, followed by Photobleaching. (2–4) Multi-cyclic imaging: Rounds of 2D-IF staining with FITC, PE and APC coupled antibody fluorochrome-conjugate, image acquisition of respective FITC, PE and APC-channels and signal erasure by photobleaching.

Article Snippet: Timing: 7 days This step describes 3D-IF staining of target cells by passive diffusion at elevated temperatures and defined antibody-conjugate concentrations to improve homogeneous staining within large tissue samples., Note: Following steps have been optimized for Alexa Fluor 647 labeled anti-GFP nanobodies (see ) or 3D-IF antibodies provided by Miltenyi Biotec.

Techniques: Imaging, Staining

Journal: STAR Protocols

Article Title: Protocol for 3D-guided sectioning and deep cell phenotyping via light sheet imaging and 2D spatial multiplexing

doi: 10.1016/j.xpro.2025.104296

Figure Lengend Snippet: 3D light sheet and 2D multi-cyclic imaging data comparison (Mouse Glioblastoma) (A) Imaris 3D surface rendering of autofluorescence (cyan) and glioblastoma target cells stained with anti-GFP-Alexa Fluor 647 nanobody (red). (B) Imaris 3D surface rendering of autofluorescence (cyan) and glioblastoma target cells stained with anti-GFP-Alexa Fluor 647 nanobody (red) with target plane in yellow. (C) Optical section of target plane of interest. (D) Fluorescence image of physical cryosection. (E) MICS image of section shown in D. (F) MICS image indicating anti-GFP-Alexa Fluor 647 nanobody (red) staining. (G) Magnified merged four color multiparameter MICS image with anti-EGFR (magenta), anti-GFAP (green), anti-NeuN (blue), anti-CD146 (yellow). (H–P) Nine exemplary MICS images with merges of anti-GFP-Alexa Fluor 647 nanobody staining (red) and antibody-conjugates against EGFR (H), Neurofilament (I), Nestin (J), GFAP (K), CD44 (L), CD146 (M), NeuN (N), EphA2 (O) and GLAST (P) (gray) (see “Antibodies”). Scale bars: (A–F) 500 μm; (G) 50 μm; (H–P) 500 μm.

Article Snippet: Timing: 7 days This step describes 3D-IF staining of target cells by passive diffusion at elevated temperatures and defined antibody-conjugate concentrations to improve homogeneous staining within large tissue samples., Note: Following steps have been optimized for Alexa Fluor 647 labeled anti-GFP nanobodies (see ) or 3D-IF antibodies provided by Miltenyi Biotec.

Techniques: Imaging, Comparison, Staining, Fluorescence

Journal: STAR Protocols

Article Title: Protocol for 3D-guided sectioning and deep cell phenotyping via light sheet imaging and 2D spatial multiplexing

doi: 10.1016/j.xpro.2025.104296

Figure Lengend Snippet: 3D light sheet and 2D multi-cyclic imaging data comparison (Human OvCa) (A) Imaris 3D surface rendering of autofluorescence (cyan) and CD326 positive cells (red). (B) Imaris 3D surface rendering of autofluorescence (cyan) with target plane in yellow. (C) Light sheet guided target plane selection representing CD326 positive cell (purple), CD45 positive cells (red), and CD3 positive cells (green). (D) DAPI overview image of selected tissue slice for 2D MACSima™ imaging. (E) Magnified merged six color multiparameter MICS image with CD45 (green), CD326 (cyan), FOLR1 (purple), Collagen III (red), Collagen IV (red), and CD31 (yellow). (F–L) Single staining MICS images (white) of DAPI (F), CD45 (G), CD326 (H), FOLR1 (I), Collagen III (J), Collagen IV (K), and CD31 (L) (gray) (see “Antibodies”). Scale bars: (A–F) 1 mm; (E) 250 μm; (F–L) 500 μm.

Article Snippet: Timing: 7 days This step describes 3D-IF staining of target cells by passive diffusion at elevated temperatures and defined antibody-conjugate concentrations to improve homogeneous staining within large tissue samples., Note: Following steps have been optimized for Alexa Fluor 647 labeled anti-GFP nanobodies (see ) or 3D-IF antibodies provided by Miltenyi Biotec.

Techniques: Imaging, Comparison, Selection, Staining

Journal: Genes & Diseases

Article Title: UBR5 regulates the progression of colorectal cancer cells through Snail-induced epithelial–mesenchymal transition

doi: 10.1016/j.gendis.2025.101679

Figure Lengend Snippet: UBR5 promoted the degradation and polyubiquitination of Snail. (A) UBR5 promoted the proteasomal degradation of Snail. HEK293T cells were transfected with Snail-Flag, Snail 6SA-Flag, UBR5-Myc, GFP, or empty vector and treated with DMSO, chloroquine, MG132, or CT99021 as indicated. The expression of Snail and GFP was assessed by western blotting. (B) UBR5 degraded Snail protein in a concentration-dependent manner. HEK293T cells were transfected with Snail-Flag, GFP, or in combination with different concentrations of wild-type and truncated UBR5-Myc for 48 h. Cell lysates were immunoblotted with anti-Snail antibodies. (C) UBR5 promoted K48 polyubiquitinated chain generation of Snail protein. In cellular ubiquitination assays, UBR5-Myc were co-transfected with Snail-Flag plasmids or with HA-Ub-K63 and HA-Ub-K48 plasmids. Western blotting was performed on cell lysates immunoprecipitated with an anti-Flag antibody, followed by the detection of polyubiquitination levels using an anti-Ub antibody. (D) UBR5 accelerated the Snail protein turnover through the HECT domain. HEK293T cells were transfected with corresponding plasmids. Cells were treated with cycloheximide (CHX) and harvested at indicated time points for immunoblotting with anti-Snail or anti-GFP antibody. The graph shows the quantification of Snail protein levels (based on the band intensity from the gels) normalized to those of GFP over the time course. Snail protein expression at the 0 h time point of treatment with CHX was set as 100 %. Experiments were performed in triplicate, and a representative experiment is presented.

Article Snippet: The membranes were probed with primary antibodies, including Flag (Proteintech, Wuhan, China, 66008-4-Ig), Myc (Proteintech, 60003-2-Ig), UBR5 (Proteintech, 66937-1-Ig), Snail (Santa Cruz Biotechnology, Oregon, USA, 166476), phosphorylated Snail (Biodragon, BD-PP0568), Slug (Santa Cruz Biotechnology, 271977), E-cadherin (Proteintech, 20874-1-AP), N-cadherin (BD Transduction Laboratories, Franklin Lakes, USA, 610920), GSK3β (Proteintech, 82061-1-RR), pGSK3β (Proteintech, 67558-1-Ig), green fluorescent protein (GFP; Proteintech, 66002-1-Ig), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Bioss, Woburn, USA, 0978M).

Techniques: Transfection, Plasmid Preparation, Expressing, Western Blot, Concentration Assay, Ubiquitin Proteomics, Immunoprecipitation

Journal: Genes & Diseases

Article Title: UBR5 regulates the progression of colorectal cancer cells through Snail-induced epithelial–mesenchymal transition

doi: 10.1016/j.gendis.2025.101679

Figure Lengend Snippet: UBR5 C2768S mutation abrogated the interaction with Snail. (A) His pull-down assays showed the abolished interactions between Snail and the UBR5 C2768S. A schematic representation of the UBR5 wild-type and C2768S mutation. (B) Co-immunoprecipitation assay showed that the interaction between the Snail and the UBR5 C2768S mutation was eliminated. HEK293T cells were transfected with UBR5-Myc, UBR5 C2768S-Myc, and Snail-Flag as indicated. Cell lysates were immunoprecipitated with either anti-Myc or anti-Flag antibodies and immunoblotted with anti-Snail and anti-UBR5 antibodies. (C) UBR5 C2768S abolished the UBR5-mediated degradation of Snail. HEK293T cells were transfected with Snail-Flag, UBR5-Myc, and UBR5 C2768S-Myc as indicated. Cell lysates were subjected to western blotting analysis with anti-Snail and anti-GFP antibodies. (D) UBR5 C2768S did not accelerate Snail protein turnover. HEK293T cells were transfected with Snail-Flag, UBR5-Myc, and UBR5 C2768S-Myc and treated with cycloheximide (CHX) as indicated. Cell lysates were subjected to western blotting analysis with anti-Snail and anti-GFP antibodi.

Article Snippet: The membranes were probed with primary antibodies, including Flag (Proteintech, Wuhan, China, 66008-4-Ig), Myc (Proteintech, 60003-2-Ig), UBR5 (Proteintech, 66937-1-Ig), Snail (Santa Cruz Biotechnology, Oregon, USA, 166476), phosphorylated Snail (Biodragon, BD-PP0568), Slug (Santa Cruz Biotechnology, 271977), E-cadherin (Proteintech, 20874-1-AP), N-cadherin (BD Transduction Laboratories, Franklin Lakes, USA, 610920), GSK3β (Proteintech, 82061-1-RR), pGSK3β (Proteintech, 67558-1-Ig), green fluorescent protein (GFP; Proteintech, 66002-1-Ig), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Bioss, Woburn, USA, 0978M).

Techniques: Mutagenesis, Co-Immunoprecipitation Assay, Transfection, Immunoprecipitation, Western Blot

Journal: bioRxiv

Article Title: Investigation of autophagy-activating molecules in a glia-specific Spinocerebellar ataxia type 1 model

doi: 10.64898/2026.02.23.707351

Figure Lengend Snippet: A) To assess autophagy activity, GFP-mCherry-Atg8a was expressed from a UAS promoter under repo-Gal4 control (a glial-specific Gal4 driver). The number of GFP puncta did not increase in any sample, indicating that autophagy was not blocked after Atg8a lipidation. Panel A shows repo-Gal4-driven expression in a dissected adult Drosophila brain. B–B′) The number of mCherry-positive vesicles (autolysosomes; red) increased significantly in the glial cells of AUTEN-99-treated brains. C–C′) GFP-2xFYVE (green) binds phosphatidylinositol 3-phosphate (PI3P). MTM14/EDTP is an antagonist of the Vps34 complex; inhibiting it elevates PI3P levels. AUTEN-99 significantly increased the abundance of GFP-2xFYVE structures C’). Nuclei were stained with Hoechst (blue) in the confocal images. D–D′) Protein extracts were prepared from AUTEN-67-, AUTEN-99-treated, and control (DMSO) animals expressing repo-Gal4-driven GFP-mCherry-Atg8a in glia. To determine autophagic flux, cleaved GFP and GFP-mCherry fragments were compared using anti-GFP immunoblotting. AUTEN-99-treated glia exhibited elevated levels of free GFP and GFP-mCherry fragments. αTub84B was used as an internal control. Together, these results suggest that AUTEN-99 increases autophagy in glial cells of the adult Drosophila brain.

Article Snippet: Our primary antibodies were anti-GFP (rat, 1:2500, Developmental Studies Hybridoma Bank, DSHB-GFP-1D2), anti-Tub84B (Merck SA, Darmstadt, Germany, mouse, 1:1000, T6199 [used as an internal control]).

Techniques: Activity Assay, Control, Expressing, Staining, Western Blot