Journal: bioRxiv

Article Title: Targeted protein degradation in lysosome utilizing naturally produced bifunctional antibodies with high levels of mannose 6-phosphate glycans

doi: 10.1101/2024.09.03.611037

Figure Lengend Snippet: A . Schematic showing the procedure for detection of FLAG-mCherry and NCA-FLAG or PNCA-FLAG complexes binding toward CI-MPR. B . Bound mCherry fluorescence from (A) for NCA-FLAG (gray) or PNCA-FLAG (red). N=3 or 4. C . HepG2 cells were incubated with FLAG-mCherry and NCA-FLAG or PNCA-FLAG overnight with or without 2 mM M6P and cells were examined by fluorescent microscopy to detect mCherry signal (red). Lysosomes were stained with LysoTracker (green) and Hoechst (blue) for nuclei. D-H . Cell lines (D. HEK293T, E. Tay-Sachs Patient Fibroblasts, F. Daoy, G. HepG2, or H. MDA-MB-231) were incubated with FLAG-HexM enzyme with NCA-FLAG or PNCA-FLAG or with addition of 2 mM M6P. HexM internalization was determined by performing a HexM activity assay and activity graphed – HexM alone (gray), NCA-FLAG with HexM (Green), PNCA-FLAG with HexM (Blue), M6P added (black striped bars). Background HexM activity in untreated group wa subtracted. Dot represents each independent experiment. N=3-6. I . HEK293T CI-MPR -/- cells were generated by CRISPR-Cas9. Western blot shows CI-MPR deficiency in the cells. J . Graphs depict internalized HexM activity in CI-MPR -/- cells. Dot represents each independent experiment. N=3. Data are represented as mean values; error bars represent the standard deviation of biological replicates.

Article Snippet: HepG2 (ATCC, HB-8065), Daoy (ATCC, HTB-186) and A431 (Sigma, 85090402) cells were cultured in MEM medium (Corning, 10-009-CV) with 10% FBS.

Techniques: Binding Assay, Fluorescence, Incubation, Microscopy, Staining, Activity Assay, Generated, CRISPR, Western Blot, Standard Deviation

Journal: bioRxiv

Article Title: Targeted protein degradation in lysosome utilizing naturally produced bifunctional antibodies with high levels of mannose 6-phosphate glycans

doi: 10.1101/2024.09.03.611037

Figure Lengend Snippet: A . Schematic demonstrating production of Ab-TNFα and PNCA-TNFα in CHO cells. B . Western blotting to examine the Ab-TNFα and PNCA-TNFα heavy chain N-glycosylation after Endo-H and PNGase-F treatment. C-D . CI-MPR affinity chromatograph to determine the binding of Ab-TNFα and PNCA-TNFα. To visualize the binding signal, Alexa Fluor 594 conjugated to Ab-TNFα (C) or PNCA-TNFα (D) was used for the assay. Red dash line indicates the concentration of free M6P for antibody elution from the CI-MPR column. E . Schematic depicting internalization and degradation of extracellular TNFα b PNCA-TNFα through CI-MPR to the lysosome. F . HepG2 cells were incubated with mCherry-TNFα and Ab-TNFα or PNCA-TNFα overnight with or without 2 mM M6P and cells were examined by fluorescent microscopy to detect mCherry-TNFα signal (red). Lysosomes were stained with LysoTracker (green) and Hoechst for Nuclei (blue). G . Internalized mCherry-TNFα protein was analyzed by western blot for cell lysates using antibodies to detect mCherry, TNFα, and GAPDH as a loading control. H . Western blot analysis for internalized mCherry-TNFα with the addition of 1% or 0.5% of lysosomal protease inhibitors (PI). Antibodies are used to detect mCherry, TNFα and GAPDH, as a loading control.

Article Snippet: HepG2 (ATCC, HB-8065), Daoy (ATCC, HTB-186) and A431 (Sigma, 85090402) cells were cultured in MEM medium (Corning, 10-009-CV) with 10% FBS.

Techniques: Western Blot, Glycoproteomics, Binding Assay, Concentration Assay, Incubation, Microscopy, Staining, Control

Journal: bioRxiv

Article Title: PHF19 drives PRC2 sub-nuclear compartmentalization to promote motility in TNBC cells

doi: 10.1101/2025.03.13.642950

Figure Lengend Snippet: ( A-B ) Representative confocal fluorescence microscopy images of endogenous EZH2 (A) or SUZ12 (B) immunostaining in MDA-MB-231 and BoM-1833 cells. Insets highlight exemplary nuclear bodies of EZH2 or SUZ12 accumulation (arrows) in the BoM-1833 cells. Scale bar: 10 µm. Images were acquired and are displayed with identical settings. ( C ) Violin plot quantifying PRC2 body diameter in BoM-1833 cells. Each dot represents a single PRC2 body; data from 3 biological replicates (N = 16–32 cells). ( D ) Quantification of percentage of cell nuclei with PRC2 bodies in MDA-MB-231 and BoM-1833 cells, based on the images representatively shown in A-B. Data represent measurements from n = 3 biological replicates. Biological repeats are color coded. Statistical significance was determined via unpaired t-test, p=0.0102. Error bars indicate mean ±SEM. ( E ) Representative confocal fluorescence microscopy image of BoM-833 cells stained for endogenous PRC2 (SUZ12, green) and H3K27me3 (magenta) immunostaining in BoM-1833 cells. The arrow indicates an exemplary area of co-localization at a PRC2 body. Scale bar: 5 µm. ( F ) Schematic representation of the 3D photo-biotinylation approach used to map the proteome of endogenous PRC2 bodies. Total EZH2 (green) is spatially distributed within the cell and selectively photo-biotinylated at defined regions of interest (magenta) upon light activation. Following cell lysis, biotinylated proteins are captured using avidin-based immunoprecipitation and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The figure was created using Biorender. ( G ) Volcano plot illustrating the proteomic content of PRC2 bodies in BoM-1833 cells. Analysis was performed on the 1384 proteins identified as enriched in the labeled versus control condition in all 4 biological repeats, with unique peptides ≥ 2, fold change ≥ 1.5; and t-test significance ≤ 0.05. The x-axis represents the log 2 enrichment ratio (2P/CTL), and the y-axis represents the -log 10 p-value, indicating statistical significance. The dotted horizontal line corresponds to the p-value threshold (p < 0.05). Members of the core PRC2 complex are labeled in green. ( H ) Representative confocal fluorescence microscopy images of endogenous PHF19 immunostaining in MDA-MB-231 and BoM-1833 cells. The arrow highlights exemplary accumulations of PHF19 within nuclear bodies in BoM-1833 cells. Scale bar: 20 µm. The images were acquired and are displayed with identical settings. ( I ) Violin plot showing the quantification of endogenous PHF19 body diameter in BoM-1833 cells based on the images representatively shown in (H). Data represent measurements from N = 14–17 cells across n = 3 biological replicates, with each dot representing the diameter of a single PHF19 body. Biological repeats are color coded. ( J ) Quantification of percentage of cell nuclei with PHF19 bodies in MDA-MB-231 and BoM-1833 cells, based on the images representatively shown in (I). Data represent measurements from n = 3 biological replicates. Biological repeats are color coded. Statistical significance was determined via unpaired t-test, p=0.003. Error bars indicate mean ±SEM. ( K ) Representative confocal fluorescence microscopy image of endogenous PHF19 (green) and H3K27me3 (magenta) immunostaining in BoM-1833 cells. The arrow indicates an exemplary area of co-localization at a PHF19 body. Scale bar: 5 µm. ( L ) Representative confocal fluorescence microscopy images of BoM-1833 cells, 24 h post transfection with a GFP-PHF19 (green) expression plasmid and immunostained for endogenous core PRC2 subunits (SUZ12, purple). The arrow indicates an exemplary area of co-localization. Scale bar: 10 µm.

Article Snippet: The cells were then incubated with the rabbit anti-EZH2 antibody (5246, Cell signaling, USA) for 4 hours at RT, washed 3 times with PBST for 5 min and then incubated with Alexa Fluor™ 647 secondary antibody (A-21245, ThermoFisher, USA) for 2 hours.

Techniques: Fluorescence, Microscopy, Immunostaining, Staining, Activation Assay, Lysis, Avidin-Biotin Assay, Immunoprecipitation, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Labeling, Control, Transfection, Expressing, Plasmid Preparation

Journal: bioRxiv

Article Title: PHF19 drives PRC2 sub-nuclear compartmentalization to promote motility in TNBC cells

doi: 10.1101/2025.03.13.642950

Figure Lengend Snippet: ( A-B ) Representative confocal fluorescence microscopy images of BoM-1833 cells transfected with the indicated siRNAs. Cells were fixed 96 hours post-transfection and immunostained for endogenous EZH2 (A) or SUZ12 (B). Regions of interest (ROIs) are highlighted, with inset images showing magnified views of the immunostained cells. Scale bar: 10 µm. Images that are to be directly compared where imaged and are displayed with identical settings. ( C ) Quantification of the percentage of nuclei exhibiting PRC2 bodies in BoM-1833 cells treated as in (A-B) and immunostained for PRC2 core subunits. Data represent measurements from N = 50–60 cells across n = 3 biological replicates. Biological repeats are color coded. Statistical significance was determined via one-way ANOVA testing, *** = 0.0003, ns= not significant. Error bars indicate mean ±SD. ( D ) BoM-1833 cells were transfected with the indicated siRNAs and lysed 96 hours later for Western blot analysis using the specified antibodies. GAPDH was used as loading control. ( E-I ) Densitometric analysis of PHF19 (E), EZH2 (F), SUZ12 (G), PHF1 (H) and MTF2 (I) protein levels in cell lysates obtained from BoM-1833 cells treated as described in (D). GAPDH was used for relative normalization of the chemiluminescence signal obtained for the different PRC2 subunits. Data represent measurements from n = 3 biological replicates, whereby the values for siPHF19 are reported relative to the mean value of the control (siNT) within each biological replicate. Biological repeats are color coded. Statistical significance was determined via one-way ANOVA testing, **** < 0.0001, ns = not significant. Error bars indicate mean ±SD.

Article Snippet: The cells were then incubated with the rabbit anti-EZH2 antibody (5246, Cell signaling, USA) for 4 hours at RT, washed 3 times with PBST for 5 min and then incubated with Alexa Fluor™ 647 secondary antibody (A-21245, ThermoFisher, USA) for 2 hours.

Techniques: Fluorescence, Microscopy, Transfection, Western Blot, Control

Journal: bioRxiv

Article Title: PHF19 drives PRC2 sub-nuclear compartmentalization to promote motility in TNBC cells

doi: 10.1101/2025.03.13.642950

Figure Lengend Snippet: ( A ) PHF19 gene expression analysis across a TCGA BRCA cohort sorted by molecular subtype subtype. Box plots display the expression levels of PHF19 in normal (grey) and tumor (green) tissue for the indicated breast cancer subtypes. Data are derived from TCGA/GTEx datasets and visualized using GEPIA2. Statistical significance between tumor and normal samples was determined by unpaired t-test (*p < 0.05). n= 291 (Normal), 194 (Luminal B), 415 (Luminal A), 66 (HER2), 135 (Basal-like). ( B-C ) Representative confocal microscopy images of EZH2 (B) and SUZ12 (C) immunostaining in the indicated cell lines. Scale bar: 20 µm. Images that are to be directly compared were recorded and are displayed using identical settings. ( D ) Quantification of the percentage of cell nuclei with PRC2 bodies in the indicated cell lines based on confocal microscopy images as shown in (B-C). Data represent measurements from N = 35– 55 cells across n = 3 biological replicates. Biological repeats are color coded. ( E ) Representative immunoblot analysis of full cell lysates prepared from the indicated cell lines and using the annotated antibodies. GAPDH was used as the loading control. ( F-G ) Densitometric quantification of EZH2, SUZ12 (F) and PCL family (G) subunit protein expression in the TNBC cell line panel used in this work. GAPDH was used for normalization of the chemiluminescence signal of the PRC2 subunits across cell lines. The data for siPHF19 are reported relative to the mean values for the siNT control. Data represent measurements from n = 3 biological replicates, error bars are mean ±SD. Measurements stemming from cell lines forming detectable PRC2 bodies by Airyscan microscopy were highlighted in red. ( H-I ) Representative confocal fluorescence microscopy images showing co-immunostaining of H3K27me3 with the endogenous PRC2 core subunit SUZ12 (H) and PHF19 (I) in MDA-MB-436 cells. Arrows indicate exemplary regions of colocalization. Scale bar: 10 µm (H), 5 µm (I). ( J ) Violin plot showing the quantification of PRC2 core and PHF19 protein body diameter as based on the images representatively shown in (F-G). Data represent measurements from N = 14–29 (core PRC2 subunits) and N= 19-22 (PHF19) cells across n = 3 biological replicates, with each dot representing the diameter of a single protein body. Biological repeats are color coded. ( K ) Representative confocal fluorescence microscopy images of MDA-MB-436 cells, 24 h post transfection with GFP-PHF19 (green) and immunostained for endogenous SUZ12 (purple). The arrow indicates an exemplary area of co-localization. Scale bar: 5 µm. ( L-M ) MDA-MB-436 cells were transfected with the indicated siRNAs followed by fixation 96 h later and immunostaining for endogenous EZH2 (L) or SUZ12 (M). The bottom row shows magnified views of the cropped fields of view. Images that are to be directly compared were acquired and are displayed using identical settings. Scale bar: 10 µm ( N ) Quantification of percentage of cell nuclei with PRC2 bodies in MDA-MB-436 cells transfected with the indicated siRNAs and imaged as representatively shown in (L-M). Data represent measurements from n = 3 biological replicates. Biological repeats are color coded. Statistical significance was determined via one-way ANOVA, ****= 0.001, ns= not significant. Error bars indicate mean ±SD. ( O ) MDA-MB-436 were treated as described in (L-M), followed by cell lysis. The material was analyzed by Western blot using the indicated antibodies. See also Figure S4. ( P , S ) Representative confocal microscopy images and ( R , T ) quantification of HS578T (P, R) and BT549 (S, T) fixed 24 h after transfection with a plasmid encoding for GFP-PHF19 (magenta) and immunostained for endogenous SUZ12 (PRC2 core). ROIs (Regions of Interest) are highlighted and magnified, showing the endogenous localization of SUZ12 in cells transfected with GFP-PHF19 (ROI 1) versus un-transfected cells (ROI 2). Scale bar: 20 µm. The bar diagrams show the endogenous SUZ12 localization phenotype in relation to the GFP-PHF19 expression status. Data represent measurements from N = 7–30 cells from n = 3 biological replicates. Biological repeats are color coded. Statistical significance was determined via unpaired t-test, * = 0.0123, **= 0.0038. Error bars indicate mean ±SD.

Article Snippet: The cells were then incubated with the rabbit anti-EZH2 antibody (5246, Cell signaling, USA) for 4 hours at RT, washed 3 times with PBST for 5 min and then incubated with Alexa Fluor™ 647 secondary antibody (A-21245, ThermoFisher, USA) for 2 hours.

Techniques: Gene Expression, Expressing, Derivative Assay, Confocal Microscopy, Immunostaining, Western Blot, Control, Microscopy, Fluorescence, Transfection, Lysis, Plasmid Preparation