EMBO/soda-nel huggingface dataset:
O-glycan initiation directs distinct biological pathways and controls epithelial differentiation (2020)
Figure 1

Figure 1. Phenotypic characterization.

(A) O-GalNAc type O-glycosylation pathway. Biosynthesis of Core 1 type structures is shown.

(B) Strategy for generation and characterization of GALNT isoform knock outs in HaCaT keratinocytes.

(C) Expression of GALNT isoforms in primary keratinocytes and HaCaT cell line. The scatter plot depicts individual RPKM values of 2 biological replicates.

(D) Expression of GalNAc-T1, -T2, and -T3 in human skin (upper panel) and HaCaT keratinocyte organoids (lower panel). Frozen human skin or HaCaT keratinocyte organotypic skin models were stained using antibodies for the GalNAc-T isoforms. Scale bar - 25 μm.

(E) Phenotypic characterization of organotypic models made with HaCaT WT or GALNT1/2/3 KO keratinocytes. IHC of tissue sections stained for differentiation marker keratin 10 (upper panel) or proliferation marker Ki67 (lower panel). Scale bar - 50 μm. Red arrows - flattened cells; red asterisks - K10-negative region in suprabasal/granular layers; purple asterisks - pyknotic nuclei; green asterisks - increase in Ki67 positive cells.

(F) Quantification of epidermal thickness of skin organotypic models. Epidermal thickness was measured in 5 distinct images (4 positions/image) of 4 clones of GALNT isoform KO or WT (4 different tissues) and is presented as averages +SD. Due to high ZFN KO phenotypic inter-clonal variation and to exclude off target effects, 5 clones of ZFN and 3 clones of CRISPR KO (each targeted by a different gRNA) were used for GALNT3 KO. ANOVA followed by Dunnet's multiple comparison test was used to compare mean epidermal thicknesses of different KOs to WT. * p ≤ 0.05; **** p ≤ 0.0001.

(G) Quantification of keratin 10-negative area in skin organotypic tissue sections. Keratin 10-negative area was measured in 5 distinct images of 4 clones of GALNT isoform KO or WT (4 different tissues) and is presented as averages +SD. Due to high ZFN KO phenotypic inter-clonal variation and to exclude off target effects, 5 clones of ZFN and 3 clones of CRISPR KO (each targeted by a different gRNA) were used for GALNT3 KO. ANOVA followed by Dunnet's multiple comparison test was used to compare mean areas of different KOs to WT. **** p ≤ 0.0001.

(H) RNA Seq expression data of select differentiation markers, expressed in spinous and granular layers of the epidermis (cartoon). Expression data is presented as log2(average KO/average WT). Significantly changed transcripts, identified in R analysis, are marked with asterisks.
O-glycan initiation directs distinct biological pathways and controls epithelial differentiation (2020)
Figure 3

Figure 3. O-glycoproteomic analysis.

(A) Numbers of quantified O-glycopeptides identified with ETD fragmentation with either dimethyl labeling or TMT labeling approach in COSMC KO or WT background, respectively.

(B) Numbers of differentially glycosylated peptides in the two approaches, using select filters on quantification values.

(C) Numbers of unambiguous unique differential O-glycosites identified with the two different approaches. The stacked bar graph differentiates between glycosites identified on peptides with single and multiple HexNAc/HexHexNAc modifications.

(D) The Venn diagram represents overlap between differential single HexNAc/HexHexNAc glycosites in the three differential datasets (pooled targets from both approaches).

(E) Analysis of differential glycosites identified with the TMT labeling approach using the Qluqore Omics Explorer software. The heatmap represents O-glycosites most significantly contributing to sample variation, using ANOVA with a q value cutoff of 0.1, where each compared sample consists of three individual KO clones. Each horizontal line depicts the median quantification of glycopeptides contributing to the same glycosite. Alternative differentially glycosylated site positions are separated by semicolons on sites derived from glycopeptides with multiple HexNAc/HexHexNAc modifications.

(F, G) GO term analysis of differentially glycosylated protein identifications from both approaches (DAVID, q ≤ 0.05, Benjamini adjusted). (F) GO terms found in all differential datasets associated with subcellular location and common glycoprotein properties. (G) Differential GO terms, found in only one of the three differential datasets. p value (Benjamini multiple comparison adjusted) of 0.05 is marked with a dashed line.

(H) Top downregulated unambiguous single HexNAc/HexHexNAc targets found in all three clones of individual GALNT KO (not changed in other isoforms) with the TMT labeling approach.
O-glycan initiation directs distinct biological pathways and controls epithelial differentiation (2020)
Figure 4

Figure 4. Pathways affected by isoform specific O-glycosylation.

(A-D) GalNAc-T1 affects the organization and function of the endomembrane system. HaCaT WT and GALNT1 KO cells were stained for intracellular trafficking markers (scale bar - 10 μm). (A) Clathrin; (B) Rab5; (C) Rab11. (D) Steady state intracellular pH measurement in in HaCaT WT and GALNT1 KO cells. Data is presented as average ± SD of 2 independent experiments. ANOVA followed by Dunnet's multiple comparison test was used to compare mean intracellular pH of 3 different clones of GALNT1 KOs to WT. * p ≤ 0.05.

(E) GalNAc-T2 is important for cell to matrix adhesion. HaCaT WT and GALNT2 KO cells were used in a cell to matrix adhesion assay using plates coated with different ECM components. Data is presented as average + SD of 3 independent experiments and is normalized to WT. ANOVA followed by Dunnet's multiple comparison test was used to compare mean relative adhesion of 2 different clones of GALNT2 KOs to WT. * p ≤ 0.05; ** p ≤ 0.01.

(F) Western blot for GalNAc-T2 specific target and hemidesmosome component COL17A1.

(G) Quantification of Western blot bands for COL17A1 (full length) normalized to β actin.

(H) Depiction of COL17A1 protein, including position of GalNAc-T2 specific O-glycosite and proteolytic processing sites.

(I) GalNAc-T2 may affect tight junction formation and integrity. Upper panel, TEM of WT and GALNT2 KO organotypic tissue sections, where tight junctions are marked with asterisks. Lower panel, IF staining of WT and GALNT2 KO for tight junction marker occludin (scale bar - 50 μm). Inserts depict 3x zoomed in partial images (scale bar - 50 μm).

(J) Expression of cyclin A (green) in HaCaT WT and GALNT3 KO organotypic tissue, visualized by IF (scale bar - 20 μm).

(K) The cartoon summarizes the effects of individual GALNT isoform KOs on signaling networks and subcellular location of protein substrates based on differential phosphoproteomic and O-glycoproteomic analyses.

(L-N) Co-culture of HaCaT WT or GALNT3 KO keratinocytes with N/TERT1 keratinocytes induces cellular differentiation. Cells co-cultured on coverslips were stained for cleaved Notch 1 intracellular domain (L), cytokeratin 10 (M), or GalNAc-T3 (N; inserts depict 2x zoomed in partial images (scale bar - 20 μm)). Green - relevant staining, red - N/TERT1 cells (labeled with CMTMTR), blue - DAPI, scale bar - 20 μm. Boundaries between HaCaT and N/TERT-1 cells in the green panel of (L) are marked with a dashed line.

Expanded View Figure Legends
EDC3 Phosphorylation Regulates Growth and Invasion through controlling P-body Formation and Dynamics (2021)
Figure 1

Figure 1. EDC3 interacts strongly with the Pim protein kinase.

A Bacterially purified recombinant EDC3 (0.2μg) was incubated in the presence and absence of biotinylated recombinant Pim1 (0.5 μg) protein with Streptavidin-coated magnetic beads. The beads were precipitated and subjected to Western blotting with EDC3 and Pim1 antibodies (Abs).

B Pim1, 2, or 3 and GFP-EDC3 expression vectors were transfected into HEK-293T. GFP-Trap Nano-body beads (GFP-Trap) were used to immunoprecipitate (IP) GFP-EDC3, and the associated proteins were analyzed by Western blotting with the indicated Abs.

C Western blot of endogenous immunoprecipitation (IP) with EDC3 in MDA-MB-231 cells using Protein G Mag Sepharose with either control IgG or EDC3 primary antibody.

D Pim1 kinase dead (KD) and GFP-EDC3 plasmids were co-transfected into HEK-293T cells. The lysates were precipitated with GFP-Trap beads. The IP products were subjected to Western blotting with the indicated Abs.
EDC3 Phosphorylation Regulates Growth and Invasion through controlling P-body Formation and Dynamics (2021)
Figure 2

Figure 2. Pim1 binds to peptide sequences in the low complexity region of EDC3.

A Schematic of the functional domains of EDC3 and the fragments used to construct GFP fusion proteins. The phosphorylation site on EDC3 serine 161 is shown.

B HEK-293T cells were transfected with Pim1 and each EDC3 domain (Fig. 2A) fused to the C-terminus of the GFP-tagged vector. The individual fragments were immunoprecipitated with GFP-Trap beads and analyzed by Western blotting with the indicated Abs. EDC3 wild type (W), F1, F2, F3, F4 were EDC3 fragments vector fused to GFP. The C1 lane was only transfected with the GFP Flag vector while C2 contained EDC3 wild type fused to GFP and no Pim1 but empty vector control (pcDNA3).

C 2D [1H,15N] HSQC spectrum of EDC3104-197 (100 µM EDC3104-197; 20 mM HEPES pH 6.2, 500 mM NaCl, 0.5 mM TCEP; 298 K; 14.1 T). HSQC spectrum shows a single cross peak for each N-HN pair, i.e. one per amino acid (other than prolines). The lack of chemical shift dispersion in the 1H chemical shift dimension is typical for intrinsically disordered protein/region (IDR), as they lack the hydrogen bonding network in secondary structure elements of folded proteins.

D 2D [1H,15N] HSQC spectra of EDC3104-197 with (red; 1:2 molar ratio) and without (blue) Pim1. We titrated Pim1 into 15N-labeled EDC3, and the 2D [1H,15N] HSQC spectrum of EDC3 (residues 104-197) when bound to Pim1 was measured. Direct comparison with the unbound 2D [1H,15N] HSQC spectrum of EDC3 showed that while the majority of EDC3 peaks are unchanged, ~30 peaks become either broadened beyond detectability demonstrating they directly bind to Pim1 or show small chemical shift perturbations. Upon completion of the sequence-specific backbone assignment of the EDC3 IDR, we identified that EDC3 residues 137 to 167 are necessary for the interaction with Pim1.
EDC3 Phosphorylation Regulates Growth and Invasion through controlling P-body Formation and Dynamics (2021)
Figure 3

Figure 3. EDC3 is a substrate of the Pim and AKT protein kinases.

A In vitro kinase assays were performed as outlined in Materials and Methods with purified recombinant EDC3 (0.2μg) and Pim1 (0.2μg). Samples were analyzed by Western blotting with the indicated Abs.

B In vitro kinase assay with EDC3 (0.2μg) and AKT (0.1μg). Samples were analyzed by Western blotting with the indicated Abs.

C PC3-LN4 cells containing a doxycycline (DOX) inducible Tripz-Pim1 vector were treated with 50 or 100 ng/ml DOX for 48 h and subsequently incubated with DMSO or PIMi (PIM447, 3 µM) for 24 h. Western blots were probed with the indicated Abs.

D PC3-LN4 cells was treated with PIMi (PIM447) at increasing doses (0, 3, 5, and 10μM) for 24 h. Extracts were subjected to Western blotting with the indicated Abs. Relative values of phospho-signals were indicated below p-EDC3 blot by quantification of intensity for p-EDC3 S161 levels using ImageJ (NIH). p-EDC3 S161 levels were normalized to levels of total EDC3 within the respective samples and compared to the p-EDC3 S161 levels in the untreated samples.

E PC3-LN4 was treated with PIMi (PIM447, 3μM), AKTi (GSK690693, 5μM), or the combination for 24 h. Extracts were subjected to Western blotting with the indicated Abs.

F PC3-LN4 CRISPR-Ctr and Pim1 KO cells were treated with PIMi (PIM447, 3μM), AKTi (GSK690693, 5μM), or the combination for 24 h. Extracts were subjected to Western blotting with the indicated Abs.
EDC3 Phosphorylation Regulates Growth and Invasion through controlling P-body Formation and Dynamics (2021)
Figure 4

Figure 4. EDC3 phosphorylation reduces its P-body localization.

A Immunofluorescence (IF) staining of PC3-LN4 cells following 6 h treatment with DMSO (Control), PIMi (PIM447, 3µM), AKTi (GSK690693, 5μM), and both inhibitors (AKTi + PIMi) and stained with anti-EDC3 and anti-DCP1a antibodies as well as DAPI.

B Quantification of EDC3/DCP1a foci number from Control (Ctrl), PIMi, AKTi and the AKTi + PIMi combination in (A).

C Immunofluorescence (IF) staining of PC3-LN4 EDC3 knock out cells following 6 h treatment with DMSO (Control), PIMi (PIM447, 3µM), AKTi (GSK690693, 5μM), and both inhibitors (AKTi + PIMi) and stained with anti-EDC3, and anti-DCP1a antibodies (Abs) as well as DAPI.

D Quantification of DCP1a foci number for Control (Ctrl), PIMi, AKTi, and AKTi + PIMi combination in (C).

Data information: IF Images were subsequently merged using FIJI image analysis software. The yellow box shows a zoom of the merged image. Images were captured under 40x magnification; scale bar: 10µm. P-body number was counted by FIJI. The quantification data of (B and D) are presented as mean +/- SEM (n = 30 cells/treatment group). p values<0.05, calculated using unpaired student t-test, are considered significant. p values>0.05 not significant (n.s.). The analyses done included multiple independent fields.
EDC3 Phosphorylation Regulates Growth and Invasion through controlling P-body Formation and Dynamics (2021)
Figure 5

Figure 5. Phospho-EDC3 (S161) status in P-bodies

A, B Western blot of whole-cell (WCL) and P-body (PB) purified from PC3-LN4 cells stably expressing GFP-DCP1a used as a P-body tag. The P-bodies (PB) were isolated with GFP-Trap beads and analyzed by Western blotting with the indicated Abs.

C Immunofluorescence (IF) of PC3LN4 cells under normal culture condition with anti-phopsho-EDC3, anti-EDC3 Abs, and DAPI. Images captured under 40x magnification; scale bar: 10µm.

D Immunofluorescence of PC3-LN4-EDC3 S161A (SA) cells under normal culture condition with anti-DCP1a and anti-EDC3 Abs as well as DAPI. Images captured under 40x magnification; scale bar: 10µm.
EDC3 Phosphorylation Regulates Growth and Invasion through controlling P-body Formation and Dynamics (2021)
Figure 7

Figure 7. S161A Mutation in EDC3 suppresses tumor cell growth, migration, and invasion.

A PC3-LN4 Naïve and CRISPR/Cas9 genome-edited EDC3 S161A (SA), S161D (Fredriksson et al.), and EDC3 knock-out (KO) cell lines were plated (4000 cells/well in 24 well plates) and imaged every 12 h using phase contrast in the IncuCyte ZOOM platform. The graph shown was generated with IncuCyte Basic Software following 5 days of growth. The experiments were done in triplicates. Values are mean +/- S.E.M. p values<0.05, calculated using unpaired student t-test.

B Quantification of transwell migration assays performed for PC3-LN4 EDC3 wild type, EDC3 S161A, EDC3 KO, and EDC3 S161D cell lines. The number of migrated cells (40 h) was counted as outlined in Materials and Methods.

C Quantification of transwell migration assays performed for PC3-LN4 EDC3 KO cells containing Doxycycline (DOX)-inducible EDC3-S161A with and without DOX (100ng/ml) induction. The number of migrated cells (40 h) was counted as outlined in Materials and Methods.

D Quantification of transwell invasion assay comparing PC3-LN4 EDC3 wild type, EDC3 S161A, EDC3 KO, and EDC3 S161D cell lines. Cells were plated in trans-wells coated with Matrigel and incubated for 72 h. The cells that invaded the matrix were counted as outlined in Materials and Methods.

E Quantification of transwell invasion assay performed for PC3-LN4 EDC3 KO cells containing DOX- inducible EDC3-S161A were either treated with or without DOX (100 ng/ml). The number of cells that invaded were counted as outlined in Materials and Methods.

F Relative mRNA expression of ITGA6, ITGB1, and KLF4 in PC3-LN4 WT and EDC3 S161A mutant cell lines.

G Western blot analysis of proteins extracted from PC3-LN4 EDC3 wild type, EDC3 S161A, EDC3 knockout (KO, C60 cell line), and EDC3 S161D cell lines using the specified antibodies.

H Relative mRNA expression of ITGA6, ITGB1, and KLF4 in PC3-LN4 EDC3 KO-C60 cells, containing reexpressed EDC3 WT or EDC3 S161A mutant cDNAs

I Western blot analysis of proteins extracted from PC3-LN4-EDC3-KO cells expressing GFP, EDC3 WT or the EDC3 S161A mutant using the specified antibodies.

J Western blot analysis of proteins extracted from PC3-LN4 cells treated with DMSO or the combination of Pim447 and AZD5363 for 96 h using the specified antibodies.

Data information: All invasion and migration assays were performed in triplicates and 6 different fields at 10x magnification were counted in 3 separate inserts. The data are presented as the mean +/- S.D. p values<0.05, calculated using unpaired student's t-test. Real-time PCR data (F & H) are presented as mean ± SEM, (n = 3/group). Unpaired student's t-test was used for statistical analysis. *P < 0.05, **P < 0.01 and NS, not significant.
EDC3 Phosphorylation Regulates Growth and Invasion through controlling P-body Formation and Dynamics (2021)
Figure 8

Figure 8. P-EDC3 levels regulate tumor growth and are elevated in multiple breast cancer cell lines.

A Two independently derived CRISPR/Cas9 PC3-LN4 EDC3 S161A mutants (EDC3 SA#1, #2) and wild type cells were injected subcutaneously in the flank (5X106) of SCID mice (n=8/group), and tumor growth was monitored until the control group reached 1000 mm3. The subcutaneous tumor volume measurement (mm3) was done as outlined in Materials and Methods. Data are presented as mean ± SEM, (n = 8/group).

B Phosphorylated EDC3 levels were examined in normal immortalized breast cell lines, MCF10A and MCF12A, human breast cancer cell lines HCC38, HCC1954, MDA-MB-231, MDA-MD-468, BT-549, MRKNU1, and mouse 4T1 by Western blotting with the indicated Abs.

C Dual immunohistochemistry staining of human breast tumor with p-EDC3 Ab (Fredriksson et al.) and total EDC3 (brown). Scale bar: 50µm.

D Dual immunohistochemistry staining of human normal resected breast tissue sections with p-EDC3 Ab (Fredriksson et al.) and total EDC3 (brown). Scale bar: 50µm.

Figure EV Legends
Arginine methylation of the DDX5 helicase RGG/RG motif by PRMT5 regulates resolution of RNA/DNA hybrids (2019)
Figure 1

Figure 1. DDX5 unwinds R-loops in vitro and repress cellular R-loop accumulation.

(A) Coomassie Blue staining of recombinant human DDX5 purified in bacteria. M denotes the molecular mass markers in kDa.

(B) R-loop unwinding assay in presence of increasing DDX5. The top panel shows a typical image obtained after the assay. The bar graph (bottom) shows the quantification. The average is expressed as percentage unwinding, and standard error of the mean (S.E.M.), n=4.

(C) S9.6 signal in the nucleoplasm of U2OS cells. An average from 3 independent experiments performed in triplicate. In total, ~90 images for each condition (10 images/slide and 9 slides for each condition) were taken and two cells per image were quantified. Statistical significance was assessed using one-way ANOVA t-test. *: p < 0.05; ****: p < 0.0001.

(D) U2OS cells transfected with siCTL, or siDDX5 were subjected to DRIP-qPCR analysis with anti-IgG, and anti-S9.6 antibodies with or without RNaseH treatment. The gene location and genomic qPCR amplification region are shown at the top of each panel. B, E, H, S and X denote the location of the BsrGI, EcoRI, HindIII, SspI, and XbaI. The identified R-loop peaks were extracted from the R-loop database (R-loop DB) for each region. The bar graphs are the average and S.E.M. from 3 independent experiments. Statistical significance was assessed using t-test. *: p < 0.05.
Arginine methylation of the DDX5 helicase RGG/RG motif by PRMT5 regulates resolution of RNA/DNA hybrids (2019)
Figure 2

Figure 2. DDX5 is arginine-methylated in U2OS cells and PRMT5 deficiency causes cellular R-loops to accumulate.

(A) U2OS cells were lysed and the lysates were incubated with anti-PRMT5 antibody or immunoglobulin G (IgG) as control followed by protein A Sepharose bead addition. The immunoprecipitated (IP) proteins or whole cell extract (WCE) were separated by SDS-PAGE and Western blotted (WB) with the indicated antibodies.

(B) Same as panel (A) except anti-DDX5 antibody was used instead of anti-PRMT5 antibody for immunoprecipitation.

(C) The mass spectrum of R502 monomethylation from Flag-DDX5 expressed in U2OS.

(D) HEK293 cells transfected with Flag-DDX5 were lysed and immunoprecipitated with anti-Flag antibodies. The bound proteins were along with WCE from untransfected (-) and Flag-DDX5 (+) transfected cells were Western blotted with anti-FLAG, anti-methylarginine antibodies (MeR).

(E and F) The knockdown using siRNAs of the indicated gene in U2OS cells was confirmed by Western blotting and duplicate cell cultures were analyzed by immunofluorescence with S9.6 and anti-nucleolin antibodies. A typical image is shown. The scale bar represents 10 μm. Graphs show the average of 3 independent experiments performed in triplicates. Statistical significance was assessed using one-way ANOVA t-test. **: p < 0.01; ****: p < 0.0001 and n.s.: not significant.
Arginine methylation of the DDX5 helicase RGG/RG motif by PRMT5 regulates resolution of RNA/DNA hybrids (2019)
Figure 3

Figure 3. Depletion of DDX5 increase R-loops at transcribed regions with local ROS-induced DNA damage.

(A) Western blotting of PRMT5 and DDX5 knockdowns.

(B) Representative images of S9.6 staining in siCTL, siDDX5, or siPRMT5 knockdown at transcription on (TA-KR) or off (TetR-KR) genomic loci in U2OS TRE cells. The nuclei are visualized by DAPI.

(C) Quantification of the S9.6 foci intensity in the indicated conditions. Three independent experiments were performed and 50 cells were analyzed in each experiment. Error bars represent mean of S9.6 foci intensity quantification with s.e.m. The statistical analysis was performed by two-tailed Student's t-test (Mann-Whitney test U-test). ****, p < 0.0001.
Arginine methylation of the DDX5 helicase RGG/RG motif by PRMT5 regulates resolution of RNA/DNA hybrids (2019)
Figure 4

Figure 4. PRMT5 deficient cells accumulate R-loops at specific loci.

(A) U2OS cells transfected with siCTL or siPRMT5 were subjected to DRIP-qPCR analysis. The average and S.E.M. from 3 independent experiments is shown. Statistical significance was assessed using Student's t-test. *: p < 0.05; and **: p < 0.01.

(B) Western blotting of whole cell extracts from siCTL, siPRMT5, siDDX5 or siDDX5/ siPRMT5 transfected cells.

(C) U2OS cells transfected with siCTL, siPRMT5, siDDX5 or siDDX5/ siPRMT5 were subjected to DRIP-qPCR analysis. The average and S.E.M. from 3 independent experiments is shown. Statistical significance was assessed using Student's t-test. *: p < 0.05; and **: p < 0.01.