Fig 2: O-GlcNAc protects GATAD2B from ITCH-mediated proteasomal degradation. (A)-TNBC cells MDA-MB-231 were treated with DMSO or OGT inhibitor OSMi-1 (100 µM) for 24 h, then subsequently treated with DMSO or proteasome inhibitor MG-132 (10 µM) for an additional 24 h before collecting for immunoblot using indicated antibodies (left). Quantified graph of blots from MDA-MB-231 cells treated with DMSO, OSMi-1 or MG-132 (right). Two-way ANOVA with Sidak test is reported as mean ± SEM, * p < 0.05. (B)-HEK-293T cells, transfected with HA-tagged Ubiquitin, were treated with DMSO or with OGT inhibitor OSMi-1 (100 µM) for 24 h prior to lysate collection. Lysate from each sample was immunoprecipitated using anti-GATAD2B antibody or IgG and was subsequently analyzed by immunoblot using indicated antibodies. (C)-Volcano plot of mass spectrometry analysis showing ITCH being co-IPed together with GATAD2B in TNBC cells MDA-MB-231 by anti-GATAD2B antibody. (D)-TNBC cells MDA-MB-231 were treated with DMSO or OGT inhibitor OSMi-1 (100 µM) for 24 h, then were collected for immunoprecipitation using anti-GATAD2B antibody or IgG. Immunoprecipitated proteins were analyzed by Western blot using indicated antibodies (left). Quantified graph shows increase ITCH level being co-immunoprecipitated with GATAD2B in the presence of OGT inhibitor OSMi-1 (100 µM) compared to control of DMSO (right). One sample t-test against hypothetical value is reported as mean ± SEM, * p < 0.05. (E)-TNBC cells MDA-MB-231, transduced with scramble or ITCH shRNAs, were treated with DMSO or OGT inhibitor OSMi-1 (100 µM) for 48 h prior to being collected for immunoblot analysis using indicated antibodies. (F)-Quantified graph of (E) shows increase in the level of GATAD2B in the presence of ITCH shRNAs, compared to scramble shRNA, in TNBC cells MDA-MB-231 treated with DMSO or OGT inhibitor OSMi-1. Two-way ANOVA with Sidak test is reported as mean ± SEM, * p < 0.05, ** p < 0.01. *** p < 0.001. (G)-TNBC cells MDA-MB-231, transduced with scramble or ITCH shRNAs, were treated with DMSO or OGT inhibitor OSMi-1 (100 µM) for 48 h prior to being plated in mammosphere condition. Mammosphere formation efficiency from each condition was determined and graphed. Two-way ANOVA with Sidak test is reported as mean ± SEM, * p < 0.05, ** p < 0.01. *** p < 0.001. (H)-Quantified graph showing ALDH+ CSC from TNBC cells MDA-MB-231, transduced with scramble or ITCH shRNA and treated with DMSO or OGT inhibitor OSMi (100 µM). Two-way ANOVA with Sidak test is reported as mean ± SEM, * p < 0.05, *** p < 0.001.

Fig 3: GATAD2B O-GlcNAcylation at the C-terminus is critical for protein stability and CSC function. (A)-TNBC cells MDA-MB-231 were treated with DMSO or OGA inhibitor Thiamet-G (2 µM) for 3 h prior to being collected for lysate. 250 µg of total protein from each condition was incubated with washed sWGA agarose bead overnight. The bead was then washed and analyzed by Western blot using indicated antibodies. For negative control we washed beads with incubated beads with GlcNAc to remove any O-GlcNAcylated proteins (lane 1). Sp1 was used as positive control. (B)-HEK-293T overexpressing GATAD2B was collected for immunoprecipitation using anti-GATAD2B antibody. Immunoprecipitated GATAD2B was subsequently analyzed using mass spectrometry. Mass spectrometry result shows detected O-GlcNAc modification at the C-terminus of GATAD2B. (C)-HEK-293T cells were transfected with HA-tagged wild-type (WT) GATAD2B or GATAD2B with S584/586/588/590A mutations (Mut) and were subsequently collected for lysate. Lysate from HEK-293T cells, control plasmid or overexpression WT or Mutant GATAD2B, were incubated with sWGA agarose bead overnight before analyzing by Western blot using indicated antibodies. For negative control we washed beads with incubated beads with GlcNAc to remove any O-GlcNAcylated proteins (lane 1). (D)-HEK-293T cells were transfected with WT or Mutant GATAD2B and were subsequently collected for immunoprecipitation using anti-GATAD2B antibodies. Immunoprecipitated proteins were analyzed by Western blot using indicated antibodies (left). Quantified graph shows increase in the level of poly-Ubiquitination co-immunoprecipitated with Mutant GATAD2B, compared to WT GATAD2B (right). One sample t-test against hypothetical value is reported as mean ± SEM, * p < 0.05. (E)-HEK-293T cell were transfected with control plasmid (lane 1), HA-tagged WT or Mutant GATAD2B and were subsequently treated with Cycloheximide (100 µM) for 0, 2 and 4 h before being collected for Western blot using indicated antibodies (left). Quantified graph shows decrease in the level of HA-tagged Mutant GATAD2B compared to WT GATAD2B overtime (right). Two-way ANOVA with Sidak test is reported as mean ± SEM, ** p < 0.01. (F)-TNBC cells transduced with control or GATAD2B-specific gRNA were infected with lentiviral vector containing WT or Mutant GATAD2B and were subsequently collected for Western blot using indicated antibodies (left). Quantified graph shows mammosphere formation efficiency of control or transduced with GATAD2B gRNA MDA-MB-231 cells overexpressing WT or Mutant GATAD2B (right). Two-way ANOVA with Sidak test is reported as mean ± SEM, ns—not significant, ** p < 0.01, *** p < 0.001, **** p < 0.001.

Fig 4: GATAD2B function downstream of OGT/O-GlcNAc in regulating CSCs in breast cancer. (A)-Lysates from control or OGT overexpressing MDA-MB-231 cells, transduced with scramble or GATAD2B shRNAs, were collected and analyzed by immunoblot using indicated antibodies (left). Control or OGT overexpressing MDA-MB-231 cells, transduced with scramble or GATAD2B shRNAs, were grown in mammosphere formation assay. The mammosphere formation efficiency in each condition was determined and presented in the quantified graph (right). Two-way ANOVA with Sidak test is reported as mean ± SEM, * p < 0.05. (B)-Quantified graph showing ALDH+ CSCs from control or OGT overexpressing MDA-MB-231 cells, transduced with scramble or GATAD2B shRNA. Two-way ANOVA with Sidak test is reported as mean ± SEM, * p < 0.05, ** p < 0.01. *** p < 0.001. (C)-Lysates from control or transduced with GATAD2B shRNAs MDA-MB-231 cells, treated with DMSO or OGA inhibitor Thiamet-G (2 µM) for 48 h, were collected and analyzed by immunoblot using indicated antibodies (left). Control or transduced with GATAD2B shRNAs MDA-MB-231 cells, treated with DMSO or OGA inhibitor Thiamet-G (2 µM) for 48 h, were grown in mammosphere formation assay. The mammosphere formation efficiency in each condition was determined and presented in the quantified graph (right). Two-way ANOVA with Sidak test is reported as mean ± SEM, * p < 0.05, ** p < 0.01. *** p < 0.001.

Fig 5: NuRD complex protein GATAD2B is regulated by OGT/O-GlcNAc in breast cancer cells. (A)-Mass spectrometric analysis of TNBC cells MDA-MB-231, treated with DMSO or OGA inhibitor Thiamet-G for 24 h, shows changes in protein level with 157 proteins down-regulated and 142 protein up-regulated (left). GO-analysis of up-regulated proteins (ShinyGO 0.75: GO-cellular components) shows enrichment of NuRD complex in Thiamet-G treatment (right). (B)-Immunoblot analysis of different TNBC cell lines shows elevated level of GATAD2B in TNBC cells compared to control of immortalized mammary gland cells MCF-10A (top and quantified graph shows significantly higher level of GATAD2B in TNBC compared to control of MCF-10A cells (bottom). One sample t-test against hypothetical value is reported as mean ± SEM, * p < 0.05. (C)-Immunoblots of TNBC cells MDA-MB-231 and SUM159, treated with DMSO or OGA inhibitor Thiamet-G for 24 h, show elevated level of GATAD2B in Thiamet-G treatment compared to control of DMSO (left and middle), and quantified graph showing significant increase in GATAD2B level in the presence of Thiamet-G compared to DMSO (right). One sample t-test against hypothetical value is reported as mean ± SEM, * p < 0.05. (D)-Immunoblots of TNBC MDA-MB-231 cells, control or with OGT overexpression, show increase in GATAD2B level in OGT overexpression, compared to control (left), and quantified graph shows significant increase in GATAD2B level in OGT overexpression, compared to control (right). One sample t-test against hypothetical value is reported as mean ± SEM, * p < 0.05. (E)-Immunoblots of TNBC MDA-MB-231 cells, transduced with control or OGT shRNA, show decrease in GATAD2B level in OGT knockdown, compared to control (left), and quantified graph shows significant decrease in GATAD2B level in OGT knockdown, compared to control (right). One sample t-test against hypothetical value is reported as mean ± SEM, * p < 0.05.