Fig 2: An APR within GATAD2 proteins co‐ordinates CDK2AP1 and CHD4 association with NuRD. (A) TANGO analysis shows β‐sheet aggregation tendency for the APR within GATAD2A and GATAD2B (B) A close‐up schematic of the β‐sheet aggregation tendency of the APRs in GATAD2A (384‐390) and GATAD2B (388‐394) and gatekeeper residues (boxed). (C) Graph representing the MTT assay performed in 2 biological and three technical replicates (n = 6). Cell viability of K562 cells treated with a range of APR peptide concentrations was measured after 48 h. Data are presented as a per cent of untreated cell viability measured using the MTT assay. (D) Western blots of endogenous GATAD2 proteins after treatment of K562 cell lysates with APR‐tat peptides. APR peptidomimetics (0–150 μm) were added to the lysates before sonication. Total lysates (TL, 5%) were taken as input before separation of the soluble and insoluble fractions by centrifugation. Cleared lysate (5% of soluble fraction) was run on SDS‐PAGE and probed for GATAD2 proteins. Black arrows indicate the canonical isoform of GATAD2A and GATAD2B proteins, and dotted rectangular box shows loss of soluble proteins post‐APR treatment. (E) Volcano plot comparing the interactors of overexpressed GATAD2B in the presence of 50 μm APRB (left panel) or CTRL (right panel) peptides. (F) The number of unique peptides of each subunit of the NuRD complex detected by MS after FLAG‐GATAD2B PD‐MS in the presence of APRB and CTRL peptides.

Fig 3: Stoichiometry of the NuRD subunits. MBD2/3 and GATAD2B were used as bait. Bait proteins were excluded from calculations, because they are in excess amount and introduce a bias in stoichiometry calculations. Stoichiometry of the subunits where (A) MBD2/3 and (B) GATAD2B were used as bait. (C) Scatter plot showing log2 of iBAQ intensities for HDAC1 and HDAC2 in all six replicates of CDK2AP1 PD‐MS. (D) Schematic representation of the possible new subcomplexes independent of the NuRD complex that can be formed via PPIs occurring with bridging subunits.

Fig 4: GATAD2B is critical to maintain CSCs phenotype of breast cancer cells. (A)-TNBC MDA-MB-231 cells were transduced with control (scramble) or with GATAD2B-specific shRNAs. Lysates from control or transduced with GATAD2B shRNAs MDA-MB-231 cells growing in monolayer (top) or in mammosphere (bottom) were collected for immunoblot analysis using indicated antibodies. Control or transduced with GATAD2B shRNAs MDA-MB-231 cells were allowed to grow in mammosphere formation assay. Mammospheres were counted and primary mammosphere formation efficiency from each condition was determined and graphed (bottom). Primary mammospheres were collected and regrown in the same mammosphere culture condition to form secondary mammosphere. The number of mammospheres and secondary mammosphere formation efficiency from each condition was determined and graph (right). Two-way ANOVA with Sidak test is reported as mean ± SEM, ** p < 0.01, *** p < 0.001. (B)-Quantified graph of ALDH+ CSCs population from TNBC cells MDA-MB-231, SUM159 and PDX cells HCI-10, which were transduced with scramble or GATAD2B shRNAs. One-way ANOVA with Sidak test is reported as mean ± SEM, * p < 0.05. (C)-Lysates from MDA-MB-231 cells transduced with scramble or GATAD2B shRNAs were collected for immunoblot analysis using indicated antibodies (left). Quantified graph of blots from MDA-MB-231 cells with control or GATAD2B shRNAs (right). Multiple one sample t-test against hypothetical value with Holm-Sidak correction is reported as mean ± SEM, * p < 0.05, ** p < 0.01, *** p < 0.001. (D)-Total RNA of MDA-MB-231 cells transduced with scramble or GATAD2B shRNAs were collected and mRNA level of indicated genes were analyzed by qRT-PCR. The level of PPIA was used as internal control. Multiple one sample t-test against hypothetical value with Holm-Sidak correction is reported as mean ± SEM, ** p < 0.01, *** p < 0.001.

Fig 5: GATAD2B promotes CSCs phenotype of breast cancer cells. (A)-Lysates from control or GATAD2B overexpressing MDA-MB-231 cells growing in monolayer (top) or in mammosphere (right) were collected for immunoblot analysis using indicated antibodies. Control or GATAD2B overexpressing MDA-MB-231 cells were allowed to grow in mammosphere formation assay. Mammospheres were counted and primary mammosphere formation efficiency from each condition was determined and graphed (right). Primary mammospheres were collected and regrown in the same mammosphere culture condition to form secondary mammosphere. The number of mammospheres and secondary mammosphere formation efficiency from each condition was determined and graph (right). Two-way ANOVA with Sidak test is reported as mean ± SEM, * p < 0.05, ** p < 0.01. (B)-Quantified graph of ALDH+ CSCs population from control or GATAD2B overexpressing TNBC cells MDA-MB-231. Paired t-test is reported as mean ± SEM, ** p < 0.01. (C)-Quantified graph of SORE-GFP+ CSCs population from control or GATAD2B overexpressing TNBC cells MDA-MB-231. Paired t-test is reported as mean ± SEM, * p < 0.05. (D)-Lysates from control or GATAD2B overexpressing MDA-MB-231 cells were collected for immunoblot analysis using indicated antibodies (left). Quantified graph of blots from control or GATAD2B overexpressing MDA-MB-231 cells (right). Multiple one sample t-test against hypothetical value with Holm-Sidak correction is reported as mean ± SEM, ** p < 0.01, * p < 0.05. (E)-Total RNA of control or GATAD2B overexpressing MDA-MB-231 cells were collected and mRNA level of indicated genes were analyzed by qRT-PCR. The level of PPIA was used as internal control. Multiple one sample t-test against hypothetical value with Holm-Sidak correction is reported as mean ± SEM, * p < 0.05.