Fig 2: Effects of MYST2 overexpression on the proliferation, migration, and invasion of lung cancer cells.A. Western blot analysis illustrating the expression of MYST2 in three lung cancer cells, with β-actin serving as a loading control. B. Western blot analysis demonstrating the overexpression of MYST2 in lung cancer cells, using β-actin as a loading control. C. CCK8 assay evaluating the impact of MYST2 overexpression on the proliferation of lung cancer cells. D. Quantitative analysis of the CCK8 assay results from Figure C using ImageJ. E-F. Wound-healing assay assessing the effect of MYST2 overexpression on cell migration, with Figure F representing the quantitative analysis of Figure E. G-H. Transwell assay measuring the effect of MYST2 overexpression on the migration of lung cancer cells, with Figure H showing the quantitative analysis of Figure G. I-J. Transwell assay investigating the impact of MYST2 overexpression on the invasion of lung cancer cells, with Figure J providing the quantitative analysis of Figure I.

Fig 3: Models for ORFIUS complex function at replication origins in different settings. (A) Model for ORFIUS complex function in normal and non-HGSC ovarian cancer cells. At baseline BRCA1 regulates BRD1-HBO1 localization to and promotion of ORC2 localization at replication origins. The complex is responsive to CDC7 kinase regulation of origins. In the setting of DNA damage and/or replication stress (RS) induced by agents like PARP inhibitors (PARPi), ATR signaling is activated, and in response the ORFIUS complex dissociates from replication origins and no longer promotes ORC2 localization there. (B) Model for ORFIUS complex function in BRCA1 mutant and sporadic (BRCA1 wildtype) HGSC cells. In BRCA1 mutant HGSC cells, either a BRCA1 truncation or point mutant or no BRCA1 may be expressed, or in sporadic HGSC cells full length BRCA1 may be expressed but signaling may be altered. In each case, BRCA1 control of HBO1, BRD1, and ORC2 at origins may be dysfunctional. There is more ORFIUS complex associated with origins than in non-malignant or non-HGSC ovarian cancer cells promoting ORC2 localization there at baseline. In the setting of DNA damage and/or RS, the ORFIUS complex is not responsive to ATR signaling and remains associated with origins promoting ORC2 localization there.

Fig 4: MYST2 influences the phosphorylation of p38 and ERK, but not AKT and p65.A. Western blot analysis showing the effects of MYST2 knockout on p-p38, total p38, p-AKT, total AKT, p-ERK, and total ERK, p-p65, and total p65 levels, with β-actin serving as a loading control. B. Western blot analysis demonstrating the effects of MYST2 overexpression on p-p38, total p38, p-AKT, total AKT, p-ERK, and total ERK, p-p65, and total p65 levels, with β-actin as a loading control. C. GEPIA database analysis showing the correlation between MYST2 and p38 expression levels. D. Co-IP assay in HEK293T cells overexpressing Myc-MYST2 and Flag-p38, demonstrating the interaction between MYST2 and p38. E. Co-IP assay in HEK293T cells overexpressing Myc-MYST2 and Flag-ERK, demonstrating the interaction between MYST2 and ERK. F. GST pull-down assay using a prokaryotic expression system to express GST-p38 fusion protein, demonstrating the interaction between MYST2 and p38. G. Schematic diagram of the truncation strategy for MYST2 truncated constructs. H. Transfection of Flag-p38 and Myc-MYST2 wild-type and various truncated constructs in HEK293T cells, followed by co-IP to detect the interaction domains between p38 and MYST2.

Fig 5: BRCA1, BRD1, HBO1, and BARD1 form the ORFIUS complex. (A) Proximity ligation assay (PLA) was performed on asynchronous FT194, OVCAR8, SKOV3, and CaOV3 cells for BRCA1 and BRD1, BRCA1 and HBO1, or BRCA1 and ORC2. Representative images of PLA foci with DAPI to mark nuclei for no antibody control, BRCA1 alone, BRD1 alone, or BRCA1-BRD1 co-stain are shown for FT194 cells on the left. Scale bars = 10 μm. Please note that these FT194 PLA signal+DAPI images are also shown in Supplementary Figure S7A along with the corresponding PLA signal alone photos, as well as representative images for all lines with this antibody pair. Violin plots from one representative experiment comparing the number of PLA foci per nucleus from at least 100 nuclei are shown for the no primary antibody control compared to the co-stain for each cell line for BRCA1-BRD1, BRCA1-HBO1, or BRCA1-ORC2 on the right. *P< 0.05 comparing the co-stain to the no primary antibody control by a one-way ANOVA with Tukey's multiple comparisons test. (B–E) 293T cells were transfected with empty vector or GST tagged full length BRD1 or HBO1 in (B), GST tagged BARD1 in (C), GST tagged full length or fragments of BRD1 in (D), or GST tagged full length or fragments of HBO1 in (E). 48 h later GST immunoprecipitations (IPs) were performed on the various lysates, and western blots were performed on the IPs. Inputs and GST IPs are shown with the western blot antibody indicated on the left and the IP target on the bottom of the blots. (Term = Terminus) For all of the experiments, expression of the GST-tagged bait is shown in a GST western for the input and IP in each panel. A green bar on top of the blots indicates input and a blue bar indicates GST IP. For westerns in which an endogenous and a GST-tagged protein may appear in the same blot, we indicate on the left side of the blot with an arrow the endogenous and the GST-tagged protein. For the fragment blots in (D) and (E), on top of the blots is a map for the respective full length and fragment proteins with key domains marked. For BRD1, domains include the N-terminus known to interact with HBO1 (HBO1 interaction), the plant homeodomain finger domain (PHD), the Bromodomain (Bromo), and the proline-tryptophan-tryptophan-proline (PWWP) domain. For HBO1, the domains include the C terminal MYST domain. FL = full length. AA = amino acids. (F) Schematic of ORFIUS complex interactions. On the bottom of the cartoon is a key to each protein with key functional domains indicated. For BRCA1, the N-terminal RING domain is highlighted. For BRD1, 1–205 represents the N-terminal amino acids one through 205 where HBO1 interacts. In the larger cartoon, the interactions of the four complex members are indicated at key domains in each protein based on GST IP or previously known results.