Fig 2: ORC1 overexpression partially reduces the inhibitory effect of PLCD1 overexpression on invasion, migration and EMT of lung adenocarcinoma cells. (A) Cell migration was detected using a wound-healing assay (magnification, ×100). (B) Cell invasion was detected using a Transwell assay (magnification, ×100). (C) The protein expression levels of MMP2 and MMP9 were detected by western blot analysis. (D) EMT-related protein expression levels, including E-cadherin, N-cadherin, vimentin and Snail, were detected by western blot analysis. *P<0.05, **P<0.01 and ***P<0.001. ORC1, origin recognition complex 1; PLCD1, phospholipase Cδ1; EMT, epithelial to mesenchymal transition; oe, overexpression; NC, negative control.

Fig 3: High expression of ORC1 in LUAD tissues and negative regulation of PLCD1. According to GEPIA database, ORC1 expression was (A) upregulated in LUAD, (B) associated with LUAD stage and (C) negatively correlated with PLCD1 expression in LUAD. The non-log scale was used for calculation and the log-scale axis for visualization. (D) Protein and (E) mRNA expression levels of ORC1 in cells. (F) Binding site of ORC1 to PLCD1 promoter. *P<0.05 and ***P<0.001. ORC1, origin recognition complex 1; PLCD1, phospholipase Cδ1; LUAD, lung adenocarcinoma; TPM, transcripts per million; num, number; T, tumor; N, normal; TSS, transcription start site.

Fig 4: ORC1 overexpression attenuates the inhibitory effect of PLCD1 overexpression on lung adenocarcinoma cell proliferation. (A) Results of cell proliferation assay. (B) Results of colony formation assay in different groups. (C) Ki67 expression level in cells determined via immunofluorescence (scale bar, 50 µm). ***P<0.001 vs. oe-NC; #P<0.05, ##P<0.01 and ###P<0.001 vs. oe-PLCD1. ORC1, origin recognition complex 1; PLCD1, phospholipase Cδ1; oe, overexpression; NC, negative control.

Fig 5: c-MYC G1/S phase RNAs interact with ORC1.a. (Upper panel). Schematic of the c-MYC locus and respective primer sets indicated by numbers. The red bar indicates the c-MYC replication origin (region 9–10). (Bottom panel) ORC1 and H2A.Z enrichment at the c-MYC origin detected by chromatin immunoprecipitation (ChIP). The qPCR analysis was performed using distinct sets of primers designed as depicted in the schematic diagram provided above. The bars indicate the mean ± S.D of three independent experiments (n=3), Student’s t-test, **: p value 0.0085 (ORC1) and **: p value 0.0055 (H2A.Z); b Expression levels of RNAs originating within the promoter of c-MYC, G1 and later stages of the S phase: S1 (2hr), S2 (5hr). The bars represent the mean ± S.D of three independent experiments (n=3), Student’s t-test, *: p value <0.0001 (G1 vs WT), p value <0.0001 (S1 vs WT) and p value 0.00189 (S1 vs S2) c. RNA immunoprecipitation (RIP) qRT-PCR performed using two separate antibodies against human ORC1 (ab85830 and ab60). The association between c-MYC S-Phase RNAs anchoring ORC1 – ANCOR is shown d-f. In vitro RNA/DNA electrophoresis mobility shift assay (EMSA) and RNA pull-down assays. d. RNA secondary structures of the RNA probes corresponding to the c-MYC origin (RM9) and unrelated to the origin (MYC1, MYC2, and MYC3) calculated using RNAfold; e. RNA EMSA showing the preferential binding of ORC1 to RM9 (RNA) versus the unrelated MYC1 sequence and the homologous DNA sequence (dRM9); f. RNA pull down assay. Bound proteins were purified on streptavidin beads and immunoblotted with antibodies against ORC1 and H2A.Z. The experiment was repeated twice with similar results; g. Molecular dynamics simulations: frontal views of the representative structures of ORC1a, ORC1b and ORC1c states in complex with either dRM9 (DNA) or RM9 (RNA). RNA and DNA structures were derived using a RMSD-based clustering approach in the last half of simulation time. All molecules are color-coded as follows: orange: ORC1-BAH domain (residues 1–200); marine: ORC1 disordered region and AAA+ domain (residues 201–782); gray: orc1-WHD domain (residues 783–861); red and yellow: RM9 and dRM9, respectively; h. The figure shows a zoomed-in view of both the top and frontal perspectives, illustrating the preferred binding state of the ORC1-RM9 complex; i. Zoomed-in view showing the ORC1 amino-acid residues interacting with RM9.