HeLa and A549 cell lines were treated with wogonin or acidic pH over three weeks and analyzed for SNVs. In either instance, using the HeLa or A549 genome in the preceding time frame as control, duplex bases on the control genome were recorded as MM, mm, or Mm loci, where M and m referred to a major or minor allele on the human reference genome hg19. On this basis, the SNVs of mutated residues from MM/mm to Mm were referred to as GOH mutations, whereas the SNVs of mutated residues from Mm to MM/mm were referred to as LOH mutations. Upon tracking the GOH and LOH occurrences over the three weeks, the HeLa cells were found to engage in a forward–reverse mutation cycle during treatment with wogonin (Fig. 1a, left panel), generating 250 GOH mutations from its 711,547 MM residues recorded on Day 14 via the G1-mutation step; all of these GOHs back mutated to MM via the L1-step, and none mutated to mm via the L2-step through an LOH on Day 16. As a result, the LOHs formed from the 250 GOHs were totally biased in favor of restoring the pre-mutated control MM loci (Fig. 2a). Likewise, there were 21 GOH mutations from its 196 mm residues recorded on Day 14; 20 of them back mutated to mm via the L4-step, and none mutated to MM via the L3-step on Day 16, which again favored restoring the pre-mutated control mm loci relative to any novel MM loci. Therefore, there was a strong propensity for the DNA residues mutated during the forward stage of the cycle to restore the original residues in the reverse stage, as highlighted by yellow triangles in the figure. The A549 cells under wogonin treatment also exhibited comparable mutations (Fig. 1a, right panel; Fig. 2b). On the other hand, when the HeLa and A549 cells were exposed to acidic pH, forward–reverse mutation cycles were observed on Days 1–3, 10–14, and 16–18 for HeLa cells, and on Days 9–11 and 30–32 for A549 cells (Fig. 1b). The chromosomal positions of the individual SNVs observed among the forward–reverse mutations are shown in Fig. 3. In accordance with Fig. 1, the forward mutations of MM and mm residues contained more GOHs, whereas their reversals contained more LOHs in Fig. 2. Moreover, when the forward and reverse SNV mutations are plotted in Fig. 4, where the Total Forward SNVs and Total Reverse SNVs are profiled to reveal the signatures of human cancers [23], the mutations were enriched in the C to T (blue) or T to C (pink) bars while the GOH in the forward stage is slightly more than the LOH in the reverse stage.

A. GOH and LOH mutations of control MM, mm or Mm residues in HeLa (left diagram) or A549 cells (right diagram) under wogonin treatment. B. GOH and LOH mutations of control MM, mm or Mm residues in HeLa or A549 cells under acidic treatment. In each instance, GOHs outnumbered LOHs during the forward stage (red arrow), whereas LOHs outnumbered GOHs during the reverse stage (blue arrow). (***P < 0.005; **P < 0.01; *P < 0.05)

Mutations of MM, mm, and Mm residues during forward and reverse stages. The MM, mm, and Mm residues of HeLa (A) and A549 (B) cells under wogonin treatment from day14 to day18 were estimated. G1 to G6 or L1 to L6 represent gains or losses of heterozygosity respectively. LOHs or GOHs showing large lineage effects were highlighted by yellow triangles. The red and blue highlighted dates were indicative of the forward and reverse stages respectively. The figures under each of the G1-G6 mutation stage represent the mutations observed from that stage

Chromosomal position of SNV mutations during the cyclic forward and reverse mutations. A. Mutations in HeLa cells under wogonin treatment. B. Mutations in A549 cells under wogonin treatment. The red and blue arrows above each vertical panel show the dates of the forward and reverse mutations respectively, and the small color-coded rectangles are indicative of the major homozygous (MM), heterozygous (Mm), and minor homozygous (mm) sites respectively

Profiles of SNVs in the forward and reverse stages. Top row: the total SNVs displayed by MM residues of HeLa and A549 cells under wogonin and acidic treatments during the forward and reverse stages; middle and bottom rows: GOH mutations and LOH mutations are shown in terms of their mutated triplets among the 16 × 6 kinds of triplets, with the second base of each triplet representing the mutated base, respectively. The pink columns highlight the intensively mutated T > C triplets, and the blue columns highlight the intensively mutated C > T triplets. There were more C to T and T to C GOHs in the forward stage than the reverse stage, but more C to T and T to C LOHs in the reverse stage than the forward stage

Within each of the eight vertical panels, when plotted according to the replicating period of the DNA, i.e., in G1b, S1, S2, S3, S4, or G2 phase (Fig. 5), the HeLa and A549 cell lines under wogonin treatment showed more CN-losses during the forward stage on Day 16 and more CN-gains during the reverse stage on Day 18. A majority of the CNVs occurring in the forward–reverse cycle involved DNA in the late S3-G2 replication phases. The phase distribution of each day in accordance with SNV of HeLa and A549 cells under wogonin or acidic pH is shown in Supplementary Fig. 1. This was also the case when the HeLa and A549 cells were exposed to acidic pH (Figs. S1c and S1d). The human genome has been classified into three types of sequence zones: Genic, Proximal, and Distal, based on the pairwise co-localizations of forty-two genomic features [26]. Figure 6a shows the relative distribution of CNVs from 10 to 500 kb windows: there were comparable ratios of Genic zone sequence: Proximal zone sequence: Distal zone sequence in the DNA fragments devoid of CNV and found in the windows of DNAs of varied sizes (upper panel). In contrast, among the DNA fragments with CNV, the smaller 10–50 kb fragments contained proportionately more Genic zone sequence compared to Proximal zone sequence or Distal zone sequence, while in the larger 100–500 kb fragments, the proportion of Proximal zone sequence or Distal zone sequence was higher (lower panel).

Distribution of the observed CNVs among the DNA-replication phases G1b to G2. The L/G ratios indicated the ratio between CNVL and CNVG on different dates from the start of wogonin treatment of the HeLa or A549 cells. The total number of base pairs mutated included are shown on a scale of 0–4 on the y-axis. The open and hatched color bars represent CNVG and CNVL respectively, while the red and blue shaded dates represent the forward and reverse stages. Both type of cells yielded higher L/G ratios in Day 16 of the forward stage relative to the reverse stage. (***P < 0.005; **P < 0.01; *P < 0.05)

Association of CNVs in genomic feature under wogonin treatment. A. Relative association of CNVs with three types of genomic sequence zones. Upper panel shows abundance of genomic zones in sequences without CNVs, while lower panel shows sequences with CNVs, in fourteen different window sizes (10–500 kb). The abundance of sequences belonging to the three types of genomic zones, Genic (blue), Proximal (green), Distal (red) zones measure in numbers of base pairs, showing prominent increment of genic zone sequences in small windows of CNVs. Amounts of base pairs belong to the three types of genomic sequence zones in the human reference genome hg19 are shown on the right-hand side of panel A. (***P < 0.005; **P < 0.01; *P < 0.05). B. Relative enrichment of various genomic features in CNVs occurred during forward stage (Upper panel) and reverse stage (Lower panel). Genomic features are labeled in their abbreviations (refer to Table S1 for full names) and arranged in four groups based on their relative enrichment in the three types of sequence zones (Genic, Proximal, Distal) of human genome and their use as genetic makers (Marker). CNVs were captured with fourteen different window sizes arranging from 10 to 500 kb, including copy number gains or copy number losses. Relative enrichment of genomic features at different window sizes were measured against genome averages and quantified in log-2 of fold change as illustrated in red-blue thermal scale. Red color illustrates enrichment while blue color illustrates deficiency of genomic features captured in CNVs. C. Distributions of 10 kb (in red) or 500-kb (in blue) CNVs and representative histone modification sites H3k27me3 (in green) and H3k79me2 (in orange) in 10-kb windows on the region spanning from 120,000 to 121,000 kb on chromosome 1. The red dashed lines represent the genome averages of H3k27me3 (1.64) and H3k79me2 (2.46), respectively

In addition, analysis of the association between genomic features and CNVs from the forward and reverse stages in 10-kb to 500-kb sized windows [27] revealed that the enrichment of CNVGs or CNVLs in H3K79me2 was reduced relative to other histone binding sites. Among the observed changes in genomic features affected by wogonin (Fig. 6b), the evolutionarily CpG rich regions (CpGe), segmental duplications (SDP), single-nucleotide polymorphism mapped to multiple locations (SNPM), and cluster (where different kinds of genetic-variant hotspots overlap) were also found to be enriched in CNVs; notably, these enrichments were more evident in CNVLs rather than CNVGs in the CNVs, which occurred in more than 25% of samples (Figure S2). The enrichment of genomic features in CNVs under acidic pH treatment was shown in Figure S3, which was similar to the enrichment profile of wogonin treatment. The results based on recurrent CNVs from wogonin or acidic treatment are shown in Fig. S4.

During cancer development, cells typically undergo both morphological and genomic alterations. Upon exposure of HeLa and A549 cells to wogonin or acidic pH, the morphologies of both cell lines exhibited evident variations over time. For HeLa cells, the sum of cells with types 1 and 2 morphologies increased in some time-frames, while the sum of cells with types 3 and 4 morphologies decreased during the same time-frames, and vice versa. These cell type changes in combination with the SNV findings in Fig. 1 and the CNV findings in Fig. 5 suggest that the types 1 and 2 morphologies were correlated with GOH increases and CN-losses pertaining to the MM and mm residues, whereas the types 3 and 4 morphologies were correlated with their LOH changes. For A549 cells, the sum of types 1 and 2 morphologies likewise trended to vary in the opposite direction to those of types 3 and 4 morphologies (Fig. 7). Therefore, the types 1 and 2 morphologies in HeLa and A549 cells were enhanced under conditions suboptimal for cell growth, while the types 3 and 4 morphologies were enhanced under conditions optimal for cell growth.

Diversity of cell morphology. A. Different types of cell morphology of HeLa and A549 cells under wogonin treatment. B. Different types of cell morphology of HeLa and A549 cells under acidic treatment. The red arrows above the charts for HeLa and A549 cells mark the typical dates for the forward phases, while the blue arrows mark the typical dates for the reverse phases, of the cyclic SNV mutations