Prism 9

Data were expressed as the mean ± SEM. Parametric and non-parametric quantitative variables were compared using the Student’s t-test and the Mann–Whitney U test, respectively. The least significant difference (LSD) method in one-way ANOVA was used for pairwise comparisons between different groups. P-values < 0.05 were statistically significant. All figures were generated using GraphPad Prism 9.0 and Adobe Illustrator CC 2015.

Data are expressed as the mean ± SEM. Parametric and non-parametric quantitative variables were compared using the Student t test and Mann–Whitney U-test, respectively. The least significant difference (LSD) method in one-way ANOVA was used for pairwise comparisons between different groups. Statistical significance was set at P < .05. All figures were generated using GraphPad Prism 9.0 and Adobe Illustrator CC 2015.

Statistical analysis was performed on qRT-PCR data using SPSS 22.0. The data were analyzed using a t-test and one-way ANOVA method, and the results were expressed as mean ± standard deviation (X ± SD). Statistical charts were drawn using GraphPad Prism 9.0 and Adobe Illustrator. p < 0.05 indicated significant differences, while p < 0.01 indicated extremely significant differences. The sequencing data were organized and summarized using an Excel spreadsheet, followed by calculation of the genotype frequencies of different mutation sites using the Excel spreadsheet, including expected heterozygosity (He), effective allele number (Ne), polymorphism information content (PIC), and testing whether they were in a Hardy Weinberg equilibrium state.

Comparative core genome analysis at branch points on the phylogenetic tree was performed to examine the differences in genetic characteristics among each subgroup. Increased and decreased core gene families at each branch point were extracted and annotated by the KEGG database (https://www.kegg.jp/); the following two different classification methods counted results: “gene function” and “pathway” and are shown in a bar chart. Based on the gene data above, relative enrichment ratio (RER) was introduced to describe the correlation between gene enrichment and pathway based on the following formula (shown in a line chart): RER = (number of genes annotated in this pathway) × 100/(total number of genes in this pathway); RER results were screened with 8 as the threshold (maximum number of peaks that can be retained), and the pathway corresponding to each peak was extracted. All figures were made by GraphPad Prism 9.0, R packages, and Adobe Illustrator CS6.

Samples were treated with CCCP or BX795 at different time points as independent biological samples. Statistical tests between two independent datasets were done by Student’s t-test, each data point within a dataset is from an independent culture well or cell (Figs.1c–f, h, 1m; j, l, 2c, f, 3a–d, f–i, 4b–d, f–i, 5b, 6b, e, h–l, Supplementary Figs. 3b, 4b–c). We used t-test rather than ANOVA because we did not want to assume that each group has the same variance. For non-normal data distribution, we performed Mann–Whitney U test to compare between two independent data sets (Fig. 5c–e, Supplementary Fig. 5b). Graphs were made using GraphPad Prism 9.0 software and figures were made using Adobe Illustrator.

The comparative core-genomic and pan-genomic analysis was conducted to investigate the genetic characteristics among different groups. Increased and decreased core-gene families were extracted and annotated by the KEGG database (database resolution through Python 3.8, based on public information from the KEGG database).Two different classification methods counted results of “Gene function” and “Pathway” and shown in a bar chart. Based on gene data above, Relative Enrichment Ratio (RER) was introduced to describe the correlation between gene enrichment and pathway (Xu and Yuan, 2022 (link)). The RER was calculated using the formula: RER = (number of genes annotated in this pathway)/(total number of genes involved in this pathway) and shown in a line chart; the RER results were screened by 0.1 and 0.2 as the threshold (maximum number of peaks can be retained), and the pathway corresponding to each peak was extracted. All figures were made by GraphPad prism 9.0, R packages, and Adobe Illustrator CS6.