Extended Data Fig. 8 |. Propionate and vitamin B₁₂ supplementation prevent PDIM loss in Mtb.

a, Schematic overview of in vitro evolution experiments. Triplicate inkwells containing standard 7H9/OADC/glycerol/tyloxapol or media supplemented with propionate or vitamin B₁₂ were inoculated with frozen Mtb culture stock (P0) and incubated for 7-10 days (P1). Cultures were then diluted into fresh media every 7 days for serial passage (P2 to PX). Selected passages were input into VAN10-P assays at the time of passage to assess PDIM production over the course of the experiment. For TLC lipid analysis, frozen stocks were first outgrown in media without propionate or vitamin B₁₂ for a single passage to allow the strains to recover before ¹⁴C-labelling. Figure created with BioRender.com. b, TLC lipid analysis of H37Rv-B before and after six serial passages in ± 0.1 or 1.0 mM propionate. This figure shows the full TLC plate from Fig. 4a with results for both biological replicates analysed by TLC (R2 is shown in Fig. 4a). c, VAN10-P assays for H37Rv-SC [PDIM(+) H37Rv wildtype] passaged in ± 0.1 mM propionate. d, H37Rv-A and e, H37Rv-B passaged in ± 7.4 μM vitamin B₁₂. Mean ± SD for n = 3 biological replicates, each assayed in triplicate. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; two-way ANOVA with šidák’s (c) or Tukey’s (d,e) multiple comparison test. Significant differences between conditions are indicated in (c) and between timepoints in (d,e).