Additional file 1 of Dietary-derived vitamin B12 protects Caenorhabditis elegans from thiol-reducing agents

Additional file 1: Figure S1. B12 supplementation alleviates DTT toxicity in a mmcm-1 mutant but not metr-1 mutant. Embryos of (A-C) wild type (N2), (D-F) metr-1(ok521), (G-I) mmcm-1(ok1637), and (J-L) rips-1(ij109) were added to plates supplemented with 0 (top row) or 5 mM DTT (middle and bottom rows), in the presence (bottom row) or absence (top and middle rows) of 64 nM vitamin B12. Development to adult stage was assessed 4 days later and representative images are shown in panels (A-L). Scale bars denote 1 mm. The number of animals used in this experiment are as follows: (A) (n = 118), (B) (n = 136), (C) (n = 73); (D) (n = 197), (E) (n = 147), (F) (n = 197); (G) (n = 59), (H) (n = 93), (I) (n = 56); (J) (n = 109), (K) (n = 62), (L) (n = 94). (M) Plotted development to adult stage in percentage under the above treatments. p-values were determined from Fisher’s exact test. NS not significant, *** p < 0.001. For all panels, purple significance marks indicate comparison of mutant worm strains to N2 wild type for each treatment group and blue significance marks indicate comparison of treatment groups (i.e., DTT or DTT+B12) to no DTT groups for each worm strain. Figure S2. DTT resistance mutants map to a single SAM methyltransferase gene. (A-B) HA mapping output from DTT resistance screen for (A) rips-1 allele ij109 (strain TP193) and (B) rips-1 allele ka14 (strain TP251). Clear peak visible on Chromosome V. (C) Protein sequence of RIPS-1 SAM methyltransferase highlighting location of mutation generated via EMS DTT resistance screen. Underlined residues exon/exon junctions and position and nature of mutation highlighted in colour and allele designation in brackets. Location of mutation relative to methyltransferase domain highlighted in cartoon (Pfam (PF13847) Methyltransf_31 residues 176-285; InterPro domain (IPR025714) Methyltranfer_dom residues 176-285. Figure S3. The loss of rips-1 causes DTT resistance phenotype. (A) High degree of identity between rips-1 (R08E5.3) and its closest homologue R08E5.1 that will account for potential RNAi cross-reaction. (B) N2 wild type [a], rips-1(ij109) mutant [TP193] [b], or wild type worms fed on E. coli expressing RNAi targeting rips-1 (R08E5.3) [c] or its homologues (R08E5.1 [d], R08F11.4 [e], or K12D9.1 [f]) was treated with 5 mM DTT for confirmation of causative gene for DTT resistance phenotype. Worms harbouring rips-1(ij109) allele or fed on rips-1 RNAi or R08E5.1 RNAi survived and reached adult stage on DTT while