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. 2021 Dec 8;21(1):e13518. doi: 10.1111/acel.13518

FIGURE 7.

FIGURE 7

Expression of Phosphoethanolamine methyltransferase pmt‐2 is transcriptionally downregulated in flr‐4(n2259) on high Vitamin B12 diet. (a) An abridged representation of the regulation of one‐carbon cycle highlighting the B12 mechanisms I and II, as described by the Walhout lab (Giese et al., 2020). Lower vitamin B12 levels in OP50 would lead to increased expression of methionine/SAM cycle genes. We hypothesized that this will also increase the expression of pmt‐1 and/or pmt‐2 (grey box). (b) Representative experiment to show that the expression of flr‐4(−);Pcyp‐35B1::gfp is increased on pmt‐1 or pmt‐2 (OP50) RNAi. Quantification provided in Figure S7a,b. (c) RT‐PCR showing the expression levels of pmt‐1 and pmt‐2. The expression of pmt‐2 is sensitive to vitamin B12 levels. Average of three biological replicates ± SEM. Unpaired two‐tailed t‐test. ns, non‐significant. (d,e) The osmotic stress tolerance of flr‐4(−) grown on control (HT115) RNAi is higher compared to control (OP50) RNAi. However, knocking down (d) pmt‐1 or (e) pmt‐2 using (OP50) RNAi leads to better osmotic stress tolerance of the mutant even on the OP50 feed. (f,g) The life span of flr‐4(−) grown on control (HT115) RNAi is higher compared to control (OP50) RNAi. However, knocking down (d) pmt‐1 or (e) pmt‐2 using (OP50) RNAi leads to increased life span of the mutant even on the OP50 feed. (h) Representative western blot of wild‐type and flr‐4(−) worms grown on control (OP50) RNAi or pmt‐1 (OP50) RNAi and probed with anti‐phospho‐PMK‐1 (P‐PMK‐1), anti‐PMK‐1 or anti‐Actin antibodies. (i) Representative western blot of wild‐type and flr‐4(−) worms grown on control (OP50) RNAi or pmt‐2 (OP50) RNAi and probed with anti‐phospho‐PMK‐1 (P‐PMK‐1), anti‐PMK‐1 or anti‐Actin antibodies. One of three biologically independent replicates shown for western blots, gfp, osmo‐tolerance and life span experiments. All experiments were performed at 20°C. Life span summary is provided in Table S1. Summary of osmotic tolerance assay is provided in Table S2. Source data is provided as a source data file