Supplementary Figure 2: Mettl14 regulates self-renewal of cortical NSCs in vitro

(a) Western blots showing Mettl14 depletion in Mettl14 KO NSCs. Similar results were obtained from three independent experiments. For uncropped images, see Supplementary Fig. 6b. (b) Growth curve of cortical NSCs isolated from E17.5 brain. Two-way ANOVA (n = 3 cell cultures for all experimental groups; P = 3E-15, F (2, 18) = 357.5) followed by Bonferroni's post hoc test (WT vs. KO, P = 2.5E-14, 95% C.I. = 0.09616 to 0.1196, WT vs. Het, P = 0.4346, 95% C.I. = −0.01786 to 0.005595). (c,d) Assessment of apoptosis in Mettl14 KO and nondeleted control NSCs. The number of apoptotic cells was determined by FACS analysis via Annexin V-FITC and PI-staining. Representative results are shown in (c) and results from 3 independent experiments are summarized in (d). One-way ANOVA (n = 3 cell cultures for all experimental groups; P = 0.6882, F (2, 6) = 0.3979) followed by Bonferroni's post hoc test (WT vs. KO, P = 0.8321, 95% C.I. = −1.999 to 3.666, WT vs. Het, P = 0.9999, 95% C.I. = −2.266 to 3.399). (e) Quantification of TUNEL assays in Mettl14 KO and control NSCs. One-way ANOVA (n = 3 fields for all experimental groups; P = 0.7572, F (2, 6) = 0.2915) followed by Bonferroni's post hoc test (WT vs. KO, P = 0.9999, 95% C.I. = −50.16 to 32.35, WT vs. Het, P = 0.9999, 95% C.I. = −40.71 to 41.81). (f) Western blots showing expression of Flag-tagged Mettl14 in WT and Mettl14 KO NSCs transduced by lentivirus containing empty or Flag-Mettl14 vectors. Similar results were obtained from three independent experiments. For uncropped images, see Supplementary Fig. 6c. (g) m⁶A dot-blots of Ribo- polyA RNAs isolated from Mettl14 KO and nondeleted NSCs transduced with lentivirus containing empty or Flag-Mettl14 vectors. Similar results were obtained from three independent experiments. (h) RT-qPCR of Alkbh5 transcripts in NSCs expressing scramble (scr) shRNA or shRNAs against Alkbh5, one-way ANOVA (n = 3 independent experiments for all experimental groups; P = 7.511E-08, F (2, 6) = 708) followed by Bonferroni's post hoc test (Scr vs. shAlkbh5-1, P = 1.06113E-07, 95% C.I. = 0.8453 to 1.013, Scr vs. shAlkbh5-2, P = 1.164E-07, 95% C.I. = 0.831 to 0.9991). (i) Western blots showing Alkbh5 depletion in NSCs expressing scramble (scr) shRNA or shRNAs against Alkbh5. Similar results were obtained from two independent experiments. For uncropped images, see Supplementary Fig. 6d. (j) Growth curve of NSCs expressing scr shRNA or shRNAs against Alkbh5. Two-way ANOVA (n = 4 cell cultures for all experimental groups; P = 0.0626, F (2, 27) = 3.075) followed by Bonferroni's post hoc test (Scr vs. shAlkbh5-1, P = 0.0928, 95% C.I. = −0.2273 to 3.548, Scr vs. shAlkbh5-2, P = 0.0726, 95% C.I. = −0.1353 to 3.64). (k) RT-qPCR of Fto transcripts in NSCs expressing scramble (scr) shRNA or shRNAs against Fto, one-way ANOVA (n = 3 independent experiments for all experimental groups; P = 1.324E-06, F (2, 6) = 270.2) followed by Bonferroni's post hoc test (Scr vs. shFto-1, P = 2.396E-06, 95% C.I. = 0.6521 to 0.887, Scr vs. shFto-2, P = 1.629E-06, 95% C.I. = 0.6521 to 0.887). (l) Western blots showing Fto depletion in NSCs expressing scramble (scr) shRNA or shRNAs against Fto. Similar results were obtained from two independent experiments. For uncropped images, see Supplementary Fig. 6e. (m) Growth curve of NSCs expressing scr shRNA or shRNAs against Fto. Two-way ANOVA (n = 4 cell cultures for all experimental groups; P = 0.0005, F (2, 27) = 10.1) followed by Bonferroni's post hoc test (Scr vs. shFto-1, P = 0.0538, 95% C.I. = −1.604 to 0.01121, Scr vs. shFto, P = 0.0809, 95% C.I. = −0.07525 to 1.54). Graphs represent mean ± SD. Dots represent data from individual data points. ns = non-significant. **** P < 0.0001.