Fig. 8: Enhancing global protein synthesis suppresses cell survival and developmental defects within AIRIMV190G organoids.
From: A programmed decline in ribosome levels governs human early neurodevelopment
a, Quantification of bright-field images at day 15. V190G TSC1+/− neural tissue is enlarged relative to V190G TSC1+/+; **P < 0.01; ****P < 0.0001; Dunnett’s multiple comparison, unpaired two-sided t-test with Welch’s correction. WT TSC1+/+, n = 11; V190G TSC1+/+, n = 12; WT TSC1+/−, n = 37; V190G TSC1+/−, n = 35, from two independent batches; P = 1.734489 × 10−6 (TSC1+/+ WT versus TSC1+/+ V190G); P = 4.751388 × 10−6 (TSC1+/+ V190G versus TSC1 +/− V190G); and P = 0.9354378 (TSC1+/− WT versus TSC1+/− V190G). Error bars show s.e.m. b, Representative images showing the TUNEL signal in control TSC1+/+, V190G TSC1+/+, control TSC1+/− and V190G TSC1+/− organoids at day 15. Scale bar, 50 μm. c, Quantification of TUNEL signal of individual NE bud. TUNEL counts were normalized to the area of the imaged bud. ****P < 0.0001, unpaired, two-sided t-test with Welch’s correction. WT TSC1+/+, n = 21; V190G TSC1+/+, n = 22; WT TSC1+/−, n = 17; V190G TSC1+/−, n = 20 NE buds from two independent batches; P = 2.310684 × 10−7 (TSC1+/+ V190G versus TSC1+/− V190G); and P = 0.1644 (TSC1+/− WT versus TSC1 +/− V190G). Error bars show s.e.m. d, IF image of day 15 control (WT TSC1+/−) and mutant (V190G TSC1+/−) organoids of transitioning NE marker ZEB2 and committed RG marker BLBP. Scale bar, 50 μm. Two independent batch were performed. e, IF image of day 15 control (WT TSC1+/−) and mutant (V190G TSC1+/−) organoids of VIM (green) and mitochondrial matrix HSP60 (red). Scale bar, 20 μm. Two independent batch were performed. f, Quantification of size of day 15 mutant (V190G) organoids treated with vehicle (DMSO) or 1 μM PI3Kα activator UCL-TRO-1938. *P < 0.05. Unpaired two-sided t-test with Welch’s correction. WT + DMSO, n = 7; V190G + DMSO, n = 12; V190G + 1938 1 μM, n = 11, organoids from two independent batches; P = 0.037447. Error bars show s.e.m. g, Quantification of size of day 15 mutant (R72W) organoids treated with vehicle (DMSO) or 1 μM or 2 µM PI3Kα activator UCL-TRO-1938. *P = 0.0373, **P = 0.0015, ****P = 8.67138 × 10−7. Dunnett’s multiple comparison, unpaired t-test with Welch’s correction (two-sided). Control + DMSO, n = 7; R72W + DMSO, n = 6; V190G + 1938 1 μM, n = 6; V190G + 1938 2 μM, n = 6 organoids from two independent batches. Error bars show s.e.m. h, Quantification of TUNEL signal of individual NE bud. TUNEL counts were normalized to the area of the imaged bud. ***P < 0.001, Dunnett’s T3 multiple comparisons test (two-sided). WT + DMSO, n = 9; V190G + DMSO, n = 11; WT + 1938, n = 7; V190G + 1938, n = 13 from two independent batches. P = 0.0008 (V190G + DMSO versus V190G + 1938). Error bars show s.e.m. i, Representative IF images of day 15 control and mutant (V190G, 1938 1 μM and 1938 2 μM) organoids of BLBP (red). Scale bar, 20 μm. j, Quantification of mean BLBP IF intensity of day 15 control and mutant (V190G, 1938 1 μM and 1938 2 μM) organoids. ****V190G versus control, P = 1.60703 × 10−5; ****V190G versus 1938 1 μM, P = 1.39136 × 10−6; ****V190G versus 1938 2 μM, P = 2.35979 × 10−7. Dunnett’s multiple comparison unpaired t-test with Welch’s correction (two-sided). Control + DMSO, n = 6; V190G + DMSO, n = 6; control + 1938, n = 6; V190G + 1938, n = 6 imaged regions from two independent batches, error bars are s.e.m. k, Representative IF images of day 15 control and mutant (R72W, 1938 1 μM and 1938 2 μM) organoids of BLBP (red). Scale bar, 20 μm. l, Quantification of mean BLBP immunofluorescence intensity of day 15 control and mutant (R72W, 1938 1 μM and 1938 2 μM) organoids. **R72W versus 1938 1 μM, P = 0.0027; **R72W versus 1938 2 μM, P = 0.0037; ****R72W versus control, P = 1.49216 × 10−6. Dunnett’s multiple comparison, unpaired two-sided t-test with Welch’s correction. Control + DMSO, n = 6; R72W + DMSO, n = 6; R72W + 1938, n = 5; R72W + 1938, n = 6 imaged regions from two independent batches. Error bars show s.e.m. m, IF images of day 15 control (V190G and DMSO 0.1%) and treated (V190G, UCL-TRO-1938 1 μM) organoids stained for VIM (green) and mitochondrial matrix HSP60 (red). Scale bar, 20 μm. n, Quantification of the nuclear/ cytoplasm ratio of RSL24D1 IF of control + DMSO, AIRIMV190G + DMSO, AIRIMV190G + 1938 2 μM, AIRIMV190G TSC1+/− and control + DMSO, AIRIMR72W + DMSO, AIRIMR72W + 1938 2 μM day 15 cerebral organoids. ****AIRIMV190G versus control, P = 1.26963 × 10−5; *AIRIMV190G versus 1938 2 μM, P = 0.024; **AIRIMV190G versus AIRIMV190G TSC1+/−, P = 0.0018; ****AIRIMR72W versus control, P = 1.0138 × 10−5; *AIRIMR72W versus 1938 2 μM, P = 0.0491. Dunnett’s multiple comparison, unpaired two-sided t-test with Welch’s correction. Control (V190G + DMSO), n = 12; V190G + DMSO, n = 2; V190G + 1938 2 μM, n = 6; V190G TSC1+/−, n = 12; control (R72W + DMSO), n = 8; R72W + DMSO, n = 11; V190G + 1938 2 μM, n = 12 organoids from two independent batches. Error bars show s.e.m. o, Model describing a potential explanation for the observed suppression of AIRIMV190G phenotypes by UCL-TRO-1938.
