Fig. 6

RMC-6272 rescues early neurodevelopmental phenotypes in TSC1^−/− NPCs. A Immunoblotting for indicated proteins in TSC1^+/+ and TSC1^−/− NPCs treated with 10 nM RMC-6272 or 50 nM rapamycin (2 or 24 h, n = 3). β-tubulin serves as a loading control. Images have been cropped for clarity and conciseness and entire blots are shown in Additional file 9. B Proliferation rate for NPCs after treatment with DMSO or 10 nM RMC-6272 was quantified at Day 0 (D0, cell number at seeding), and live cell numbers were assessed at D1-4 using trypan blue exclusion. Data represent average fold change normalized to TSC1^+/+ NPCs at D0 (n = 3 ± SD). C Representative flow cytometry plots indicate Ki-67-positive populations upon 72 h treatment with DMSO, 50 nM rapamycin or 10 nM RMC-6272 for TSC1^+/+ NPCs (left) and TSC1^−/− NPCs (right). Graphs were generated by plotting forward scatter-area (FSC-A) versus FITC-area (representing cells positive for Ki-67-Alexa488). Boxed regions represent gating of Ki-67 positively stained cells with percentages shown. D Representative images are shown for immunofluorescence staining of TSC1^−/− NPCs treated with DMSO (left), 50 nM rapamycin (middle) or 10 nM RMC-6272 (right) using the neuronal marker microtubule associated protein 2 (MAP2, grayscale, top) as well as traced images generated employing HCA-Vision image quantitation software (bottom), which traces the cell body along branching layers of the neurite arborization. Scale bar = 100 µm. E Quantitation of neurite number (left), length (middle) and extremities (right) in TSC1^−/− NPCs treated with 10 nM RMC-6272 (RMC) or 50 nM rapamycin (Rap) for 48 h are shown. Data represent analysis of 8 non-overlapping field images/treatment generated using GraphPad Prism9 with relative fold change normalized to DMSO-treated NPCs (mean, ± SD). *p < 0.05, **p < 0.01, ***p < 0.001, ns not significant, p values were calculated using Student’s t test (B, E)