anti isl1 2  (Developmental Studies Hybridoma Bank)

Journal: Communications Biology

Article Title: Metabolic costs and trade-offs of hypermetabolism in human motor neurons with ATP synthase deficiency

doi: 10.1038/s42003-025-09149-7

Figure Lengend Snippet: A Schematic cartoon illustrating the experimental design. Patient skin cells with approximately 30% heteroplasmy were previously reprogrammed into iPSC, resulting in clones with varying levels of heteroplasmy . We selected two control lines (0% heteroplasmy) and two lines with ~50% heteroplasmy for differentiation into motor neurons. Created in BioRender. Torregrosa, R. (2025), https://BioRender.com/rijw3bk . B Representative cropped immunoblot of Complex V assembly determined by Blue-Native PAGE. Complex V (CV) was detected using anti-ATP5FA1, targeting the F1 subunit (facing the matrix) of ATP synthase; and Complex II (CII) was detected using anti-SDHA, which served as the loading control. n = 2 technical replicates (independent wells), sc subcomplexes. C iPSC-derived motor neurons on day 30. Upper panels, representative bright field microscope images showing similar neural morphologies, scale bar 200 µm. Lower panels, representative immunocytochemistry images of neuronal markers MAP2 (green), Hb9 (red), TUJ1 (green), ISL1/2 (red) and nucleus (Dapi, blue). Scale bar 50 µm. D , E Differentiation efficiency measured as the % of ISL1/2- or HB9-positive nuclei relative to DAPI positive cells from Fig. 1C. Data represent the mean of ~1000 cells quantified from 15 images taken across three independent frames ( n = 3), obtained from two separate differentiation experiments. D % of ISL1/2-positive nuclei relative to DAPI positive cells from Fig. 1C. E % Hb9-positive nuclei relative to DAPI positive cells from Fig. 1C. F Basal mitochondrial respiration determined before oligomycin injection (Seahorse XF Mito Stress test). G Proton leak was calculated as the remaining mitochondria respiration rate after inhibition of complex I and II by rotenone and antimycin (Seahorse XF Mito Stress test). H % Coupling efficiency was determined as the fraction of basal ATP-linked OCR/basal OCR and indicates the proportion of O 2 consumed to fuel ATP synthesis (Seahorse XF Mito Stress test). Expressed as %. For ( F – H ) n = 13–21 wells per cell line per experiment, from three independent differentiations. Data was normalized to total DNA per well. I Glycolytic rate (glycoPER) determined after subtracting mitochondrial acidification from total proton efflux rate (Seahorse XF Glycolytic Rate Assay). n = 9–11 wells per cell line per experiment, from two independent experiments. Seahorse data was normalized to total DNA per well. J Total ATP production rate (ATP-OxPhos + ATP-glycolysis) computed from the basal OCR and ECAR (Seahorse XF Mito Stress test). K Mitochondrial membrane potential based on the intensity of TMRM (membrane potential) normalized to Mitotracker (mitochondrial mass). Each dot represents an independent motor neuron differentiation experiment (n_exp = 3 independent differentiations) with an average of 2 to 11 wells per cell line per experiment. For all graphs, data shown as mean ± standard deviation, * p < 0.05, ** p < 0.01, *** p < 0.001, or ns non-significant by One-Way ANOVA followed by Bonferroni´s (Seahorse XF) or Tukey´s (membrane potential) multiple comparison post-hoc test.

Article Snippet: Cells were blocked with 5% protease-free BSA (Biowest # P6154) in 0.1% Tween20 (Sigma, #P1379) in PBS-T for 2 h in RT, and then were incubated overnight at +4 °C in blocking buffer containing 5% bovine serum and primary antibodies: HB9 (DSHB #81.5C10-s, 1:50), ISL1 (DSHB #39.4D5-s, 1:50), MAP2 (Abcam #5392, 1:1000), TUJ1 (Biolegend #801201, 1:1000), total H3 (Cell Signaling #4499, 1:200) and PanKAc H3 (Abcam #ab47915, 1:400).

Techniques: Clone Assay, Control, Western Blot, Blue Native PAGE, Derivative Assay, Microscopy, Immunocytochemistry, Injection, Inhibition, Membrane, Standard Deviation, Comparison