Fig 1: In vitro expression of NGLY1 in neuronal cultures and reduction of GNA in NGLY1-/- HEK293 cells after GS-100 transduction(A) In vitro expression of NGLY1 protein in rat primary neurons following GS-100 transduction. Rat primary neurons were transduced with GS-100 or AAV9-GFP control at 40,000 or 80,000 multiplicity of infection (MOI) 5 days after plating. The neurons were lysed 5 days (day 10 of the experiment) or 10 days (day 15 of the experiment) after transduction, and human NGLY1 protein was probed by western blot. GAPDH was used as the loading control. (B) NGLY1-/- ReNcell VM neurons were transduced with GS-100 at 0, 200,000, or 400,000 MOI 5 days after differentiation. Cells were fixed and probed for NGLY1 (red) or MAP2 (green) expression using immunofluorescence 7 days after transduction. Cell nuclei were stained with Hoechst 33342 blue dye. (C) Reduction of GNA in NGLY1-/- HEK293 cells after GS-100 transduction. NGLY1-/- HEK293 cells were transduced with GS-100 at 300,000 MOI and cell lysates were harvested 72 h after transduction. GNA levels in the lysate were measured and normalized to the number of cells. Each data point represents an experiment and error bars show the standard deviation. AAV9, adeno-associated virus 9; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GFP, green fluorescent protein; GNA, GlcNAc-Asn; KO, knockout; MAP2, microtubule-associated protein 2; MOI, multiplicity of infection; NGLY1, N-glycanase 1.
Fig 2: Muscle structure morphology. Representation of ngly1 (+/+) (A,B) and ngly1 (-/-) (C,D) Phalloidin staining for actin (A-X4 and B-X20). Af = Actin filaments. (E) Quantification analysis of the Phalloidin fluorescence. t-test (n = 9 in each group, **p < 0.01). (F) Mean size of somite (µm). t-test (ngly1 (+/+), n = 65. ngly1 (-/-), n = 79, ***p < 0.001). Scale bars—50 µm.
Fig 3: A model for the generation of GNA in NGLY1 deficiency. (A) Normal degradation of misfolded proteins through the ERAD pathway. (B) NGLY1 deficiency leads to generation and accumulation of GNA. N = Asn; D = Asp
Fig 4: Characterization of NGLY1-deficient K562 lines. (A) Western blot analysis of K562 cell lines used in this paper. (B) Flow readout and gating for the analysis of K562 cell lines used in this paper. Data were used to calculate geometric means for the Venus to mCherry ratio. (C) Average geometric mean of the Venus to mCherry signal for all lines used in the paper. (D) Dose response of NGLY1-deficient K562 lines to Bortezomib. 95% confidence interval shown as shading on the graph.
Fig 5: Drug treatment of NGLY1-deficient and WT K562 cell lines. (A) Treatment of WT K562 cells with 48 compounds, plotted by assay and concentration to visualize compounds that were not toxic but still inhibited ddVenus fluorescence. Each point represents a single concentration and are labelled by compound if they decreased ATPlite signal by more than 50% or if they reduced ddVenus signal to the level of the NGLY1 KD line control. (B) Dose response curve for NVP-BEZ235 and PAC-1 treatment of WT and KD NGLY1 K562 cells, exemplative of a positively confirmed hit from the screen. (C) ATP measurement of the dose response curve for NVP-BEZ235 and PAC-1 treatment of WT and KD NGLY1 K562 cells. (D) Western blot analysis of RTA?-V5 expression levels after NVP-BEZ235 and PAC-1 treatment of WT and KD NGLY1 K562 cells for 5 hr and 24 hr treatment at 15 µM for PAC-1 and 0.5 µM for NVP-BEZ235. Exemplative blot from 3 repeated experiments. (E) Western blot analysis of autophagic flux due to NVP-BEZ235 and PAC-1. Time course treatment of WT K562 cells in the presence of compound. (F) Fluorescent signal of Venus and ddVenus at multiple time points in WT and KD lines.
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