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. 2023 Oct 27;14:6774. doi: 10.1038/s41467-023-42342-y

Fig. 5. UBR-dependent NatC knockout phenotypes include abnormal mitochondrial morphology, increased lysosomal content and cell granularity.

Fig. 5

a NatC KO cells have abnormal mitochondrial morphology. HAP1 cells were stained with anti-COX IV and analyzed by immunofluorescence (IF). Cells were grouped into four bins based on mitochondrial morphology: normal, fragmented, elongated, and elongated + fragmented (n = 100 per cell line). b NAA30-KO and NAA35-KO cells have increased lysosomal content. Lysosomes were stained with LysoView 488 and analyzed by flow cytometry. Median FITC values were normalized to cell size (FSC-A) and expressed relative to WT sample. Data are shown as mean ± SD (n = 3 independent experiments). ***p = 0.001, ****p < 0.0001; one-way ANOVA with Dunnett’s correction. c NatC KO cells display increased granularity. Median side scatter area (SSC-A) indicating cell granularity or internal complexity was determined by flow cytometry. Data are shown as mean ± SD (n = 3 independent experiments). **p = 0.0069, ****p < 0.0001; one-way ANOVA with Dunnett’s correction. d NatC regulates protein neddylation. Immunoblot analysis of neddylation pathway components using total cell extract (n = 3 biologically independent samples). All proteins have NatA-type N-termini, except CUL4B which has a NatC/E-type N-terminus (MM-). The upper cullin band represents the neddylated form and * indicates saturated bands. e Increased p62/SQSTM1 levels in NatC KO cells. HAP1 cells were stained with anti-p62 and analyzed by IF. Scale bar, 10 μm. (see Supplementary Fig. 10 for quantification): f NatC affects components of the autophagy pathway. Immunoblot analysis of autophagy markers using the indicated HAP1 cell extracts. EEA1 (ML-) has a NatC-type N-terminus while BCL2, p62 and LC3B has NatA-type N-termini (n = 3 biologically independent samples). g–j HAP1 WT and NAA30-KO cells were transfected with siCtrl or siUBR1/UBR2/UBR4 for 72 h (n = 3 independent experiments). g Immunoblot analysis of endogenous p62 and CUL5 protein levels. Arrowhead indicates NEDD8-CUL5. h Mitochondrial morphology was assessed by IF like in a. i Lysosome levels and j cell granularity (SSC-A) was determined by flow cytometry as in b and c, respectively (f, g). i, j Data are shown as mean ± SD (n = 3 independent experiments). *p = 0.0228, **p = 0.0051, ****p < 0.0001; one-way ANOVA with Šídák’s correction. ns not significant. Source data are provided as a Source Data file.