Fig. 4: NAA60 knockdown but not NAA60 knockout cells have Golgi fragmentation. | Nature Communications

Fig. 4: NAA60 knockdown but not NAA60 knockout cells have Golgi fragmentation.

From: Biallelic NAA60 variants with impaired N-terminal acetylation capacity cause autosomal recessive primary familial brain calcifications

Fig. 4

a NAA60-FLAG-positive compartments localized close to compartments stained by the cis-Golgi marker GM130 but did not perfectly colocalize. HeLa cells were transfected with FLAG-NAA60, fixed, and stained with anti-FLAG and anti-GM130 followed by imaging using an OMX 3D-SIM microscope. Images are shown as z-stack max intensity projection and are representative of <100 cells inspected. b Golgi fragmentation was not observed in fibroblast cells derived from Family 1. Cells were fixed and stained for the cis-Golgi marker GM130 and with DAPI for visualization of the nucleus and phalloidin for F-actin. Imaging was performed with a Leica SP8 using the Lightning module for deconvolution and optimized resolution. Representative of at least three independent experiments. The scale bar is 20 µm for all except for the magnified frames. c Schematic overview of the predicted protein resulting from the frameshift introduced in HAP1 NAA60 KO cells. Compare to the PFBC variants shown in Fig. 2a. d Golgi fragmentation was not observed in HAP1 NAA60 knockout cells. Ploidy-controlled HAP1 CTRL, NAA60 knockout, and NAA80 knockout cells were fixed and stained for the cis-Golgi marker GM130 and with DAPI for visualization of the nucleus. NAA80 knockout was used as a positive control for Golgi fragmentation in the HAP1 cell line. The scale bar is 25 µm for all images on top and 5 µm for all images at the bottom. e Dermal fibroblast healthy control cells were transfected with shNAA60+RFP plasmid 72 h prior to fixation. Images were acquired with a Leica TCS SP8 confocal microscope at 100x using a zoom factor of 0.75 and a z-step size of 0.25 μm. The scale bar is 5 µm for both images shown. The degree of Golgi intactness was evaluated by microscopy using a Leica DMI6000 B wide-field fluorescence microscope with an HCX PL APO 100 × 1.4 NA oil objective. Images are representative of at least 100 inspected cells (HAP1) and at least 30 transfection-positive dermal fibroblast cells where near all transfected cells had the phenotype.

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