Fig 1: C9ORF72 localizes to lysosomes.(A) Immunofluorescence of endogenous C9ORF72 stained with GTX632041 in U2OS cells expressing LAMP1-RFP. The large images on the left show a maximum projection to highlight intracellular structures. Insets 1 and 2 are single focal plane, higher magnification images of the boxed regions. Merge and grayscale images of C9ORF72 and LAMP1-RFP are shown. C9ORF72 localization was observed under basal conditions (top panels) or under starvation for 2 hr (bottom panels). Cells are outlined with white dashed lines. Scale bars = 20 µm for large images and 2 µm for insets. (B) Quantification of LAMP1 structures positive for C9ORF72 in basal growing condition (fed) or 2 hr starved condition (starved) in U2OS cells. Between 228–262 lysosomes were counted from at least nine different cells from three independent experiments. A Student’s t-test reveals that the percentage of LAMP1 structures positive for C9ORF72 is not significantly different between the fed and starved conditions (C) Lysates were prepared from HEK-293 cells expressing the Tmem192-3xHA (HA-Lyso cells) or the Tmem192-2xFlag (Control-Lyso cells). Lysosomes were immunoprecipitated using anti-HA magnetic beads. Quantitative immunoblotting for the indicated proteins was performed for starting material (SM), purified lysosomes and control immunoprecipitates (anti-HA IP). C9ORF72 was detected using GTX634482.
Fig 2: HRE + C9ORF72 KO Neurons Show Increased Apoptosis without Increasing DPR Protein Levels(A) Immunostaining for the indicated markers. Images were segmented using CellProfiler to quantify cleaved caspase-3 (CC3), a marker of apoptosis. Scale bar, 50 µm.(B) C9 + KO MNs show more apoptosis compared with gene corrected C9GC (n = 3 biological replicates).(C) Quantification of endogenous poly-GP DPR levels using an ELISA assay (n = 7 biological replicates).(D) Immunostaining for the indicated markers, including MAP2 and poly-GP DPR proteins. Poly-GP peptides could be detected in the cytoplasm of C9 and C9 + KO MNs (indicated by arrows) but not in gene corrected controls. Isogenic cell lines set 1 (left) and set 2 (right). Scale bars, 10 µm.All values are presented as mean ± SEM. One-way ANOVA followed by Tukey's post-test for multiple comparisons was performed (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).
Fig 3: Generation of Isogenic iPSC Lines(A) Strategy for targeting C9ORF72 to knockout C9ORF72 protein production in WT iPSCs (wtKO). Quadruple Cas9-nickase (Cas9n) introduced two double-strand breaks (yellow arrows).(B) PCR confirmed the deletion in KO iPSC lines.(C) Strategy for gene correction by reducing HRE to WT length of three repeats.(D) Repeat-primed PCR confirmed absence of HRE in C9GC lines.(E) Scheme of KO deletion in iPSCs with HRE in C9ORF72 (C9 + KO).(F) Capillary electrophoresis confirmed loss of C9ORF72 protein in KO cells. Note that no significant differences between C9-1 and C9-1 in comparison with WT were not significant. N = 4 biological replicates. All values are presented as mean ± SEM. One-way ANOVA showed p < 0.05. Tukey's post-test for multiple comparisons was performed (**p < 0.01, ***p < 0.001).See also Figures S1–S3.
Fig 4: Macrophages express C9ORF72 at high levels.(A) Lysates were prepared from rat cortical neurons, rat hippocampal neurons, mouse bone marrow-derived macrophages (BMDMs) and mouse whole brain, and processed for quantitative immunoblot using antibody GTX634482 with the LI-COR Odyssey Imaging System that utilizes fluorescent secondary antibodies to allow for quantitative blots. The total protein stained transfers are shown as a loading control. The C9ORF72 protein signal as a ratio to total protein was determined, normalized to parental HEK-293 cells (red), and presented as fold change (blue numbers). C9ORF72 KO mouse brain (KO) was included as a specificity control. (B) Lysates were prepared from human monocyte-derived macrophages (MDMs) treated with either IFN-? and LPS or with TGF-ß. Immunoblot and C9ORF72 quantification were performed as in (A).
Fig 5: C9orf72 immunoreactivity in the hippocampus of Alzheimer's disease brains. Expression of chromosome 9 open reading frame 72 (C9orf72) studied in the hippocampus of Alzheimer's disease brains by immunohistochemistry using sc-138763 and HPA023873 antibodies. (a) sc-138763, CA1 overview. (b) sc-138763, CA1, dystrophic neurites accumulated on senile plaque. (c) sc-138763, CA1, dystrophic neurites with stick and rugby ball shapes. (d) HPA023873, CA4, dystrophic neurites accumulated on senile plaques.
Supplier Page from MilliporeSigma for Anti-C9orf72 antibody produced in rabbit