Extended Data Fig. 8: ATR-dependent Lamin B1 loss and increased autophagic flux in Er1Lyz2/− BMDMs.
From: DNA damage in macrophages drives immune autoreactivity via nuclear antigen presentation
(A.) Cell cycle analysis of wt and Er1Lyz2/− BMDMs stained with DAPI-ki67. The rectangle gates in the representative scatter plots and the bar chart shows the percentages of cells in the indicated cell cycle phases (n = 4). (B.) Western blotting of whole-cell extracts derived from wt and Er1Lyz2/− untreated cells and cells treated with ATM and ATR inhibitors for the quantification of Lamin B1. Actin was used as a loading control (n = 4, pval=0.0487 and pval= 0.0025). (C.) Immunofluorescence staining of γ-Η2AX and 53BP1 in Er1Lyz2/− untreated, 3-MA treated and CQ-treated BMDMs. Cells with over 2 colocalized foci of the two proteins were considered positive and the percentage of positive cells is plotted. (At least 5 independent optical fields were counted from n = 3 biological replicates.) Single-channel images of Extended Data Fig. 8C are shown in Supplementary file 3C. (D.) Immunofluorescence detection of cGAS in wt untreated, Er1Lyz2/− untreated and Er1Lyz2/− BMDMs that underwent FBS-starvation (Er1Lyz2/− +starv). The bar plot shows the MFI of cytoplasmic cGAS in the corresponding conditions. (At least 4 independent optical fields were counted from n = 3 biological replicates, pval=0.0095 and pval=0.0069). Single-channel images of Extended Data Fig. 8D are shown in Supplementary file 3B(E.) Autophagic flux measurement after lentiviral infection of wt and Er1Lyz2/− BMDMs with a construct for the expression of LC3-GFP-mCherry. Green fluorescence corresponds to autophagosomes, while red fluorescence represents the autophagosomes and lysosomes in each cell. The graph shows the ratio of average red to green fluorescence per cell. (At least 3 independent optical fields were counted from n = 4 biological replicates, pval=0.0170) (F-G.) An overlay of the fluorescence intensity of (F.) LysoTracker Red (n = 5, pval=0.0289) and (G.) LysoSensor Green (n = 7-8, pval=0.0302) in Er1Lyz2/− BMDMs and wt corresponding controls, measured by flow cytometry analysis. The graphs show the respective MFI values. Error bars indicate S.E.M. among replicates. Asterisk indicates the significance set at p-value: *≤0.05, **≤0.01 (two-tailed Student’s t-test). Scale bars: 10μm.
