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. 2022 Mar 1;132(5):e139828. doi: 10.1172/JCI139828

Figure 4. NR-regulated autophagy controls monocytic IFN-β production.

Figure 4

(A) Autophagy DE gene heatmap comparing LPS-activated placebo and NR-supplemented groups. (B) qRT-PCR analysis of monocytic autophagy-related genes from placebo and NR-supplemented groups (n = 8 individuals/group). (C) Representative immunoblot and quantification of LC3 (I and II) in monocytes preincubated overnight with vehicle or NR followed by LPS stimulation (10 ng/mL for 2 hours). (n = 6, 3 experiments). (D) Representative confocal images showing LC3 puncta (green) and Lamp1 puncta (red) in monocytes preincubated overnight with vehicle or NR followed by LPS stimulation. Arrows indicate cytoplasmic LC3 puncta. Scale bar: 2 μM. Quantification of total number and area of LC3 puncta in vehicle- or NR-incubated monocytes (n = 14 cells/group). (E) Representative immunoblot of LC3-II accumulation in response to chloroquine comparing vehicle or NR-supplemented monocytes in response to LPS and quantification of autophagic flux (n = 8, 4 experiments). (F) LPS-stimulated (10 ng/mL for 6 hours) IFN-β production from monocytes preincubated with autophagosome inhibitors (3-MA or nocodazole) or lysosome inhibitor chloroquine (CQ) in combination with vehicle or NR for 16 hours (representative results from 3 experiments). (G) IFN-β production measured by ELISA from monocytes transfected with control siRNA or siRNAs against multiple autophagy genes (representative results from 3 experiments). (H) IFN-β production in monocytes preincubated overnight with vehicle or NR or NR + VH (autophagy activator) followed by LPS stimulation (representative results from 3 experiments). (I) Representative immunoblot and quantification of LC3-II in human monocytes pretreated with vehicle or NR or NR + VH overnight followed by LPS stimulation. (n = 4–6, 3 experiments). Data analyzed using unpaired 2-tailed Student’s t test (B, D and E) or 1-way ANOVA followed by Dunnett’s multiple-comparison test (C and I) or 2-way ANOVA followed by Tukey’s multiple-comparison test (F to H). All data represented as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.