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. Author manuscript; available in PMC: 2022 Jan 11.
Published in final edited form as: Nat Metab. 2020 Nov 16;2(11):1284–1304. doi: 10.1038/s42255-020-00298-z

Figure 7. Regulation of NAD+ and NMN levels by CD38 in macrophages.

Figure 7.

(a-b) NAD+ levels in BMDM from 3 month-old WT (a) and CD38 KO (b) mice. Macrophages were treated with vehicle (Ctrl) or LPS (100 ng/mL), with or without 78c (0.5 μM) (n=7; with exception of WT LPS+78c where n=6 biologically independent samples). (c-k) BMDM isolated from 18 month-old WT mice. (c) NAD+ levels in macrophages treated with LPS (100 ng/mL), Ab68 (5 μg/mL), 78c (0.5 μM), and olaparib (olap, 5 μM) (Ctrl, LPS n=11; LPS+78c n=4; LPS+78c, LPS+olap, LPS+78c+olap n=9 biologically independent samples). (d) Macrophages were treated with LPS (100 ng/mL), Ab68 (5 μg/mL), 78c (0.5 μM), and olaparib (olap, 5 μM). 3 hours later, 300 μM NMN was added and cells were collected 20 hours later for NAD+ measurements (n=7 except for NMN+LPS+olap and NMN+LPS+olap+78c where n=6 biologically independent samples). (e) Representative immunoblotting of three biologically independent samples of M0 or M1 (LPS-treated) macrophages and AML12 lysates showing CD38 and Slc12a8. (f) NAD+ levels in AML 12 cells cocultured with macrophages. Macrophages were treated with LPS (100 ng/mL), Ab68 (10 μg/mL), and NMN (200 μM) before incubation with AML 12 cells (n=7 biologically independent samples except for LPS+Ab68 where n=5). (g) Extracellular NMN in culture media after treatment of macrophages with 100 μM NMN, LPS (100 ng/mL), and Ab68 (5 μg/mL). LPS was given 9 hours before NMN, and Ab68 was given 3 hours before NMN. Cells were incubated with NMN for 12 hours before media was collected (n=5 biologically independent samples). (h) NAD+ levels in AML 12 cells cocultured with macrophages. Macrophages were treated with LPS (100 ng/mL), Ab68 (10 μg/mL), and NR (200 μM) before incubation with AML 12 cells (n=5 biologically independent samples). (i) NAD+ levels in macrophages treated with CM from non-senescent (CM-NS) or senescent mouse pre-adipocytes (CM-SEN or CM-SEN + 78c (0.5 μM)) (n=6 biologically independent samples). (j-k) NAD+ (n=8 biologically independent samples except for CM SEN+NMN+Ab68 where n=7) and NMN levels (n=4 biologically independent samples) in AML 12 cells cocultured with macrophages. Macrophages were treated with CM-NS or CM-SEN from mouse preadipocytes, Ab68 (10 μg/mL), and NMN (200 μM) before incubation with AML 12 cells. All levels are relative to their respective control (Ctrl or CM-NS). Data are mean ± SEM, analyzed by unpaired two-sided t-test.