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. Author manuscript; available in PMC: 2023 Sep 13.
Published in final edited form as: Immunity. 2022 Aug 10:S1074-7613(22)00340-5. doi: 10.1016/j.immuni.2022.07.007

Figure 5. SPARC-mediated ISG induction is through TLR4 activation.

Figure 5.

(A) Immunoblot analysis for activated interferon signaling proteins (p-TBK1, p-IRF3, and p-STAT1) in M2 BMDMs with the treatment of 20 μg/ml SPARC for 1, 3, 6, 12, and 24 hours as well as non-treated control. (B) ELISA assay for IFN-β detection in supernatants of SPARC treated M2 BMDMs in (A) (n=3). (C) Solid-phase binding assay to detect the interaction between SPARC and human TLR4 (hTLR), SPARC and human TLR2 (hTLR2), SPARC and human MD-2 (MD-2), SPARC and BSA (BSA). The indicated amount of TLR4s or TLR2s were coated on plates, and 20 μg/ml SPARC, MD-2, or BSA were added for interaction (n=3). Nonlinear regression analyses were performed for the values in the graph. (D, E) Q-PCR analysis for Irf7, Ifit2, Stat1 (D), and pro-inflammatory genes (E) in M2 polarized BMDMs from WT, Tlr4−/−, Myd88−/−, and Ifnar1−/− mice with or without SPARC (20 μg/ml) treatment (n=3). (F) Expression pattern of glycolysis pathway genes in RNA-sequencing analysis of peritoneal macrophages induced by IL-4c (IL-4) and thioglycollate (TG) injection with or without SPARC treatment ex vivo (n=3). (G) Oxygen consumption rate (OCR) for M1 and M2 BMDMs with or without SPARC (20 μg/ml) treatment measured by seahorse mitostress assay (n=3). (H) Oxygen consumption rate (OCR) of M1, M2, and 20 μg/ml SPARC treated M2 BMDMs with or without pre-treatment of TLR4 inhibitor, TAK-242 (2.5 μM). (I, J) Gene expression analysis of Irf7, ISGs, and Stat1 in non-teated and SPARC (20 μg/ml, 24 hours) treated M2 BMDMs with or without pre-treatment of glycolysis inhibitor (2-DG, 10 mM) (n=3). (K) Immunoblot analysis for activated interferon signaling proteins (p-TBK1, p-IRF3, and p-STAT1) in M2 polarized BMDMs treated with SPARC (20 μg/ml) for indicated time with or without pre-treatment of glycolysis inhibitor (2-DG, 10 mM) (n=3). All in vitro or ex vivo experiments were repeated independently at least twice. Error bars represent the mean ± S.E.M. Two-tailed paired and unpaired t-tests were performed for statistical analysis. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. Please also see Figure S5.