LY2874455 suppressed inflammation though autophagy pathway.

(A) Autophagy inhibitor reversed the effect of LY2874455 on ROS-reduction in LPS-stimulated macrophages. RAW264.7 cells were stained with ROS-probe DCFH-DA after treatment with LPS (20 ng/ml), LY2874455 (2 μm), and wortmannin (100 nM) as indicated for 24 h. Quantitative measurement of cellular ROS was performed by flow cytometry. (B, C) Autophagy inhibitor reversed the effect of LY2874455 in LPS-stimulated macrophages. The expression of inflammatory cytokine genes (iNOS and IL-6) was checked by qRT-PCR in RAW264.7 cells treated as (A). (D, E) The expression of inflammatory cytokine IL-6 and iNOS was checked by qRT-PCR in PMs as treated as in (A). (F) Autophagy deficiency reversed the effect of LY2874455 in inflammatory macrophages. WT or ATG7 knock out (ATG7-KO) RAW264.7 cells were treated with LPS (20 ng/ml) and LY2874455 (2 μm) for 24 h. The expression of iNOS and IL-6 was checked by qRT-PCR and expressed as fold changes compared to values in WT cells. (G) p62 knock down reversed the effect of LY2874455 in inflammatory macrophages. Control (sh-CTL, control shRNA) or p62 knockdown (sh-p62, p62-targeting shRNA) RAW264.7 cells were treated with LPS (20 ng/ml) and LY2874455 (2 μm) for 24 h. The expression of iNOS and IL-6 was checked by qRT-PCR and expressed as fold changes compared to values in control cells. (H–J) The enhanced production of proinflammatory factors in autophagy-deficient macrophages was suppressed by immunoproteasome inhibitor ONX-0914. WT or ATG7 knockout (ATG7-KO) RAW264.7 cells treated with LPS (20 ng/ml) and ONX-0914 (1 μm). The expression of iNOS and inflammatory cytokine genes (IL-6 and TNF-α) was checked by qRT-PCR. *: P P P P S1 Data. iNOS, inducible nitric oxide synthase; LPS, lipopolysaccharide; PM, peritoneal macrophage; qRT-PCR, quantitative real-time polymerase chain reaction; ROS, reactive oxygen species; WT, wild type.</p

LY287445 suppressed viral infection- and aging-induced inflammation.

(A–C) LY2874455 inhibited viral infection-induced inflammation. Polyinosinic-polycytidylic acid (poly(I:C)) (50 μg/ml) and LY2874455 (2 μm) were applied to BMDM for 24 h and expression levels of inflammatory cytokines were analyzed qRT-PCR. (D–F) LY2874455 inhibited the bacteria (E. coli) infection-induced inflammation. E. coli and LY2874455 (2 μm) were applied to BMDM for 24 h and expression levels of inflammatory cytokines were analyzed qRT-PCR. (G–K) LY2874455 inhibited H2O2-induced cell aging. NIH3T3 cells were treated with H2O2 (400 μm for 2 h) for induction of cell aging followed by LY2874455 (2 μm) treatment. Representative images of β-GAL staining of the cells were shown (G) and the expression levels of p16 (H) and inflammatory cytokines (I–K) were analyzed by qRT-PCR. (L) The protein expression of IL1β, IL18, and GAPDH were detected by western blot. And the protein expression was quantified with densitometric values and statistical significance was analyzed. *: P P P P S1 Data. (PDF)</p

LY2874455 induced autophagy in macrophages.

(A) Inhibitors MG132, chloroquine, or wortmannin were used at different concentrations to analyze their effects on macrophage viabilities in the dose- and time-dependent cell death assays. (B) Inhibitor of constitutive proteasome, MG132, could not block the reduction of immunoproteasome subunits induced by LY2874455. RAW264.7 cells were treated with LPS (20 ng/ml), LY2874455 (2 μm), and proteasome inhibitor MG132 (5 μm). Protein levels of LMP2, LMP7, and GAPDH were detected by western blot. The blots were quantified with densitometric values and statistical significance was analyzed. (C) Autophagy inhibitor wortmannin blocked the reduction of immunoproteasome subunits induced by LY2874455. Protein levels of LMP2, LMP7, and GAPDH were analyzed in RAW264.7 cells treated with LPS (20 ng/ml), LY2874455 (2 μm), and autophagy inhibitor wortmannin (100 nM). The blots were quantified with densitometric values and statistical significance was analyzed. (D) Model scheme of LY2874455 effect on activation of autophagy. FGFRs are transmembrane RTKs and activated by FGF to form dimers, leading to the activation of downstream FRS2 complex and subsequent activation of PI3K/AKT/mTOR signaling pathway. Activated mTOR then phosphorylates and inhibits autophagy initiating ULK1 kinase complex, leading to suppression of autophagy. LY2874455 suppresses the FGFR activities and the downstream PI3K/AKT/mTOR signaling pathway, leading to the release and activation of autophagy. (E) NIH3T3 cells were treated with LPS (20 ng/ml) and LY2874455 (0.2, 0.5, 1, 2, 5 μm) for 24 h and protein levels of p62, LC3, and GAPDH were analyzed by western blot. The blots were quantified with densitometric values and statistical significance was analyzed. (F) AZD4547, an inhibitor of FGFR, was used to detect the effect on autophagy and immunoproteasome. RAW264.7 cells were treated with LPS (20 ng/ml) and ADZ4547 (10, 50, 100 nM) for 24 h and protein levels of p62, LC3, and GAPDH were analyzed by western blot. The blots were quantified with densitometric values and statistical significance was analyzed. (G–I) AZD4547 also has effects on inflammation suppression in RAW264.7 cells. RAW264.7 cells were treated with LPS (20 ng/ml) and ADZ4547 (100 nM) for 24 h, and the expression of IL-6, TNF-α, IL1β, and iNOS was checked by qRT-PCR. (J) Chemicals (Fenticonazole, Domiphen, and Cetylpyridinium) were used to detect their effects on autophagy activation. RAW264.7 cells were treated with Fenticonazole, Domiphen, and Cetylpyridinium (0.5, 1, 15 μm) for 24 h and protein levels of p62 and GAPDH were analyzed by western blot. The blots were quantified with densitometric values and statistical significance was analyzed. (K–M) The protein expression of LMP2 and LMP7 in Fig 4A–4C were quantified with densitometric values and statistical significance was analyzed. (N) The number of LC3 puncta were quantified in Fig 4D. (O) Quantification of autophagosomes in macrophages treated with LY2874455 for results in Fig 4E. (P) The protein expression of p62 and the ratio of LC3II/LC3I in Fig 4F were quantified with densitometric values and statistical significance was analyzed. (Q–S) The protein expression of p62 in Fig 4G–4I were quantified with densitometric values and statistical significance was analyzed. (T–W) The protein expression of p-mTOR (Ser248), mTOR, p-AKT(Ser473), AKT in Fig 4J were quantified with densitometric values and statistical significance was analyzed. (X–AA) The protein expression of LMP2 and LMP7 in S4B and S4C Fig were quantified with densitometric values and statistical significance was analyzed. (AB–AC) The protein expression of p62 and the ratio of LC3II/LC3I in S4E Fig were quantified with densitometric values and statistical significance was analyzed. (AD–AF) The protein expression of LMP2, LMP7, and p62 in S4F Fig were quantified with densitometric values and statistical significance was analyzed. (AG–AI) The protein expression of p62 in S4J Fig was quantified with densitometric values and statistical significance was analyzed. *: P P P P S1 Data. (PDF)</p

Proteins analyzed by mass spectrometry in RAW264.7 macrophages treated with LPS and LY2874455.

Proteins analyzed by mass spectrometry in RAW264.7 macrophages treated with LPS and LY2874455.</p

LY287445 suppressed inflammation through autophagy.

(A, B) Autophagy inhibitor wortmannin reversed the effect of LY2874455 in LPS-stimulated macrophages. BMDMs were treated with LPS (20 ng/ml), LY2874455 (2 μm), and wortmannin (100 nM) as indicated for 24 h. The expression of iNOS and inflammatory cytokine genes IL-6 was checked by qRT-PCR. (C) RAW264.7 cells and used for western blot analysis of protein levels of p65, phosphorylated p65 (p-p65), and IKK-β. The blots were quantified with densitometric values and statistical significance was analyzed. (D) RAW264.7 cells were stimulated with LPS (20 ng/ml) for 2 h with or without LY2874455 (2 μm). Representative images of double immunostaining for p65 and nucleus (DAPI) were shown. Scale bars: 10 μm. (D, F) Deficiency of autophagy factor ATG7 or receptor p62 caused blockage of LY2874455 function in suppression of NF-κB factor p65 (phosphorylation and nucleus translocation). (E, G) Total proteins were extracted from ATG7 knockout or p62 knockdown RAW264.7 cells and used for western blot analysis of protein levels of p65, phosphorylated p65 (p-p65), and IKK-β. The blots were quantified with densitometric values and statistical significance was analyzed. (F, H) ATG7 knockout or p62 knockdown RAW264.7 cells were stimulated with LPS (20 ng/ml) for 2 h with or without LY2874455 (2 μm). Representative images of double immunostaining for p65 and nucleus (DAPI) were shown. Scale bars: 10 μm. (I) The enhanced inflammatory capacity in autophagy-deficient macrophages was suppressed by immunoproteasome inhibitor ONX-0914. Wild-type (WT) or ATG7 knockout (ATG7-KO) RAW264.7 cells treated with LPS (20 ng/ml) and ONX-0914 (1 μm). The protein levels of iNOS and inflammatory cytokine genes (IL-6 and TNF-α) were checked. The blots were quantified with densitometric values and statistical significance was analyzed. (J, K) Autophagy inhibitors (ULK1-IN-2 and chloroquine (CQ)) reversed the effect of LY2874455 in LPS-stimulated macrophages. RAW264.7 cells were harvest after treatment with LPS (20 ng/ml), LY2874455 (2 μm) and ULK1-IN-2 (5 μm) or chloroquine (20 μm) as indicated for 24 h. The expression of inflammatory cytokine genes (iNOS and IL-6) was checked by qRT-PCR in RAW264.7 cells. (L) siRNA was used to knock down the expression of lmp7. The gene expression of lmp7 was detected by qRT-PCR. (M) RAW264.7 cells were transfected with siRNA for 48 h and then treated with ONX-0914 for 24 h. The gene expression of IL6 was detected by qRT-PCR. (N) The protein expression of p65, phosphorylated p65 (p-p65), and IKK-β in S6C Fig were quantified with densitometric values and statistical significance was analyzed. (O) The protein expression of p65, phosphorylated p65 (p-p65), and IKK-β in S6D Fig were quantified with densitometric values and statistical significance was analyzed. (P) The protein expression of p65, phosphorylated p65 (p-p65), and IKK-β in S6G Fig were quantified with densitometric values and statistical significance was analyzed. (Q) The protein expression of iNOS, TNF-α, and IL-6 in S6I Fig were quantified with densitometric values and statistical significance was analyzed. (R) Diagrammatic sketch of selective autophagy of the immunoproteasome suppresses inflammation regulation. *: P P P P S1 Data. (PDF)</p

LY2874455 activated the autophagy process.

(A) Proteasome inhibitor MG132 cannot block the reduction of immunoproteasome subunits induced by LY2874455. Protein levels of LMP2, LMP7, and GAPDH were analyzed in RAW264.7 cells treated with LPS (20 ng/ml), LY2874455 (2 μm), and MG132 (5 μm). The blots were quantified with densitometric values and statistical significance was analyzed. (B, C) Autophagy inhibitors blocked the reduction of immunoproteasome subunits induced by LY2874455. Protein levels of LMP2, LMP7, and GAPDH were analyzed in RAW264.7 cells treated with LPS (20 ng/ml), LY2874455 (2 μm), and autophagy inhibitors chloroquine (CQ, 20 μm) or wortmannin (100 nM). The blots were quantified with densitometric values and statistical significance was analyzed. (D) LY2874455 enhanced autophagy flux. RAW264.7 cells transfected with mCherry-EGFP-LC3 were stimulated with LPS (20 ng/ml) or/and LY2874455 for another 24 h, and the puncta of LC3 in cells was monitored after fixation under a confocal fluorescent microscope. Higher level of mCherry-LC3 signal and lower level of EGFP-LC3 signal (showing red in merge) indicated the stimulated autophagy flux, while the similar signal levels of mCherry-LC3 and GFP-L3 (showing yellow in merge) indicated the blocked autophagy flux. Representative images from 3 replicated experiments were shown. Scale bars: 10 μm. (E) LY2874455 promoted the formation of autophagosomes. RAW264.7 cells treated with control or LY2874455 (2 μm) for 24 h were analyzed by transmission electron microscopy. Autophagosomes were pointed by arrows and representative images from 3 replicated experiments were shown. Scale bars: 1 μm. (F) LY2874455 promoted the autophagic degradation of substrate receptor p62. RAW264.7 cells were treated with LPS (20 ng/ml) and LY2874455 (0.5, 2, 4 μm) with or without Bafilomycin A1 (20 nM) for 24 h and protein levels of p62, LC3, and GAPDH were analyzed by western blot. The blots were quantified with densitometric values and statistical significance was analyzed. (G) Protein levels of p62 and GAPDH were detected by western blot in RAW264.7 cells treated with LY2874455 (2 μm) and LPS (20 ng/ml) and for 24 h. The blot was quantified with densitometric values and statistical significance was analyzed. (H) Protein levels of p62 and GAPDH were detected by western blot in PMs from C57BL/6J mice treated as in (G). The blot was quantified with densitometric values and statistical significance was analyzed. (I) Protein levels of p62 and GAPDH were detected by western blot in BMDMs from C57BL/6J mice treated as in (G). The blot was quantified with densitometric values and statistical significance was analyzed. (J) LY2874455 inhibited the AKT-mTOR signaling. Protein levels of p-mTOR (Ser248), mTOR, p-AKT (Ser473), AKT, and GAPDH were detected by western blot in RAW264.7 cells treated as in (G). The blots were quantified with densitometric values and statistical significance was analyzed. *: P P P P < 0.0001, NS: no statistical difference. BMDM, bone marrow-derived macrophage; LPS, lipopolysaccharide; PM, peritoneal macrophage.</p

LY2874455 suppressed inflammation in LPS-stimulated macrophages.

(A) RAW264.7 cells were incubated with 27 chemicals and stimulated with LPS (20 ng/ml) for 24 h. NO concentration in culture supernatant was measured and shown as fold change. (B) The names of the 27 chemicals. (C, D) Different concentrations of LY2874455 were applied to LPS (20 ng/ml)-treated RAW264.7 cells and cell viabilities and levels of NO were measured. (E–G) The expression of proinflammatory genes (IL-6, TNF-α) and iNOS was checked by qRT-PCR treated with LY2874455 (2 μm) and LPS (20 ng/ml) for 24 h. (H) Protein levels of IL-6, TNF-α, iNOS, and β-actin were detected by western blot in RAW264.7 cells. The blot was quantified with densitometric values and statistical significance was analyzed. (I–K) The expression of proinflammatory genes (IL-6, TNF-α) and iNOS was checked by qRT-PCR in PMs treated with LY2874455 (2 μm) and LPS (20 ng/ml) for 24 h. (L) Protein levels of iNOS and GAPDH were detected by western blot in PM. (M) The blot was quantified with densitometric values and statistical significance was analyzed. (N–P) The expression of proinflammatory genes (IL-6, TNF-α) and iNOS was checked by qRT-PCR in BMDMs treated with LY2874455 (2 μm) and LPS (20 ng/ml) for 24 h. (Q, R) Expression levels of iNOS were detected by in BMDM. The blot was quantified with densitometric values and statistical significance was analyzed. (S, T) The expression of proinflammatory genes (IL-6 and TNF-α) was checked by qRT-PCR in human THP-1 monocytes treated with LY2874455 (2 μm) and LPS (20 ng/ml) for 24 h. Phorbol12-myristate13-acetate (PMA) was used to induce THP-1 monocyte to macrophages. (U, V) The cytokine of IL-6 and TNF-α was detected by elisa in in RAW264.7 cells treated with LY2874455 (2 μm) and LPS (20 ng/ml) for 24 h. (W–Y) The protein expression of IL-6, TNF-α, and iNOS in Fig 1H were quantified with densitometric values and statistical significance was analyzed. (Z–AB) The protein expression of IL-6, TNF-α, and iNOS in S1H Fig were quantified with densitometric values and statistical significance was analyzed. (AC) The protein expression of IKK-β in Fig 1I was quantified with densitometric values and statistical significance was analyzed. (AD–AE) The protein expression of p65 and p-p65 in Fig 1J were quantified with densitometric values and statistical significance was analyzed. (AF) The immune fluorescence micrographs of p65 were quantified in Fig 1K. *: P P P P S1 Data. (PDF)</p

Uncropped images for all relevant figures in this article.

Uncropped images for all relevant figures in this article.</p

LY2874455 attenuated the inflammatory reactions in LPS-stimulated macrophages.

(A) The chemical structure of LY2874455. (B–K) Macrophage RAW264.7 cells were stimulated with LPS (20 ng/ml) for 24 h together with or without LY2874455 (2 μm). (B) LY2874455 abolished the release of NO from LPS-stimulated RAW264.7 cells. NO concentration in culture supernatant was measured and shown as fold change compared to the control group. Similar results were obtained from 3 independent replicated experiments. **P P (C) LY2874455 abolished the generation of ROS in LPS-stimulated RAW264.7 cells. After treatment with LPS or/and LY2874455, RAW264.7 cells were washed and labeled with DCFH-DA, a ROS probe. Quantitative measurement of cellular DCFH-DA staining was performed by flow cytometry. Similar results were obtained from 3 independent experiments. (D) A set of representative images showing fluorescent staining of ROS by DCFH-DA probe were shown. Scale bars: 10 μm. (E–G) LY2874455 suppressed the expression of proinflammatory cytokines and NO synthetase in macrophages stimulated with LPS. The expression of proinflammatory cytokine genes (IL-6, TNF-α) and NO synthetase (iNOS) was checked by qRT-PCR. (H–J) Total proteins were extracted from RAW264.7 cells and used for western blot analysis of protein levels of IL6, TNF-α, iNOS, IKK-β, p65, and phosphorylated p65 (p-p65). For the similar experiments shown by immunoblots, 3 independent experiments were conducted and representative images were shown. The blots were quantified with densitometric values and statistical significance was analyzed (S1W–S1AE Fig). (K) LY2874455 suppressed the nucleus translocation of phosphorylated p65 in macrophages stimulated with LPS. RAW264.7 cells were stimulated with LPS (20 ng/ml) for 2 h with or without LY2874455 (2 μm). Representative images of double immunostaining for p65 and nucleus (DAPI) were shown. Scale bars: 10 μm. *: P P P P S1 Data. iNOS, inducible nitric oxide synthase; LPS, lipopolysaccharide; NO, nitric oxide; qRT-PCR, quantitative real-time polymerase chain reaction; ROS, reactive oxygen species.</p

LY287445 induced the degradation of immunoproteasome subunits through autophagy.

(A) Confirmation of ATG7 gene knock out in macrophages. ATG7 gene was deleted through CRISPR-CAS9 assay in RAW264.7 cells. Clones of gene deletion cells were cultured and detected for protein levels of p62, LC3, ATG7, and intrinsic control GAPDH. The blots were quantified with densitometric values and statistical significance was analyzed. (B) Confirmation of the decrease of p62 protein levels in p62 knock down RAW264.7 cells. The blot was quantified with densitometric values and statistical significance was analyzed. (C) The protein expression of LMP2 and LMP7 in Fig 5A were quantified with densitometric values and statistical significance was analyzed. (D) The protein expression of LMP2, LMP7, and p62 in Fig 5B were quantified with densitometric values and statistical significance was analyzed. (E) The protein expression of LMP2, LMP7, and p62 in Fig 5C were quantified with densitometric values and statistical significance was analyzed. (F) The immune fluorescence micrographs in Fig 5D was quantified by ImageJ. (G) The protein expression of NRB1, Tollip, and p62 in Fig 5E were quantified with densitometric values and statistical significance was analyzed. (H) The protein expression of LMP2 and LMP7 in Fig 5F were quantified with densitometric values and statistical significance was analyzed. *: P P P P S1 Data. (PDF)</p