Af5002

MedChemExpress (New Jersey, USA) provided the high purity (≥99.0%) SAHA used in this study. Dimethyl sulfoxide (DMSO) (Sigma–Aldrich, Sydney, NSW, Australia) was employed to prepare SAHA to a concentration of 10 mM for storage at − 20 °C before being diluted to working concentrations with culture medium. Dulbecco’s modified Eagle’s medium (DMEM/high glucose) along with fetal bovine serum (FBS) were acquired from HyClone (Logan, UT, USA), whereas recombinant murine RANKL was supplied by R&D system (Minneapolis, MN, USA). FITC-phalloidin was procured from Thermo Fisher Scientific (Scoresby, VIC, Australia) and DAPI staining solution was bought from Beyotime (Shanghai, China). Primary antibodies against β-Actin (AC006), NFATc1 (A1539), CTSK (A5871), MMP9 (A11147), phospho-P38 (AP0057), phospho-ERK (AP0485), ERK (A4782), and phosphor-P65 (AP0475) were obtained from ABclonal (Wuhan, China). Primary antibody against IκB-α (AF5002) was obtained from Affinity Biosciences (Jiangsu, China). Primary antibodies for P38 (ab170099), phospho-JNK (ab76572), JNK (ab208035), and TRAF6 (ab40675) were purchased from Abcam (Cambridge, UK). The corresponding secondary antibodies were purchased from Beyotime (Shanghai, China). Yangming Biotechnology (Hangzhou, China) supplied bovine bone slices.

Total liver tissue homogenates and whole cell lysates were used for Western blotting to determine (i) protein levels of proinflammation cytokines: TNF-α (Proteintech, 60291-1-Ig, 1:1000) and IL-1β (Abcam, ab234437, 1:1000); (ii) the activation state of NF-κB p50/p65 signals including NF-κB p105 (Proteintech, 66992-1-Ig, 1:1000), p-NF-κB p50 (Abclonal, AP0125, 1:1000)/NF-κB p50 (Proteintech, 66992-1-Ig, 1:1000), p-NF-κB p65 (Affinity,AF3388, 1:2000)/NF-κB p65 (Abclonal, A19653, 1:500), and p-IκB (Affinity,AF2002, 1:2000)/IκB (Affinity,AF5002, 1:2000); and (iii) protein levels of Sema7a (Proteintech, 67397-1-Ig, 1:1000) and integrin β1 (Abcam, ab183666, 1:5000). Quantification of Western blotting results was determined by densitometric scanning using ImageJ software. Raw data of Western blotting results were included in Additional file 4.

For western blot analysis, proteins were extracted from femoral tissues using RIPA Buffer (20101ES60, Yeasen, China). We used the BCA kit (BI-WB005, SBJBIO, China) for protein quantification. After electrophoresis, the protein was loaded onto PVDF membranes (PW0034, Leagene, China). Next, 5% bovine serum albumin (BL-082, SBJBIO, China) was added to seal the membranes (37 °C, 60 min). The membranes were then immersed in primary antibodies (4 °C, overnight) and subsequently in anti-rabbit secondary antibodies (31,466, Invitrogen, USA) or anti-mouse secondary antibodies (S0002, Affinity, USA) at 37 °C for 60 min. Protein visualization was performed using an ECL reagent (GK10008, GlpBio, USA) on an eZwest Lite Auto Imaging System (Genscript, USA). The primary antibodies of Nrf2 (1:2000, AF0639), heme oxygenase 1 (HO-1) (1:2000, AF5393), phospho-IKB alpha (Ser32/Ser36, 1:2000, AF2002), IKB alpha (1:2000, AF5002), phospho-NF-kB p65 (Ser536, 1:2000, AF2006), NF-kB p65 (1:2000, AF5006), phospho-extracellular regulated protein kinases ½ (ERK1/2) (Tyr204, 1:2000, AF1014), ERK1/2 (1:2000, AF0155), phospho-p38 mitogen-activated protein kinase (MAPK) (Thr180/Tyr182, 1:500, AF4001), p38 MAPK (1:1000, AF6456), and GAPDH (1:20,000, AF7021) were obtained from Affinity (USA). GAPDH was used as the loading control.

The BV2 or SH-SY5Y cells or midbrain tissue lysates were subjected to Western blotting to detect NFATc2, STAT1, p-STAT1, IRF9, BAX, BCL-2, p65, p-p65, IκBα, p-IκBα and GAPDH. The following antibodies were used for protein detection by western blotting. NFATc2 (22023–1-AP, 1:1000, Proteintech), STAT1 (66545–1-lg, 1:2000, Proteintech), p-STAT1 (ab109461, 1:1000, Abcam), IRF9 (14167–1-AP, 1:1000, proteintech), BAX (A19684, 1:1000, ABclonal), BCL-2 (26593–1-AP, 1:1000, Proteintech), NF-kB p65 (AF5006, 1:1000, Affinity), Phospho-NF-kB p65(Ser536) (AF2006, 1:1000, Affinity), IKB alpha (AF5002, 1:1000, Affinity), Phospho-IKB alpha (Ser32/Ser36) (AF2002, 1:1000, Affinity), and GAPDH (#5174, 1:1000, Cell Signaling Technology).

Western blot assay was applied as we previously reported [31 (link)]. The following antibodies were used in the Western blot assay including CXCL1 antibody (AF5403, Affinity), β-actin antibody (4970S, CST), p65(10745-I-AP, Proteintech), FOXP3 (22228–1-AP, Proteintech), GAPDH (5174S), IκBα (AF5002, Affinity), p-IκBα (AF2002, Affinity), α-Tubulin (11224–1-AP, Proteintech), Lamin B1 (12987–1-AP, Proteintech), iNOS (18985–1-AP, Proteintech), and ARG1 (DF6657, Affinity).

Cell lysates were prepared and Western blot analysis was performed as previously described [19 , 40 (link)]. Following antibodies were used: anti-G6PD antibody (ab133525, Abcam, Cambridge, U.K.); anti-STAT3 antibody (4904, Cell Signaling Technology), anti-phospho-STAT3 antibody (Ser 727) (ab30647), anti-CyclinD1 antibody (ab16663, Abcam), Anti-CDK4 antibody (ab108357), Anti-p105/50 antibody (3035, Cell Signaling Technology), anti-p65 antibody (ab32536), anti-pIκBα (Ser32 + Ser36) antibody (AF2002, Affinity, USA), anti-IκBα antibody (AF5002, Affinity, USA), anti-β-actin antibody (4967, Cell Signaling Technology), and anti-GAPDH antibody (2118, Cell Signaling Technology).