Actinβ

The THP-1 macrophages were treated with 25 nM of S and S2 proteins alone or in combination with 25 µM COB-187 or 1% DMSO (carrier control for COB-187). After a 6-h incubation, the THP1 macrophages were harvested, and the mRNA was isolated via RNeasy Mini Kit (74,104, Qiagen; Germantown, MD) according to the manufacturer’s protocol. The mRNA was also isolated from non-treated THP-1, DRTHP1, and KO-DRTHP1 macrophages to determine if they express TLR2. Preparation of cDNA was achieved using the high-capacity cDNA reverse transcription kit with RNase Inhibitor (Applied Biosystems/ThermoFisher). SYBR Green biochemistry [iTaq Universal SYBR Green Supermix (1,725,121), Bio-Rad Laboratories, Inc.; Hercules, CA] was used to perform qPCR to quantify gene expression. Taqman Gene Expression Assays that were used as qPCR primers to determine the levels of CXCL10 and TLR2 gene expression include: CXCL10 (Hs00171042_m1, ThermoFisher) and TLR2 (Hs00610101_m1, ThermoFisher). The CFX96 Touch Real-Time PCR Detection System (Bio Rad; Hercules, CA) was used to perform the qPCR. Housekeeping genes for CXCL10 evaluation were Actinβ (Hs99999903_m1, ThermoFisher), and HPRT1 (Hs02800695_m1, ThermoFisher) and for TLR2 were Actinβ and GAPDH​ (Hs99999905_m1, ThermoFisher). The ΔΔCT method was used to determine the level of expression of CXCL10 versus media control.

Antibodies used for immunoblots were purchased from the indicated companies: MST1 (BD-611052 and H00006789-M02), GAPDH (CS-2118), P-LATS (CS-8654), LATS (SC-398560), Histone H3 (CS-9715), cl-CAS3 (CS-9661), cl-CAS9 (CS-9509), Actin-β (SC-69879), SIRT3 (SC-365175 and CS-5490), Vinculin (sc-25336), H2B (CS-12364), P_ser14-H2B(CS-6959), Tri-Methyl-Histone (Lys27) H3 (CS-9733), Cardiac Troponin T (Thermo MA5-12,960), COX IV (CS- 4850) [BD = Bio-Rad; CS = Cell Signaling; H = Invitrogen; SC = Santa Cruz]. Anti-mouse and anti-rabbit HRP-linked antibodies were purchased from Bio-Rad and Cell Signaling Technology. All antibodies were diluted in 5% non-fat dry milk powder in 0.05% Tween 20 Tris-buffered saline (TBST) or 3% BSA in TBST.