Supplementary Figure Legend

Supplementary figure legend

S1. Bacterial LPS induces early expression of SAG mRNA and protein in J774 cells. (A) P. aeruginosa LPS induces SAG mRNA (upper panel) and protein (lower panel) levels to peak at 4 h and 6-12 h, respectively. (B) Treatment of cells with E. coli LPS showed consistent profile of expression of SAG mRNA and protein.

S2. SAG-knockdown in J774, RAW and BMDM cells. To examine SAG RNAi efficacy in infected (A) J774, (B) RAW264.7 or (C) BMDM cells, SAG-sequence specific siRNAs were transfected into the macrophages for 16 h, followed by poly I:C treatment for 12 h, then a Western blot and real-time PCR for SAG was processed. β–actin was used as a loading control. Quantitative results are shown as mean ±SD (n=3).

S3. FACS analysis of apoptosis after PAMP-treatments of J774 cells with and without SAG-knockdown. (A) J774 apoptosis data (Annexin V and 7AAD double staining), including controls (PAMP+SAG siRNA or PAMP+control siRNA, SAG siRNA only, and PAMP only controls). (B) TMRE staining assay for mitochondrial membrane permeability after P.a LPS- or poly I:C- treatment. Control cells treated with SAG siRNA or PBS only (without PAMP treatment) are provided. Quantitative results show % of cells stained with TMRE (n=3). (C) Representative histograms of caspase-9 and caspase-3 activity assay.

S4. Expression profiles of SAG, Bcl-2, Bax, SARM, Noxa and Bcl-xL in J774 cells after PAMP treatments (with and without SAG-knockdown). Real-time PCR was carried out to determine the mRNA levels of apoptotic factors. (A) Dynamic changes in the mRNA levels of Bax, SARM, SAG and Bcl-2. (B) Transcriptional levels of Noxa and Bcl-xL remained unchanged. Data are presented as means ±SD (n=3). (C) Western blot of Bax, SARM, SAG, Bcl-2, Noxa and Bcl-xL. Noxa and Bcl-xL are indicated with a red arrow. Actin was used as a loading control. SAG siRNA approach was used to study the potential modulatory role of SAG on Bax, SARM, Bcl-2, Noxa and Bcl-xL.

S5. FACS histograms of SAG, Bcl-2, Bax and SARM in J774 cells after PAMP treatments (with and without SAG-knockdown). Intracellular staining of: (A) SAG (B) Bcl-2 (C) Bax and (D) SARM was conducted in LPS and poly I:C challenged J774 cells. SAG siRNA approach was used to study its role in modulating Bcl-2, Bax and SARM.

S6. SAG-knockdown did not affect the level of ubiquitinated Bcl-2 in J774, RAW and BMDM cells. To confirm the effect of SAG on Bcl-2 ubiquitination, we performed a Co-IP using Bcl-2 as bait, followed by an ubiquitin immunoblotting. The assay was tested in (A) J774, (B) BMDM and (C) RAW264.7 cells. Unlike Bax and SARM, reduction in SAG levels showed a similar pattern of HMW /ubiquitinated Bcl-2, compared to control siRNA treatment. These results suggest that Bcl-2 ubiquitination is independent of SAG. To confirm that the observed ubiquitination is due to Bcl-2 proteins, an immunoprecipitation with ubiquitin followed by immunoblotting with Bcl-2 was performed. Immunoprecipitation with control mouse IgG demonstrates the specificity of the assay.

S7. A control IgG tested in J774 and BMDM cells demonstrates the specificity of immunoprecipitation. A control IgG is used as a non-specific IgG control (corresponding to the host species). Immunoprecipitation with control rabbit IgG was used in (A) J774 cells and (B) cytosolic fraction of BMDM cells to demonstrate the specificity of the assay.

S8. RAW264.7 cells challenged with PAMPs show induction of SAG which ubiquitinates Bax and SARM. (A) SAG protein is increased at the early stage (3-12 h) of stimulations with bacterial /viral PAMPs compared to PBS control. P. aeruginosa LPS (P.a. LPS at 10 ng/ml), viral mimics (poly I:C at 10 μg/ml or R848 at 25 ng/ml). P.a. LPS and poly I:C, but not R848, induced SAG. (B) Cytochrome c release indicates the level of intrinsic apoptosis, which is increased in SAG-knockdown RAW cells. (C) Western blot analysis of whole cell lysates shows the presence of Bax, SARM, SAG and Bcl-2 in the RAW cells (before IP). Actin was used as a loading control. (D) To investigate the association between SAG and Bax (or SARM) in RAW cells, the total cell lysates were immunoprecipitated with anti-Bax (or anti-SARM) antibody, separated by 12 and 10 % SDS-PAGE, respectively, immunoblotted and probed with anti-SAG, anti-ubiquitin, anti-Bax or anti-SARM antibodies. SAG-knockdown attenuated the high-molecular-weight (HMW) ubiquitinated /modified forms of Bax and SARM but not that of Bcl-2 (see Fig. S6C). Arrows indicate unmodified (un-ubiquitinated) Bax or SARM. A densitometric analysis of ubiquitinated Bax or SARM, relative to total Bax or SARM is plotted (mean ±SD, n=3). To confirm that observed ubiquitination is due to Bax (or SARM) proteins, immunoprecipitation was performed with anti-ubiquitin antibody, followed by immunoblot detection with antibodies against Bax (or SARM). Immunoprecipitation with control rabbit IgG demonstrates the specificity of the assay.

S9. SAG is an evolutionarily conserved protein. Comparative alignment of SAG from human (AAD25962), mouse (XP_921660), zebra fish (NP_001012516), chicken (NP_001026478), platypus (XP_001509995), fruit fly (XP_001986494), spruce (ABK21786), soybean (XP_003527486) and yeast (EGA60630) shows conservation of residues among all species (blue underline). There is 84% sequence identity (shaded). T-Coffee (Tree based Consistency Objective Function For AlignmEnt Evaluation) was used for multiple sequence alignment and Gblocks was used for alignment curation.

S10. SAG overexpression in primary BMDM cells. HA-SAG was transiently transfected and overexpressed in BMDM cells for 24 h, followed by Western blot analysis using anti-HA. GAPDH was tracked as a loading control.