Fig 2: FACS analyses of FAMIN expression in PBMCs and granulocytes.PBMCs and granulocytes from human healthy donors were co-stained for different cell markers and FAMIN. Neutrophils, monocytes and DCs were shown to be FAMIN+. The events in the displayed graphs were first identified and gated by forward and side scatter parameters. (A) Gating strategy for FAMIN+ monocytes (CD14+, black) and CD14- neutrophils (CD15+, purple and CD16+, orange). (B) Gating strategy for FAMIN+ myeloid DCs (CD11c+, brown) and plasmacytoid DCs (BDCA2+, green) was obtained from gating the CD3-CD19- population followed by gating of CD14-HLADR+ cells. (C) Gating strategy for FAMIN+ T-cells (CD3+, purple) and B-cells (CD19+, orange). Numbers in the outlined areas indicate percent cells in each cell subtype. Data are representative of three independent experiments.

Fig 3: Characterization of anti-FAMIN antibodies and FAMIN expression in HeLa and THP-1 cell lines.(A) Anti-FAMIN mouse monoclonal antibodies (H-6, E-7 and E-12) were used in WB analyses of cell extracts from HeLa cells untransfected (mock) or transfected with FAMIN, GFP-FAMIN or GFP (control vector). (B) FAMIN expression is induced upon PMA differentiation of THP-1 cells (lanes 1–2 cells transfected with scrambled-control siRNA). Complete knockdown of FAMIN expression was obtained upon transfection of THP-1 cells with siRNA targeting LACC1 transcript (lanes 3–4). ß-actin was used for equal loading control. Data are representative of three independent experiments.

Fig 4: Characterization of FAMIN subcellular localization.PMA differentiated THP-1 cells were co-stained with a panel of antibodies directed towards different organelle markers and anti-FAMIN antibodies. Magnifications are shown for each staining on the right side of the pictures, together with indication of the target protein and corresponding compartment or cell organelle. Co-localization of FAMIN with PMP70 and catalase were previously shown and serve as positive control, Some additional co-localization could also be detected for endomembrane structure proteins detected at the level of endoplasmic reticulum (ER; calnexin, calreticulin and PDI), lysosomes (LAMP-1) and mitochondrion (COX-IV). Data are representative of six independent experiments.

Fig 5: LPS stimulation of THP-1 cells results in FAMIN upregulation.(A) qRT-PCR analysis of LACC1 mRNA expression in THP-1 cells stimulated with PMA and LPS. (B) WB analysis of FAMIN protein expression in THP-1 cells stimulated with PMA and LPS. Relative intensity (RI) from densitometric analysis is reported. Data are representative of three independent experiments. Abbreviation; UN: unstimulated control.