Figure 3. LYSMD3 mediates the expression of pro-inflammatory products upon stimulation with chitin or chitin containing fungi.
(A) ELISA of cytokines in supernatants of BEAS-2B cells stimulated for 24 h with 500 μM chitin oligomers.
(B) Immunoblot of LYSMD3 in membrane protein fraction of BEAS-2B cells 48 h after transfection with control siRNA or a siRNA pool specific for LYSMD3. NaK ATPase is loading control. See also Figure S2C.
(C) ELISA of cytokines in supernatants of BEAS-2B cells transfected with siRNAs as in (B), then stimulated for 24 h with chitin DP7 (500 μg/mL), chitin particles (500 μg/mL), Alternaria spores (2 × 105/mL), FLA-ST (300 ng/mL), or Poly(I:C) (3 μg/mL). See also Figures S2D and S2E.
(D) Immunoblot of LYSMD3 in cell lysates of NHBE cells 48 h after transfection with control siRNA or siRNA specific for LYSMD3. β-Actin is loading control.
(E) ELISA of IL-8 in supernatants of NHBE cells transfected with siRNA as in (D) and then stimulated for 24 h with chitin DP7 (500 μg/mL) or A. alternata spores (2 × 105/mL).
(F) Immunoblot of LYSMD3 in membrane protein fraction of RAW 264.7 cells 48 h after transfection with control siRNA or siRNA specific for LYSMD3.
(G) ELISA of IL-6 in supernatants of RAW 264.7 cells transfected with siRNA as in (F) and then stimulated for 24 h with chitin DP7 (100 μg/mL) or Pam3CSK4 (100 ng/mL).
(H) MTT assay 48 h after transient transfection of siRNA asin(B)in BEAS-2B cells. Cell growth rate is expressed as absorbance at 570 and was normalized to the value with control siRNA as 1.
(I) LDH cytotoxicity assay 48 h after transient transfection of siRNAs as in (B) in BEAS-2B cells.
(J) Expression of LYSMD3 protein in BEAS-2B cell with CRISPR-Cas9 knockout of LYSMD3 (sgD3). Hsp90 is the loading control.
(K) ELISA of IL-8 in supernatants of BEAS-2B cell knockout of LYSMD3 as in (J), unstimulated or stimulated for 24 h with chitin DP7 (500 μg/mL) or FLA-ST (300 ng/mL). See also Figure S2F.
(L) ELISA of IL-33 in supernatants of hBE33 cells transfected with siRNA as in (D) and then stimulated for 1 h with Alternaria extract (200 μg/mL).
*p < 0.05, **p < 0.01, and ***p < 0.001 (Student’s t test). Data are representative of three experiments (mean and SD of three samples per group in A, C, and E, or four samples per group in G, K, and I, or eight samples per group in H and L).