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. 2023 Mar 15;24(6):5623. doi: 10.3390/ijms24065623

Figure 1.

Figure 1

Development of inducible system to express MyD88 wt or L265P in lymphoma cell lines. (A) Schematic representation of MyD88 protein domains. The missense mutation (L265P) is located at the TIR domain at the C terminus. (B) Schematic representation of the tetracycline-inducible lentiviral gene expression system, pLVX-TetOne-Puro, used for the overexpression of MyD88 (wt/L265P) in stable cell lines. The Tet-On 3G transactivator is constitutively expressed under the human PGK promoter. The MyD88 gene (NM_002468.5) is under the TRE3GS promoter in the opposite orientation. In the presence of doxycycline (DOX), the Tet-On 3G transactivator binds and activates the TRE3GS inducible promoter that controls MyD88 expression. The gene sequence encoding cell selection marker puromycin N-acetyltransferase (Puro) under the simian virus 40 (SV40) promoter confers puromycin resistance. (C) MyD88 inducible expression analyzed with qPCR after 24 h of DOX (250 ng/mL) treatment in U2932 cell lines (ns, not significant for p > 0.05, *** for p ≤ 0.001). (D) Western blot analysis of MyD88 inducible expression after 24 h of DOX (250 ng/mL) treatment in U2932 cell lines. IkBα staining was used as a marker for NF-kB pathway activation. Actin was used as a loading control.