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. 2021 Apr 9;218(6):e20200944. doi: 10.1084/jem.20200944

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© 2021 Singh et al.

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Figure 8. — TNFα and/or TGFβ1 from HNSCC suppress DMBT1 in adjacent histologically normal epithelium via E-cadherin (E-cad), MMP9, and ZEB1 modulation. (A) Schematic of procedure for depletion of TNFα and TGFβ1 in CM from HNSCC cells. (B) Immunoblot verification of antibody (ab) depletion of TNFα and TGFβ1 in CM from HNSCC cells (n = 2). (C) CM was collected from UM-SCC-29 (n = 2). CM with or without depletion, or with depletion and add-back of TNFα or TGFβ1, was incubated with HOK16B for 72 h. Cell lysates were immunoblotted with DMBT1 and actin antibodies. DUs were calculated by normalizing to corresponding controls. (D) Schematic of siRNA-mediated depletion of TNFα or TGFβ1 in CM from HNSCC cells. (E–H) Validation of down-regulation of TNFα (E) and TGFβ1 (G) in UM-SCC-29 lysate (top) and CM (bottom), respectively. HOK16B were incubated with TNFα-depleted (F) and TGFβ1-depleted (H) CM, and lysates were immunoblotted for DMBT1, E-cad, MMP9, ZEB1, and actin. DUs were calculated by normalizing to corresponding controls (n = 2).