Extended Data Fig. 7: Knockdown of the clathrin adaptor AP-1B impacts the TLR3 mediated polarized immune response. | Nature Microbiology

Extended Data Fig. 7: Knockdown of the clathrin adaptor AP-1B impacts the TLR3 mediated polarized immune response.

From: Asymmetric distribution of TLR3 leads to a polarized immune response in human intestinal epithelial cells

Extended Data Fig. 7

(a) Knock-down efficiency AP-1B was evaluated by q-RT-PCR. Results show the mean +/- sd. n=3. (b-d) The integrity of the barrier function of the AP-1B knock down T84 cells was evaluated by (b) indirect immunofluorescence of the tight junction protein Z-O1 (red). Representative image for immunofluorescence is shown. Scale bar =100 μm. (c) Dextran diffusion assay. Results show the mean +/- sd. n=3. and (d) TEER measurement. Results show the mean +/- sd. n=6. (e) Polarized WT and AP-1B knock-down T84 cells were infected with MRV in a side specific manner. Supernatants were collected at indicated time points and subjected to ELISA to monitor the amount of secreted type III IFNs. Results show the mean +/- sd. n=3. (f) AP-1B knock-down T84 cells were seeded on transwells and treated with indicated TLR agonists: TLR1/2 (PAM3CSK4, 1 μg/mL); TLR3 (1:1 HMW and LMW poly(I:C), 10 μg/mL); TLR4 (LPS-EK, 0.01 μg/mL); TLR5 (Flagellin-ST, 1 μg/mL); TLR7/8 (R848, 1 μg/mL), TLR9 (ODN 2395 5 μM). 6 h post-treatment RNA was harvested and evaluated for the upregulation of IL-6 by q-RT-PCR. Results are normalized to mock-treated samples and show the mean +/- s.d. n=3. (g) Same as f except type III IFN (IFNλ2/3) was evaluated. Results are normalized to mock-treated samples and show the mean +/- s.d. n=3. (e-g) P values were calculated from a two-tailed unpaired t test using GraphPad Prizm.

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