Figure 1. UVB upregulates NKG2D ligand expression via EGFR.

(A) Confluent HaCat cells were untreated (control) or exposed to UVB (60mJ/cm²) or heat shock (HS, 90mins 42°C), labelled with ³⁵S-methionine/cysteine immediately after HS and 24h post-UVB, and lysed. MICA immunoprecipitates were analysed relative to known size markers (kD) by SDS-PAGE and autoradiography. (B) Confluent HaCat (top) or Int407 (bottom) cells were untreated or exposed to UVB. RNA was extracted 24h later and MICA quantified relative to GAPDH by real-time qPCR. Data were compiled from 9 (HaCat) and 4 (Int407) independent experiments. Numbers in italic indicate p values (paired t-test). (C) Primary human keratinocytes were untreated (Mock) or UVB-irradiated (60mJ/cm²) and MICA/B and ULBP2 expression monitored by flow cytometry 24h post-treatment. Numbers in italic indicate respective MFI values. (D) Confluent HaCat cells were exposed to UVB or treated with doxorubicin (Dox, 1μg/mL), hydroxyurea (HUrea, 1mM) or 4NQO (1μg/mL). MICA expression was monitored as in B. Each dot represents the analysis of an individual sample. Numbers in italic indicate p values relative to control (unpaired t-test). (E) Confluent HaCat cells were untreated (control) or treated with the EGFR inhibitor AG1478 (AG) alone or 30mins prior to UVB irradiation (left); or serum-deprived overnight and incubated +/− EGF (500ng/mL, right), and mRNA levels quantified as in B. Results from 5 independent experiments are shown. Numbers in italic indicate p values (paired t-test). (F) Confluent primary murine keratinocytes in serum free medium (SFM) were untreated or stimulated with EGF (100ng/mL) for 24h (left). As a positive control for Rae1b and H60a upregulation, cells were UVB irradiated (right). Expression was monitored by real-time qPCR. Data were normalized to cyclophilin and expressed as the mean of three treatments +/− SD. Numbers in italic indicate p values (unpaired t-test).