Fig. 3

Spike protein receptor binding domain inhibits ARSB activity and expression by activation of phospho-p38 MAPK and phospho-(S249/T252)-RB-E2F1 interaction. a ARSB mRNA expression was unaffected by NSC23766, but following exposure to the p38 MAPK inhibitor SB, mRNA expression was restored to baseline control values. b In contrast, to the observed effects of p38α inhibitors on expression of CHST11 and CHST15, p38α siRNA and PH797804 reversed the SPRBD-induced decline in ARSB expression. TAB1 siRNA had no effect. c SIS3 had no impact on the SPRBD- or SPRBD + IFN-β- induced decline in ARSB expression. d ARSB promoter activation was reduced by SPRBD and by SPRBD + IFN-β. These declines were reversed by exposure to SB, but not by SIS3. e Following treatment with SPRBD or SPRBD + IFN-β, C-terminal phospho-(Ser807/811)-Rb declined as shown by ELISA. These declines were reversed by SB, but not by SIS3. f In contrast to the decline in C-terminal Rb phosphorylation, exposure to SPRBD + IFN-β increased N-terminus phospho-(S249/T252)-Rb, as detected by Western blot and shown in detail in Supplementary Fig. 4a–d. This increase was inhibited by SB, and total Rb was unchanged. g Densitometry confirms the impression of Western blot and shows the ratio of phospho-(S249)-Rb to total Rb following SPRBD + IFN-β has increased to 3.89 times the baseline. h Following exposure to SPRBD and SPRBD + IFN-β, E2F-DNA binding declined significantly, and SB reversed the declines. i %DNA input declined following SPRBD + IFN-β and increased following inhibition of p38-MAPK by SB. j Agarose gel of chromatin immunoprecipitation (ChIP) indicated no effect of the IgG negative control on E2F1 binding to the ARSB promoter at baseline, increased binding following IFN-β, reduced binding following SPRBD + IFN-β, and reversal of this decline following treatment with SB. P values were determined by unpaired t tests, two-tailed with unequal variance, with n of at least 3 independent experiments. Error bars show one standard deviation. * represents p ≤ 0.05; ** is for p ≤ 0.01, *** for p ≤ 0.001, and **** for p ≤ 0.0001. ARSB arylsulfatase B = N-acetylgalactosamine-4-sulfatase, IFN interferon, ND no difference, NSC = NSC23766, Rb retinoblastoma protein, SB = SB203580, SIS3 specific inhibitor of Smad3, SPRBD spike protein receptor-binding domain