Fig. 2: MPXV infection increases Rubicon levels.
From: The monkeypox virus suppresses autophagy by modulating Rubicon expression
A Calu-3 cells were infected with MPXV at an MOI of 0.5 or 1 for 24 h and 48 h. Two hours before lysis, cells were incubated with E64d/Pep.A as indicated (+). Rubicon and L1R levels were analyzed by western blot. HSP90 and Tubulin were included as loading controls. The graphs represent mean ± SEM of Rubicon:HSP90 values from three independent experiments. B Real-time PCR analysis of Rubicon mRNA levels in Calu-3 cells infected with MPXV at MOI of 0.5 or 1 for 24 or 48 h. Expression levels were normalized based on HSP90 values. A.U. arbitrary unit. Graph reports mean ± SEM of values from 3 independent experiments. C Calu-3 cells were treated for 4 h with the proteasomal inhibitor MG132 at a final concentration of 10 or 5 µM. Rubicon levels were analyzed by western blot. Actin was included as a loading control. The graphs represent mean ± SEM of Rubicon:Actin values from three independent experiments. D Calu-3 cells were treated for 4 h with the proteasomal inhibitor MG132 at a final concentration of 5 µM or infected with MPXV at MOI of 0.5 48 h. p21, p53, K48-linked ubiquitin (ubq) chains, and Rubicon levels were analyzed by western blot. L1R levels were monitored to verify MPXV replication. Actin and Tubulin were included as loading controls. The graphs represent mean ± SEM of p21:Actin, p53:Actin, L1R:Actin K48-Ubiquitin:Tubulin, Rubicon:Actin values from three independent experiments.
