MOAP-1-mediated dissociation of p62/SQSTM1 bodies releases Keap1 and suppresses Nrf2 signaling (2020)
Figure 2

Figure 2 - MOAP-1 regulates levels of p62 bodies.

A MOAP-1 downregulates the levels of the diethylnitrosamine (DEN)-induced p62 bodies in a time-dependent manner. LO2 cells were transfected with plasmid encoding Myc-MOAP-1. 14 hours later, cells were treated with DEN (200 µM) for the indicated period of times, before being subjected to IF with anti-Myc (in green) and anti-p62 (in red) antibodies. Cells expressing Myc-MOAP-1 were marked by dashed lines.

B MOAP-1 downregulates the p62 bodies in the resting HepG2 liver cancer cells. HepG2 were transfected with plasmid encoding Myc-MOAP-1. At the indicated time post-transfection, transfected cells were subjected to IF as in (A).

C Loss of MOAP-1 leads to spontaneous elevation of the basal levels of p62 bodies in the LO2 cells. WT, MOAP-1 KO and p62 KO LO2 cells were subjected to IF with anti-p62 antibody (in red). p62 KO cells were included as a negative control. Insets in the upper panels are enlarged in the lower panels.

D Quantification of the p62 bodies in the WT and MOAP-1 KO LO2 cells. (Left panel) number of p62 bodies per cell. n = 7 independently plated samples. (Right panel) size of p62 bodies. n = 442 and 1014 bodies detected in the WT and MOAP-1 KO LO2 cells, respectively, from three independently plated samples.

E Re-expression of MOAP-1 reduces levels of p62 bodies in the MOAP-1 deficient LO2 cells. MOAP-1 KO LO2 cells were transfected with plasmid encoding Myc-MOAP-1 for 24 hours and subjected to IF with anti-p62 (in red) and anti-Myc (green) antibodies. Cells expressing Myc-MOAP-1 were marked by dashed lines.

F Quantification of the p62 bodies in the MOAP-1 KO LO2 cells re-introduced with Myc-MOAP-1. (Left panel) number of p62 bodies per cell. n = 7 independently plated samples of MOAP-1 KO cells transfected with empty vector control (Ctrl) or plasmid encoding Myc-MOAP-1. (Right panel) size of p62 bodies. n = 178 and 69 bodies detected in the Ctrl and Myc-MOAP-1 expressing cells, respectively, from three independently plated samples.

G Increased level of p62 bodies in MOAP-1 KO MEFs upon treatment with arsenic trioxide. WT and MOAP-1 KO MEFs were treated with arsenic trioxide (As2O3, 10 µM) for the indicated durations and subjected to IF with anti-p62 antibody (in red).

H Quantification of the p62 bodies in the WT and MOAP-1 KO MEFs treated with arsenic trioxide. (Left panel) number of p62 bodies per cell. n ≥ 6 independently plated samples. (Right panel) size of p62 bodies. n = 10 (WT, basal), 17 (MOAP-1 KO, basal), 104 (WT, As2O3 2h), 137 (MOAP-1 KO, As2O3 2h), 605 (WT, As2O3 4h), 977 (MOAP-1 KO, As2O3 4h), 721 (WT, As2O3 6h) and 487 bodies (MOAP-1 KO, As2O3 6h) from three independently plated samples.

I MOAP-1 deficiency does not alter total protein levels of p62 in the LO2 cells. Western blotting analysis of p62 protein levels in the WT, MOAP-1 KO and p62 KO LO2 cells lysed in 1x Laemmli sample buffer containing 2% SDS. Actin as loading control.

J MOAP-1 deficiency does not affect total protein levels of p62 in MEFs under basal and As2O3-treated conditions. Western blotting analysis of p62 and MOAP-1 in the WT and MOAP-1 KO MEFs treated with As2O3 as indicated. MOAP-1 was immunoprecipitated from the lysates. Actin as loading control.

K Loss of MOAP-1 does not alter protein turnover of p62 in the LO2 cells. WT and MOAP-1 KO LO2 cells stably expressing Halo-tagged p62 were subjected to TMR labelling and harvested at the time points post-labelling as indicated. TMR signals of Halo-p62 were visualized by a Bio-rad Imager at 565 nM wavelength, whereas the total Halo-p62 protein was detected by Western blotting using anti-Halo or anti-p62 antibodies.

L Quantification of the TMR signals in the WT and MOAP-1 KO LO2 cells by densitometric analysis. Average integrated density values of the TMR signals from three independent experiments were presented, relative to the levels at 0 hour.

Data information: In (A, B, C, E, G), nuclei were counterstained with DAPI (blue), scale bar: 5 µm. In (D, F, H, L), error bars represent S.E.M. **P<0.01, ***P<0.001, ns, not significant, student's t-test.