Figure 3 ( D ) Validation by western blot of changes in specific phosphorylation events on kinases . Left , Immunoblots of cortical lysates of CTL and micTNFα - KO at ZT6 for : ( top ) phosphorylated GSK3α / β ( pGSK3α at Ser21 and pGSK3β at Ser9 ) and total GSK3α ; and ( bottom ) phosphorylated MAPK ( pMAPK , MAPK3 at Thr203 / Tyr205 and MAPK1 at Thr183 / Tyr185 ) and total MAPK . Right , Ratio between phosphorylated and total kinase normalized to CTL at ZT18 and ZT6 . n = 5 mice per group . * * * p = 0 . 000009 ( GSK3α ) , multiple t - test with Benjamini - Hochberg correction . ( E ) Kinases with altered activity between CTL and micTNF - KOs predicted by robust kinase activity inference ( RoKAI ) at ZT18 and ZT6 . Graph shows the z - score value of each kinase assigned by RoKAI . Kinases above and below the dotted line represent prediction of up - and downregulated activity in micTNFα - KO , respectively . Kinases are color - coded according to kinase group as described in ( C ) . ( F Validation by western blot of altered activity of predicted kinases between CTL and micTNF - KOs . ( F ) Left , Immunoblots of cortical lysates of CTL and micTNFα - KO at ZT6 using antibodies specific to target phosphorylation motifs of MAPK / CDK ( PXS * P , S * PX ( K / R ) ) and PKC ( ( K / R ) XS * X ( K / R ) ) . Dashed boxes indicate quantified signal . Right , Ratio between phosphorylated substrates and loading control normalized to CTL at ZT18 and ZT6 . ( F , n = 5 mice per group . * p = 0 . 022 ( MAPK / CDK phospho sub ) , * p = 0 . 014 ( PKC phospho sub ) multiple t - test with Benjamini - Hochberg correction . G ) Validation by western blot of altered activity of predicted kinases between CTL and micTNF - KOs . ( G ) Left , Immunoblots show signal of phosphorylated threonine in MARKs activation loop . Right , Ratio between phosphorylated MARK activation loop and loading control normalized to CTL at ZT18 and ZT6 . G ) n = 5 mice per group . * * p = 0 . 0091 ( MARK ) , multiple t - test with Benjamini - Hochberg correction .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(D) Validation by western blot of changes in specific phosphorylation events on kinases. Left, Immunoblots of cortical lysates of CTL and micTNFα-KO at ZT6 for: (top) phosphorylated GSK3α/β (pGSK3α at Ser21 and pGSK3β at Ser9) and total GSK3α; and (bottom) phosphorylated MAPK (pMAPK, MAPK3 at Thr203/Tyr205 and MAPK1 at Thr183/Tyr185) and total MAPK. Right, Ratio between phosphorylated and total kinase normalized to CTL at ZT18 and ZT6. n=5 mice per group. ***p=0.000009 (GSK3α), multiple t-test with Benjamini-Hochberg correction.(E) Kinases with altered activity between CTL and micTNF-KOs predicted by robust kinase activity inference (RoKAI) at ZT18 and ZT6. Graph shows the z-score value of each kinase assigned by RoKAI. Kinases above and below the dotted line represent prediction of up- and downregulated activity in micTNFα-KO, respectively. Kinases are color-coded according to kinase group as described in (C).(F Validation by western blot of altered activity of predicted kinases between CTL and micTNF-KOs. (F) Left, Immunoblots of cortical lysates of CTL and micTNFα-KO at ZT6 using antibodies specific to target phosphorylation motifs of MAPK/CDK (PXS*P, S*PX(K/R)) and PKC ((K/R)XS*X(K/R)). Dashed boxes indicate quantified signal. Right, Ratio between phosphorylated substrates and loading control normalized to CTL at ZT18 and ZT6. (F, n=5 mice per group. *p=0.022 (MAPK/CDK phospho sub), *p=0.014 (PKC phospho sub) multiple t-test with Benjamini-Hochberg correction.G) Validation by western blot of altered activity of predicted kinases between CTL and micTNF-KOs. (G) Left, Immunoblots show signal of phosphorylated threonine in MARKs activation loop. Right, Ratio between phosphorylated MARK activation loop and loading control normalized to CTL at ZT18 and ZT6. G) n=5 mice per group. **p=0.0091 (MARK), multiple t-test with Benjamini-Hochberg correction.


Figure 1 A MOAP - 1 co - localizes with p62 in the cytoplasmic inclusion bodies upon proteasome inhibition . HCT116 cells were transfected with plasmid encoding GFP - MOAP - 1 or GFP only control . 14 hours later , cells were treated with the proteasome inhibitor MG132 ( 5 µM ) for 8 hours and analyzed by immunofluorescence ( IF ) with anti - GFP ( in green ) and anti - p62 ( in red ) antibodies . Data information : , nuclei were counterstained with DAPI ( blue ) B Endogenous MOAP - 1 resides at the p62 bodies upon proteasome inhibition . HCT116 cells were treated with MG132 ( 5 µM ) for 8 hours and analyzed by IF with anti - MOAP - 1 ( in green ) and anti - p62 ( in red ) antibodies . Data information : nuclei were counterstained with DAPI ( blue ) C MOAP - 1 is recruited to the p62 bodies upon exposure to cellular stresses . LO2 hepatocytes were transfected with plasmid encoding Myc - MOAP - 1 . 14 hours later , cells were then treated with MG132 ( 5 µM ) , arsenic trioxide ( As2O3 , 10 µM ) or diethylnitrosamine ( DEN , 200 µM ) for 8 hours each . Cells were then processed for IF analysis with anti - Myc ( in green ) and anti - p62 ( in red ) antibodies . Data information : nuclei were counterstained with DAPI ( blue ) D MOAP - 1 spontaneously localizes to the p62 bodies at resting state in the liver cancer cell lines , HepG2 , Huh - 1 , JHH5 and JHH7 . The liver cancer cell lines were transfected with the plasmid encoding Myc - MOAP - 1 . 14 hours later , transfected cells were subjected to IF analysis with anti - Myc ( in green ) and anti - p62 ( in red ) antibodies . Data information : nuclei were counterstained with DAPI ( blue ) E Absence of the aggregated patterns of MOAP - 1 in the p62 deficient cells . WT and p62 KO HepG2 cells were transfected with plasmid encoding Myc - MOAP - 1 for 14 hours and the cells were subjected to IF analysis as in ( D ) . Data information : , nuclei were counterstained with DAPI ( blue ) F Western blotting analysis of p62 and Myc - MOAP - 1 protein levels in the WT and p62 KO HepG2 cells as described in ( E ) . Actin as loading control .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1 A MOAP-1 co-localizes with p62 in the cytoplasmic inclusion bodies upon proteasome inhibition. HCT116 cells were transfected with plasmid encoding GFP-MOAP-1 or GFP only control. 14 hours later, cells were treated with the proteasome inhibitor MG132 (5 µM) for 8 hours and analyzed by immunofluorescence (IF) with anti-GFP (in green) and anti-p62 (in red) antibodies. Data information: , nuclei were counterstained with DAPI (blue)  B Endogenous MOAP-1 resides at the p62 bodies upon proteasome inhibition. HCT116 cells were treated with MG132 (5 µM) for 8 hours and analyzed by IF with anti-MOAP-1 (in green) and anti-p62 (in red) antibodies. Data information: nuclei were counterstained with DAPI (blue)  C MOAP-1 is recruited to the p62 bodies upon exposure to cellular stresses. LO2 hepatocytes were transfected with plasmid encoding Myc-MOAP-1. 14 hours later, cells were then treated with MG132 (5 µM), arsenic trioxide (As2O3, 10 µM) or diethylnitrosamine (DEN, 200 µM) for 8 hours each. Cells were then processed for IF analysis with anti-Myc (in green) and anti-p62 (in red) antibodies. Data information: nuclei were counterstained with DAPI (blue)  D MOAP-1 spontaneously localizes to the p62 bodies at resting state in the liver cancer cell lines, HepG2, Huh-1, JHH5 and JHH7. The liver cancer cell lines were transfected with the plasmid encoding Myc-MOAP-1. 14 hours later, transfected cells were subjected to IF analysis with anti-Myc (in green) and anti-p62 (in red) antibodies. Data information: nuclei were counterstained with DAPI (blue)  E Absence of the aggregated patterns of MOAP-1 in the p62 deficient cells. WT and p62 KO HepG2 cells were transfected with plasmid encoding Myc-MOAP-1 for 14 hours and the cells were subjected to IF analysis as in (D). Data information: , nuclei were counterstained with DAPI (blue)  F Western blotting analysis of p62 and Myc-MOAP-1 protein levels in the WT and p62 KO HepG2 cells as described in (E). Actin as loading control.


Figure 2 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 . Data information : nuclei were counterstained with DAPI ( blue ) 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 ) . Data information : nuclei were counterstained with DAPI ( blue ) 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 . Data information : nuclei were counterstained with DAPI ( blue ) 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 . Data information : , nuclei were counterstained with DAPI ( blue ) 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 . Data information : nuclei were counterstained with DAPI ( blue ) 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2 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. Data information: nuclei were counterstained with DAPI (blue)  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). Data information: nuclei were counterstained with DAPI (blue)  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. Data information: nuclei were counterstained with DAPI (blue) 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. Data information: , nuclei were counterstained with DAPI (blue) 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. Data information: nuclei were counterstained with DAPI (blue) 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.


Figure 3 A Overexpression of MOAP - 1 downregulates levels of p62 bodies in the ATG5 - depleted cells . HepG2 cells were infected with lentivirus encoding short hairpin RNA for ATG5 ( shATG5 ) or scramble control ( shCtrl ) for 48 hours before being transfected with Myc - MOAP - 1 or empty vector ( EV ) control for 24 hours and subjected to IF analysis using anti - p62 ( in red ) and anti - Myc ( in green ) antibodies . Cells expressing Myc - MOAP - 1 were marked by dashed lines . B Quantification of the p62 bodies in the ATG5 - depleted cells transfected with Myc - MOAP - 1 or empty vector . ( Upper panel ) number of p62 bodies per cell . n ≥ 3 independently plated samples . ( Lower panel ) size of p62 bodies . n = 135 , 133 and 65 bodies in the shCtrl + EV , shATG5 + EV and shATG5 + Myc - MOAP - 1 cells , respectively , from three independently plated samples . Data information : nuclei were counterstained with DAPI ( blue ) C Western blotting of the lysates from HepG2 cells infected with lentivirus encoding shATG5 or shCtrl and transfected with Myc - MOAP - 1 . D Inhibition of the auto - lysosomal degradation pathway does not prevent MOAP - 1 - mediated reduction of the p62 bodies . MOAP - 1 KO LO2 cells were pre - treated with auto - lysosome inhibitors chloroquine ( CQ ) ( 10 µM ) or BafA1 ( 1 µM ) for 3 hours , before being transfected with Myc - MOAP - 1 for 24 hours in the presence of CQ or BafA1 and subjected to immunofluorescence using anti - p62 ( in red ) and anti - Myc ( in green ) antibodies . Cells expressing Myc - MOAP - 1 were marked by dashed lines . E Quantification of the p62 bodies in the MOAP - 1 KO LO2 cells pre - treated with CQ or BafA1 before being transfected with Myc - MOAP - 1 or empty vector control . ( Upper panel ) number of p62 bodies per cell . n = 5 independently plated samples . ( Lower panel ) size of p62 bodies . n = 362 ( Ctrl , BafA1 ) , 226 ( Myc - MOAP - 1 , BafA1 ) , 544 ( Ctrl , CQ ) and 266 bodies ( Myc - MOAP - 1 , CQ ) , from three independently plated samples . Data information : nuclei were counterstained with DAPI ( blue ) , F Western blotting of lysates from MOAP - 1 KO LO2 cells pre - treated with CQ or BafA1 and transfected with Myc - MOAP - 1 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3 A Overexpression of MOAP-1 downregulates levels of p62 bodies in the ATG5-depleted cells. HepG2 cells were infected with lentivirus encoding short hairpin RNA for ATG5 (shATG5) or scramble control (shCtrl) for 48 hours before being transfected with Myc-MOAP-1 or empty vector (EV) control for 24 hours and subjected to IF analysis using anti-p62 (in red) and anti-Myc (in green) antibodies. Cells expressing Myc-MOAP-1 were marked by dashed lines. B Quantification of the p62 bodies in the ATG5-depleted cells transfected with Myc-MOAP-1 or empty vector. (Upper panel) number of p62 bodies per cell. n ≥ 3 independently plated samples. (Lower panel) size of p62 bodies. n = 135, 133 and 65 bodies in the shCtrl+EV, shATG5+EV and shATG5+Myc-MOAP-1 cells, respectively, from three independently plated samples. Data information: nuclei were counterstained with DAPI (blue) C Western blotting of the lysates from HepG2 cells infected with lentivirus encoding shATG5 or shCtrl and transfected with Myc-MOAP-1.  D Inhibition of the auto-lysosomal degradation pathway does not prevent MOAP-1-mediated reduction of the p62 bodies. MOAP-1 KO LO2 cells were pre-treated with auto-lysosome inhibitors chloroquine (CQ) (10 µM) or BafA1 (1 µM) for 3 hours, before being transfected with Myc-MOAP-1 for 24 hours in the presence of CQ or BafA1 and subjected to immunofluorescence using anti-p62 (in red) and anti-Myc (in green) antibodies. Cells expressing Myc-MOAP-1 were marked by dashed lines. E Quantification of the p62 bodies in the MOAP-1 KO LO2 cells pre-treated with CQ or BafA1 before being transfected with Myc-MOAP-1 or empty vector control. (Upper panel) number of p62 bodies per cell. n = 5 independently plated samples. (Lower panel) size of p62 bodies. n = 362 (Ctrl, BafA1), 226 (Myc-MOAP-1, BafA1), 544 (Ctrl, CQ) and 266 bodies (Myc-MOAP-1, CQ), from three independently plated samples. Data information: nuclei were counterstained with DAPI (blue),  F Western blotting of lysates from MOAP-1 KO LO2 cells pre-treated with CQ or BafA1 and transfected with Myc-MOAP-1.


Figure 4 A Larger RFP - p62 bodies in the MOAP - 1 deficient cells . WT and MOAP - 1 KO LO2 cells were transfected with expression vector encoding RFP - tagged p62 . 16 hours later , cells were subjected to live imaging analysis . Z - stacks of the confocal images were flattened as maximal intensity projections . Scale bar : 5 µm . B Quantification of size of RFP - p62 bodies in WT and MOAP - 1 KO LO2 cells . n = 129 and 161 bodies in the WT and MOAP - 1 KO cells , respectively , from three independently plated samples . * * * P < 0 . 001 , student ' s t - test . C Western analysis of lysates of the WT and MOAP - 1 KO LO2 cells transfected with expression vector encoding RFP - tagged p62 as in ( A ) . D , E RFP - p62 bodies exhibited fusion ( D ) and fission ( E ) characteristics of liquid droplets . WT and MOAP - 1 KO LO2 cells were transfected with expression vector encoding RFP - tagged p62 and 14 hours later , cells were analyzed by live - imaging to visualize the dynamics of RFP - p62 bodies . Scale bar : 2 µm F Loss of MOAP - 1 leads to slower exchange of p62 in the RFP - p62 bodies . WT and MOAP - 1 KO LO2 cells transfected with expression vector encoding RFP - tagged p62 . 14 hours later , cells were subjected to the fluorescence recovery after photobleaching ( FRAP ) assay . Scale bar : 2 µm . G Quantification of the rate of fluorescence recovery of RFP - p62 in WT and MOAP - 1 KO cells . n = 5 samples . H Overexpression of MOAP - 1 but not GFP leads to increased exchange of p62 in the RFP - p62 bodies . HepG2 cells were transfected with expression vector encoding RFP - tagged p62 and GFP - tagged MOAP - 1 or GFP only control and 14 hours later , cells were subjected to FRAP assay . Scale bar : 2 µm . I Quantification of the rate of fluorescence recovery of RFP - p62 in HepG2 cells co - expressing GFP - MOAP - 1 or GFP . n = 5 samples . J Re - introduction of MOAP - 1 disrupts the spherical structure of p62 bodies in the MOAP - 1 deficient LO2 cells . MOAP - 1 KO LO2 cells were transfected with expression vector encoding Myc - MOAP - 1 and harvested at 14 - or 24 - hours post - transfection for IF analysis using anti - p62 ( in red ) and anti - Myc ( in green ) antibodies . Insets were enlarged in the lower panels . Scale bar : 2 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4 A Larger RFP-p62 bodies in the MOAP-1 deficient cells. WT and MOAP-1 KO LO2 cells were transfected with expression vector encoding RFP-tagged p62. 16 hours later, cells were subjected to live imaging analysis. Z-stacks of the confocal images were flattened as maximal intensity projections. Scale bar: 5 µm. B Quantification of size of RFP-p62 bodies in WT and MOAP-1 KO LO2 cells. n = 129 and 161 bodies in the WT and MOAP-1 KO cells, respectively, from three independently plated samples. ***P<0.001, student's t-test.  C Western analysis of lysates of the WT and MOAP-1 KO LO2 cells transfected with expression vector encoding RFP-tagged p62 as in (A).  D, E RFP-p62 bodies exhibited fusion (D) and fission (E) characteristics of liquid droplets. WT and MOAP-1 KO LO2 cells were transfected with expression vector encoding RFP-tagged p62 and 14 hours later, cells were analyzed by live-imaging to visualize the dynamics of RFP-p62 bodies. Scale bar: 2 µm  F Loss of MOAP-1 leads to slower exchange of p62 in the RFP-p62 bodies. WT and MOAP-1 KO LO2 cells transfected with expression vector encoding RFP-tagged p62. 14 hours later, cells were subjected to the fluorescence recovery after photobleaching (FRAP) assay. Scale bar: 2 µm. G Quantification of the rate of fluorescence recovery of RFP-p62 in WT and MOAP-1 KO cells. n = 5 samples.  H Overexpression of MOAP-1 but not GFP leads to increased exchange of p62 in the RFP-p62 bodies. HepG2 cells were transfected with expression vector encoding RFP-tagged p62 and GFP-tagged MOAP-1 or GFP only control and 14 hours later, cells were subjected to FRAP assay. Scale bar: 2 µm. I Quantification of the rate of fluorescence recovery of RFP-p62 in HepG2 cells co-expressing GFP-MOAP-1 or GFP. n = 5 samples.  J Re-introduction of MOAP-1 disrupts the spherical structure of p62 bodies in the MOAP-1 deficient LO2 cells. MOAP-1 KO LO2 cells were transfected with expression vector encoding Myc-MOAP-1 and harvested at 14- or 24-hours post-transfection for IF analysis using anti-p62 (in red) and anti-Myc (in green) antibodies. Insets were enlarged in the lower panels. Scale bar: 2 µm.


Figure 5 A MOAP - 1 associates with p62 and their interaction is enhanced by DEN - mediated stress . LO2 cells transfected with Myc - MOAP - 1 were treated with or without diethylnitrosamine ( DEN ) ( 200 µM ) for 12 h and subjected to Duolink proximity ligation assay using anti - p62 and anti - Myc antibodies . Red dots represent MOAP - 1 / p62 interaction . B Alanine substitution of positively charged residues in the KYKKLR sequence of MOAP - 1 abolishes its interaction with p62 . Replacement of the positively charged residues in the KYKKLR sequence of MOAP - 1 with alanine ( referred to as the M3 mutant ) disrupts MOAP - 1 / p62 interaction , whereas alanine substitution in the neighboring region of positively charged KRRR ( M1 mutant ) and negatively charged EEE ( M2 mutant ) has no effect . LO2 cells were transfected with plasmid encoding Myc - MOAP - 1 or the indicated alanine mutants together with plasmid encoding Flag - p62 . The cells were then subjected to co - IP assay with anti - Myc antibody . D MOAP - 1 , but not its p62 - binding defective mutant , MOAP - 1 - M3 , localizes at the p62 bodies . MOAP - 1 KO LO2 cells were transfected with plasmid encoding Myc - MOAP - 1 or Myc - MOAP - 1 - M3 mutant . The transfected cells were harvested at 14 hours post - transfection , which is the time point that localization of MOAP - 1 at the p62 bodies is readily detected . IF analysis was performed on these cells using anti - p62 ( red ) and anti - Myc ( green ) antibodies . Arrowheads indicate colocalization between Myc - MOAP - 1 and p62 bodies . E MOAP - 1 but not the MOAP - 1 - M3 - mutant promotes the exchange of RFP - p62 in the p62 bodies . WT LO2 cells transfected with expression vector encoding RFP - tagged p62 and GFP - tagged MOAP - 1 or MOAP - 1 - M3 . At 14 hours post - transfection , cells were subjected to FRAP assay . Scale bar : 2 µm . F Quantification of the rate of fluorescence recovery of RFP - p62 in cells co - expressing GFP - MOAP - 1 or GFP - MOAP - 1 - M3 mutant . n = 6 samples . Error bars represent S . E . M . * P < 0 . 05 , * * P < 0 . 01 , ns , not significant , two - way ANOVA . G Western analysis of lysates of the WT LO2 cells transfected with expression vector encoding RFP - tagged p62 and GFP - tagged MOAP - 1 or MOAP - 1 - M3 as described in ( E ) . H Re - expression of MOAP - 1 , but not the MOAP - 1 - M3 mutant , reduces the levels of p62 bodies . MOAP - 1 KO LO2 cells were transfected with plasmid encoding Myc - MOAP - 1 or Myc - MOAP - 1 - M3 . The transfected cells were harvested at 24 hours post - transfection , which is the time point that MOAP - 1 was found to effectively downregulate p62 bodies , followed by IF analysis as in ( D ) . Cells expressing Myc - MOAP - 1 or Myc - MOAP - 1 - M3 were marked by dashed lines . I Quantification of the p62 bodies in the MOAP - 1 KO LO2 cells expressing Myc - MOAP - 1 and Myc - MOAP - 1 - M3 mutant . ( Left panel ) number of p62 bodies per cell . n = 7 independently plated samples . ( Right panel ) size of p62 bodies . n = 107 and 267 bodies detected in the MOAP - 1 KO LO2 cells expressing Myc - MOAP - 1 and Myc - MOAP - 1 - M3 mutant , respectively , from three independently plated samples . Error bars represent S . E . M . * * * P < 0 . 001 , Student ' s t - test . J Western blotting of MOAP - 1 KO LO2 cells transfected with plasmid encoding Myc - MOAP - 1 or Myc - MOAP - 1 - M3 mutant as described in ( H ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5 A MOAP-1 associates with p62 and their interaction is enhanced by DEN-mediated stress. LO2 cells transfected with Myc-MOAP-1 were treated with or without diethylnitrosamine (DEN) (200 µM) for 12 h and subjected to Duolink proximity ligation assay using anti-p62 and anti-Myc antibodies. Red dots represent MOAP-1/p62 interaction.  B Alanine substitution of positively charged residues in the KYKKLR sequence of MOAP-1 abolishes its interaction with p62. Replacement of the positively charged residues in the KYKKLR sequence of MOAP-1 with alanine (referred to as the M3 mutant) disrupts MOAP-1/p62 interaction, whereas alanine substitution in the neighboring region of positively charged KRRR (M1 mutant) and negatively charged EEE (M2 mutant) has no effect. LO2 cells were transfected with plasmid encoding Myc-MOAP-1 or the indicated alanine mutants together with plasmid encoding Flag-p62. The cells were then subjected to co-IP assay with anti-Myc antibody.  D MOAP-1, but not its p62-binding defective mutant, MOAP-1-M3, localizes at the p62 bodies. MOAP-1 KO LO2 cells were transfected with plasmid encoding Myc-MOAP-1 or Myc-MOAP-1-M3 mutant. The transfected cells were harvested at 14 hours post-transfection, which is the time point that localization of MOAP-1 at the p62 bodies is readily detected. IF analysis was performed on these cells using anti-p62 (red) and anti-Myc (green) antibodies. Arrowheads indicate colocalization between Myc-MOAP-1 and p62 bodies.  E MOAP-1 but not the MOAP-1-M3-mutant promotes the exchange of RFP-p62 in the p62 bodies. WT LO2 cells transfected with expression vector encoding RFP-tagged p62 and GFP-tagged MOAP-1 or MOAP-1-M3. At 14 hours post-transfection, cells were subjected to FRAP assay. Scale bar: 2 µm. F Quantification of the rate of fluorescence recovery of RFP-p62 in cells co-expressing GFP-MOAP-1 or GFP-MOAP-1-M3 mutant. n = 6 samples. Error bars represent S.E.M. *P<0.05, **P<0.01, ns, not significant, two-way ANOVA. G Western analysis of lysates of the WT LO2 cells transfected with expression vector encoding RFP-tagged p62 and GFP-tagged MOAP-1 or MOAP-1-M3 as described in (E).  H Re-expression of MOAP-1, but not the MOAP-1-M3 mutant, reduces the levels of p62 bodies. MOAP-1 KO LO2 cells were transfected with plasmid encoding Myc-MOAP-1 or Myc-MOAP-1-M3. The transfected cells were harvested at 24 hours post-transfection, which is the time point that MOAP-1 was found to effectively downregulate p62 bodies, followed by IF analysis as in (D). Cells expressing Myc-MOAP-1 or Myc-MOAP-1-M3 were marked by dashed lines. I Quantification of the p62 bodies in the MOAP-1 KO LO2 cells expressing Myc-MOAP-1 and Myc-MOAP-1-M3 mutant. (Left panel) number of p62 bodies per cell. n = 7 independently plated samples. (Right panel) size of p62 bodies. n = 107 and 267 bodies detected in the MOAP-1 KO LO2 cells expressing Myc-MOAP-1 and Myc-MOAP-1-M3 mutant, respectively, from three independently plated samples. Error bars represent S.E.M. ***P<0.001, Student's t-test.  J Western blotting of MOAP-1 KO LO2 cells transfected with plasmid encoding Myc-MOAP-1 or Myc-MOAP-1-M3 mutant as described in (H).


Figure 6 A Overexpression of MOAP - 1 inhibits dimerization of p62 . p62 KO LO2 hepatocytes were transfected with the indicated plasmids . 24 hours later , cell lysates were subjected to co - IP assay with anti - Myc antibody . PB1 deletion ( ΔPB1 ) mutant , which is impaired in p62 oligomerization , is included as a control . TCL , total cell lysates . B Dimerization of p62 is enhanced in the MOAP - 1 deficient cells . WT and MOAP - 1 KO LO2 cells were transfected with plasmids encoding Flag - tagged and Myc - tagged p62 or empty vector . 24 h post - transfection , cells were harvested , lysed and the cell lysates were subjected to co - IP assay with anti - Myc antibody . C Quantification of the levels of Flag - p62 bound to Myc - p62 in the co - IP experiment as described in ( B ) by densitometric analysis . D Re - expression of MOAP - 1 , but not the p62 - binding defective mutant , MOAP - 1 - M3 , reduces p62 oligomerization in the MOAP - 1 KO LO2 hepatocytes . WT and MOAP - 1 KO LO2 cells were transfected with plasmids encoding His - tagged p62 and Myc - MOAP - 1 or Myc - MOAP - 1 - M3 and 24 h later , subjected to His - pull down and cross - linking using DSSO . E Quantification of the levels of p62 oligomer depicted in ( D ) by densitometric analysis . F The region of p62 spanning the PB1 and ZZ domains are required for mediating its interaction with MOAP - 1 . p62 KO LO2 cells were used in the analysis . Cells were transfected with plasmid encoding Myc - p62 or the indicated deletion mutants and Flag - MOAP - 1 for 24 hours . The transfected cells were subjected to co - IP assay with anti - Myc antibody . ( Lower panel ) Schematics depicting the domains in p62 . PB1 , Phox and Bem1p ; ZZ , Zinc Finger ; TB , TRAF6 - binding domain ; KIR , Keap1 interacting region ; LIR , LC3 interacting region ; UBA , ubiquitin - associated domain . G The fragment encompassing the PB1 and ZZ domains of p62 is sufficient for mediating its interaction with MOAP - 1 . p62 KO LO2 cells were transfected with plasmid encoding Myc - p62 or the indicated fragments and Flag - MOAP - 1 . 24 hours later , the transfected cells were subjected to co - IP assay with anti - Myc antibody . H MOAP - 1 binds and inhibits homodimerization of the PB1 - ZZ fragment . p62 KO LO2 cells were transfected with plasmid encoding Myc and Flag - tagged PB1 - ZZ in the absence of presence of HA - tagged MOAP - 1 for 24 h and subjected to co - IP assay with anti - Myc antibody .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6 A Overexpression of MOAP-1 inhibits dimerization of p62. p62 KO LO2 hepatocytes were transfected with the indicated plasmids. 24 hours later, cell lysates were subjected to co-IP assay with anti-Myc antibody. PB1 deletion (ΔPB1) mutant, which is impaired in p62 oligomerization, is included as a control. TCL, total cell lysates.  B Dimerization of p62 is enhanced in the MOAP-1 deficient cells. WT and MOAP-1 KO LO2 cells were transfected with plasmids encoding Flag-tagged and Myc-tagged p62 or empty vector. 24 h post-transfection, cells were harvested, lysed and the cell lysates were subjected to co-IP assay with anti-Myc antibody. C Quantification of the levels of Flag-p62 bound to Myc-p62 in the co-IP experiment as described in (B) by densitometric analysis.  D Re-expression of MOAP-1, but not the p62-binding defective mutant, MOAP-1-M3, reduces p62 oligomerization in the MOAP-1 KO LO2 hepatocytes. WT and MOAP-1 KO LO2 cells were transfected with plasmids encoding His-tagged p62 and Myc-MOAP-1 or Myc-MOAP-1-M3 and 24 h later, subjected to His-pull down and cross-linking using DSSO. E Quantification of the levels of p62 oligomer depicted in (D) by densitometric analysis.  F The region of p62 spanning the PB1 and ZZ domains are required for mediating its interaction with MOAP-1. p62 KO LO2 cells were used in the analysis. Cells were transfected with plasmid encoding Myc-p62 or the indicated deletion mutants and Flag-MOAP-1 for 24 hours. The transfected cells were subjected to co-IP assay with anti-Myc antibody. (Lower panel) Schematics depicting the domains in p62. PB1, Phox and Bem1p; ZZ, Zinc Finger; TB, TRAF6-binding domain; KIR, Keap1 interacting region; LIR, LC3 interacting region; UBA, ubiquitin-associated domain.  G The fragment encompassing the PB1 and ZZ domains of p62 is sufficient for mediating its interaction with MOAP-1. p62 KO LO2 cells were transfected with plasmid encoding Myc-p62 or the indicated fragments and Flag-MOAP-1. 24 hours later, the transfected cells were subjected to co-IP assay with anti-Myc antibody.  H MOAP-1 binds and inhibits homodimerization of the PB1-ZZ fragment. p62 KO LO2 cells were transfected with plasmid encoding Myc and Flag-tagged PB1-ZZ in the absence of presence of HA-tagged MOAP-1 for 24 h and subjected to co-IP assay with anti-Myc antibody.


Figure 7 A Increased recruitment of Keap1 to p62 bodies in the MOAP - 1 deficient LO2 cells . WT and MOAP - 1 KO LO2 cells were subjected to IF analysis with anti - Keap1 ( green ) and anti - p62 ( red ) antibodies . Insets in the upper panels are enlarged in the lower panels . Nuclei were counterstained with DAPI ( in blue ) . Scale bar : 5 µm . B MOAP - 1 deficiency promotes p62 / Keap1 interaction . WT , MOAP - 1 KO and p62 KO LO2 cells were subjected to co - IP assay using anti - p62 antibody . TCL , total cell lysates . C Quantification of the levels of Keap1 bound to p62 in the co - IP experiment as described in ( B ) by densitometric analysis . D Diminished Keap1 / Nrf2 interaction in the absence of MOAP - 1 . WT and MOAP - 1 KO LO2 cells were subjected to Duolink PLA using anti - Keap1 and anti - Nrf2 antibodies . Red dots represent Keap1 / Nrf2 interaction . Nuclei were counterstained with DAPI ( in blue ) . Scale bar : 10 µm . E Quantification of the Duolink signals depicted in ( D ) . Duolink signals , which appear as red dots , were quantified by ImageJ analysis and normalized to the number of nuclei . F Loss of MOAP - 1 results in marked elevation of nuclear Nrf2 . WT and MOAP - 1 KO LO2 cells were subjected to IF analysis with anti - Nrf2 ( in green ) and anti - p62 ( in red ) antibodies . Scale bar : 10 µm . G Quantification of nuclear levels of Nrf2 in ( F ) . The mean gray values of Nrf2 fluorescence signals were quantified by ImageJ analysis . H MOAP - 1 deficiency promotes activation of Nrf2 signaling . Transcript levels of downstream targets of Nrf2 ( i . e . HMOX - 1 , NQO1 , SLC7A11 , GCLC and G6PD ) in WT and MOAP - 1 KO LO2 cells were determined by RT - PCR and normalized to GAPDH . I Elevated transcriptional activity of Nrf2 in the MOAP - 1 deficient cells is mediated through p62 . Transcript levels of Nrf2 target genes ( HMOX - 1 and NQO1 ) in WT , MOAP - 1 KO and MOAP - 1 / p62 DKO LO2 cells were determined by RT - PCR and normalized to GAPDH . J Re - expression of MOAP - 1 and MOAP - 1 - L120E , but not the p62 binding defective mutant of MOAP - 1 , MOAP - 1 - M3 , inhibits p62 / Keap1 interaction in the MOAP - 1 deficient cells . MOAP - 1 KO LO2 cells were transfected with plasmid encoding Myc - tagged MOAP - 1 , L120E or M3 . The transfected cells were then subjected to co - IP assay with anti - p62 antibody . K Re - introduction of MOAP - 1 and MOAP - 1 - L120E , but not the p62 binding defective MOAP - 1 - M3 , reduces Nrf2 transcriptional activity in the MOAP - 1 KO cells . MOAP - 1 KO LO2 cells were transfected with Myc - tagged MOAP - 1 , MOAP - 1 - L120E or MOAP - 1 - M3 and ARE - Nrf2 - firely and renilla luciferase reporter constructs for overnight and subjected to luciferase assay .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7 A Increased recruitment of Keap1 to p62 bodies in the MOAP-1 deficient LO2 cells. WT and MOAP-1 KO LO2 cells were subjected to IF analysis with anti-Keap1 (green) and anti-p62 (red) antibodies. Insets in the upper panels are enlarged in the lower panels. Nuclei were counterstained with DAPI (in blue). Scale bar: 5 µm.  B MOAP-1 deficiency promotes p62/Keap1 interaction. WT, MOAP-1 KO and p62 KO LO2 cells were subjected to co-IP assay using anti-p62 antibody. TCL, total cell lysates. C Quantification of the levels of Keap1 bound to p62 in the co-IP experiment as described in (B) by densitometric analysis.  D Diminished Keap1/Nrf2 interaction in the absence of MOAP-1. WT and MOAP-1 KO LO2 cells were subjected to Duolink PLA using anti-Keap1 and anti-Nrf2 antibodies. Red dots represent Keap1/Nrf2 interaction. Nuclei were counterstained with DAPI (in blue). Scale bar: 10 µm. E Quantification of the Duolink signals depicted in (D). Duolink signals, which appear as red dots, were quantified by ImageJ analysis and normalized to the number of nuclei.  F Loss of MOAP-1 results in marked elevation of nuclear Nrf2. WT and MOAP-1 KO LO2 cells were subjected to IF analysis with anti-Nrf2 (in green) and anti-p62 (in red) antibodies. Scale bar: 10 µm. G Quantification of nuclear levels of Nrf2 in (F). The mean gray values of Nrf2 fluorescence signals were quantified by ImageJ analysis.  H MOAP-1 deficiency promotes activation of Nrf2 signaling. Transcript levels of downstream targets of Nrf2 (i.e. HMOX-1, NQO1, SLC7A11, GCLC and G6PD) in WT and MOAP-1 KO LO2 cells were determined by RT-PCR and normalized to GAPDH.  I Elevated transcriptional activity of Nrf2 in the MOAP-1 deficient cells is mediated through p62. Transcript levels of Nrf2 target genes (HMOX-1 and NQO1) in WT, MOAP-1 KO and MOAP-1/p62 DKO LO2 cells were determined by RT-PCR and normalized to GAPDH.  J Re-expression of MOAP-1 and MOAP-1-L120E, but not the p62 binding defective mutant of MOAP-1, MOAP-1-M3, inhibits p62/Keap1 interaction in the MOAP-1 deficient cells. MOAP-1 KO LO2 cells were transfected with plasmid encoding Myc-tagged MOAP-1, L120E or M3. The transfected cells were then subjected to co-IP assay with anti-p62 antibody.  K Re-introduction of MOAP-1 and MOAP-1-L120E, but not the p62 binding defective MOAP-1-M3, reduces Nrf2 transcriptional activity in the MOAP-1 KO cells. MOAP-1 KO LO2 cells were transfected with Myc-tagged MOAP-1, MOAP-1-L120E or MOAP-1-M3 and ARE-Nrf2-firely and renilla luciferase reporter constructs for overnight and subjected to luciferase assay.


Figure 8 A High level of p62 bodies in the liver of the MOAP - 1 KO mice subjected to acute diethylnitrosamine ( DEN ) treatment . Eight - week - old male WT and MOAP - 1 KO mice were injected with an acute dose of DEN ( 100 µg / g body weight ) . Mice were sacrificed at 48 hours post - injection . Livers were harvested for immunohistochemistry ( IHC ) analysis with anti - p62 antibody ( in red ) . Nuclei were counterstained with DAPI ( in blue ) . Insets in the upper panels are enlarged in the lower panels . Scale bar : 20 µm . B Loss of MOAP - 1 results in higher levels of Nrf2 and its downstream target protein , HMOX - 1 , in liver upon acute DEN treatment . Lysates of the livers harvested from mice subjected to acute DEN treatment as described in ( A ) were prepared in RIPA lysis buffer containing 1 % SDS and analyzed by Western blotting . Actin as loading control . C MOAP - 1 deficiency elevates levels of p62 protein in the detergent - insoluble fractions of the liver lysates from mice subjected to acute DEN treatment . Livers from mice injected as described in ( A ) were lysed in 1 % Triton - X lysis buffer , separated into detergent - soluble ( S ) and insoluble ( I ) fractions and analyzed by Western blotting . D High levels of p62 bodies detected in the tumors of the MOAP - 1 KO mice . Tumor sections from livers dissected from the 8 . 5 - month - old male WT and MOAP - 1 KO mice injected with a single dose of DEN ( 25 µg / g body weight ) at 15 - day - old were subjected to IHC analysis with anti - p62 antibody ( in red ) . Nuclear were stained with DAPI ( in blue ) . Insets were enlarged in the bottom right . Scale bar : 20 µm . E Quantification of the p62 bodies in the WT and MOAP - 1 KO tumors . ( Left panel ) number of p62 bodies per cell . n = 7 tumors from the livers of WT and MOAP - 1 KO mice . ( Right panel ) size of p62 bodies . n = 456 and 1211 bodies detected in the WT and MOAP - 1 KO tumors , respectively , from three independent samples . Error bar represents S . E . M . * P < 0 . 05 , * * * P < 0 . 001 , student ' s t - test . F Increased levels of Nrf2 and its downstream target protein , HMOX - 1 , in the MOAP - 1 deficient tumors . While p62 protein is upregulated in the tumors compared to the adjacent non - tumor tissues in both WT and MOAP - 1 KO livers , no significant difference is observed in the p62 protein levels between the WT and MOAP - 1 KO tumors . Lysates of the tumors and non - tumor parts of the livers from mice injected with DEN as described in ( D ) were prepared in RIPA lysis buffer containing 1 % SDS and subjected to Western blotting . Actin as loading control . P , PBS injected control ; N , non - tumor ; T , tumor . G Upregulation of mRNA levels of HMOX - 1 and NQO1 in the MOAP - 1 deficient tumors . Livers harvested from the mice injected with PBS or DEN as described in ( D ) were subjected to RT - PCR analysis . n = 3 WT and 3 MOAP - 1 KO mice injected with the PBS control , and 8 WT and 9 MOAP - 1 KO mice injected with DEN . Error bar represents S . E . M . * * P < 0 . 01 , student ' s t - test . H MOAP - 1 deficient mice bear higher tumor burden in the DEN - mediated liver cancer model . Representative images of liver tumors dissected from the 8 . 5 - month - old male WT and MOAP - 1 KO mice injected with a single dose of DEN ( 25 µg / g body weight ) at 15 - day - old . Scale bar : 5 mm . I Measurements of the number , maximum diameter of the tumor nodules and total liver weight from WT and MOAP - 1 KO mice ( n = 13 and 14 respectively ) injected with DEN as described in ( H ) . Error bar represents S . E . M . * * P < 0 . 01 , * * * P < 0 . 001 , student ' s t - test . MOAP - 1 deficiency promotes tumor initiation . measurements of the number , diameter of the largest nodules and total liver weight of the 4 . 5 - month - old WT and MOAP - 1 KO mice ( n = 18 and 23 respectively ) injected with a single dose of DEN ( 25 µg / g body weight ) at 15 - day - old .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8 A High level of p62 bodies in the liver of the MOAP-1 KO mice subjected to acute diethylnitrosamine (DEN) treatment. Eight-week-old male WT and MOAP-1 KO mice were injected with an acute dose of DEN (100 µg/g body weight). Mice were sacrificed at 48 hours post-injection. Livers were harvested for immunohistochemistry (IHC) analysis with anti-p62 antibody (in red). Nuclei were counterstained with DAPI (in blue). Insets in the upper panels are enlarged in the lower panels. Scale bar: 20 µm.  B Loss of MOAP-1 results in higher levels of Nrf2 and its downstream target protein, HMOX-1, in liver upon acute DEN treatment. Lysates of the livers harvested from mice subjected to acute DEN treatment as described in (A) were prepared in RIPA lysis buffer containing 1% SDS and analyzed by Western blotting. Actin as loading control.  C MOAP-1 deficiency elevates levels of p62 protein in the detergent-insoluble fractions of the liver lysates from mice subjected to acute DEN treatment. Livers from mice injected as described in (A) were lysed in 1% Triton-X lysis buffer, separated into detergent-soluble (S) and insoluble (I) fractions and analyzed by Western blotting.  D High levels of p62 bodies detected in the tumors of the MOAP-1 KO mice. Tumor sections from livers dissected from the 8.5-month-old male WT and MOAP-1 KO mice injected with a single dose of DEN (25 µg/g body weight) at 15-day-old were subjected to IHC analysis with anti-p62 antibody (in red). Nuclear were stained with DAPI (in blue). Insets were enlarged in the bottom right. Scale bar: 20 µm. E Quantification of the p62 bodies in the WT and MOAP-1 KO tumors. (Left panel) number of p62 bodies per cell. n = 7 tumors from the livers of WT and MOAP-1 KO mice. (Right panel) size of p62 bodies. n = 456 and 1211 bodies detected in the WT and MOAP-1 KO tumors, respectively, from three independent samples. Error bar represents S.E.M. *P<0.05, ***P<0.001, student's t-test.  F Increased levels of Nrf2 and its downstream target protein, HMOX-1, in the MOAP-1 deficient tumors. While p62 protein is upregulated in the tumors compared to the adjacent non-tumor tissues in both WT and MOAP-1 KO livers, no significant difference is observed in the p62 protein levels between the WT and MOAP-1 KO tumors. Lysates of the tumors and non-tumor parts of the livers from mice injected with DEN as described in (D) were prepared in RIPA lysis buffer containing 1% SDS and subjected to Western blotting. Actin as loading control. P, PBS injected control; N, non-tumor; T, tumor.  G Upregulation of mRNA levels of HMOX-1 and NQO1 in the MOAP-1 deficient tumors. Livers harvested from the mice injected with PBS or DEN as described in (D) were subjected to RT-PCR analysis. n = 3 WT and 3 MOAP-1 KO mice injected with the PBS control, and 8 WT and 9 MOAP-1 KO mice injected with DEN. Error bar represents S.E.M. **P<0.01, student's t-test.  H MOAP-1 deficient mice bear higher tumor burden in the DEN-mediated liver cancer model. Representative images of liver tumors dissected from the 8.5-month-old male WT and MOAP-1 KO mice injected with a single dose of DEN (25 µg/g body weight) at 15-day-old. Scale bar: 5 mm. I Measurements of the number, maximum diameter of the tumor nodules and total liver weight from WT and MOAP-1 KO mice (n=13 and 14 respectively) injected with DEN as described in (H). Error bar represents S.E.M. **P<0.01, ***P<0.001, student's t-test.  MOAP-1 deficiency promotes tumor initiation. measurements of the number, diameter of the largest nodules and total liver weight of the 4.5-month-old WT and MOAP-1 KO mice (n=18 and 23 respectively) injected with a single dose of DEN (25 µg/g body weight) at 15-day-old.


Figure 1 ( B ) Confocal images of HeLa cells transfected with EGFP - ORP5 or EGFP - ORP8 showing variable expression levels . Arrows indicate ORP5 and ORP8 localization to cortical ER ( cER ) . Scale bar , 10 μm . ( C - D ) Electron microscopy of ultrathin cryosections of HeLa cells transfected with EGFP - ORP5 or EGFP - ORP8 and immunogold stained with anti - GFP ( 15 nm gold ) to detect ORP5 or ORP8 and anti - PDI ( 10 nm gold ) to label the ER lumen . ORP5 and ORP8 localize at ER - PM ( arrows ) and ER - mitochondria contact sites ( arrowheads ) . m , mitochondria ; cER , cortical ER . Scale bar 200nm . ( E - F ) Quantification of the IEM labelling for EGFP - ORP5 , EGFP - ORP8 ( E ) and GFP - Sec61β ( F ) in transfected HeLa cells . Results are presented as the percentage of the total number of gold particles ( 800 per condition , n = 35 - 45 cells ) for ORP5 , ORP8 and Sec61β in the indicated compartments ( non - cER , non - cortical ER ; ER - PM , ER - PM contact sites ; ER - mito , ER - mitochondria contact sites . % gold particles ± SEM . Data are the mean of three independent replicates . * P < 0 , 001 compared to non - cER and ER - mito [ EGFP - ORP5 ] and to ER - PM and ER - mito [ EGFP - ORP8 and GFP - Sec61β ] , # P < 0 , 001 compared to ER - PM and ER - mito [ GFP - Sec61β ] ) . ( G ) Representative electron microscopy of mitochondria and ( H ) quantification of the percentage of ER - mitochondria contact sites ( ER - mito contact ) per mitochondria of HeLa cells expressing EGFP - ORP5 , EGFP - ORP8 together with HRP - KDEL , to visualize the ER . Control ( Ctrl ) consisted of HeLa cells overexpressing only HRP - KDEL . ( I ) Crude mitochondria , mitochondria and MAM fractions were purified from HeLa cells and equal amount of protein ( 25g ) from each fraction were loaded on a 4 - 20 % gradient SDS - PAGE gel and immunoblotted using anti - ORP5 , anti - ORP8 and anti - IP3R - 3 ( MAM protein ) , anti - PDI ( ER protein ) and anti - cytochrome c ( mitochondrial protein ) . TC , Total cell ; MAM , mitochondria - associated ER membrane .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(B) Confocal images of HeLa cells transfected with EGFP-ORP5 or EGFP-ORP8 showing variable expression levels. Arrows indicate ORP5 and ORP8 localization to cortical ER (cER). Scale bar, 10 μm.(C-D) Electron microscopy of ultrathin cryosections of HeLa cells transfected with EGFP-ORP5 or EGFP-ORP8 and immunogold stained with anti-GFP (15 nm gold) to detect ORP5 or ORP8 and anti-PDI (10 nm gold) to label the ER lumen. ORP5 and ORP8 localize at ER-PM (arrows) and ER-mitochondria contact sites (arrowheads). m, mitochondria; cER, cortical ER. Scale bar 200nm. (E-F) Quantification of the IEM labelling for EGFP-ORP5, EGFP-ORP8 (E) and GFP-Sec61β (F) in transfected HeLa cells. Results are presented as the percentage of the total number of gold particles (800 per condition, n=35-45 cells) for ORP5, ORP8 and Sec61β in the indicated compartments (non-cER, non-cortical ER; ER-PM, ER-PM contact sites; ER-mito, ER-mitochondria contact sites. % gold particles ±SEM. Data are the mean of three independent replicates. *P<0,001 compared to non-cER and ER-mito [EGFP-ORP5] and to ER-PM and ER-mito [EGFP-ORP8 and GFP-Sec61β], #P<0,001 compared to ER-PM and ER-mito [GFP-Sec61β]).(G) Representative electron microscopy of mitochondria and (H) quantification of the percentage of ER-mitochondria contact sites (ER-mito contact) per mitochondria of HeLa cells expressing EGFP-ORP5, EGFP-ORP8 together with HRP-KDEL, to visualize the ER. Control (Ctrl) consisted of HeLa cells overexpressing only HRP-KDEL.(I) Crude mitochondria, mitochondria and MAM fractions were purified from HeLa cells and equal amount of protein (25g) from each fraction were loaded on a 4-20% gradient SDS-PAGE gel and immunoblotted using anti-ORP5, anti-ORP8 and anti-IP3R-3 (MAM protein), anti-PDI (ER protein) and anti- cytochrome c (mitochondrial protein). TC,Total cell; MAM, mitochondria-associated ER membrane .


Figure 2 ( A ) Confocal images of HeLa cells transfected with EGFP - ORP5 , EGFP - ORP8 , or GFP - Sec61β together with PTPIP51 - HA and immunostained using HA antibody to detect PTPIP51 . Scale bar , 10 μm . ( B ) Electron microscopy of ultrathin cryosections of HeLa cells co - transfected with EGFP - ORP5 and PTPIP51 - HA and immunogold stained with anti - GFP ( 15 nm gold ) and anti - HA ( 10 nm gold ) showing EGFP - ORP5 localization at ER - mitochondria contacts ( arrows ) . Arrowheads indicate the highly expanded ER membrane juxtaposed to mitochondria . Scale bar 200 nm . ( C ) Quantification of the IEM labelling for EGFP - ORP5 , EGFP - ORP8 and GFP - Sec61β in HeLa cells co - transfected with PTPIP51 - HA . Results are presented as the percentage of the total number of gold particles ( 800 per condition , n = 30 - 35 cells ) for ORP5 , ORP8 and Sec61β in the indicated compartments ( ER , reticular ER ; ER - mito MCS , ER - mitochondria membrane contact sites ; ER - PM MCS , ER - PM membrane contact sites ) . % gold particles ± SEM . Data are representative of three independent replicates . * * P < 0 , 001 . ( D ) Confocal images of HeLa cells transfected with EGFP - ORP5 or EGFP - ORP8 together with PTPIP51 - HA and RFP - Sec61β and immunostained using HA antibody to detect PTPIP51 . Scale bar , 10 μm . ( E ) Representative EM images of ER - mitochondria contacts in the indicated overexpression conditions . ( F ) Quantification of the percentage of ER - mitochondria contact sites ( ER - mito contact ) per mitochondria of HeLa cells transfected with HRP - KDEL ( Ctrl ) or co - transfected with PTPIP51 - HA and EGFP or PTPIP51 - HA and EGFP - ORP5 or PTPIP51 - HA and EGFP - ORP8 ( n = 30 cells and 1082 - 1223 mitochondria ) . % ER - mitochondria contact sites ± SEM . Data are representative of three independent replicates . * * P < 0 , 001 , * P < 0 , 05 . ( G ) EGFP , EGFP - ORP5 or EGFP - ORP8 were immuno - precipitated from lysates of HeLa cells co - expressing PTPIP51 - HA and treated with control ( siCtrl ) or VAPA and VAPB ( SiVAPA / B ) siRNAs and analyzed by western blot using antibodies against GFP ( ORPs ) , HA ( PTPIP51 ) , VAPA , VAPB and tubulin .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(A) Confocal images of HeLa cells transfected with EGFP-ORP5, EGFP-ORP8, or GFP-Sec61β together with PTPIP51-HA and immunostained using HA antibody to detect PTPIP51. Scale bar, 10 μm.(B) Electron microscopy of ultrathin cryosections of HeLa cells co-transfected with EGFP-ORP5 and PTPIP51-HA and immunogold stained with anti-GFP (15 nm gold) and anti-HA (10 nm gold) showing EGFP-ORP5 localization at ER-mitochondria contacts (arrows). Arrowheads indicate the highly expanded ER membrane juxtaposed to mitochondria. Scale bar 200 nm. (C) Quantification of the IEM labelling for EGFP-ORP5, EGFP-ORP8 and GFP-Sec61β in HeLa cells co-transfected with PTPIP51-HA. Results are presented as the percentage of the total number of gold particles (800 per condition, n=30-35 cells) for ORP5, ORP8 and Sec61β in the indicated compartments (ER, reticular ER; ER-mito MCS, ER-mitochondria membrane contact sites; ER-PM MCS, ER-PM membrane contact sites). % gold particles ±SEM. Data are representative of three independent replicates. **P<0,001.(D) Confocal images of HeLa cells transfected with EGFP-ORP5 or EGFP-ORP8 together with PTPIP51-HA and RFP-Sec61β and immunostained using HA antibody to detect PTPIP51. Scale bar, 10 μm.(E) Representative EM images of ER-mitochondria contacts in the indicated overexpression conditions. (F) Quantification of the percentage of ER-mitochondria contact sites (ER-mito contact) per mitochondria of HeLa cells transfected with HRP-KDEL (Ctrl) or co-transfected with PTPIP51-HA and EGFP or PTPIP51-HA and EGFP-ORP5 or PTPIP51-HA and EGFP-ORP8 (n=30 cells and 1082-1223 mitochondria). % ER-mitochondria contact sites ±SEM. Data are representative of three independent replicates. **P<0,001, *P<0,05.(G) EGFP, EGFP-ORP5 or EGFP-ORP8 were immuno-precipitated from lysates of HeLa cells co-expressing PTPIP51-HA and treated with control (siCtrl) or VAPA and VAPB (SiVAPA/B) siRNAs and analyzed by western blot using antibodies against GFP (ORPs), HA (PTPIP51), VAPA, VAPB and tubulin.


Figure 3 ( A ) Confocal images of HeLa cells transfected with EGFP - ORP5 , EGFP - ORP5PH or EGFP - ORP5ORD and immunostained using anti - TOM20 antibody to visualize mitochondria . Scale bar , 10 μm . ( B ) Confocal images of HeLa cells transfected with the EGFP - tagged ORD domain of ORP5 ( EGFP - ORD ORP5 ) and immunostained using anti - TOM20 antibody ( upper panels ) or co - transfected with PTPIP51 - HA and immunostained using HA antibody to detect PTPIP51 . Scale bar , 10 μm . ( C - D ) Confocal images of HeLa cells transfected with EGFP - ORP5PH or EGFP - ORP5ORD ( C ) together with PTPIP51 - HA and immunostained using HA antibody to detect PTPIP51 . Scale bar , 10 μm . ( C - D ) Confocal images of HeLa cells transfected with EGFP - ORP5 , EGFP - ORP5L389D or EGFP - ORP8L425D ( D ) together with PTPIP51 - HA and immunostained using HA antibody to detect PTPIP51 . Scale bar , 10 μm . ( E ) EGFP - ORP5 , EGFP - ORP8 , EGFP - ORP5L389D , EGFP - ORP8L425D , GFP - Sec61β or EGFP alone were immunoprecipitated from lysates of HeLa cells co - expressing PTPIP51 - HA and analyzed by western blot with antibodies against GFP and HA .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(A) Confocal images of HeLa cells transfected with EGFP-ORP5, EGFP-ORP5PH or EGFP-ORP5ORD and immunostained using anti-TOM20 antibody to visualize mitochondria. Scale bar, 10 μm.(B) Confocal images of HeLa cells transfected with the EGFP-tagged ORD domain of ORP5 (EGFP-ORD ORP5) and immunostained using anti-TOM20 antibody (upper panels) or co-transfected with PTPIP51-HA and immunostained using HA antibody to detect PTPIP51. Scale bar, 10 μm.(C-D) Confocal images of HeLa cells transfected with EGFP-ORP5PH or EGFP-ORP5ORD (C) together with PTPIP51-HA and immunostained using HA antibody to detect PTPIP51. Scale bar, 10 μm.(C-D) Confocal images of HeLa cells transfected with EGFP-ORP5, EGFP-ORP5L389D or EGFP-ORP8L425D (D) together with PTPIP51-HA and immunostained using HA antibody to detect PTPIP51. Scale bar, 10 μm.(E) EGFP-ORP5, EGFP-ORP8, EGFP-ORP5L389D, EGFP-ORP8L425D, GFP-Sec61β or EGFP alone were immunoprecipitated from lysates of HeLa cells co-expressing PTPIP51-HA and analyzed by western blot with antibodies against GFP and HA.


Figure 4 ( A ) ORP5 , ORP8 and actin levels of HRP - KDEL - expressing HeLa cells treated with Ctrl siRNAs ( siCtrl ) or siRNAs against ORP5 ( siORP5 ) or ORP8 ( siORP8 ) . ( B ) Electron microscopy of HRP - KDEL - expressing HeLa cells treated with Ctrl siRNAs or siRNAs against ORP5 or ORP8 . Red arrows indicate ER - PM contact sites . Scale bar , 2 μm . Insets show representative mitochondria and red arrows in the insets indicate ER - mitochondria contacts . Inset scale bar , 500nm . ( C ) Representative EM micrographs showing the morphology of mitochondria in siCtrl , siORP5 and siORP8 - treated cells . Scale bar , 1 μm . ( D ) Quantifications of the number of mitochondria with altered morphology in the indicated siRNA conditions . % altered mitochondria ± SEM , n = 25 cells and 632 - 710 mitochondria . * P < 0 , 001 compared to siCtrl . ( E ) Mitochondrial oxygen consumption rate ( OCR ) measured in Ctrl and ORP5 siRNA transfected cells . OCR trace was obtained by sequential measurement of basal OCR ( OCRBAS ) , OCR after the addition of oligomycin ( OM ) and OCR after addition of antimycin A ( AA ) . Note the reduced basal OCR ( OCRBAS ) compared to ctrl siRNA cells . Error bars denote ± SEM . Data are the mean of 4 independent repeats ( n = 4 ) , * P < 0 , 05 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(A) ORP5, ORP8 and actin levels of HRP-KDEL-expressing HeLa cells treated with Ctrl siRNAs (siCtrl) or siRNAs against ORP5 (siORP5) or ORP8 (siORP8).(B) Electron microscopy of HRP-KDEL-expressing HeLa cells treated with Ctrl siRNAs or siRNAs against ORP5 or ORP8. Red arrows indicate ER-PM contact sites. Scale bar, 2 μm. Insets show representative mitochondria and red arrows in the insets indicate ER-mitochondria contacts. Inset scale bar, 500nm.(C) Representative EM micrographs showing the morphology of mitochondria in siCtrl, siORP5 and siORP8-treated cells. Scale bar, 1 μm. (D) Quantifications of the number of mitochondria with altered morphology in the indicated siRNA conditions. % altered mitochondria ±SEM, n=25 cells and 632-710 mitochondria. *P<0,001 compared to siCtrl.(E) Mitochondrial oxygen consumption rate (OCR) measured in Ctrl and ORP5 siRNA transfected cells. OCR trace was obtained by sequential measurement of basal OCR (OCRBAS), OCR after the addition of oligomycin (OM) and OCR after addition of antimycin A (AA). Note the reduced basal OCR (OCRBAS) compared to ctrl siRNA cells. Error bars denote ±SEM. Data are the mean of 4 independent repeats (n=4), *P<0,05.


Figure 1 ( C ) Representative traces obtained with ADP • BeFx . The upper FRET trace was calculated from the middle traces obtained by exciting the donor fluorophore and measuring both donor ( green ) and acceptor ( red ) fluorescence . The lowest trace was obtained by exciting the acceptor fluorophore directly . The arrow indicates a bleaching event . ( D ) Distribution of FRET values determined from 97 traces as in ( C ) fit with a Gaussian model ( black curve ) . ( E ) As in ( C ) , but in the presence of ATP . Periods in which a fluorescently labeled SecA molecule is bound are indicated by grey shading . ( F ) As in ( D ) , but with ATP ( 257 traces ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(C) Representative traces obtained with ADP•BeFx. The upper FRET trace was calculated from the middle traces obtained by exciting the donor fluorophore and measuring both donor (green) and acceptor (red) fluorescence. The lowest trace was obtained by exciting the acceptor fluorophore directly. The arrow indicates a bleaching event. (D) Distribution of FRET values determined from 97 traces as in (C) fit with a Gaussian model (black curve).(E) As in (C), but in the presence of ATP. Periods in which a fluorescently labeled SecA molecule is bound are indicated by grey shading. (F) As in (D), but with ATP (257 traces).


Figure 2 ( A ) A representative FRET trace ( blue line ) was obtained as in Fig 1 , and fit with a hidden Markov model ( black dashed line ) . ( B ) Traces as in ( A ) obtained in the presence of different nucleotides , were used to determine the number of states best fit by the Markov model . ( C ) Transition density plot of idealized ATP FRET states obtained in ( B ) . ( D ) The distributions of dwell times of the low FRET states observed in ATP were fit with a single exponential ( 1500 low FRET states ) . The inset shows average dwell time and error , defined as the standard error based on the number of traces . ( E ) As in ( D ) , but with high FRET ( 1656 high FRET states ) . ( F ) Representative traces obtained with ATPγS . The upper FRET trace was calculated from the middle traces obtained by exciting the donor fluorophore and measuring both donor ( green ) and acceptor ( red ) fluorescence . The lowest trace was obtained by exciting the acceptor fluorophore directly . The arrow indicates a bleaching event . ( G ) Distribution of FRET values determined from 168 traces as in ( D ) fit with a Gaussian model ( black curve ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(A) A representative FRET trace (blue line) was obtained as in Fig 1, and fit with a hidden Markov model (black dashed line).(B) Traces as in (A) obtained in the presence of different nucleotides, were used to determine the number of states best fit by the Markov model. (C) Transition density plot of idealized ATP FRET states obtained in (B).(D) The distributions of dwell times of the low FRET states observed in ATP were fit with a single exponential (1500 low FRET states). The inset shows average dwell time and error, defined as the standard error based on the number of traces. (E) As in (D), but with high FRET (1656 high FRET states).(F) Representative traces obtained with ATPγS. The upper FRET trace was calculated from the middle traces obtained by exciting the donor fluorophore and measuring both donor (green) and acceptor (red) fluorescence. The lowest trace was obtained by exciting the acceptor fluorophore directly. The arrow indicates a bleaching event. (G) Distribution of FRET values determined from 168 traces as in (D) fit with a Gaussian model (black curve).


Figure 3 ( C ) Representative traces obtained with ADP • BeFx . The upper FRET trace was calculated from the middle traces obtained by exciting the donor fluorophore and measuring both donor ( green ) and acceptor ( red ) fluorescence . The lowest trace was obtained by exciting the acceptor fluorophore directly . The arrow indicates a bleaching event . ( D ) Distribution of FRET values determined from 197 traces as in ( B ) fit with a Gaussian model ( black curve ) . ( E ) As in ( C ) , but with ATP . Periods in which a fluorescently labeled SecA molecule is bound are indicated by grey shading . ( F ) As in ( D ) , but with ATP ( 228 traces ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(C) Representative traces obtained with ADP•BeFx. The upper FRET trace was calculated from the middle traces obtained by exciting the donor fluorophore and measuring both donor (green) and acceptor (red) fluorescence. The lowest trace was obtained by exciting the acceptor fluorophore directly. The arrow indicates a bleaching event. (D) Distribution of FRET values determined from 197 traces as in (B) fit with a Gaussian model (black curve).(E) As in (C), but with ATP. Periods in which a fluorescently labeled SecA molecule is bound are indicated by grey shading. (F) As in (D), but with ATP (228 traces).


Figure 4 ( A ) FRET traces of clamp movements in the presence of different nucleotides were used to determine the number of states best fit by a Markov model . ( B ) Transition density plot of idealized ATP FRET states obtained in ( A ) . ( C ) Representative traces obtained with ATPγS . The upper FRET trace was calculated from the middle traces obtained by exciting the donor fluorophore and measuring both donor ( green ) and acceptor ( red ) fluorescence . The lowest trace was obtained by exciting the acceptor fluorophore directly . The arrow indicates a bleaching event . ( D ) Distribution of FRET values determined from 315 traces as in ( C ) fit with a Gaussian model ( black curve ) . ( E ) Comparison of high and low FRET state occupancy in ADP • Pi and ADP • Vi for the clamp and THF . ( F ) The distributions of dwell times of the low FRET states observed in ATP were fit with a single exponential ( 1539 low FRET states ) . The inset shows average dwell time and error , defined as the standard error based on the number of traces . ( G ) As in ( F ) , but with high FRET ( 1773 high FRET states ) . ( H ) Comparison of dwell times of the high FRET states for the two - helix finger ( THF ) and clamp for different fluorophore positions . Errors as in ( G ) with significance based on two sample Kolmogorov - Smirnov tests with a 1 % threshold . n . s . P = 0 . 012 ( left ) , n . s . P = 0 . 681 ( right ) ; * P < 1 * 10 - 35 ( between each pair of THF / clamp mutants ) . 809C / 394C : 1656 high FRET states ; 809C / 103C : 1349 states ; 233C / 103C : 1773 states ; 233C / 336C : 1778 states .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(A) FRET traces of clamp movements in the presence of different nucleotides were used to determine the number of states best fit by a Markov model. (B) Transition density plot of idealized ATP FRET states obtained in (A).(C) Representative traces obtained with ATPγS. The upper FRET trace was calculated from the middle traces obtained by exciting the donor fluorophore and measuring both donor (green) and acceptor (red) fluorescence. The lowest trace was obtained by exciting the acceptor fluorophore directly. The arrow indicates a bleaching event. (D) Distribution of FRET values determined from 315 traces as in (C) fit with a Gaussian model (black curve).(E) Comparison of high and low FRET state occupancy in ADP•Pi and ADP•Vi for the clamp and THF.(F) The distributions of dwell times of the low FRET states observed in ATP were fit with a single exponential (1539 low FRET states). The inset shows average dwell time and error, defined as the standard error based on the number of traces. (G) As in (F), but with high FRET (1773 high FRET states).(H) Comparison of dwell times of the high FRET states for the two-helix finger (THF) and clamp for different fluorophore positions. Errors as in (G) with significance based on two sample Kolmogorov-Smirnov tests with a 1% threshold. n.s.P = 0.012 (left), n.s.P = 0.681 (right); *P < 1*10-35 (between each pair of THF/clamp mutants). 809C/394C: 1656 high FRET states; 809C/103C: 1349 states; 233C/103C: 1773 states; 233C/336C: 1778 states.


Figure 1A - B : Percentage of pDCs ( A ) and mDCs ( B ) from total PBMCs collected at D1 was quantified by flow cytometry and grouped to self - reported duration of symptoms prior to D1 ( A - B ; 0 - 4 n = 22 ; 5 - 8 n = 39 ; 9 - 12 n = 39 ; ≥ 13 n = 16 ) . Data information : Each dot represents a patient , lines with error bars show the median values with interquartile ranges Statistical significance was determined using the Kruskal - Wallis test * < p0 . 05 , * * < p0 . 01 * * * < p0 . 001 , ns = not significant . PBMCs and plasma samples were collected from SARS - CoV - 2 infected individuals at time of hospitalization ( D1 ) and after 5 days ( D5 ) . C - D : Percentage of pDCs ( C ) and mDCs ( D ) at D1 and D5 matched samples from all patients independent of symptom duration ( n = 93 ) . Data information : Each dot represents a patient , lines with error bars show the median values with interquartile ranges Statistical significance was determined using the Wilcoxon matched pairs signed rank test * < p0 . 05 , * * < p0 . 01 * * * < p0 . 001 , ns = not significant . PBMCs and plasma samples were collected from SARS - CoV - 2 infected individuals at time of hospitalization ( D1 ) and after 5 days ( D5 ) . E - J : Matched D1 and D5 plasma samples were available from 55 donors and protein levels were determined for IFNα2a ( E , n = 30 ) , IL - 3 ( F , n = 38 ) , IL - 8 ( G , n = 44 ) , IL - 6 ( H , n = 51 ) , TNFα ( I , n = 42 ) and CXCL10 ( J , n = 46 ) , note that some samples could not be quantified and were excluded from the analysis . Data information : Each dot represents a patient , lines with error bars show the median values with interquartile ranges Statistical significance was determined using the Wilcoxon matched pairs signed rank test * < p0 . 05 , * * < p0 . 01 * * * < p0 . 001 , ns = not significant . K - L : Percentage of pDCs grouped by COVID - 19 severity , as determined by oxygen supplementation ( no oxygen n = 43 , nasal oxygen n = 65 and high flow oxygen n = 5 ) . Percentage of pDCs ( K ) was correlated to COVID - 19 disease severity ( L ) using simple linear regression . Data information : Each dot represents a patient , lines with error bars show the median values with interquartile ranges mean values with the standard error of the mean Statistical significance was determined using the Kruskal - Wallis test and simple linear regression * < p0 . 05 , * * < p0 . 01 * * * < p0 . 001 , ns = not significant .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A-B: Percentage of pDCs (A) and mDCs (B) from total PBMCs collected at D1 was quantified by flow cytometry and grouped to self-reported duration of symptoms prior to D1 (A-B; 0-4 n= 22; 5-8 n=39; 9-12 n= 39; ≥13 n=16). Data information: Each dot represents a patient, lines with error bars show the median values with interquartile ranges Statistical significance was determined using the Kruskal-Wallis test *<p0.05, **<p0.01 ***<p0.001, ns= not significant.PBMCs and plasma samples were collected from SARS-CoV-2 infected individuals at time of hospitalization (D1) and after 5 days (D5). C-D: Percentage of pDCs (C) and mDCs (D) at D1 and D5 matched samples from all patients independent of symptom duration (n=93). Data information: Each dot represents a patient, lines with error bars show the median values with interquartile ranges Statistical significance was determined using the Wilcoxon matched pairs signed rank test *<p0.05, **<p0.01 ***<p0.001, ns= not significant.PBMCs and plasma samples were collected from SARS-CoV-2 infected individuals at time of hospitalization (D1) and after 5 days (D5). E-J: Matched D1 and D5 plasma samples were available from 55 donors and protein levels were determined for IFNα2a (E, n=30), IL-3 (F, n=38), IL-8 (G, n=44), IL-6 (H, n=51), TNFα (I, n=42) and CXCL10 (J, n=46), note that some samples could not be quantified and were excluded from the analysis. Data information: Each dot represents a patient, lines with error bars show the median values with interquartile ranges Statistical significance was determined using the Wilcoxon matched pairs signed rank test *<p0.05, **<p0.01 ***<p0.001, ns= not significant.K-L: Percentage of pDCs grouped by COVID-19 severity, as determined by oxygen supplementation (no oxygen n=43, nasal oxygen n=65 and high flow oxygen n=5). Percentage of pDCs (K) was correlated to COVID-19 disease severity (L) using simple linear regression. Data information: Each dot represents a patient, lines with error bars show the median values with interquartile ranges mean values with the standard error of the mean Statistical significance was determined using the Kruskal-Wallis test and simple linear regression *<p0.05, **<p0.01 ***<p0.001, ns= not significant.


Figure 2C - J : The FR2020 strain was used in subsequent experiments where pDC were either mock treated ( mock , grey ) , exposed to SARS - CoV - 2 at 1 MOI ( SARS - 2 , purple ) , TLR7 ( 2 . 5 μg / mL R837 , blue ) or TLR3 agonist ( 800 ng / mL poly ( I : C ) , pink ) . Supernatants were collected after 24 hrs and analyzed for type I IFNα ( C ) , IFNβ ( D ) , type II IFNγ ( E ) , type III IFNλ1 ( F ) , IL - 6 ( G ) , IL - 8 ( H ) , CXCL10 ( I ) and TNFα ( J ) expression by ELISA . Data information : Bars represent mean values with standard error of the mean and symbols represent individual pDC donors ( n = 3 - 4 ) . Equal symbols represent equal donors Statistical significance was determined using the ratio paired student T test and compared the treated condition with the time point - matched mock condition * < p0 . 05 , * * < p0 . 01 * * * < p0 . 001 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2C- J: The FR2020 strain was used in subsequent experiments where pDC were either mock treated (mock, grey), exposed to SARS-CoV-2 at 1 MOI (SARS-2, purple), TLR7 (2.5 μg/mL R837, blue) or TLR3 agonist (800 ng/mL poly(I:C), pink). Supernatants were collected after 24 hrs and analyzed for type I IFNα (C), IFNβ (D), type II IFNγ (E), type III IFNλ1 (F), IL-6 (G), IL-8 (H), CXCL10 (I) and TNFα (J) expression by ELISA. Data information: Bars represent mean values with standard error of the mean and symbols represent individual pDC donors (n=3-4). Equal symbols represent equal donors Statistical significance was determined using the ratio paired student T test and compared the treated condition with the time point-matched mock condition *<p0.05, **<p0.01 ***<p0.001.


Figure 4Using CRISPR / Cas9 , MyD88 knock - out ( KO ) and AAVS1KO ( control ) pDCs were generated . MyD88 protein levels in KO and control pDCs were analyzed by western blottingC - D : MyD88KO and control pDCs were either mock treated ( mock ) or exposed to SARS - CoV - 2 ( SARS - 2 , 1 MOI ) , supernatants were collected at indicated time points and analyzed for type I IFNα ( C ) and CXCL10 ( D ) . Done with n = 4 Data information : Bars represent mean values and equal symbols represent equal donors . Statistical significance was determined using Mann - Whitney one - tailed T test in ( C ) as MyD88 - KO data had no variance and values of 0 . For ( D ) a Mann - Whitney two - tailed T test was used for comparison between conditions of mock and MyD88 KO donors . E - F : Type I IFNα production was then determined in cell culture supernatant from SARS - CoV - 2 exposed TRIFKO or TRIG + MyD88KO ( E ) and RIG - IKO or RIG - I + MyD88KO ( F ) pDCs . Done with n =

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4Using CRISPR/Cas9, MyD88 knock-out (KO) and AAVS1KO (control) pDCs were generated. MyD88 protein levels in KO and control pDCs were analyzed by western blottingC-D: MyD88KO and control pDCs were either mock treated (mock) or exposed to SARS-CoV-2 (SARS-2, 1 MOI), supernatants were collected at indicated time points and analyzed for type I IFNα (C) and CXCL10 (D). Done with n=4 Data information: Bars represent mean values and equal symbols represent equal donors. Statistical significance was determined using Mann-Whitney one-tailed T test in (C) as MyD88-KO data had no variance and values of 0. For (D) a Mann-Whitney two-tailed T test was used for comparison between conditions of mock and MyD88 KO donors.E-F: Type I IFNα production was then determined in cell culture supernatant from SARS-CoV-2 exposed TRIFKO or TRIG+MyD88KO (E) and RIG-IKO or RIG-I+MyD88KO (F) pDCs. Done with n=2


Figure 4 ( A ) Surface plasmon resonance ( SPR ) KAP1 - KRAB binding assay . MBP - KRAB was immobilized on the chip . WT or R311E KAP1 RBCC were flowed over the chip . Data points are shown in dark red or blue ; fits are shown as light red or blue lines . ( C ) LINE - 1 reporter repression with single point mutants and a previously described KRAB - binding deficient KAP1 variant containing four mutations in the CC domain ( CCmut ; V293S / K296A / M297A / L300S ) . ( D ) SVA reporter repression with the same set of mutants as in ( B ) . Data were normalized to KAP1 KO cells transfected with an empty vector ( EV ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(A) Surface plasmon resonance (SPR) KAP1-KRAB binding assay. MBP-KRAB was immobilized on the chip. WT or R311E KAP1 RBCC were flowed over the chip. Data points are shown in dark red or blue; fits are shown as light red or blue lines.(C) LINE-1 reporter repression with single point mutants and a previously described KRAB-binding deficient KAP1 variant containing four mutations in the CC domain (CCmut; V293S/K296A/M297A/L300S).(D) SVA reporter repression with the same set of mutants as in (B). Data were normalized to KAP1 KO cells transfected with an empty vector (EV).