F Loss of MOAP - 1 leads to slower exchange of p62 in RFP - p62 bodies . WT MOAP - 1 KO LO2 transfected with expression vector encoding RFP - tagged p62 . 14 hours later , were subjected to recovery after photobleaching ( FRAP ) . Scale bar : 2 µm .

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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.


H Overexpression of MOAP - 1 but not GFP leads to increased exchange of p62 in RFP - p62 bodies . HepG2 were transfected with expression vector encoding RFP - tagged p62 GFP - tagged MOAP - 1 or GFP only control 14 hours later , were subjected to FRAP . Scale bar : 2 µm . I of rate of recovery of RFP - p62 in HepG2 co - expressing GFP - MOAP - 1 or GFP . n = 5 samples .

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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.


H Overexpression of MOAP - 1 but not GFP leads to increased exchange of p62 in RFP - p62 bodies . HepG2 were transfected with expression vector encoding RFP - tagged p62 GFP - tagged MOAP - 1 or GFP only control 14 hours later , were subjected to FRAP . Scale bar : 2 µm .

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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.


J Re - introduction of MOAP - 1 disrupts spherical structure of p62 bodies in MOAP - 1 deficient LO2 . MOAP - 1 KO LO2 were transfected with expression vector encoding Myc - MOAP - 1 harvested at 14 - or 24 - hours post - transfection for IF using anti - p62 ( in red ) anti - Myc ( in green ) antibodies . Insets were enlarged in lower panels . Scale bar : 2 µm .

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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.


A MOAP - 1 associates with p62 their interaction is enhanced by DEN - mediated stress . LO2 transfected with Myc - MOAP - 1 were treated with or without diethylnitrosamine ( DEN ) ( 200 µM ) for 12 h subjected to Duolink proximity ligation using anti - p62 anti - Myc antibodies . Red dots represent MOAP - 1 / p62 interaction .

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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 KYKKLR of MOAP - 1 abolishes its interaction with p62 . Replacement of positively charged residues in KYKKLR of MOAP - 1 with alanine ( referred to as M3 mutant ) disrupts MOAP - 1 / p62 interaction , whereas alanine substitution in neighboring region of positively charged KRRR ( M1 mutant ) negatively charged EEE ( M2 mutant ) has no effect . LO2 were transfected with plasmid encoding Myc - MOAP - 1 or indicated alanine mutants together with plasmid encoding Flag - p62 . were then subjected to co - IP with anti - Myc antibody .

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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 p62 bodies . MOAP - 1 KO LO2 were transfected with plasmid encoding Myc - MOAP - 1 or Myc - MOAP - 1 - M3 mutant . transfected were harvested at 14 hours post - transfection , which is time point that localization of MOAP - 1 at p62 bodies is readily detected . IF was performed on these using anti - p62 ( red ) anti - Myc ( green ) antibodies . Arrowheads indicate colocalization between Myc - MOAP - 1 p62 bodies .

O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O B-MEASURED_VAR O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O

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 MOAP - 1 - M3 - mutant promotes exchange of RFP - p62 in p62 bodies . WT LO2 transfected with expression vector encoding RFP - tagged p62 GFP - tagged MOAP - 1 or MOAP - 1 - M3 . At 14 hours post - transfection , were subjected to FRAP . Scale bar : 2 µm .

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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 of rate of recovery of RFP - p62 in 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 .

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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 of lysates of WT LO2 transfected with expression vector encoding RFP - tagged p62 GFP - tagged MOAP - 1 or MOAP - 1 - M3 as described in ( E ) .

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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 MOAP - 1 - M3 mutant , reduces of p62 bodies . MOAP - 1 KO LO2 were transfected with plasmid encoding Myc - MOAP - 1 or Myc - MOAP - 1 - M3 . transfected were harvested at 24 hours post - transfection , which is time point that MOAP - 1 was found to effectively downregulate p62 bodies , followed by IF as in ( D ) . expressing Myc - MOAP - 1 or Myc - MOAP - 1 - M3 were marked by dashed lines . I of p62 bodies in MOAP - 1 KO LO2 expressing Myc - MOAP - 1 Myc - MOAP - 1 - M3 mutant . ( Left panel ) number of p62 bodies per . n = 7 independently plated samples . ( Right panel ) of p62 bodies . n = 107 267 bodies detected in MOAP - 1 KO LO2 expressing Myc - MOAP - 1 Myc - MOAP - 1 - M3 mutant , respectively , from three independently plated samples . Error bars represent S . E . M . * * * P < 0 . 001 , Student ' s t - .

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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.


H Re - expression of MOAP - 1 , but not MOAP - 1 - M3 mutant , reduces of p62 bodies . MOAP - 1 KO LO2 were transfected with plasmid encoding Myc - MOAP - 1 or Myc - MOAP - 1 - M3 . transfected were harvested at 24 hours post - transfection , which is time point that MOAP - 1 was found to effectively downregulate p62 bodies , followed by IF as in ( D ) . expressing Myc - MOAP - 1 or Myc - MOAP - 1 - M3 were marked by dashed lines .

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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.


J Western blotting of MOAP - 1 KO LO2 transfected with plasmid encoding Myc - MOAP - 1 or Myc - MOAP - 1 - M3 mutant as described in ( H ) .

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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).


A Overexpression of MOAP - 1 inhibits dimerization of p62 . p62 KO LO2 hepatocytes were transfected with indicated plasmids . 24 hours later , lysates were subjected to co - IP with anti - Myc antibody . PB1 deletion ( ΔPB1 ) mutant , which is impaired in p62 oligomerization , is included as a control . TCL , total lysates .

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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 MOAP - 1 deficient . WT MOAP - 1 KO LO2 were transfected with plasmids encoding Flag - tagged Myc - tagged p62 or empty vector . 24 h post - transfection , were harvested , lysed lysates were subjected to co - IP with anti - Myc antibody . C of of Flag - p62 bound to Myc - p62 in co - IP experiment as described in ( B ) by densitometric .

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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.


B Dimerization of p62 is enhanced in MOAP - 1 deficient . WT MOAP - 1 KO LO2 were transfected with plasmids encoding Flag - tagged Myc - tagged p62 or empty vector . 24 h post - transfection , were harvested , lysed lysates were subjected to co - IP with anti - Myc antibody .

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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.


D Re - expression of MOAP - 1 , but not p62 - binding defective mutant , MOAP - 1 - M3 , reduces p62 oligomerization in MOAP - 1 KO LO2 hepatocytes . WT MOAP - 1 KO LO2 were transfected with plasmids encoding His - tagged p62 Myc - MOAP - 1 or Myc - MOAP - 1 - M3 24 h later , subjected to His - pull down cross - linking using DSSO . E of of p62 oligomer depicted in ( D ) by densitometric .

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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.


D Re - expression of MOAP - 1 , but not p62 - binding defective mutant , MOAP - 1 - M3 , reduces p62 oligomerization in MOAP - 1 KO LO2 hepatocytes . WT MOAP - 1 KO LO2 were transfected with plasmids encoding His - tagged p62 Myc - MOAP - 1 or Myc - MOAP - 1 - M3 24 h later , subjected to His - pull down cross - linking using DSSO .

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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.


F region of p62 spanning PB1 ZZ domains are required for mediating its interaction with MOAP - 1 . p62 KO LO2 were used in . were transfected with plasmid encoding Myc - p62 or indicated deletion mutants Flag - MOAP - 1 for 24 hours . transfected were subjected to co - IP with anti - Myc antibody . ( Lower panel ) Schematics depicting domains in p62 . PB1 , Phox Bem1p ; ZZ , Zinc Finger ; TB , TRAF6 - binding domain ; KIR , Keap1 interacting region ; LIR , LC3 interacting region ; UBA , ubiquitin - associated domain .

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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 fragment encompassing PB1 ZZ domains of p62 is sufficient for mediating its interaction with MOAP - 1 . p62 KO LO2 were transfected with plasmid encoding Myc - p62 or indicated fragments Flag - MOAP - 1 . 24 hours later , transfected were subjected to co - IP with anti - Myc antibody .

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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 inhibits homodimerization of PB1 - ZZ fragment . p62 KO LO2 were transfected with plasmid encoding Myc Flag - tagged PB1 - ZZ in absence of presence of HA - tagged MOAP - 1 for 24 h subjected to co - IP with anti - Myc antibody .

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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.


A Increased recruitment of Keap1 to p62 bodies in MOAP - 1 deficient LO2 . WT MOAP - 1 KO LO2 were subjected to IF with anti - Keap1 ( green ) anti - p62 ( red ) antibodies . Insets in upper panels are enlarged in lower panels . Nuclei were counterstained with DAPI ( in blue ) . Scale bar : 5 µm .

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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 p62 KO LO2 were subjected to co - IP using anti - p62 antibody . TCL , total lysates . C of of Keap1 bound to p62 in co - IP experiment as described in ( B ) by densitometric .

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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.


B MOAP - 1 deficiency promotes p62 / Keap1 interaction . WT , MOAP - 1 KO p62 KO LO2 were subjected to co - IP using anti - p62 antibody . TCL , total lysates .

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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.


D Diminished Keap1 / Nrf2 interaction in absence of MOAP - 1 . WT MOAP - 1 KO LO2 were subjected to Duolink PLA using anti - Keap1 anti - Nrf2 antibodies . Red dots represent Keap1 / Nrf2 interaction . Nuclei were counterstained with DAPI ( in blue ) . Scale bar : 10 µm . E of Duolink signals depicted in ( D ) . Duolink signals , which appear as red dots , were quantified by ImageJ normalized to number of nuclei .

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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.


D Diminished Keap1 / Nrf2 interaction in absence of MOAP - 1 . WT MOAP - 1 KO LO2 were subjected to Duolink PLA using anti - Keap1 anti - Nrf2 antibodies . Red dots represent Keap1 / Nrf2 interaction . Nuclei were counterstained with DAPI ( in blue ) . Scale bar : 10 µm .

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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.


F Loss of MOAP - 1 results in marked elevation of Nrf2 . WT MOAP - 1 KO LO2 were subjected to IF with anti - Nrf2 ( in green ) anti - p62 ( in red ) antibodies . Scale bar : 10 µm . G of of Nrf2 in ( F ) . mean gray values of Nrf2 signals were quantified by ImageJ .

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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.


F Loss of MOAP - 1 results in marked elevation of Nrf2 . WT MOAP - 1 KO LO2 were subjected to IF with anti - Nrf2 ( in green ) anti - p62 ( in red ) antibodies . Scale bar : 10 µm .

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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.


H MOAP - 1 deficiency promotes activation of Nrf2 signaling . Transcript of downstream targets of Nrf2 ( i . e . HMOX - 1 , NQO1 , SLC7A11 , GCLC G6PD ) in WT MOAP - 1 KO LO2 were determined by RT - PCR normalized to GAPDH .

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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 MOAP - 1 deficient is mediated through p62 . Transcript of Nrf2 target genes ( HMOX - 1 NQO1 ) in WT , MOAP - 1 KO MOAP - 1 / p62 DKO LO2 were determined by RT - PCR normalized to GAPDH .

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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 MOAP - 1 - L120E , but not p62 binding defective mutant of MOAP - 1 , MOAP - 1 - M3 , inhibits p62 / Keap1 interaction in MOAP - 1 deficient . MOAP - 1 KO LO2 were transfected with plasmid encoding Myc - tagged MOAP - 1 , L120E or M3 . transfected were then subjected to co - IP with anti - p62 antibody .

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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 MOAP - 1 - L120E , but not p62 binding defective MOAP - 1 - M3 , reduces Nrf2 transcriptional activity in MOAP - 1 KO . MOAP - 1 KO LO2 were transfected with Myc - tagged MOAP - 1 , MOAP - 1 - L120E or MOAP - 1 - M3 ARE - Nrf2 - firely renilla luciferase reporter constructs for overnight subjected to luciferase .

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.


A High level of p62 bodies in liver of MOAP - 1 KO mice subjected to acute diethylnitrosamine ( DEN ) treatment . Eight - week - old WT MOAP - 1 KO mice were injected with an acute dose of DEN ( 100 µg / g ) . Mice were sacrificed at 48 hours post - injection . Livers were harvested for immunohistochemistry ( ) with anti - p62 antibody ( in red ) . Nuclei were counterstained with DAPI ( in blue ) . Insets in upper panels are enlarged in lower panels . Scale bar : 20 µm .

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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 of Nrf2 its downstream target , HMOX - 1 , in liver upon acute DEN treatment . Lysates of livers harvested from mice subjected to acute DEN treatment as described in ( A ) were prepared in RIPA lysis buffer containing 1 % SDS analyzed by Western blotting . Actin as loading control .

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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 of p62 in detergent - insoluble fractions of 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 ) insoluble ( I ) fractions analyzed by Western blotting .

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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 of p62 bodies detected in tumors of MOAP - 1 KO mice . sections from livers dissected from 8 . 5 - month - old WT MOAP - 1 KO mice injected with a single dose of DEN ( 25 µg / g ) at 15 - day - old were subjected to with anti - p62 antibody ( in red ) . were stained with DAPI ( in blue ) . Insets were enlarged in bottom right . Scale bar : 20 µm . E of p62 bodies in WT MOAP - 1 KO tumors . ( Left panel ) number of p62 bodies per . n = 7 tumors from livers of WT MOAP - 1 KO mice . ( Right panel ) of p62 bodies . n = 456 1211 bodies detected in WT MOAP - 1 KO tumors , respectively , from three independent samples . Error bar represents S . E . M . * P < 0 . 05 , * * * P < 0 . 001 , student ' s t - .

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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.


D High of p62 bodies detected in tumors of MOAP - 1 KO mice . sections from livers dissected from 8 . 5 - month - old WT MOAP - 1 KO mice injected with a single dose of DEN ( 25 µg / g ) at 15 - day - old were subjected to with anti - p62 antibody ( in red ) . were stained with DAPI ( in blue ) . Insets were enlarged in bottom right . Scale bar : 20 µm .

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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.


F Increased of Nrf2 its downstream target , HMOX - 1 , in MOAP - 1 deficient tumors . While p62 is upregulated in tumors compared to adjacent non - tissues in both WT MOAP - 1 KO livers , no significant difference is observed in p62 between WT MOAP - 1 KO tumors . Lysates of tumors non - parts of livers from mice injected with DEN as described in ( D ) were prepared in RIPA lysis buffer containing 1 % SDS subjected to Western blotting . Actin as loading control . P , PBS injected control ; N , non - ; T , .

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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 of HMOX - 1 NQO1 in MOAP - 1 deficient tumors . Livers harvested from mice injected with PBS or DEN as described in ( D ) were subjected to RT - PCR . n = 3 WT 3 MOAP - 1 KO mice injected with PBS control , 8 WT 9 MOAP - 1 KO mice injected with DEN . Error bar represents S . E . M . * * P < 0 . 01 , student ' s t - .

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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 burden in DEN - mediated liver cancer model . Representative of liver tumors dissected from 8 . 5 - month - old WT MOAP - 1 KO mice injected with a single dose of DEN ( 25 µg / g ) at 15 - day - old . Scale bar : 5 mm . I Measurements of number , maximum diameter of nodules total liver from WT MOAP - 1 KO mice ( n = 13 14 respectively ) injected with DEN as described in ( H ) . Error bar represents S . E . M . * * P < 0 . 01 , * * * P < 0 . 001 , student ' s t - .

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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.


H MOAP - 1 deficient mice bear higher burden in DEN - mediated liver cancer model . Representative of liver tumors dissected from 8 . 5 - month - old WT MOAP - 1 KO mice injected with a single dose of DEN ( 25 µg / g ) at 15 - day - old . Scale bar : 5 mm .

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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.


MOAP - 1 deficiency promotes initiation . measurements of number , diameter of largest nodules total liver of 4 . 5 - month - old WT MOAP - 1 KO mice ( n = 18 23 respectively ) injected with a single dose of DEN ( 25 µg / g ) at 15 - day - old .

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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.


( B ) Confocal of HeLa transfected with EGFP - ORP5 or EGFP - ORP8 showing variable expression . Arrows indicate ORP5 ORP8 localization to cortical ER ( cER ) . Scale bar , 10 μm .

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(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 transfected with EGFP - ORP5 or EGFP - ORP8 immunogold stained with anti - GFP ( 15 nm gold ) to detect ORP5 or ORP8 anti - PDI ( 10 nm gold ) to label ER lumen . ORP5 ORP8 localize at ER - PM ( arrows ) ER - mitochondria contact sites ( arrowheads ) . m , mitochondria ; cER , cortical ER . Scale bar 200nm . ( E - F ) of IEM labelling for EGFP - ORP5 , EGFP - ORP8 ( E ) GFP - Sec61β ( F ) in transfected HeLa . Results are presented as of total number of gold particles ( 800 per condition , n = 35 - 45 ) for ORP5 , ORP8 Sec61β in indicated compartments ( non - cER , non - cortical ER ; ER - PM , ER - PM contact sites ; ER - mito , ER - mitochondria contact sites . % gold particles ± . Data are mean of three independent replicates . * P < 0 , 001 compared to non - cER ER - mito [ EGFP - ORP5 ] to ER - PM ER - mito [ EGFP - ORP8 GFP - Sec61β ] , # P < 0 , 001 compared to ER - PM ER - mito [ GFP - Sec61β ] ) .

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(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 ( H ) of of ER - mitochondria contact sites ( ER - mito contact ) per mitochondria of HeLa expressing EGFP - ORP5 , EGFP - ORP8 together with HRP - KDEL , to visualize ER . Control ( Ctrl ) consisted of HeLa overexpressing only HRP - KDEL .

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(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 MAM fractions were purified from HeLa equal amount of ( 25g ) from each fraction were loaded on a 4 - 20 % gradient SDS - PAGE gel immunoblotted using anti - ORP5 , anti - ORP8 anti - IP3R - 3 ( MAM ) , anti - PDI ( ER ) anti - cytochrome c ( mitochondrial ) . TC , Total ; MAM , mitochondria - associated ER .

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(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 .


( A ) Confocal of HeLa transfected with EGFP - ORP5 , EGFP - ORP8 , or GFP - Sec61β together with PTPIP51 - HA immunostained using HA antibody to detect PTPIP51 . Scale bar , 10 μm .

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(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 co - transfected with EGFP - ORP5 PTPIP51 - HA immunogold stained with anti - GFP ( 15 nm gold ) anti - HA ( 10 nm gold ) showing EGFP - ORP5 localization at ER - mitochondria contacts ( arrows ) . Arrowheads indicate highly expanded ER juxtaposed to mitochondria . Scale bar 200 nm . ( C ) of IEM labelling for EGFP - ORP5 , EGFP - ORP8 GFP - Sec61β in HeLa co - transfected with PTPIP51 - HA . Results are presented as of total number of gold particles ( 800 per condition , n = 30 - 35 ) for ORP5 , ORP8 Sec61β in indicated compartments ( ER , reticular ER ; ER - mito MCS , ER - mitochondria contact sites ; ER - PM MCS , ER - PM contact sites ) . % gold particles ± . Data are representative of three independent replicates . * * P < 0 , 001 .

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(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 of HeLa transfected with EGFP - ORP5 or EGFP - ORP8 together with PTPIP51 - HA RFP - Sec61β immunostained using HA antibody to detect PTPIP51 . Scale bar , 10 μm .

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(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 of ER - mitochondria contacts in indicated overexpression conditions . ( F ) of of ER - mitochondria contact sites ( ER - mito contact ) per mitochondria of HeLa transfected with HRP - KDEL ( Ctrl ) or co - transfected with PTPIP51 - HA EGFP or PTPIP51 - HA EGFP - ORP5 or PTPIP51 - HA EGFP - ORP8 ( n = 30 1082 - 1223 mitochondria ) . % ER - mitochondria contact sites ± . Data are representative of three independent replicates . * * P < 0 , 001 , * P < 0 , 05 .

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(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 co - expressing PTPIP51 - HA treated with control ( siCtrl ) or VAPA VAPB ( SiVAPA / B ) siRNAs analyzed by western blot using antibodies against GFP ( ORPs ) , HA ( PTPIP51 ) , VAPA , VAPB tubulin .

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(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.


( A ) Confocal of HeLa transfected with EGFP - ORP5 , EGFP - ORP5PH or EGFP - ORP5ORD immunostained using anti - TOM20 antibody to visualize mitochondria . Scale bar , 10 μm .

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(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 of HeLa transfected with EGFP - tagged ORD domain of ORP5 ( EGFP - ORD ORP5 ) immunostained using anti - TOM20 antibody ( upper panels ) or co - transfected with PTPIP51 - HA immunostained using HA antibody to detect PTPIP51 . Scale bar , 10 μm .

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(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 of HeLa transfected with EGFP - ORP5PH or EGFP - ORP5ORD ( C ) together with PTPIP51 - HA immunostained using HA antibody to detect PTPIP51 . Scale bar , 10 μm .

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(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 of HeLa transfected with EGFP - ORP5 , EGFP - ORP5L389D or EGFP - ORP8L425D ( D ) together with PTPIP51 - HA immunostained using HA antibody to detect PTPIP51 . Scale bar , 10 μm .

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(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 co - expressing PTPIP51 - HA analyzed by western blot with antibodies against GFP HA .

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(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.


( A ) ORP5 , ORP8 actin of HRP - KDEL - expressing HeLa treated with Ctrl siRNAs ( siCtrl ) or siRNAs against ORP5 ( siORP5 ) or ORP8 ( siORP8 ) .

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(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 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 red arrows in insets indicate ER - mitochondria contacts . Inset scale bar , 500nm .

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(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 showing of mitochondria in siCtrl , siORP5 siORP8 - treated . Scale bar , 1 μm . ( D ) Quantifications of number of mitochondria with altered in indicated siRNA conditions . % altered mitochondria ± , n = 25 632 - 710 mitochondria . * P < 0 , 001 compared to siCtrl .

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(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 ORP5 siRNA transfected . OCR trace was obtained by sequential measurement of basal OCR ( OCRBAS ) , OCR after addition of oligomycin ( OM ) OCR after addition of antimycin A ( AA ) . Note reduced basal OCR ( OCRBAS ) compared to ctrl siRNA . Error bars denote ± . Data are mean of 4 independent repeats ( n = 4 ) , * P < 0 , 05 .

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(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.


( C ) Representative traces obtained with ADP • BeFx . upper FRET trace was calculated from middle traces obtained by exciting donor fluorophore measuring both donor ( green ) acceptor ( red ) . lowest trace was obtained by exciting acceptor fluorophore directly . arrow indicates a bleaching event . ( D ) Distribution of FRET values determined from 97 traces as in ( C ) fit with a Gaussian model ( black curve ) .

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(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 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 ) .

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(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).


( A ) A representative FRET trace ( blue ) was obtained as in Fig 1 , fit with a hidden Markov model ( black dashed ) .

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(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 presence of different nucleotides , were used to determine number of states best fit by Markov model . ( C ) Transition density plot of idealized ATP FRET states obtained in ( B ) .

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(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 ) distributions of dwell times of low FRET states observed in ATP were fit with a single exponential ( 1500 low FRET states ) . inset shows average dwell time error , defined as standard error on number of traces . ( E ) As in ( D ) , but with high FRET ( 1656 high FRET states ) .

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(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 . upper FRET trace was calculated from middle traces obtained by exciting donor fluorophore measuring both donor ( green ) acceptor ( red ) . lowest trace was obtained by exciting acceptor fluorophore directly . arrow indicates a bleaching event . ( G ) Distribution of FRET values determined from 168 traces as in ( D ) fit with a Gaussian model ( black curve ) .

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(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).


( C ) Representative traces obtained with ADP • BeFx . upper FRET trace was calculated from middle traces obtained by exciting donor fluorophore measuring both donor ( green ) acceptor ( red ) . lowest trace was obtained by exciting acceptor fluorophore directly . arrow indicates a bleaching event . ( D ) Distribution of FRET values determined from 197 traces as in ( B ) fit with a Gaussian model ( black curve ) .

(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 ) .

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(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).


( A ) FRET traces of clamp movements in presence of different nucleotides were used to determine number of states best fit by a Markov model . ( B ) Transition density plot of idealized ATP FRET states obtained in ( A ) .

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(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 . upper FRET trace was calculated from middle traces obtained by exciting donor fluorophore measuring both donor ( green ) acceptor ( red ) . lowest trace was obtained by exciting acceptor fluorophore directly . arrow indicates a bleaching event . ( D ) Distribution of FRET values determined from 315 traces as in ( C ) fit with a Gaussian model ( black curve ) .

(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 low FRET state occupancy in ADP • Pi ADP • Vi for clamp THF .

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(E) Comparison of high and low FRET state occupancy in ADP•Pi and ADP•Vi for the clamp and THF.


( F ) distributions of dwell times of low FRET states observed in ATP were fit with a single exponential ( 1539 low FRET states ) . inset shows average dwell time error , defined as standard error on number of traces . ( G ) As in ( F ) , but with high FRET ( 1773 high FRET states ) .

(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 high FRET states for two - helix finger ( THF ) clamp for different fluorophore positions . Errors as in ( G ) with significance on two sample Kolmogorov - Smirnov 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 .

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(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.


A - B : of pDCs ( A ) mDCs ( B ) from total PBMCs collected at D1 was quantified by flow cytometry 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 , lines with error bars show median values with interquartile ranges Statistical significance was determined using Kruskal - Wallis * < p0 . 05 , * * < p0 . 01 * * * < p0 . 001 , ns = not significant .

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A-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 plasma samples were collected from SARS - CoV - 2 infected individuals at time of hospitalization ( D1 ) after 5 days ( D5 ) . C - D : of pDCs ( C ) mDCs ( D ) at D1 D5 matched samples from independent of symptom duration ( n = 93 ) . Data information : Each dot represents a , lines with error bars show median values with interquartile ranges Statistical significance was determined using Wilcoxon matched pairs signed rank * < p0 . 05 , * * < p0 . 01 * * * < p0 . 001 , ns = not significant .

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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 plasma samples were collected from SARS - CoV - 2 infected individuals at time of hospitalization ( D1 ) after 5 days ( D5 ) . E - J : Matched D1 D5 plasma samples were available from 55 donors 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 ) CXCL10 ( J , n = 46 ) , note that some samples could not be quantified were excluded from . Data information : Each dot represents a , lines with error bars show median values with interquartile ranges Statistical significance was determined using Wilcoxon matched pairs signed rank * < p0 . 05 , * * < p0 . 01 * * * < p0 . 001 , ns = not significant .

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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 : of pDCs grouped by COVID - 19 severity , as determined by oxygen supplementation ( no oxygen n = 43 , nasal oxygen n = 65 high flow oxygen n = 5 ) . of pDCs ( K ) was correlated to COVID - 19 disease severity ( L ) using simple linear regression . Data information : Each dot represents a , lines with error bars show median values with interquartile ranges mean values with standard error of mean Statistical significance was determined using Kruskal - Wallis simple linear regression * < 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.


C - J : FR2020 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 analyzed for type I IFNα ( C ) , IFNβ ( D ) , type II IFNγ ( E ) , type III IFNλ1 ( F ) , IL - 6 ( G ) , IL - 8 ( H ) , CXCL10 ( I ) TNFα ( J ) expression by ELISA . Data information : Bars represent mean values with standard error of mean symbols represent pDC donors ( n = 3 - 4 ) . Equal symbols represent equal donors Statistical significance was determined using paired student T compared treated condition with time point - matched mock condition * < p0 . 05 , * * < p0 . 01 * * * < p0 . 001 .

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C- 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.


Using CRISPR / Cas9 , MyD88 knock - out ( KO ) AAVS1KO ( control ) pDCs were generated . MyD88 in KO control pDCs were analyzed by western

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Using CRISPR/Cas9, MyD88 knock-out (KO) and AAVS1KO (control) pDCs were generated. MyD88 protein levels in KO and control pDCs were analyzed by western blotting


C - D : MyD88KO control pDCs were either mock treated ( mock ) or exposed to SARS - CoV - 2 ( SARS - 2 , 1 MOI ) , supernatants were collected at indicated time points analyzed for type I IFNα ( C ) CXCL10 ( D ) . Done with n = 4 Data information : Bars represent mean values equal symbols represent equal donors . Statistical significance was determined using Mann - Whitney one - T in ( C ) as MyD88 - KO data had no variance values of 0 . For ( D ) a Mann - Whitney two - T was used for comparison between conditions of mock MyD88 KO donors .

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C-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 culture supernatant from SARS - CoV - 2 exposed TRIFKO or TRIG + MyD88KO ( E ) RIG - IKO or RIG - I + MyD88KO ( F ) pDCs . Done with n =

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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


( A ) Surface plasmon resonance ( SPR ) KAP1 - KRAB binding . MBP - KRAB was immobilized on chip . WT or R311E KAP1 RBCC were flowed over chip . Data points are shown in red or blue ; fits are shown as red or blue lines .

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(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 ) - 1 reporter repression with single point mutants a previously described KRAB - binding deficient KAP1 variant containing four mutations in CC domain ( CCmut ; V293S / K296A / M297A / L300S ) .

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(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 same set of mutants as in ( B ) . Data were normalized to KAP1 KO transfected with an empty vector ( EV ) .

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(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).


( A ) BMDMs were infected with Mtb - WT , Mtb - Δeis , or Mtb - c - eis ( MOI = 10 ) for 4 h ( as described in Materials Methods ) , then incubated for 24 h ( left ) or indicated periods of time ( right ) . were fixed , stained with DAPI to visualize nuclei ( blue ) , immunolabeled with an anti - LC3 antibody . antibody was detected using an Alexa Fluor 488 - conjugated goat anti - rabbit IgG ( green ) . Left : representative immunofluorescence of LC3 ; right : of data ( LC3 - punctated were counted manually ) . * * * p < 0 . 001 , vs . Mtb - WT - infected condition . Scale bars , 5 µm . (

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(A) BMDMs were infected with Mtb-WT, Mtb-Δeis, or Mtb-c-eis (MOI = 10) for 4 h (as described in the Materials and Methods), and then incubated for 24 h (left) or the indicated periods of time (right). Cells were fixed, stained with DAPI to visualize nuclei (blue), and immunolabeled with an anti-LC3 antibody. Primary antibody was detected using an Alexa Fluor 488-conjugated goat anti-rabbit IgG (green). Left: representative immunofluorescence images of LC3 punctae; right: quantification of data (LC3-punctated cells were counted manually). ***p<0.001, vs. Mtb-WT-infected condition. Scale bars, 5 µm. (


( B ) BMDMs were infected with Mtb - Δeis in absence or presence of 3 - methyladenine ( 3 - MA ; 10 mM ) subjected to confocal as described in Figure 1A . LC3 - punctated were counted manually . Each condition was assayed in triplicate , at least 250 were counted in each well . * * * p < 0 . 001 , vs . SC .

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(B) BMDMs were infected with Mtb-Δeis in the absence or presence of 3-methyladenine (3-MA; 10 mM) and subjected to confocal analysis as described in Figure 1A. LC3-punctated cells were counted manually. Each condition was assayed in triplicate, and at least 250 cells were counted in each well. ***p<0.001, vs. SC.


( C ) Immunoblot analyses performed using Abs raised to LC3 or β - actin . Experimental conditions were identical to those outlined in panel A . Gel representative of three experiments are shown .

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(C) Immunoblot analyses performed using Abs raised to LC3 or β-actin. Experimental conditions were identical to those outlined in panel A. Gel images representative of three experiments are shown.


( D ) Electron of Mtb - Δeis - infected BMDMs under low ( left ) high ( right ) magnification show accumulation of autophagic vesicles ( black arrow , initial autophagic vacuoles ; arrow , degradative autophagic vacuoles ) . Scale bars : 2 µm ( left ) , 0 . 5 µm ( right ) .

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(D) Electron micrographs of Mtb-Δeis-infected BMDMs under low (left) and high (right) magnification show the accumulation of autophagic vesicles (black arrow, initial autophagic vacuoles; white arrow, degradative autophagic vacuoles). Scale bars: 2 µm (left), 0.5 µm (right).


( E ) Immunoblot analyses performed using Abs raised to LC3 or β - actin . BMDMs were infected with Mtb - Δeis in presence or absence of 3 - MA ( 10 mM ) or bafilomycin A1 ( Baf - A1 ; 100 nM ) . Gel representative of three experiments are shown . of intensities of LC3 - II / LC3 - I β - actin bands is indicated below each lane ( C E ) . UI , uninfected ; SC , solvent control ( 0 . 1 % distilled water ( B ) , 0 . 1 % DMSO ( E ) ) .

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(E) Immunoblot analyses performed using Abs raised to LC3 or β-actin. BMDMs were infected with Mtb-Δeis in the presence or absence of 3-MA (10 mM) or bafilomycin A1 (Baf-A1; 100 nM). Gel images representative of three experiments are shown. The ratio of the intensities of the LC3-II/LC3-I and β-actin bands is indicated below each lane (C and E). UI, uninfected; SC, solvent control (0.1% distilled water (B), 0.1% DMSO (E)).


( A B ) BMDMs were infected with Mtb - WT , Mtb - Δeis or Mtb - c - eis at different MOIs ( 0 . 1 , 1 10 ) for 18 h ( A ) or for indicated periods of time ( B ; MOI = 10 ) . Supernatants were assessed by ELISA for of TNF - α IL - 6 . Data ( A B ) are presented as mean ± SD of five experiments .

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(A and B) BMDMs were infected with Mtb-WT, Mtb-Δeis or Mtb-c-eis at different MOIs (0.1, 1 and 10) for 18 h (A) or for the indicated periods of time (B; MOI = 10). Supernatants were assessed by ELISA for levels of TNF-α and IL-6. Data (A and B) are presented as the mean±SD of five experiments.


( C D ) BMDMs were stimulated with Mtb - WT , Mtb - Δeis , or Mtb - c - eis for 30 min . were then incubated with 10 µM DHE or 5 µM DCFH - DA for 15 min , washed thoroughly , immediately analyzed for superoxide or H2O2 production by flow cytometry ( C , Left ) . were labeled with MitoSOX for 30 min analyzed for mitochondrial ROS by flow cytometry ( D , top ) . Quantitative of ROS generation ( C , right ; D , bottom ) . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , vs . Mtb - WT - infected condition . UI , uninfected .

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(C and D) BMDMs were stimulated with Mtb-WT, Mtb-Δeis, or Mtb-c-eis for 30 min. Cells were then incubated with 10 µM DHE or 5 µM DCFH-DA for 15 min, washed thoroughly, and immediately analyzed for superoxide or H2O2 production by flow cytometry (C, Left). Cells were labeled with MitoSOX for 30 min and analyzed for mitochondrial ROS levels by flow cytometry (D, top). Quantitative analysis of ROS generation (C, right; D, bottom). *p<0.05, **p<0.01, ***p<0.001, vs. Mtb-WT-infected condition. UI, uninfected.


( A - C ) BMDMs were infected with Mtb - Δeis ( MOI = 10 ) for 18 h in presence or absence of DPI ( 10 µM ) , NAC ( 20 mM ) , catalase ( Cat , 1 mU / mL ) , or tiron ( 5 mM ) . ( A ) Representative immunofluorescence ( top ) ; of endogenous LC3 - punctated ( bottom )

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(A-C) BMDMs were infected with Mtb-Δeis (MOI = 10) for 18 h in the presence or absence of DPI (10 µM), NAC (20 mM), catalase (Cat, 1 mU/mL), or tiron (5 mM). (A) Representative immunofluorescence images (top); percentage of endogenous LC3-punctated cells (bottom)


( B ) Immunoblot analyses of BMDMs with antibodies raised to LC3 or β - actin . Gel are representative of three experiments . of intensities of LC3 - II / LC3 - I β - actin bands is indicated below each lane .

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(B) Immunoblot analyses of BMDMs with antibodies raised to LC3 or β-actin. Gel images are representative of three experiments. The ratio of the intensities of the LC3-II/LC3-I and β-actin bands is indicated below each lane.


( C ) Experimental conditions were identical to those outlined in Figure 3A . Supernatants collected 18 h after infection were assessed for cytokine by ELISA . Data represent mean ± SD of five experiments .

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(C) Experimental conditions were identical to those outlined in Figure 3A. Supernatants collected 18 h after infection were assessed for cytokine levels by ELISA. Data represent the mean±SD of five experiments.


( D - F ) BMDMs from WT NOX2 KO mice were infected with Mtb - WT , Mtb - Δeis , or Mtb - c - eis for 18 h . ( D ) Numbers of LC3 - punctated ( counted manually ) are shown ( at least 250 were counted in each well ) .

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(D-F) BMDMs from WT and NOX2 KO mice were infected with Mtb-WT, Mtb-Δeis, or Mtb-c-eis for 18 h. (D) Numbers of LC3-punctated cells (counted manually) are shown (at least 250 cells were counted in each well).


( E ) Immunoblot analyses performed using Abs raised to LC3 or β - actin . BMDMs from WT NOX2 KO mice were infected with Mtb - Δeis for 18 h . Gel representative of three experiments are shown .

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(E) Immunoblot analyses performed using Abs raised to LC3 or β-actin. BMDMs from WT and NOX2 KO mice were infected with Mtb-Δeis for 18 h. Gel images representative of three experiments are shown.


( F ) Supernatants collected 18 h after infection were assessed for cytokine by ELISA . Data are presented as mean ± SD of at least three separate experiments , each performed in triplicate . * * * p < 0 . 001 , vs . SC ( A C ) ; WT mice ( D F ) . UI , uninfected ; SC , solvent control ( 0 . 1 % DMSO ) .

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(F) Supernatants collected 18 h after infection were assessed for cytokine levels by ELISA. Data are presented as the mean±SD of at least three separate experiments, each performed in triplicate. ***p<0.001, vs. SC (A and C); WT mice (D and F). UI, uninfected; SC, solvent control (0.1% DMSO).


( A ) BMDMs were infected with Mtb - WT , Mtb - Δeis , or Mtb - c - eis ( MOI = 10 ) for indicated periods of time , washed to remove unbound mycobacteria , then incubated in complete DMEM at 37 ° C in 5 % CO2 . viability was assessed by PI then examined by microscopy . ( B C ) Experimental conditions were identical to those outlined in panel A . BMDMs were infected with three strains of mycobacteria for 36 h .

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(A) BMDMs were infected with Mtb-WT, Mtb-Δeis, or Mtb-c-eis (MOI = 10) for the indicated periods of time, washed to remove unbound mycobacteria, and then incubated in complete DMEM at 37°C in 5% CO2. Cell viability was assessed by PI staining and then examined by fluorescence microscopy. (B and C) Experimental conditions were identical to those outlined in panel A. BMDMs were infected with the three strains of mycobacteria for 36 h.


B ) Apoptosis was assessed using a TUNEL / apoptosis detection kit , according to manufacturer ' s protocol . were then examined under a - scanning confocal microscope ( LSM 510 ; Zeiss ) . Percentages of TUNEL - positive , PI - positive , TUNEL - / PI - double - positive were calculated . Data are representative of three separate experiments .

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B) Apoptosis was assessed using a TUNEL/apoptosis detection kit, according to the manufacturer's protocol. Cells were then examined under a laser-scanning confocal microscope (LSM 510; Zeiss). Percentages of TUNEL-positive, PI-positive, and TUNEL-/PI-double-positive cells were calculated. Data are representative of three separate experiments.


( C ) Morphological changes in BMDMs infected with Mtb - WT , Mtb - Δeis , or Mtb - c - eis at MOIs of 1 10 . Representative are shown . Scale bars : 50 µm ( low magnification ) , 20 µm ( high magnification ) .

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(C) Morphological changes in BMDMs infected with Mtb-WT, Mtb-Δeis, or Mtb-c-eis at MOIs of 1 and 10. Representative images are shown. Scale bars: 50 µm (low magnification), 20 µm (high magnification).


( D ) Macrophages were infected with Mtb - WT , Mtb - Δeis , or Mtb - c - eis in presence or absence of zVAD - fmk ( 20 µM ) or 3 - MA ( 10 mM ) . Staurosporine ( STS ; 500 nM ) was used as a positive control . After 36 h , were stained with PI then examined by microscopy . Data are presented as mean ± SD of at least three separate experiments , each performed in duplicate . * p < 0 . 05 , * * * p < 0 . 001 , vs . Mtb - WT - infected condition ( A B ) ; Mtb - Δeis - infected condition without inhibitors ( D ) . UI , uninfected .

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(D) Macrophages were infected with Mtb-WT, Mtb-Δeis, or Mtb-c-eis in the presence or absence of zVAD-fmk (20 µM) or 3-MA (10 mM). Staurosporine (STS; 500 nM) was used as a positive control. After 36 h, cells were stained with PI and then examined by fluorescence microscopy. Data are presented as the mean±SD of at least three separate experiments, each performed in duplicate. *p<0.05, ***p<0.001, vs. Mtb-WT-infected condition (A and B); Mtb-Δeis-infected condition without inhibitors (D). UI, uninfected.


( A B ) BMDMs were infected with Mtb - WT , Mtb - Δeis , or Mtb - c - eis ( MOI = 1 ) for indicated periods of time , then subjected to Western blot using Abs raised to p - ERK1 / 2 , p - p38 , p - JNK , β - actin . Data shown are representative of three independent experiments that yielded similar results ( A ) . Expression of phospho - MAPK / β - actin in extracts of BMDMs was quantified densitometrically ( B ) .

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(A and B) BMDMs were infected with Mtb-WT, Mtb-Δeis, or Mtb-c-eis (MOI = 1) for the indicated periods of time, and then subjected to Western blot analysis using Abs raised to p-ERK1/2, p-p38, p-JNK, and β-actin. Data shown are representative of three independent experiments that all yielded similar results (A). Expression of phospho-MAPK/β-actin protein in cytoplasmic extracts of BMDMs was quantified densitometrically (B).


( C D ) BMDMs were pretreated with U0126 ( 5 , 10 , 20 µM ) , SB203580 ( SB ; 1 , 5 , 10 µM ) , or SP600125 ( SP ; 5 , 10 , 20 µM ) for 45 min , then infected with Mtb - Δeis for 30 min ( C ) or 36 h ( D ) . ( C ) were then incubated with DHE for 15 min , washed rapidly thoroughly , analyzed immediately for superoxide by flow cytometry . Quantitative DHE data represent mean ± SD of four experiments . ( D ) after 36 h was assessed by PI then examined by microscopy . (

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(C and D) BMDMs were pretreated with U0126 (5, 10, 20 µM), SB203580 (SB; 1, 5, 10 µM), or SP600125 (SP; 5, 10, 20 µM) for 45 min, and then infected with Mtb-Δeis for 30 min (C) or 36 h (D). (C) Cells were then incubated with DHE for 15 min, washed rapidly and thoroughly, and analyzed immediately for superoxide levels by flow cytometry. Quantitative DHE fluorescence data represent the mean±SD of four experiments. (D) Cell death after 36 h was assessed by PI staining and then examined by fluorescence microscopy. (


( E ) Raw264 . 7 were transfected with siRNA specific for JNK1 ( siJNK ) or a non - specific control siRNA ( siNS ) . At 24 h after transfection , were infected with Mtb - Δeis for 36 h . was then assessed by PI , then examined by flow cytometry . Transfection efficiency was assessed by RT - PCR ( inset ) . Data represent mean ± SD of five random fields are representative of three independent experiments ( D E ) . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , vs . Mtb - WT - infected condition ( B ) ; SC ( C D ) ; siNS ( E ) . UI , uninfected ; SC , solvent control ( 0 . 1 % DMSO ) .

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(E) Raw264.7 cells were transfected with siRNA specific for JNK1 (siJNK) or a non-specific control siRNA (siNS). At 24 h after transfection, cells were infected with Mtb-Δeis for 36 h. Cell death was then assessed by PI staining, and then examined by flow cytometry. Transfection efficiency was assessed by RT-PCR (inset). Data represent the mean±SD of five random fields and are representative of three independent experiments (D and E). *p<0.05, **p<0.01, ***p<0.001, vs. Mtb-WT-infected condition (B); SC (C and D); siNS (E). UI, uninfected; SC, solvent control (0.1% DMSO).


C57BL / 6 mice were challenged , by aerosol , with 10 - 30 CFU Mtb - WT , Mtb - Δeis , or Mtb - c - eis sacrificed 4 post - infection . ( A ) High - low - magnification electron of lung tissue sections from mice infected with Mtb - Δeis show accumulation of autophagic vesicles ( black arrow , in autophagic vacuoles ; arrow , degradative autophagic vacuoles ) . Scale bars : 2 µm ( left upper ) , 0 . 5 µm ( right ) . Numbers of autophagic vacuoles per in each section ( left lower ) ( mean ± SD ; n = 50 ) .

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C57BL/6 mice were challenged, by aerosol, with 10-30 CFU Mtb-WT, Mtb-Δeis, or Mtb-c-eis and sacrificed 4 weeks post-infection. (A) High- and low-magnification electron micrographs of lung tissue sections from mice infected with Mtb-Δeis show accumulation of autophagic vesicles (black arrow, bacteria in autophagic vacuoles; white arrow, degradative autophagic vacuoles). Scale bars: 2 µm (left upper), 0.5 µm (right). Numbers of autophagic vacuoles per cell in each TEM section (left lower) (mean±SD; n = 50).


( B ) Quantitative RT - PCR of lung tissue from Mtb - WT - , Mtb - Δeis - , Mtb - c - eis - infected mice . Total was extracted from paraffin - embedded lung tissue sections , as described in Materials Methods .

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(B) Quantitative RT-PCR analysis of lung tissue from Mtb-WT-, Mtb-Δeis-, and Mtb-c-eis-infected mice. Total RNA was extracted from paraffin-embedded lung tissue sections, as described in the Materials and Methods.