figf3 ( a ) Protein extracts from HeLa cells were immunoprecipitated using anti - ULK1 antibody or with IgG as a negative control and analysed by western blotting using anti - Lys63 specific ubiquitin and anti - ULK1 antibodies . ( b ) HeLa cells were co - transfected with ULK1 - Myc alone or in combination with wild - type ubiquitin or its mutant ( K48R or K63R ) HA - tagged constructs . Protein extracts were immunoprecipitated using an anti - Myc antibody . Purified complexes and total extracts were analysed by western blotting using anti - Myc and anti - HA antibodies . ( c ) In HeLa cells , AMBRA1 was downregulated by RNAi . Protein extracts were immunoprecipitated using anti - ULK1 antibody or with IgG as a negative control . Purified complexes and total extracts were analysed by western blotting using anti - ULK1 , anti - Lys - 63 ubiquitin and anti - AMBRA1 antibodies . Of note , ULK1 levels in the co - immunoprecipitation experiment do not change in this case in the AMBRA1 siRNA lane , owing to saturation of the anti - ULK1 antibody ( see also Fig . 4h ) . ( d ) HeLa cells were co - transfected with vectors encoding for ubiquitin Lys - 63 - HA alone or in combination with AMBRA1 . Protein extracts were immunoprecipitated using an anti - ULK1 antibody or with IgG as a negative control and Ub - Lys - 63 - HA , ULK1 and AMBRA1 were analysed by western blotting . ( e , f ) HeLa cells were cultured in complete or starvation medium for 30 min or in the presence of 100 nM of rapamycin for 3 h and then protein extracts were immunoprecipitated using anti - ULK1 antibody or with IgG as a negative control . Purified complexes and total extracts were analysed by western blotting as in a . Quantifications are shown on the right . In both panels , n = 3 extracts prepared from independent experiments . Data are presented as means ± s . d . and significance is P = 0 . 003 and P = 0 . 0015 , respectively .

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

figf3(a) Protein extracts from HeLa cells were immunoprecipitated using anti-ULK1 antibody or with IgG as a negative control and analysed by western blotting using anti-Lys63 specific ubiquitin and anti-ULK1 antibodies.(b) HeLa cells were co-transfected with ULK1-Myc alone or in combination with wild-type ubiquitin or its mutant (K48R or K63R) HA-tagged constructs. Protein extracts were immunoprecipitated using an anti-Myc antibody. Purified complexes and total extracts were analysed by western blotting using anti-Myc and anti-HA antibodies.(c) In HeLa cells, AMBRA1 was downregulated by RNAi. Protein extracts were immunoprecipitated using anti-ULK1 antibody or with IgG as a negative control. Purified complexes and total extracts were analysed by western blotting using anti-ULK1, anti-Lys-63 ubiquitin and anti-AMBRA1 antibodies. Of note, ULK1 levels in the co-immunoprecipitation experiment do not change in this case in the AMBRA1 siRNA lane, owing to saturation of the anti-ULK1 antibody (see also Fig. 4h).(d) HeLa cells were co-transfected with vectors encoding for ubiquitin Lys-63-HA alone or in combination with AMBRA1. Protein extracts were immunoprecipitated using an anti-ULK1 antibody or with IgG as a negative control and Ub-Lys-63-HA, ULK1 and AMBRA1 were analysed by western blotting.(e,f) HeLa cells were cultured in complete or starvation medium for 30 min or in the presence of 100 nM of rapamycin for 3 h and then protein extracts were immunoprecipitated using anti-ULK1 antibody or with IgG as a negative control. Purified complexes and total extracts were analysed by western blotting as in a. Quantifications are shown on the right. In both panels, n = 3 extracts prepared from independent experiments. Data are presented as means±s.d. and significance is P = 0.003 and P = 0.0015, respectively.


figf4 ( a ) HeLa cells were transfected with FLAG - TRAF6 or grown in starvation medium for 1 h . Some of them were treated with chloroquine . LC3 , p62 and actin were analysed by western blotting . Quantification of LC3II and p62 bands is shown on the right . n = 3 extracts prepared from independent experiments . Data are presented as means ± s . d . and significance is P = 0 . 017 for LC3 and P = 0 . 01 and P = 0 . 04 for p62 . ( b ) HeLa cells were transduced with AMBRA1 lentiviral shRNA and transfected or not with FLAG - TRAF6 . Quantification of LC3II and p62 bands is shown on the right . n = 3 extracts prepared from independent experiments . Data are presented as means ± s . d . and significance is P = 0 . 001 and P = 0 . 0018 for LC3 and P = 0 . 004 for p62 . ( c ) Count of LC3 puncta in HeLa cells transduced as in b . Five different confocal microscopy images for each experiment were randomly chosen and GFP - positive dots were examined on the images with identical brightness and contrast settings . The results are represented as the average number of LC3 dots with 100 cells analysed per sample . Confocal images are shown in Supplementary Fig . S3b . ( d ) HEK293 cells were transfected with AMBRA1 and FLAG - TRAF6 as indicated . Protein extracts were immunoprecipitated using an anti - FLAG antibody . ( e ) HeLa cells were transfected as in b and FLAG - TRAF6 was purified as in d . Quantification of immunoprecipitated TRAF6 is shown on the right . n = 3 extracts prepared from independent experiments . Data are presented as means ± s . d . and significance is P = 0 . 0003 . ( f ) In HeLa cells , TRAF6 was downregulated by RNAi . AMBRA1 , ULK1 , TRAF6 , ATG13 and actin were analysed by western blotting . ( g ) HeLa cells were co - transfected with vectors encoding ULK1 - Myc , FLAG - TRAF6 and Ub - Lys - 63 - HA , as indicated . Protein extracts were immunoprecipitated using an anti - Myc antibody . ( h ) HeLa cells stably interfered for AMBRA1 were transfected with FLAG - TRAF6 and Ub - Lys - 63 - HA as indicated . Protein extracts were immunoprecipitated using anti - ULK1 antibody . ( i ) Endogenous ULK1 is immunoprecipitated from HeLa cells transfected as in b . The resulting precipitates were subjected to a ULK1 kinase assay using MBP as the substrate . Purified complexes were analysed by autoradiography and by western blotting using an anti - ULK1 antibody .

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

figf4(a) HeLa cells were transfected with FLAG-TRAF6 or grown in starvation medium for 1 h. Some of them were treated with chloroquine. LC3, p62 and actin were analysed by western blotting. Quantification of LC3II and p62 bands is shown on the right. n = 3 extracts prepared from independent experiments. Data are presented as means±s.d. and significance is P = 0.017 for LC3 and P = 0.01 and P = 0.04 for p62.(b) HeLa cells were transduced with AMBRA1 lentiviral shRNA and transfected or not with FLAG-TRAF6. Quantification of LC3II and p62 bands is shown on the right. n = 3 extracts prepared from independent experiments. Data are presented as means±s.d. and significance is P = 0.001 and P = 0.0018 for LC3 and P = 0.004 for p62.(c) Count of LC3 puncta in HeLa cells transduced as in b. Five different confocal microscopy images for each experiment were randomly chosen and GFP-positive dots were examined on the images with identical brightness and contrast settings. The results are represented as the average number of LC3 dots with 100 cells analysed per sample. Confocal images are shown in Supplementary Fig. S3b.(d) HEK293 cells were transfected with AMBRA1 and FLAG-TRAF6 as indicated. Protein extracts were immunoprecipitated using an anti-FLAG antibody.(e) HeLa cells were transfected as in b and FLAG-TRAF6 was purified as in d. Quantification of immunoprecipitated TRAF6 is shown on the right. n = 3 extracts prepared from independent experiments. Data are presented as means±s.d. and significance is P = 0.0003.(f) In HeLa cells, TRAF6 was downregulated by RNAi. AMBRA1, ULK1, TRAF6, ATG13 and actin were analysed by western blotting.(g) HeLa cells were co-transfected with vectors encoding ULK1-Myc, FLAG-TRAF6 and Ub-Lys-63-HA, as indicated. Protein extracts were immunoprecipitated using an anti-Myc antibody.(h) HeLa cells stably interfered for AMBRA1 were transfected with FLAG-TRAF6 and Ub-Lys-63-HA as indicated. Protein extracts were immunoprecipitated using anti-ULK1 antibody.(i) Endogenous ULK1 is immunoprecipitated from HeLa cells transfected as in b. The resulting precipitates were subjected to a ULK1 kinase assay using MBP as the substrate. Purified complexes were analysed by autoradiography and by western blotting using an anti-ULK1 antibody.


figf5 ( a - c ) Two canonical TRAF6 - binding sites in AMBRA1 are essential for its interaction with TRAF6 . HEK293 cells were co - transfected with vectors encoding FLAG - TRAF6 together with AMBRA1WT and mutant constructs . Protein extracts were immunoprecipitated using an anti - FLAG antibody . AMBRA1 and FLAG - TRAF6 were analysed by western blotting . ( a - c ) Two canonical TRAF6 - binding sites in AMBRA1 are essential for its interaction with TRAF6 . HEK293 cells were co - transfected with vectors encoding FLAG - TRAF6 together with AMBRA1WT and mutant constructs . Protein extracts were immunoprecipitated using an anti - FLAG antibody . AMBRA1 and FLAG - TRAF6 were analysed by western blotting . ( d ) HeLa cells were transfected with AMBRA1 RNAi oligonucleotide ( siRNA AMBRA1 ) . Then , cells were co - transfected with vectors encoding ubiquitin - HA and AMBRA1WT or AMBRA1AA , respectively . Protein extracts were immunoprecipitated using anti - ULK1 antibody ; ubiquitin - HA , ULK1 and AMBRA1 were analysed by western blotting . ( e ) HeLa cells stably interfered for AMBRA1 were transduced with lentiviral vectors encoding β - galactosidase ( negative control ) AMBRA1WT or AMBRA1AA respectively ; protein extracts were analysed by western blotting using anti - ULK1 , anti - AMBRA1 anti - ATG13 , anti - pATG13 and anti - actin antibodies . ( f ) HeLa cells were transfected as in e and some of them were treated with chloroquine for 1 h . LC3 , p62 , AMBRA1 and actin were analysed by western blotting . Quantification of LC3II and p62 are shown on the right . n = 3 extracts prepared from independent experiments . Data are presented as means ± s . d . and significance is P = 0 . 03 for LC3 and P = 0 . 045 for p62 . ( g ) GFP - LC3 MEF cells from wild - type ( Ambra1 + / + ) and homozygous ( Ambra1gt / gt ) embryos were transduced with the indicated lentiviral vectors and quantification of LC3 puncta per cells is shown on the right . Bars represent mean ± s . e . m . of triplicate samples with 50 cells analysed per sample ; n = 3 independent experiments . Significance is P = 0 . 001 and P = 0 . 003 ( n = 3 ) . Scale bar , 10 μm .

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

figf5(a-c) Two canonical TRAF6-binding sites in AMBRA1 are essential for its interaction with TRAF6. HEK293 cells were co-transfected with vectors encoding FLAG-TRAF6 together with AMBRA1WT and mutant constructs. Protein extracts were immunoprecipitated using an anti-FLAG antibody. AMBRA1 and FLAG-TRAF6 were analysed by western blotting.(a-c) Two canonical TRAF6-binding sites in AMBRA1 are essential for its interaction with TRAF6. HEK293 cells were co-transfected with vectors encoding FLAG-TRAF6 together with AMBRA1WT and mutant constructs. Protein extracts were immunoprecipitated using an anti-FLAG antibody. AMBRA1 and FLAG-TRAF6 were analysed by western blotting.(d) HeLa cells were transfected with AMBRA1 RNAi oligonucleotide (siRNA AMBRA1). Then, cells were co-transfected with vectors encoding ubiquitin-HA and AMBRA1WT or AMBRA1AA, respectively. Protein extracts were immunoprecipitated using anti-ULK1 antibody; ubiquitin-HA, ULK1 and AMBRA1 were analysed by western blotting.(e) HeLa cells stably interfered for AMBRA1 were transduced with lentiviral vectors encoding β-galactosidase (negative control) AMBRA1WT or AMBRA1AA respectively; protein extracts were analysed by western blotting using anti-ULK1, anti-AMBRA1 anti-ATG13, anti-pATG13 and anti-actin antibodies.(f) HeLa cells were transfected as in e and some of them were treated with chloroquine for 1 h. LC3, p62, AMBRA1 and actin were analysed by western blotting. Quantification of LC3II and p62 are shown on the right. n = 3 extracts prepared from independent experiments. Data are presented as means±s.d. and significance is P = 0.03 for LC3 and P = 0.045 for p62.(g) GFP-LC3 MEF cells from wild-type (Ambra1+/+) and homozygous (Ambra1gt/gt) embryos were transduced with the indicated lentiviral vectors and quantification of LC3 puncta per cells is shown on the right. Bars represent mean±s.e.m. of triplicate samples with 50 cells analysed per sample; n = 3 independent experiments. Significance is P = 0.001 and P = 0.003 (n = 3). Scale bar, 10 μm.


figf6 ( a ) HEK293 cells were co - transfected with vectors encoding ULK1 - HA and ULK1 - Myc and then grown either in normal ( Ctrl ) or in starvation ( EBS ) medium for 1 h . Protein extracts were immunoprecipitated using an anti - Myc antibody , and analysed by western blotting using anti - HA , and anti - Myc antibodies . Quantification is shown on the right . n = 3 extracts prepared from independent experiments . Data are presented as means ± s . d . and significance is P = 0 . 001 . ( b ) HEK293 cells were transfected with TRAF6 RNAi oligonucleotide ( TRAF6 siRNA ) or unrelated oligonucleotides as a control ( Ctrl siRNA ) . Then , cells were co - transfected with vectors encoding ULK1 - HA and ULK1 - Myc and protein complexes were immunoprecipitated using anti - Myc antibody . Protein extracts were analysed by western blotting using anti - HA , anti - Myc and anti - TRAF6 antibodies . Quantification of ULK1 - HA relative to ULK1 - Myc is reported on the right . n = 3 extracts prepared from independent experiments . Data are presented as means ± s . d . and significance is P = 0 . 037 . ( c ) HEK293 cells were transfected with AMBRA1 RNAi oligonucleotide ( AMBRA1 siRNA ) or unrelated oligonucleotides as a control ( Ctrl siRNA ) . Then , cells were co - transfected and analysed as in a . Quantification is shown on the right . n = 3 extracts prepared from independent experiments . Data are presented as means ± s . d . and significance is P = 0 . 044 . ( d ) HEK293 cells were co - transfected with vectors encoding for ULK1 - HA , ULK1 - Myc and AMBRA1WT or AMBRA1AA respectively . Protein complexes were immunoprecipitated and analysed as in a . Quantification is shown on the right . n = 3 extracts prepared from independent experiments . Data are presented as means ± s . d . and significance is P = 0 . 014 .

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 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 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 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

figf6(a) HEK293 cells were co-transfected with vectors encoding ULK1-HA and ULK1-Myc and then grown either in normal (Ctrl) or in starvation (EBS) medium for 1 h. Protein extracts were immunoprecipitated using an anti-Myc antibody, and analysed by western blotting using anti-HA, and anti-Myc antibodies. Quantification is shown on the right. n = 3 extracts prepared from independent experiments. Data are presented as means±s.d. and significance is P = 0.001.(b) HEK293 cells were transfected with TRAF6 RNAi oligonucleotide (TRAF6 siRNA) or unrelated oligonucleotides as a control (Ctrl siRNA). Then, cells were co-transfected with vectors encoding ULK1-HA and ULK1-Myc and protein complexes were immunoprecipitated using anti-Myc antibody. Protein extracts were analysed by western blotting using anti-HA, anti-Myc and anti-TRAF6 antibodies. Quantification of ULK1-HA relative to ULK1-Myc is reported on the right. n = 3 extracts prepared from independent experiments. Data are presented as means±s.d. and significance is P = 0.037.(c) HEK293 cells were transfected with AMBRA1 RNAi oligonucleotide (AMBRA1 siRNA) or unrelated oligonucleotides as a control (Ctrl siRNA). Then, cells were co-transfected and analysed as in a. Quantification is shown on the right. n = 3 extracts prepared from independent experiments. Data are presented as means±s.d. and significance is P = 0.044.(d) HEK293 cells were co-transfected with vectors encoding for ULK1-HA, ULK1-Myc and AMBRA1WT or AMBRA1AA respectively. Protein complexes were immunoprecipitated and analysed as in a. Quantification is shown on the right. n = 3 extracts prepared from independent experiments. Data are presented as means±s.d. and significance is P = 0.014.


figf7 ( a ) Highly purified mTOR was incubated with radiolabelled ATP without substrate , with purified FLAG - AMBRA1 and purified FLAG - AMBRA1S52A and analysed by autoradiography . The lower panels show a FLAG and mTOR western blot ( WB ) . ( b ) HEK293 cells expressing FLAG - RAPTOR were subjected to FLAG immunoprecipitation . Protein complexes were analysed by western blotting using anti - FLAG and anti - AMBRA1 antibodies . ( c ) HEK293 cells were treated with Torin1 for 3 h or cultured in starvation medium for 2 h and protein extracts were analysed by western blotting using anti - pSer - 52 and anti - AMBRA1 antibodies . ( d ) HeLa cells stably interfered for AMBRA1 were transduced with the indicated lentiviral vectors encoding for β - galactosidase , AMBRA1WT or AMBRA1S52A respectively and some of them were treated with chloroquine for 1 h . Protein extracts were analysed by western blotting using anti - LC3 , anti - p62 and anti - actin antibodies . Quantification of LC3II and p62 bands is shown below . n = 3 extracts prepared from independent experiments . Data are presented as means ± s . d . and significance is P = 0 . 038 . ( e ) GFP - LC3 MEF cells from wild - type ( Ambra1 + / + ) and homozygous ( Ambra1gt / gt ) embryos were transduced with indicated lentiviral vectors and quantification of LC3 puncta per cell is shown on the right . Bars represent mean ± s . e . m . of triplicate samples and 50 cells analysed per sample . n = 3 independent experiments . Significance is P = 0 . 001 and P = 0 . 003 . Scale bar , 10 μm ( f ) HeLa cells stably interfered for AMBRA1 were co - transfected with vectors encoding Ub - Lys - 63 - HA and AMBRA1WT or AMBRA1S52A , respectively . Protein extracts were immunoprecipitated using an anti - ULK1 antibody and ubiquitin Lys - 63 - HA , ULK1 , AMBRA1 and actin were analysed by western blotting . ( g ) HeLa cells stably interfered for AMBRA1 were transduced as in d ; protein extracts were analysed by western blotting using anti - AMBRA1 , anti - ATG13 , anti - pATG13 and anti - actin antibodies . Quantification of the p - ATG13 band is shown below . ( h ) HeLa cells transfected with ULK1 - HA , ULK1 - Myc and different AMBRA1 constructs , respectively . Protein complexes were immunoprecipitated using anti - HA antibody and analysed by western blotting using anti - HA , anti - Myc and anti - AMBRA1 antibodies .

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 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 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 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 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 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 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

figf7(a) Highly purified mTOR was incubated with radiolabelled ATP without substrate, with purified FLAG-AMBRA1 and purified FLAG-AMBRA1S52A and analysed by autoradiography. The lower panels show a FLAG and mTOR western blot (WB).(b) HEK293 cells expressing FLAG-RAPTOR were subjected to FLAG immunoprecipitation. Protein complexes were analysed by western blotting using anti-FLAG and anti-AMBRA1 antibodies.(c) HEK293 cells were treated with Torin1 for 3 h or cultured in starvation medium for 2 h and protein extracts were analysed by western blotting using anti-pSer-52 and anti-AMBRA1 antibodies.(d) HeLa cells stably interfered for AMBRA1 were transduced with the indicated lentiviral vectors encoding for β-galactosidase, AMBRA1WT or AMBRA1S52A respectively and some of them were treated with chloroquine for 1 h. Protein extracts were analysed by western blotting using anti-LC3, anti-p62 and anti-actin antibodies. Quantification of LC3II and p62 bands is shown below. n = 3 extracts prepared from independent experiments. Data are presented as means±s.d. and significance is P = 0.038.(e) GFP-LC3 MEF cells from wild-type (Ambra1+/+) and homozygous (Ambra1gt/gt) embryos were transduced with indicated lentiviral vectors and quantification of LC3 puncta per cell is shown on the right. Bars represent mean±s.e.m. of triplicate samples and 50 cells analysed per sample. n = 3 independent experiments. Significance is P = 0.001 and P = 0.003. Scale bar, 10 μm(f) HeLa cells stably interfered for AMBRA1 were co-transfected with vectors encoding Ub-Lys-63-HA and AMBRA1WT or AMBRA1S52A, respectively. Protein extracts were immunoprecipitated using an anti-ULK1 antibody and ubiquitin Lys-63-HA, ULK1, AMBRA1 and actin were analysed by western blotting.(g) HeLa cells stably interfered for AMBRA1 were transduced as in d; protein extracts were analysed by western blotting using anti-AMBRA1, anti-ATG13, anti-pATG13 and anti-actin antibodies. Quantification of the p-ATG13 band is shown below.(h) HeLa cells transfected with ULK1-HA, ULK1-Myc and different AMBRA1 constructs, respectively. Protein complexes were immunoprecipitated using anti-HA antibody and analysed by western blotting using anti-HA, anti-Myc and anti-AMBRA1 antibodies.


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 ) Representative high content microscopy images of RIPosomes in THP - 1 cells infected with S . flexneri ( MOI 1 : 25 , 8 h ) . About 50000 cells were plated per well and RIPosomes were screened in 35 fields per well in 3 technical replicates . Right panel , the graph depicts an average number of RIPosomes / cell , which is calculated from 3 biological replicates , Mean ± SD . * * * p < 0 . 005 , Student ' s unpaired t - test . ( C ) Western blot analysis of soluble and insoluble fractions of S . flexneri infected THP - 1 cells ( MOI 1 : 25 , 8 h ) . Arrow indicates oligomers of RIPK2 . ( D ) Left panel , representative high content microscopy image of control and RIPK2 knockdown THP - 1 cells infected with S . flexneri ( MOI 1 : 25 , 8 h ) . Right panel , the graph depicts an average number of RIPosomes / cell , which is calculated from 3 biological replicates , Mean ± SD . * * * p < 0 . 0005 , Student ' s unpaired t - test . ( E - F ) Western blot analysis of soluble and insoluble fractions of S . flexneri infected ( E ) control siRNA and RIPK2 siRNA transfected THP - 1 cells ( F ) Control CRISPR ( RIPK2 + / + ) cells and RIPK2 CRISPR knockout ( RIPK2 - / - ) HT - 29 cells . ( G ) Representative confocal images of RIPosomes in mouse BMDMs infected with S . flexneri ( MOI 1 : 25 , 8 h ) . Line profile : co - localization analysis using line intensity profile . Scale bar , 5 µm . Zoom panels are digital magnifications . DNA is stained with DAPI ( pseudo - colored red for better contrast ) . ( I ) Representative confocal images of RIPosomes in THP - 1 cells infected with RFP expressing Salmonella typhimurium ( MOI 1 : 5 , 4 h ) . Line profile : co - localization analysis using line intensity profile . Scale bar , 8 µm . Zoom panels are digital magnifications . DNA is stained with DAPI . ( J ) Left panel , representative high - content microscopy images ( yellow masks represent software algorithms - defined cell boundaries ) of RIPosomes in THP - 1 cells infected with S . typhimurium ( MOI 1 : 5 , 4 h ) . Right panel , the graph depicts an average number of RIPosomes / cell . Mean ± SD , n = 4 ( biological replicates ) , * * * * p < 0 . 00005 , Student ' s unpaired t - test . DNA is stained with DAPI . Representative confocal images of RIPosomes in THP - 1 cells infected with ( K ) E . coli LF82 strain ( MOI 1 : 10 , 8h ) ( LPS antibody is used to stain LF82 ) Line profile : co - localization analysis using line intensity profiles . Scale bar , 5 µm . Zoom panels are digital magnifications . DNA is stained with DAPI . Representative confocal images of ( M ) GFP expressing M . tuberculosis ( MOI 1 : 10 , 8 h ) . Line profile : co - localization analysis using line intensity profiles . Scale bar , 5 µm . Zoom panels are digital magnifications . DNA is stained with DAPI . ( N ) Western blot analysis of soluble and insoluble fractions from THP - 1 cells , uninfected or infected with E . coli DH5α ( MOI 1 : 10 , 8h ) or E . coli LF82 strains ( MOI 1 : 10 , 8h ) .

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 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 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 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 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 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 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 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 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

Figure 1(B) Representative high content microscopy images of RIPosomes in THP-1 cells infected with S. flexneri (MOI 1:25, 8 h). About 50000 cells were plated per well and RIPosomes were screened in 35 fields per well in 3 technical replicates. Right panel, the graph depicts an average number of RIPosomes/cell, which is calculated from 3 biological replicates, Mean ± SD. ***p < 0.005, Student's unpaired t-test.(C) Western blot analysis of soluble and insoluble fractions of S. flexneri infected THP-1 cells (MOI 1:25, 8 h). Arrow indicates oligomers of RIPK2.(D) Left panel, representative high content microscopy image of control and RIPK2 knockdown THP-1 cells infected with S. flexneri (MOI 1:25, 8 h). Right panel, the graph depicts an average number of RIPosomes/cell, which is calculated from 3 biological replicates, Mean ± SD. ***p < 0.0005, Student's unpaired t-test.(E-F) Western blot analysis of soluble and insoluble fractions of S. flexneri infected (E) control siRNA and RIPK2 siRNA transfected THP-1 cells (F) Control CRISPR (RIPK2+/+) cells and RIPK2 CRISPR knockout (RIPK2-/-) HT-29 cells.(G) Representative confocal images of RIPosomes in mouse BMDMs infected with S. flexneri ( MOI 1:25, 8 h). Line profile: co-localization analysis using line intensity profile. Scale bar, 5 µm. Zoom panels are digital magnifications. DNA is stained with DAPI (pseudo-colored red for better contrast).(I) Representative confocal images of RIPosomes in THP-1 cells infected with RFP expressing Salmonella typhimurium (MOI 1:5, 4 h). Line profile: co-localization analysis using line intensity profile. Scale bar, 8 µm. Zoom panels are digital magnifications. DNA is stained with DAPI.(J) Left panel, representative high-content microscopy images (yellow masks represent software algorithms-defined cell boundaries) of RIPosomes in THP-1 cells infected with S. typhimurium (MOI 1:5, 4 h). Right panel, the graph depicts an average number of RIPosomes/cell. Mean ± SD, n=4 (biological replicates), ****p < 0.00005, Student's unpaired t-test. DNA is stained with DAPI.Representative confocal images of RIPosomes in THP-1 cells infected with (K) E. coli LF82 strain (MOI 1: 10, 8h) (LPS antibody is used to stain LF82) Line profile: co-localization analysis using line intensity profiles. Scale bar, 5 µm. Zoom panels are digital magnifications. DNA is stained with DAPI.Representative confocal images of (M) GFP expressing M. tuberculosis (MOI 1:10, 8 h). Line profile: co-localization analysis using line intensity profiles. Scale bar, 5 µm. Zoom panels are digital magnifications. DNA is stained with DAPI.(N) Western blot analysis of soluble and insoluble fractions from THP-1 cells, uninfected or infected with E. coli DH5α (MOI 1:10, 8h) or E. coli LF82 strains (MOI 1:10, 8h).


Figure 2 ( B ) Left panel , representative high - content microscopy images of HEK293T cells transfected with GFP - NOD1 , GFP - NOD2 , and GFP - RIPK2 plasmids for 9 h . Right panel , the graph depicts the average number of RIPosomes per GFP - positive cell . About 15000 cells were plated per well and RIPosomes were screened in 35 fields per well . Mean ± SD , n = 3 ( biological replicates ) , * * * * p < 0 . 00005 , ordinary one - way ANOVA ( Tukey ' s multiple comparisons test ) . ( C ) Western blot analysis of soluble and insoluble fractions of HEK293T cells transfected with the CARD domain - containing region of NOD1 , NOD2 , and RIPK2 . ( E ) The soluble and insoluble fractions of S . flexneri infected THP - 1 ( MOI 1 : 25 , 8 h ) cell lysates were subjected to Western blot analysis with indicated antibodies . ( F ) Representative confocal images of immunofluorescence assays conducted with THP - 1 cells infected with RFP expressing S . flexneri ( pseudo - colored , blue ) ( MOI 1 : 25 , 8h ) . Line profile : co - localization analysis using line intensity profiles . Scale bar , 5 µm . Zoom panels are digital magnifications . ( G ) Representative confocal images of immunofluorescence assays performed with HEK293T cells transfected with GFP - RIPK2 and Flag - NOD2 ( upper panel ) or Flag - NOD1 ( lower panel ) . Line profile : co - localization analysis using line intensity profiles . Scale bar , 5 or 10 µm as indicated in figures . Inset zoom panels are digital magnifications . ( H - I ) HEK293T cells were transfected with ( H ) full length and ( I ) CARD domains of NOD1 , NOD2 , and RIPK2 followed by Western blot analysis with soluble and insoluble fractions using indicated antibodies . ( J - K ) Western blot analysis of soluble and insoluble fractions of RIPK2 + / + and RIPK2 - / - HT - 29 cells infected with S . flexneri ( MOI 1 : 25 , 8 h ) with indicated antibodies . ( L - M ) Representative immunofluorescence images of doxycycline - inducible GFP - RIPK2 expressing HeLa cells . ( L ) Upper panel , uninfected . Lower panel , S . flexneri - infected MOI 1 : 25 , 4h ) . Immunostaining was performed with the p65 antibody ( red ) and DNA stained with DAPI ( Blue ) . ( M ) The graph indicates % of cells that are RIPosomes positive or negative with nuclear / cytoplasmic p65 ( 5 fields ( each group ) , Mean ± SD , n = 3 ) . * * * * p < 0 . 00005 , Student ' s unpaired t - test .

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 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 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 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 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 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

Figure 2(B) Left panel, representative high-content microscopy images of HEK293T cells transfected with GFP-NOD1, GFP-NOD2, and GFP -RIPK2 plasmids for 9 h. Right panel, the graph depicts the average number of RIPosomes per GFP-positive cell. About 15000 cells were plated per well and RIPosomes were screened in 35 fields per well. Mean ± SD, n=3 (biological replicates), ****p < 0.00005, ordinary one-way ANOVA (Tukey's multiple comparisons test).(C) Western blot analysis of soluble and insoluble fractions of HEK293T cells transfected with the CARD domain-containing region of NOD1, NOD2, and RIPK2.(E) The soluble and insoluble fractions of S. flexneri infected THP-1 (MOI 1:25, 8 h) cell lysates were subjected to Western blot analysis with indicated antibodies.(F) Representative confocal images of immunofluorescence assays conducted with THP-1 cells infected with RFP expressing S. flexneri (pseudo-colored, blue) (MOI 1:25, 8h). Line profile: co-localization analysis using line intensity profiles. Scale bar, 5 µm. Zoom panels are digital magnifications.(G) Representative confocal images of immunofluorescence assays performed with HEK293T cells transfected with GFP-RIPK2 and Flag-NOD2 (upper panel) or Flag-NOD1 (lower panel). Line profile: co-localization analysis using line intensity profiles. Scale bar, 5 or 10 µm as indicated in figures. Inset zoom panels are digital magnifications.(H-I) HEK293T cells were transfected with (H) full length and (I) CARD domains of NOD1, NOD2, and RIPK2 followed by Western blot analysis with soluble and insoluble fractions using indicated antibodies.(J-K) Western blot analysis of soluble and insoluble fractions of RIPK2 +/+ and RIPK2-/- HT-29 cells infected with S. flexneri (MOI 1:25, 8 h) with indicated antibodies.(L-M) Representative immunofluorescence images of doxycycline-inducible GFP-RIPK2 expressing HeLa cells. (L) Upper panel, uninfected. Lower panel, S. flexneri -infected MOI 1:25, 4h). Immunostaining was performed with the p65 antibody (red) and DNA stained with DAPI (Blue). (M) The graph indicates % of cells that are RIPosomes positive or negative with nuclear/cytoplasmic p65 (5 fields (each group), Mean ± SD, n=3). ****p < 0.00005, Student's unpaired t-test.