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 )

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

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)


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 .

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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.


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 )

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

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)


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 .

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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.


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 )

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

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)


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 ) .

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

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).


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 .

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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 .

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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 .

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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.


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 .

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

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.


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 )

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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)


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 .

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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.


C Western blotting of the lysates from HepG2 cells infected with lentivirus encoding shATG5 or shCtrl and transfected with Myc - MOAP - 1 .

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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 ) ,

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

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 .

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F Western blotting of lysates from MOAP-1 KO LO2 cells pre-treated with CQ or BafA1 and transfected with Myc-MOAP-1.


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 .

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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.


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 .

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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.


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 ) .

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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

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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


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

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F Loss of MOAP - 1 leads to slower exchange of p62 in the RFP - p62 bodies . WT and MOAP - 1 KO LO2 cells transfected with expression vector encoding RFP - tagged p62 . 14 hours later , cells were subjected to the fluorescence recovery after photobleaching ( FRAP ) assay . Scale bar : 2 µm . G Quantification of the rate of fluorescence recovery of RFP - p62 in WT and MOAP - 1 KO cells . n = 5 samples .

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F Loss of MOAP-1 leads to slower exchange of p62 in the RFP-p62 bodies. WT and MOAP-1 KO LO2 cells transfected with expression vector encoding RFP-tagged p62. 14 hours later, cells were subjected to the fluorescence recovery after photobleaching (FRAP) assay. Scale bar: 2 µm. G Quantification of the rate of fluorescence recovery of RFP-p62 in WT and MOAP-1 KO cells. n = 5 samples.


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 .

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


H Overexpression of MOAP - 1 but not GFP leads to increased exchange of p62 in 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 .

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


H Overexpression of MOAP - 1 but not GFP leads to increased exchange of p62 in 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 .

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


J Re - introduction of MOAP - 1 disrupts 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 .

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


A MOAP - 1 associates with p62 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 .

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


B Alanine substitution of positively charged residues in 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 .

O B-CONTROLLED_VAR O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O 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 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 .

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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 .

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E MOAP-1 but not the MOAP-1-M3-mutant promotes the exchange of RFP-p62 in the p62 bodies. WT LO2 cells transfected with expression vector encoding RFP-tagged p62 and GFP-tagged MOAP-1 or MOAP-1-M3. At 14 hours post-transfection, cells were subjected to FRAP assay. Scale bar: 2 µm.


F 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 .

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F Quantification of the rate of fluorescence recovery of RFP-p62 in cells co-expressing GFP-MOAP-1 or GFP-MOAP-1-M3 mutant. n = 6 samples. Error bars represent S.E.M. *P<0.05, **P<0.01, ns, not significant, two-way ANOVA.


G Western 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 ) .

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


H Re - expression of MOAP - 1 , but not 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 .

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H Re-expression of MOAP-1, but not the MOAP-1-M3 mutant, reduces the levels of p62 bodies. MOAP-1 KO LO2 cells were transfected with plasmid encoding Myc-MOAP-1 or Myc-MOAP-1-M3. The transfected cells were harvested at 24 hours post-transfection, which is the time point that MOAP-1 was found to effectively downregulate p62 bodies, followed by IF analysis as in (D). Cells expressing Myc-MOAP-1 or Myc-MOAP-1-M3 were marked by dashed lines. I Quantification of the p62 bodies in the MOAP-1 KO LO2 cells expressing Myc-MOAP-1 and Myc-MOAP-1-M3 mutant. (Left panel) number of p62 bodies per cell. n = 7 independently plated samples. (Right panel) size of p62 bodies. n = 107 and 267 bodies detected in the MOAP-1 KO LO2 cells expressing Myc-MOAP-1 and Myc-MOAP-1-M3 mutant, respectively, from three independently plated samples. Error bars represent S.E.M. ***P<0.001, Student's t-test.


H Re - expression of MOAP - 1 , but not 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 .

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


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

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


A Overexpression of MOAP - 1 inhibits dimerization of p62 . p62 KO LO2 hepatocytes were transfected with 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 .

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 .

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


B Dimerization of p62 is enhanced in 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 .

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


D Re - expression of MOAP - 1 , but not 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 .

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


D Re - expression of MOAP - 1 , but not 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 .

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


F 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 .

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


G 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 .

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


H MOAP - 1 binds 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 .

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


A Increased recruitment of Keap1 to p62 bodies in 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 .

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


B MOAP - 1 deficiency promotes p62 / Keap1 interaction . WT , MOAP - 1 KO and p62 KO LO2 cells were subjected to co - IP assay using anti - p62 antibody . TCL , total cell lysates . C Quantification of the levels of Keap1 bound to p62 in the co - IP experiment as described in ( B ) by densitometric analysis .

B MOAP-1 deficiency promotes p62/Keap1 interaction. WT, MOAP-1 KO and p62 KO LO2 cells were subjected to co-IP assay using anti-p62 antibody. TCL, total cell lysates. C Quantification of the levels of Keap1 bound to p62 in the co-IP experiment as described in (B) by densitometric analysis.


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

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


D Diminished Keap1 / Nrf2 interaction in 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 .

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


D Diminished Keap1 / Nrf2 interaction in 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 .

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


F Loss of MOAP - 1 results in marked elevation of 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 .

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


F Loss of MOAP - 1 results in marked elevation of 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 .

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


H MOAP - 1 deficiency promotes activation of Nrf2 signaling . Transcript 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 .

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


I Elevated transcriptional activity of Nrf2 in 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 .

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


J Re - expression of MOAP - 1 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 .

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 .

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


A High level of p62 bodies in 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 .

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


B Loss of MOAP - 1 results in higher 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 .

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


C MOAP - 1 deficiency elevates 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 .

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C MOAP-1 deficiency elevates levels of p62 protein in the detergent-insoluble fractions of the liver lysates from mice subjected to acute DEN treatment. Livers from mice injected as described in (A) were lysed in 1% Triton-X lysis buffer, separated into detergent-soluble (S) and insoluble (I) fractions and analyzed by Western blotting.


D High 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 .

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D High levels of p62 bodies detected in the tumors of the MOAP-1 KO mice. Tumor sections from livers dissected from the 8.5-month-old male WT and MOAP-1 KO mice injected with a single dose of DEN (25 µg/g body weight) at 15-day-old were subjected to IHC analysis with anti-p62 antibody (in red). Nuclear were stained with DAPI (in blue). Insets were enlarged in the bottom right. Scale bar: 20 µm. E Quantification of the p62 bodies in the WT and MOAP-1 KO tumors. (Left panel) number of p62 bodies per cell. n = 7 tumors from the livers of WT and MOAP-1 KO mice. (Right panel) size of p62 bodies. n = 456 and 1211 bodies detected in the WT and MOAP-1 KO tumors, respectively, from three independent samples. Error bar represents S.E.M. *P<0.05, ***P<0.001, student's t-test.


D High 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 .

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D High levels of p62 bodies detected in the tumors of the MOAP-1 KO mice. Tumor sections from livers dissected from the 8.5-month-old male WT and MOAP-1 KO mice injected with a single dose of DEN (25 µg/g body weight) at 15-day-old were subjected to IHC analysis with anti-p62 antibody (in red). Nuclear were stained with DAPI (in blue). Insets were enlarged in the bottom right. Scale bar: 20 µm.


F Increased 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 .

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


G Upregulation of mRNA 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 .

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G Upregulation of mRNA levels of HMOX-1 and NQO1 in the MOAP-1 deficient tumors. Livers harvested from the mice injected with PBS or DEN as described in (D) were subjected to RT-PCR analysis. n = 3 WT and 3 MOAP-1 KO mice injected with the PBS control, and 8 WT and 9 MOAP-1 KO mice injected with DEN. Error bar represents S.E.M. **P<0.01, student's t-test.


H MOAP - 1 deficient mice bear higher 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 .

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H MOAP-1 deficient mice bear higher tumor burden in the DEN-mediated liver cancer model. Representative images of liver tumors dissected from the 8.5-month-old male WT and MOAP-1 KO mice injected with a single dose of DEN (25 µg/g body weight) at 15-day-old. Scale bar: 5 mm. I Measurements of the number, maximum diameter of the tumor nodules and total liver weight from WT and MOAP-1 KO mice (n=13 and 14 respectively) injected with DEN as described in (H). Error bar represents S.E.M. **P<0.01, ***P<0.001, student's t-test.


H MOAP - 1 deficient mice bear higher 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 .

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H MOAP-1 deficient mice bear higher tumor burden in the DEN-mediated liver cancer model. Representative images of liver tumors dissected from the 8.5-month-old male WT and MOAP-1 KO mice injected with a single dose of DEN (25 µg/g body weight) at 15-day-old. Scale bar: 5 mm.


MOAP - 1 deficiency promotes 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 .

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MOAP-1 deficiency promotes tumor initiation. measurements of the number, diameter of the largest nodules and total liver weight of the 4.5-month-old WT and MOAP-1 KO mice (n=18 and 23 respectively) injected with a single dose of DEN (25 µg/g body weight) at 15-day-old.


( B ) 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 .

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(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 .

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(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 .

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(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 .

(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 ) .

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(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 .

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(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 .

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(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 .

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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 .

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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 ) .

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(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).


( 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 ) .

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(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 .

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(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 .

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(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 .

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(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 .

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(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 .

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(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 .

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(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 .

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(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.


( A - B ) Western blot analysis with cell lysate of uninfected and S . flexneri infected ( MOI 1 : 25 , 8 h ) ( A ) control and ATG5 siRNA knockdown THP - 1 cells , ( B ) wild type ( WT ) and ATG5 knockout ( ATG5 - / - ) MEF cells with indicated antibodies . Densitometric analysis was performed using Image J software . FC , fold change .

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(A-B) Western blot analysis with cell lysate of uninfected and S. flexneri infected (MOI 1:25, 8 h) (A) control and ATG5 siRNA knockdown THP-1 cells, (B) wild type (WT) and ATG5 knockout (ATG5-/-) MEF cells with indicated antibodies. Densitometric analysis was performed using Image J software. FC, fold change.


( C ) Left panel , representative high - content microscopy images of RIPosomes in HEK293T cells that are control cells or cells treated with rapamycin ( 500 nM , 4h ) or cells treated with rapamycin ( 500 nM , 4h ) and bafilomycin A1 ( BafA1 , 300 nM ) . Right panel , the graph depicts the average number of RIPosomes / 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 . 005 and , * * * p < 0 . 0005 , ordinary one - way ANOVA ( Tukey ' s multiple comparisons test ) .

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

(C) Left panel, representative high-content microscopy images of RIPosomes in HEK293T cells that are control cells or cells treated with rapamycin (500 nM, 4h) or cells treated with rapamycin (500 nM, 4h) and bafilomycin A1 (BafA1, 300 nM). Right panel, the graph depicts the average number of RIPosomes/cell. About 15000 cells were plated per well and RIPosomes were screened in 35 fields per well. Mean ± SD, n=3 (biological replicates), **p < 0.005 and, ***p < 0.0005, ordinary one-way ANOVA (Tukey's multiple comparisons test).


( D ) Left panel , representative high - content microscopy images of RIPosomes in control and ATG5 siRNA transfected THP - 1 cells infected with S . flexneri ( 8 h ) . Right panel , the graph depicts average number of RIPosomes / cell . About 50000 cells were plated per well and RIPosomes were screened in 35 fields per well . Mean ± SD , n = 3 ( biological replicates ) , * * * p < 0 . 0005 , Student ' s unpaired t - test .

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(D) Left panel, representative high-content microscopy images of RIPosomes in control and ATG5 siRNA transfected THP-1 cells infected with S. flexneri (8 h). Right panel, the graph depicts average number of RIPosomes/cell. About 50000 cells were plated per well and RIPosomes were screened in 35 fields per well. Mean ± SD, n=3 (biological replicates), ***p < 0.0005, Student's unpaired t-test.


( E ) The soluble and insoluble fractions of S . flexneri infected ( MOI 1 : 25 , 8 h ) control and ATG5 siRNA knockdown THP - 1 cells were subjected to Western blot analysis with indicated antibodies .

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(E) The soluble and insoluble fractions of S. flexneri infected (MOI 1:25, 8 h) control and ATG5 siRNA knockdown THP-1 cells were subjected to Western blot analysis with indicated antibodies.


( F ) Representative confocal images of HEK293T cells transfected with GFP - RIPK2 and immunostained with anti - FK2 antibodies . Line profile : co - localization analysis using line intensity profiles . Scale bar , 3 µm .

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(F) Representative confocal images of HEK293T cells transfected with GFP-RIPK2 and immunostained with anti-FK2 antibodies. Line profile: co-localization analysis using line intensity profiles. Scale bar, 3 µm.


( G ) Autophagy receptor screen using co - immunoprecipitation ( Co - IP ) assay to analyze the interaction between GFP - RIPK2 and Flag - p62 or Flag - NBRI or Flag - NDP52 or Flag - TAX1BP1 or Flag - OPTINEURIN in HEK293T cell lysate .

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(G) Autophagy receptor screen using co-immunoprecipitation (Co-IP) assay to analyze the interaction between GFP-RIPK2 and Flag-p62 or Flag-NBRI or Flag-NDP52 or Flag-TAX1BP1 or Flag-OPTINEURIN in HEK293T cell lysate.


( H ) Western blot analysis of soluble and insoluble fractions of control and p62 siRNA transfected and S . flexneri infected THP - 1 cells ( MOI 1 : 25 , 8 h ) .

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(H) Western blot analysis of soluble and insoluble fractions of control and p62 siRNA transfected and S. flexneri infected THP-1 cells (MOI 1:25, 8 h).


( I ) Representative confocal images of HEK293T cells expressing GFP - RIPK2 ( 6 h ) immunostained with p62 and LC3B antibodies . Zoom panels are digital magnifications .

(I) Representative confocal images of HEK293T cells expressing GFP-RIPK2 (6 h) immunostained with p62 and LC3B antibodies. Zoom panels are digital magnifications.


( J ) GST pull - down assay using purified GST or GST - RIPK2 proteins with in - vitro translated 35S radiolabeled myc - ULK1 or myc - ATG16L or myc - BECLIN1 .

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(J) GST pull-down assay using purified GST or GST-RIPK2 proteins with in-vitro translated 35S radiolabeled myc-ULK1 or myc-ATG16L or myc-BECLIN1.


Representative confocal images of HEK293T cells transfected with GFP - RIPK2 ( 9 h ) and immunofluorescence assay performed with antibodies specific to ( K ) ULK1 Line profile : co - localization analysis using line intensity profiles . Scale bar , 5 µm or 3 µm as indicated . Zoom panels are digital magnifications .

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Representative confocal images of HEK293T cells transfected with GFP-RIPK2 (9 h) and immunofluorescence assay performed with antibodies specific to (K) ULK1 Line profile: co-localization analysis using line intensity profiles. Scale bar, 5 µm or 3 µm as indicated. Zoom panels are digital magnifications.


Representative confocal images of HEK293T cells transfected with GFP - RIPK2 or mcherry - RIPK2 ( 9 h ) and immunofluorescence assay performed with antibodies specific to ( L ) ATG16L ( M ) BECLIN1 . Line profile : co - localization analysis using line intensity profiles . Scale bar , 5 µm or 3 µm as indicated . Zoom panels are digital magnifications .

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Representative confocal images of HEK293T cells transfected with GFP-RIPK2 or mcherry-RIPK2 (9 h) and immunofluorescence assay performed with antibodies specific to (L) ATG16L (M) BECLIN1. Line profile: co-localization analysis using line intensity profiles. Scale bar, 5 µm or 3 µm as indicated. Zoom panels are digital magnifications.


( N ) Luciferase assays were performed with ATG5 or p62 knockdown HEK293T cells transfected with NF - κB luciferase reporter vector pGL4 . 32 NFκB - RE , GFP - RIPK2 , and Flag - NOD1 plasmids . Mean ± SD , n = 3 ( biological replicates ) , * * * * p < 0 . 00005 , ordinary one - way ANOVA ( Tukey ' s multiple comparisons test ) .

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(N) Luciferase assays were performed with ATG5 or p62 knockdown HEK293T cells transfected with NF-κB luciferase reporter vector pGL4.32 NFκB-RE, GFP-RIPK2, and Flag-NOD1 plasmids. Mean ± SD, n=3 (biological replicates), ****p < 0.00005, ordinary one-way ANOVA (Tukey's multiple comparisons test).


( A - B ) The THP - 1 cell lysates were subjected to immunoprecipitation analysis ( A ) untreated and treated with iE - DAP ( 40 μg / ml , 6 h ) , ( B ) untreated and treated with MDP ( 40 μg / ml , 6 h ) . IP was performed with isotype control IgG or IRGM antibody and Western blotting was performed with indicated antibodies . IgG ( H ) ; IgG heavy chain .

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(A-B) The THP-1 cell lysates were subjected to immunoprecipitation analysis (A) untreated and treated with iE-DAP (40 μg/ml, 6 h), (B) untreated and treated with MDP (40 μg/ml, 6 h). IP was performed with isotype control IgG or IRGM antibody and Western blotting was performed with indicated antibodies. IgG (H); IgG heavy chain.


( C - D ) Co - IP analysis of the interactions between ( C ) GFP - RIPK2 and Flag - IRGM or ( D ) Flag - NOD1 and GFP - IRGM in HEK293T cell lysates .

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(C-D) Co-IP analysis of the interactions between (C) GFP-RIPK2 and Flag-IRGM or (D) Flag-NOD1 and GFP-IRGM in HEK293T cell lysates.


( E ) GST pull - down assay using purified GST and GST - IRGM and in - vitro translated 35S radiolabeled myc - NOD2 , myc - NOD1 , and myc - RIPK2 .

(E) GST pull-down assay using purified GST and GST-IRGM and in-vitro translated 35S radiolabeled myc-NOD2, myc-NOD1, and myc-RIPK2.


( F ) Representative confocal images of HEK293T cells expressing GFP - RIPK2 and mCherry or mCherry - IRGM . Line profile : co - localization analysis using line intensity profiles . Scale bar , 5 µm .

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(F) Representative confocal images of HEK293T cells expressing GFP-RIPK2 and mCherry or mCherry-IRGM. Line profile: co-localization analysis using line intensity profiles. Scale bar, 5 µm.


Representative confocal images of THP - 1 cells infected with RFP expressing S . flexneri , ( MOI 1 : 25 , 8 h ) and immunostained with , ( H ) IRGM and RIPK2 antibody . Line profile : co - localization analysis using line intensity profiles . Scale bar , 5 µm . Zoom panels are digital magnifications . In image H , for better contrast , RFP expressing Shigella is pseudocolored to blue .

Representative confocal images of THP-1 cells infected with RFP expressing S. flexneri, (MOI 1:25, 8 h) and immunostained with, (H) IRGM and RIPK2 antibody. Line profile: co-localization analysis using line intensity profiles. Scale bar, 5 µm. Zoom panels are digital magnifications. In image H, for better contrast, RFP expressing Shigella is pseudocolored to blue.


( J ) Representative confocal images of HEK293T cells expressing GFP - RIPK2 , Flag - NOD1 , and mCherry - IRGM . Line profile : co - localization analysis using line intensity profiles . Scale bar , 3 µm . Zoom panels are digital magnifications .

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(J) Representative confocal images of HEK293T cells expressing GFP-RIPK2, Flag-NOD1, and mCherry-IRGM. Line profile: co-localization analysis using line intensity profiles. Scale bar, 3 µm. Zoom panels are digital magnifications.


( M ) A co - IP analysis is performed with HEK293T cell lysates expressing various domains of RIPK2 and IRGM to map the domain / s of RIPK2 interacting with IRGM . Asterisk indicate the main band of overexpressed protein .

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(M) A co-IP analysis is performed with HEK293T cell lysates expressing various domains of RIPK2 and IRGM to map the domain/s of RIPK2 interacting with IRGM. Asterisk indicate the main band of overexpressed protein.


( O ) A co - IP analysis is performed with HEK293T cell lysates expressing various domains of NOD1 and IRGM to map the domain / s of NOD1 interacting with IRGM . Asterisk indicate the main band of overexpressed

(O) A co-IP analysis is performed with HEK293T cell lysates expressing various domains of NOD1 and IRGM to map the domain/s of NOD1 interacting with IRGM. Asterisk indicate the main band of overexpressed protein


( A ) Western blot analysis with the cell lysates of control CRISPR cells and CRISPR - Cas9 mediated IRGM partial knockout ( IRGM + / - ) HT - 29 cells ( 2 clones were tested ) . Densitometric analysis was performed using Image J software . FC , fold change .

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(A) Western blot analysis with the cell lysates of control CRISPR cells and CRISPR-Cas9 mediated IRGM partial knockout (IRGM+/-) HT-29 cells (2 clones were tested). Densitometric analysis was performed using Image J software. FC, fold change.


( B ) Left panel , soluble and insoluble fractions of S . flexneri ( MOI 1 : 25 , 8 h ) infected mouse BMDMs from Irgm1 + / + and Irgm1 - / - mice were subjected to immunoblot analysis with antibodies as indicated . Right panel , Western blot analysis with the colon lysates from Irgm1 + / + and Irgm1 - / - mice with indicated antibodies . The graph indicate ratio of band intensity ( measured using ImageJ ) and actin ( n = 5 , Mean ± SD , * p < 0 . 05 , * * p < 0 . 005 , and * * * * p < 0 . 00005 , Student ' s unpaired t - test )

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(B) Left panel, soluble and insoluble fractions of S. flexneri (MOI 1:25, 8 h) infected mouse BMDMs from Irgm1+/+ and Irgm1-/- mice were subjected to immunoblot analysis with antibodies as indicated. Right panel, Western blot analysis with the colon lysates from Irgm1+/+ and Irgm1-/- mice with indicated antibodies. The graph indicate ratio of band intensity (measured using ImageJ) and actin (n=5, Mean ± SD, *p < 0.05, **p < 0.005, and ****p < 0.00005, Student's unpaired t-test)


( C ) Doxycycline - inducible stable GFP - RIPK2 HeLa cells were transfected with control siRNA or IRGM siRNA followed by infection with S . flexneri ( MOI 1 : 25 , 4h ) . The cells were fixed and subjected to high - content microscopy to quantitate the number of RIPosomes formed . The graph depicts an average number of RIPosome / cell . About 10000 cells were plated per well and RIPosomes were screened in 35 fields per well . Mean ± SD , n = 4 ( biological replicates ) , * * * * p < 0 . 00005 , Student ' s unpaired t - test .

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(C) Doxycycline-inducible stable GFP-RIPK2 HeLa cells were transfected with control siRNA or IRGM siRNA followed by infection with S. flexneri (MOI 1:25, 4h). The cells were fixed and subjected to high-content microscopy to quantitate the number of RIPosomes formed. The graph depicts an average number of RIPosome/cell. About 10000 cells were plated per well and RIPosomes were screened in 35 fields per well. Mean ± SD, n=4 (biological replicates), ****p < 0.00005, Student's unpaired t-test.


Western blot analysis with the cell lysate of ( D ) HT - 29 clones stably expressing Flag - vector control or Flag - IRGM Densitometric analysis was performed using Image J software . FC , fold change .

Western blot analysis with the cell lysate of (D) HT-29 clones stably expressing Flag-vector control or Flag-IRGM Densitometric analysis was performed using Image J software. FC, fold change.


Western blot analysis with the cell lysate of ( E ) THP - 1 cells transiently transfected with GFP or GFP - IRGM for 6 h . Densitometric analysis was performed using Image J software . FC , fold change .

Western blot analysis with the cell lysate of (E) THP-1 cells transiently transfected with GFP or GFP-IRGM for 6 h. Densitometric analysis was performed using Image J software. FC, fold change.


( F ) Left panel , representative high - content microscopy images of NODo - RIPosomes in HEK293T cells transfected with GFP - RIPK2 and myc - NOD2 ( upper panel ) or GFP - RIPK2 , myc - NOD2 , and Flag - IRGM ( lower panel ) . Right panel , the graph depicts average number of NODo - RIPosome / cell . About 15000 cells plated per well and RIPosomes were screened in 35 fields per well . Mean ± SD , n = 5 ( biological replicates ) , * * * * p < 0 . 00005 , Student ' s unpaired t - test .

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(F) Left panel, representative high-content microscopy images of NODo-RIPosomes in HEK293T cells transfected with GFP-RIPK2 and myc-NOD2 (upper panel) or GFP-RIPK2, myc-NOD2, and Flag-IRGM (lower panel). Right panel, the graph depicts average number of NODo-RIPosome/cell. About 15000 cells plated per well and RIPosomes were screened in 35 fields per well. Mean ± SD, n=5 (biological replicates), ****p < 0.00005, Student's unpaired t-test.


( G ) The soluble and insoluble fractions of HEK293T cells transfected with indicated plasmids were subjected to immunoblot analysis with indicated antibodies .

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(G) The soluble and insoluble fractions of HEK293T cells transfected with indicated plasmids were subjected to immunoblot analysis with indicated antibodies.


( H ) Representative confocal images of HEK293T cells transfected with mCherry and GFP - RIPK2CARD or mCherry - IRGM and GFP - RIPK2CARD for 9 h . Line profile : co - localization analysis using line intensity profiles . Scale bar , 5 µm .

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(H) Representative confocal images of HEK293T cells transfected with mCherry and GFP-RIPK2CARD or mCherry-IRGM and GFP-RIPK2CARD for 9 h. Line profile: co-localization analysis using line intensity profiles. Scale bar, 5 µm.


( I - K ) Western blot analysis with the cell lysates of HEK293T transfected with indicated plasmids and probe with Actin , Flag , Myc , and , GFP antibodies as indicated . Densitometric analysis was performed using Image J software . FC , fold change .

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(I-K) Western blot analysis with the cell lysates of HEK293T transfected with indicated plasmids and probe with Actin, Flag, Myc, and, GFP antibodies as indicated. Densitometric analysis was performed using Image J software. FC, fold change.


( L ) Left panels , representative high - content microscopy images of HEK293T cells transfected with GFP - RIPK2 or GFP - RIPK2 and Flag - IRGM or GFP - RIPK2 and Flag - IRGM ( S47N ) . The graph depicts the average number of RIPosome / cell . Mean ± SD , n = 4 ( biological replicates ) , * * * * p < 0 . 00005 , ordinary one - way ANOVA ( Tukey ' s multiple comparisons test ) .

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(L) Left panels, representative high-content microscopy images of HEK293T cells transfected with GFP-RIPK2 or GFP-RIPK2 and Flag-IRGM or GFP-RIPK2 and Flag-IRGM (S47N). The graph depicts the average number of RIPosome/cell. Mean ± SD, n=4 (biological replicates), ****p < 0.00005, ordinary one-way ANOVA (Tukey's multiple comparisons test).


( C ) Left panel , representative pictures of colons of Irgm1 + / + and Irgm - / - mice untreated or treated with GSK583 infected with S . flexneri . Right panel , the graph depicts the average colon lengths of the mice groups . Mean ± SD , n = 6 , * * p < 0 . 005 , * * * p < 0 . 0005 , Student ' s unpaired t - test .

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(C) Left panel, representative pictures of colons of Irgm1+/+ and Irgm-/- mice untreated or treated with GSK583 infected with S. flexneri. Right panel, the graph depicts the average colon lengths of the mice groups. Mean ± SD, n=6, **p < 0.005, ***p < 0.0005, Student's unpaired t-test.


( D ) Representative microscopic images of H & E staining of colon tissues of Irgm1 + / + and Irgm - / - mice untreated or treated with GSK583 administrated with DSS . The graph depicts the combined histological scores . Mean ± SD , n = 3 ( DSS ) , * * p < 0 . 005 , Student ' s unpaired t - test . Scale bar , 200

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(D) Representative microscopic images of H&E staining of colon tissues of Irgm1+/+ and Irgm-/- mice untreated or treated with GSK583 administrated with DSS. The graph depicts the combined histological scores. Mean ± SD, n=3 (DSS), **p < 0.005, Student's unpaired t-test. Scale bar, 200 µm


( E ) The soluble and insoluble fractionations of lysates from colon tissues of Shigella - infected or DSS - treated Irgm1 + / + and Irgm - / - mice treated with GSK583 as indicated , were subjected to immunoblot analysis with indicated antibodies .

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(E) The soluble and insoluble fractionations of lysates from colon tissues of Shigella-infected or DSS-treated Irgm1+/+ and Irgm-/- mice treated with GSK583 as indicated, were subjected to immunoblot analysis with indicated antibodies.


( H - I ) Western blot analysis with the colon tissue lysates of DSS - treated or S . flexneri infected mice groups as indicated .

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(H-I) Western blot analysis with the colon tissue lysates of DSS-treated or S. flexneri infected mice groups as indicated.


A ) Quantification of the effect of individual BCL - 2 - family proteins on HCT AKO cell death , measured as percentage of cells showing released cyt c or pyknotic nuclei . Data correspond to at least three independent experiments , n > 30 cells per condition per experiment . * * * p < 0 , 001 and * p < 0 , 05 with respect to GFP condition .

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A) Quantification of the effect of individual BCL-2-family proteins on HCT AKO cell death, measured as percentage of cells showing released cyt c or pyknotic nuclei. Data correspond to at least three independent experiments, n>30 cells per condition per experiment. *** p<0,001 and * p<0,05 with respect to GFP condition.


C ) Western blot analysis of SMAC and cyt c release in GFP - BAX , tBID - GFP and GFP - BCLXL expressing cells at 12 h , 16 h and 24 h after transfection in HCT AKO . SN ( supernatant , cytosolic fraction ) , P ( pellet , membrane fraction ) and PS ( Ponceau Staining ) .

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C) Western blot analysis of SMAC and cyt c release in GFP-BAX, tBID-GFP and GFP-BCLXL expressing cells at 12 h, 16 h and 24 h after transfection in HCT AKO. SN (supernatant, cytosolic fraction), P (pellet, membrane fraction) and PS (Ponceau Staining).