B ) Profiling of the red ( UIS4 ) and green ( CD63 , left or LAMP ‐ 1 , right ) signals along the indicated line within the insets from the images in ( A ) above .

O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O

B) Profiling of the red (UIS4) and green (CD63, left or LAMP‐1, right) signals along the indicated line within the insets from the images in (A) above.


LC3 colocalizes with UIS4 on the Plasmodium liver stage PVM and TVNHepatoma cells infected with Plasmodium berghei expressing UIS4 ‐ mCherry ( top ) or IBIS1 ‐ mCherry ( bottom ) were fixed 22 h or 48 h post ‐ infection , respectively , and stained with anti ‐ LC3 to label autophagic membranes . In the right panels , fluorescence is merged , and selected areas of interest are shown enlarged . Scale bars , 10 µm .

B-GENEPROD O O B-GENEPROD O O O O O B-SUBCELLULAR O O O O O B-ORGANISM I-ORGANISM O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O

LC3 colocalizes with UIS4 on the Plasmodium liver stage PVM and TVNHepatoma cells infected with Plasmodium berghei expressing UIS4‐mCherry (top) or IBIS1‐mCherry (bottom) were fixed 22 h or 48 h post‐infection, respectively, and stained with anti‐LC3 to label autophagic membranes. In the right panels, fluorescence is merged, and selected areas of interest are shown enlarged. Scale bars, 10 µm.


A . A549 cells were transfected with 2 μg of poly ( I : C ) for 6 hrs and then infected with ZIKV ( MOI = 3 ) for 18 hrs ( Pre - treatment ) or infected with ZIKV first for 6 hrs and then transfected with poly ( I : C ) for 12 hrs ( Post - treatment ) . Levels of ZIKVgenomic RNA were determined by qRT - PCR . Values are expressed as mean ± standard error . * * * P < 0 . 001 ( Student ' s t - test ) , N =

O O B-CELL_LINE O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O B-ORGANISM B-SUBCELLULAR O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_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

A. A549 cells were transfected with 2 μg of poly(I:C) for 6 hrs and then infected with ZIKV (MOI=3) for 18 hrs (Pre-treatment) or infected with ZIKV first for 6 hrs and then transfected with poly(I:C) for 12 hrs (Post-treatment). Levels of ZIKVgenomic RNA were determined by qRT-PCR. Values are expressed as mean ± standard error. ***P<0.001 (Student's t-test), N=3


B . A549 cells infected with ZIKV ( MOI = 3 ) for 6 hrs were transfected with 2 μg poly ( I : C ) for 12 hrs after which levels of ifn -

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B. A549 cells infected with ZIKV (MOI=3) for 6 hrs were transfected with 2 μg poly(I:C) for 12 hrs after which levels of ifn-


C . A549 cells were infected with ZIKV ( MOI = 5 ) for 16 hrs and then transfected with an IFIT1 promoter - driven firefly luciferase plasmid ( pGL3B / 561 ) and a constitutively active renilla luciferase construct ( pRL - TK ) , as well as 1 μg of poly ( I : C ) . Eight hours later cell lysates were harvested and subjected to luciferase assay . Values are expressed as mean of two independent experiments , N =

O O B-CELL_LINE O O O O B-ORGANISM O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O

C. A549 cells were infected with ZIKV (MOI=5) for 16 hrs and then transfected with an IFIT1 promoter-driven firefly luciferase plasmid (pGL3B/561) and a constitutively active renilla luciferase construct (pRL-TK), as well as 1 μg of poly(I:C). Eight hours later cell lysates were harvested and subjected to luciferase assay. Values are expressed as mean of two independent experiments, N=2


D . HEK293T cells were transfected with plasmids encoding each of the 10 ZIKV proteins . Sixteen hours later they were transfected with an IFIT1 promoter - driven firefly luciferase plasmid ( pGL3B / 561 ) and a constitutively active renilla luciferase construct ( pRL - TK ) , as well as 1 μg of poly ( I : C ) . Eight hours later cell lysates were harvested and subjected to luciferase assay . C = capsid ; E = envelope . Values are expressed as mean ± standard error . * P < 0 . 05 , * * P < 0 . 01 ( Student ' s t - test ) , N =

O O B-CELL_LINE O O O O O O O O O O B-ORGANISM O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-SUBCELLULAR O O O B-SUBCELLULAR O O O O O O O O O O O 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. HEK293T cells were transfected with plasmids encoding each of the 10 ZIKV proteins. Sixteen hours later they were transfected with an IFIT1 promoter-driven firefly luciferase plasmid (pGL3B/561) and a constitutively active renilla luciferase construct (pRL-TK), as well as 1 μg of poly(I:C). Eight hours later cell lysates were harvested and subjected to luciferase assay. C = capsid; E = envelope. Values are expressed as mean ± standard error. *P<0.05, **P<0.01 (Student's t-test), N=4


E . A549 cells were infected with ZIKV ( MOI = 5 ) for 16 hrs and then transfected with the indicated promoter - driven firefly luciferase plasmids and a constitutively active renilla luciferase construct ( pRL - TK ) , as well as 1 μg of poly ( I : C ) . Eight hours later cell lysates were harvested and subjected to luciferase assay . Values are expressed as mean of two independent experiments , N =

O O B-CELL_LINE O O O O B-ORGANISM O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O

E. A549 cells were infected with ZIKV (MOI=5) for 16 hrs and then transfected with the indicated promoter-driven firefly luciferase plasmids and a constitutively active renilla luciferase construct (pRL-TK), as well as 1 μg of poly(I:C). Eight hours later cell lysates were harvested and subjected to luciferase assay. Values are expressed as mean of two independent experiments, N=2


F . HEK293T cells were transfected with plasmids encoding individual ZIKV proteins , the indicated promoter - driven firefly luciferase plasmids and a constitutively active renilla luciferase construct ( pRL - TK ) , as well as 0 . 4 μg of poly ( I : C ) . Twenty - four hours later cell lysates were harvested and subjected to luciferase assay . C = capsid ; E = envelope . Values are expressed as mean ± standard error . * P < 0 . 05 , * * P < 0 . 01 ( Student ' s t - test ) ; N =

O O B-CELL_LINE O O O O O O O B-ORGANISM O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-SUBCELLULAR O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F. HEK293T cells were transfected with plasmids encoding individual ZIKV proteins, the indicated promoter-driven firefly luciferase plasmids and a constitutively active renilla luciferase construct (pRL-TK), as well as 0.4 μg of poly(I:C). Twenty-four hours later cell lysates were harvested and subjected to luciferase assay. C = capsid; E = envelope. Values are expressed as mean ± standard error. *P<0.05, **P<0.01 (Student's t-test); N=3


A . A549 cells were treated with IFN - α ( 100 U / mL ) , IFN - λ ( 200 ng / mL ) or IFN - γ ( 10 U / mL ) for 6 hrs after which they were infected with ZIKV ( MOI = 3 ) for 18 hrs ( Pre - treatment ) . Alternatively , cells were first infected with ZIKV for 6 hrs followed by treatment with IFN for 12 hrs ( Post - treatment ) . Total RNA was isolated and levels of ZIKV genomic RNA were determined by qRT - PCR . Values are expressed as mean ± standard error . * P < 0 . 05 , * * P < 0 . 01 ( Student ' s t - test ) ; N =

O O B-CELL_LINE O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O B-ORGANISM 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-ORGANISM B-SUBCELLULAR I-SUBCELLULAR O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_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

A. A549 cells were treated with IFN-α (100 U/mL), IFN-λ (200 ng/mL) or IFN-γ (10 U/mL) for 6 hrs after which they were infected with ZIKV (MOI=3) for 18 hrs (Pre-treatment). Alternatively, cells were first infected with ZIKV for 6 hrs followed by treatment with IFN for 12 hrs (Post-treatment). Total RNA was isolated and levels of ZIKV genomic RNA were determined by qRT-PCR. Values are expressed as mean ± standard error. *P<0.05, **P<0.01 (Student's t-test); N=3


B . A549 cells were infected with ZIKV ( MOI = 3 ) for 6 hrs prior to addition of IFN - α ( 100 U / mL ) , IFN - λ ( 200 ng / mL ) or IFN - γ ( 10 U / mL ) for 12 hrs . Total RNA was isolated and levels of ifit1 RNAs were determined by qRT - PCR . Values are expressed as mean ± standard error . * P < 0 . 05 , * * P < 0 . 01 ( Student ' s t - test ) ; N =

O O B-CELL_LINE O O O O B-ORGANISM O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_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

B. A549 cells were infected with ZIKV (MOI=3) for 6 hrs prior to addition of IFN-α (100 U/mL), IFN-λ (200 ng/mL) or IFN-γ (10 U/mL) for 12 hrs. Total RNA was isolated and levels of ifit1 RNAs were determined by qRT-PCR. Values are expressed as mean ± standard error. *P<0.05, **P<0.01 (Student's t-test); N=3


C . A549 cells were infected with ZIKV ( MOI = 5 ) for 6 hrs and then transfected with ISRE - or GAS - promoter - driven firefly luciferase together with a constitutively active renilla luciferase construct . Sixteen hours later , cells were treated with IFN - α ( 100 U / ml ) or IFN - α ( 10 U / ml ) for 2 hrs . Cells lysates were harvested and relative luciferase activity from ISRE - and GAS - promoters were determined . Values are expressed as mean ± standard error . * P < 0 . 05 ( Student ' s t - test ) ; N =

O O B-CELL_LINE O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. A549 cells were infected with ZIKV (MOI=5) for 6 hrs and then transfected with ISRE- or GAS- promoter-driven firefly luciferase together with a constitutively active renilla luciferase construct. Sixteen hours later, cells were treated with IFN-α (100 U/ml) or IFN-α (10 U/ml) for 2 hrs. Cells lysates were harvested and relative luciferase activity from ISRE- and GAS-promoters were determined. Values are expressed as mean ± standard error. *P<0.05 (Student's t-test); N=3


D . HEK293T cells were transfected with ZIKV protein expression constructs together with an ISRE promoter - driven firefly luciferase plasmid ( pGL3B / 561 ) and a constitutively active renilla luciferase construct ( pRL - TK ) . After 24 hrs they were treated with IFN - α ( 100 U / mL ) for 10 hrs . Cell lysates were then harvested and subjected to luciferase assays . C = capsid ; E = envelope . Values are expressed as mean ± standard error . * P < 0 . 05 ( Student ' s t - test ) ; N =

O O B-CELL_LINE O O O O B-ORGANISM O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O B-SUBCELLULAR O O O 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. HEK293T cells were transfected with ZIKV protein expression constructs together with an ISRE promoter-driven firefly luciferase plasmid (pGL3B/561) and a constitutively active renilla luciferase construct (pRL-TK). After 24 hrs they were treated with IFN-α (100 U/mL) for 10 hrs. Cell lysates were then harvested and subjected to luciferase assays. C = capsid; E = envelope. Values are expressed as mean ± standard error. *P<0.05 (Student's t-test); N=3


A . A549 cells were infected with ZIKV ( MOI = 5 ) for 24 - 48 hrs after which cell lysates were processed by SDS - PAGE and immunoblotting . Representative panels are shown . The experiment was repeated three times .

O O B-CELL_LINE O O O O B-ORGANISM O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O O O

A. A549 cells were infected with ZIKV (MOI=5) for 24-48 hrs after which cell lysates were processed by SDS-PAGE and immunoblotting. Representative panels are shown. The experiment was repeated three times.


B . A549 cells were infected with ZIKV ( MOI = 5 ) for 24 hrs and then treated with DMSO , MG132 ( 20 µM ) or epoxomicin ( Epox ) ( 400 nM ) for 12 hrs . Cell lysates were processed by SDS - PAGE and immunoblotting . Representative panels are shown . The experiment was repeated three times .

O O B-CELL_LINE O O O O B-ORGANISM O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O O O

B. A549 cells were infected with ZIKV (MOI=5) for 24 hrs and then treated with DMSO, MG132 (20 µM) or epoxomicin (Epox) (400 nM) for 12 hrs. Cell lysates were processed by SDS-PAGE and immunoblotting. Representative panels are shown. The experiment was repeated three times.


C . A549 cells ( MOI = 2 ) or Human primary fetal astrocytes ( HFA ) ( MOI = 5 ) were infected with ZIKV for 24 hrs and 48 hrs respectively , after which IFN - α ( 100 U / mL ) was added for 2 hrs . Cells were then fixed and processed for indirect immunofluorescence . Images were acquired on a spinning disk confocal microscope with a 40X objective . Dashed line white circles indicated ZIKV - infected cells . Representative panels are shown . The experiment was repeated two times .

O O B-CELL_LINE O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O B-ORGANISM O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O 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-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O

C. A549 cells (MOI=2) or Human primary fetal astrocytes (HFA) (MOI=5) were infected with ZIKV for 24 hrs and 48 hrs respectively, after which IFN-α (100 U/mL) was added for 2 hrs. Cells were then fixed and processed for indirect immunofluorescence. Images were acquired on a spinning disk confocal microscope with a 40X objective. Dashed line white circles indicated ZIKV-infected cells. Representative panels are shown. The experiment was repeated two times.


D . Mouse embryonic fibroblasts ( MEF ) were infected with ZIKV ( MOI = 5 ) for 48 hrs after which IFN - α ( 200 U / mL ) was added for 2 hrs . Samples were processed for immunofluorescence assay as described in panel C . Representative panels are shown . The experiment was repeated two times .

O O O O O O B-CELL_LINE O O O O B-ORGANISM O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O 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. Mouse embryonic fibroblasts (MEF) were infected with ZIKV (MOI=5) for 48 hrs after which IFN-α (200 U/mL) was added for 2 hrs. Samples were processed for immunofluorescence assay as described in panel C. Representative panels are shown. The experiment was repeated two times.


E . STAT2 levels in MEFs were decreased by transfection with siRNAs for 48 hrs after which the cells were infected with ZIKV ( MOI = 5 ) for another 48 hrs . The ZIKV replication was measured by qRT - PCR .

O O B-GENEPROD O O B-CELL_LINE O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O B-ORGANISM O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O

E. STAT2 levels in MEFs were decreased by transfection with siRNAs for 48 hrs after which the cells were infected with ZIKV (MOI=5) for another 48 hrs. The ZIKV replication was measured by qRT-PCR.


E . STAT2 levels in MEFs were decreased by transfection with siRNAs for 48 hrs after which the cells were infected with ZIKV ( MOI = 5 ) for another 48 hrs . The silencing efficiency was determined by immunoblotting . Representative panels are shown . The experiment was repeated three times .

O O B-GENEPROD O O B-CELL_LINE O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O

E. STAT2 levels in MEFs were decreased by transfection with siRNAs for 48 hrs after which the cells were infected with ZIKV (MOI=5) for another 48 hrs. The silencing efficiency was determined by immunoblotting. Representative panels are shown. The experiment was repeated three times.


A . A549 cells were transfected with control plasmid pcDNA 3 . 1 or plasmids encoding FLAG - tagged WT or mutant NS5 proteins . At 48 hrs post - transfection cells were treated with IFN - α ( 100 U / mL ) for 2 hrs and then processed for indirect immunofluorescence . Images were acquired on a spinning disk confocal microscope with a 40X objective . NS5 positive cells are indicated by dashed white circles . Representative panels are shown . The experiment was repeated three times . B . Quantification of nuclearSTAT2 was performed using Volocity image analyses software . A minimum of 20 cells were counted for each sample . N . D . = not detected . Values are expressed as mean ± standard error . * P < 0 . 05 , * * P < 0 . 01 ( Student ' s t - test ) ; N =

O O B-CELL_LINE O O O O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O 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-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. A549 cells were transfected with control plasmid pcDNA 3.1 or plasmids encoding FLAG-tagged WT or mutant NS5 proteins. At 48 hrs post-transfection cells were treated with IFN-α (100 U/mL) for 2 hrs and then processed for indirect immunofluorescence. Images were acquired on a spinning disk confocal microscope with a 40X objective. NS5 positive cells are indicated by dashed white circles. Representative panels are shown. The experiment was repeated three times.B. Quantification of nuclearSTAT2 was performed using Volocity image analyses software. A minimum of 20 cells were counted for each sample. N.D.= not detected. Values are expressed as mean ± standard error. *P<0.05, **P<0.01 (Student's t-test); N=3


C . A549 cells were transfected with plasmids encoding FLAG - tagged ZIKVNS5 or FLAG alone for 24 hrs and then treated with epoxomicin ( 400 nM ) for 24 hrs . Cells were harvested and process for immunoprecipitation ( IP ) using a mouse anti - FLAG or anti - myc antibody followed by SDS - PAGE and Immunoblotting . Representative panels are shown . The experiment was repeated three times .

O O B-CELL_LINE O O O O O O B-GENEPROD O O B-ORGANISM O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O B-GENEPROD O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O O O

C. A549 cells were transfected with plasmids encoding FLAG-tagged ZIKVNS5 or FLAG alone for 24 hrs and then treated with epoxomicin (400 nM) for 24 hrs. Cells were harvested and process for immunoprecipitation (IP) using a mouse anti-FLAG or anti-myc antibody followed by SDS-PAGE and Immunoblotting. Representative panels are shown. The experiment was repeated three times.


D . A549 cells were transfected with plasmids encoding FLAG - tagged WT , mutant NS5 proteins or FLAG alone for 48 hrs . Cells were harvested and process for immunoprecipitation ( IP ) using rabbit anti - STAT2 followed by SDS - PAGE and immunoblotting . Arrows indicate co - immunoprecipitated WT NS5 and NS5 mutants that are in a complex with STAT2 . Representative panels are shown . The experiment was repeated two times .

O O B-CELL_LINE O O O O O O B-GENEPROD O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY O O O O O O O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O

D. A549 cells were transfected with plasmids encoding FLAG-tagged WT, mutant NS5 proteins or FLAG alone for 48 hrs. Cells were harvested and process for immunoprecipitation (IP) using rabbit anti-STAT2 followed by SDS-PAGE and immunoblotting. Arrows indicate co-immunoprecipitated WT NS5 and NS5 mutants that are in a complex with STAT2. Representative panels are shown. The experiment was repeated two times.


ER Proliferation under UPR - Inducing Conditions ( A ) Determination of ER abundance in control and UPR - induced cells . Representative cells are shown . The UPR was induced in wild - type cells by addition of DTT . Ultrastructure of control cells and UPR - induced cells was analyzed using ImageJ . The lower images show traces of cortical ER ( represented in magenta ) and the nuclear envelope ( NE , in blue ) . Vacuoles , nuclei , and mitochondria are indicated as V , N , and M , respectively . ( B ) Quantification of the ER proliferation during the UPR . UPR was induced and cells were collected for EM at the indicated time points . Length of the ER ( as traced in [ A ] ) was measured and divided by the area of the section . Data are plotted relative to time 0 . Measurements for each time point correspond to the mean of 25 independent cell images . ( C ) Expression of HAC1i was induced by addition of 100 μM DOC for 3 h . ER was quantified as described above in ( B ) .

B-SUBCELLULAR O O O O O O O O O O O B-SUBCELLULAR O O 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-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O O B-SUBCELLULAR O O O O O O O O O O

ER Proliferation under UPR-Inducing Conditions(A) Determination of ER abundance in control and UPR-induced cells. Representative cells are shown. The UPR was induced in wild-type cells by addition of DTT. Ultrastructure of control cells and UPR-induced cells was analyzed using ImageJ. The lower images show traces of cortical ER (represented in magenta) and the nuclear envelope (NE, in blue). Vacuoles, nuclei, and mitochondria are indicated as V, N, and M, respectively.(B) Quantification of the ER proliferation during the UPR. UPR was induced and cells were collected for EM at the indicated time points. Length of the ER (as traced in [A]) was measured and divided by the area of the section. Data are plotted relative to time 0. Measurements for each time point correspond to the mean of 25 independent cell images.(C) Expression of HAC1i was induced by addition of 100 μM DOC for 3 h. ER was quantified as described above in (B).


The ER Morphologically Changes during the UPR ( A ) Control cells and UPR - induced cells were used to analyze and follow the ER within a single cell using EM . Boxes indicate the areas magnified in ( B ) . Cells shown here correspond to the full section of the images labeled " + 140 nm " in ( B ) . ( B ) Serial section of control and UPR - induced cells . Sections are separated by 70 nm on the z - axis . ER is represented in magenta and NE in blue . ( C ) Electro micrographs from control and UPR - induced cells showing that the distance between ER membranes increases during the UPR . For a better preservation of the ultrastructure , samples for this experiment were prepared using high - pressure freezing / freeze substitution techniques ( see Material and Methods ) .

O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O B-EXP_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 B-SUBCELLULAR O O O O O B-SUBCELLULAR O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

The ER Morphologically Changes during the UPR(A) Control cells and UPR-induced cells were used to analyze and follow the ER within a single cell using EM. Boxes indicate the areas magnified in (B). Cells shown here correspond to the full section of the images labeled "+140 nm" in (B).(B) Serial section of control and UPR-induced cells. Sections are separated by 70 nm on the z-axis. ER is represented in magenta and NE in blue.(C) Electro micrographs from control and UPR-induced cells showing that the distance between ER membranes increases during the UPR. For a better preservation of the ultrastructure, samples for this experiment were prepared using high-pressure freezing/freeze substitution techniques (see Material and Methods).


Characterization of ER - Containing Autophagosomes ( ERAs ) during the UPR ( A ) Images of representative DTT - treated wild - type cells that contain ERAs . Nuclei and cytoplasm are indicated as N and C , respectively . ( B ) Enlargement of representative images of ERAs from different cells . The bottom right image is likely to show a section through a cup - shaped ERA . Note that there are no connections between the stacked cisternae and the envelope . ( C ) High magnification of the ERA double membrane envelope . ( D ) Some ERAs are found attached to or are in close proximity to ER tubules / sheets ( indicated by the arrow ) . Note that the section in ( A ) includes two such junctions . ( E ) High - pressure freezing / freeze substitution image of an ERA linked to an ER tubule / sheet . The osmium / lead staining used in this technique visualizes ribosomes and demonstrates that the outer ERA envelope membrane , but not the stacked internal cisternae , are tightly studded with ribosomes , indicating that they originate from ER membranes . ( F ) High - pressure freezing / freeze substitution image of an ER - ERA junction using an improved protocol to visualize membranes . ( G ) Using the same technique as in ( F ) , we visualized the internal membrane content of an ERA . Note that both portions of the internal membranes and of the sequestering double membrane envelope contain bound ribosomes , and hence are likely derived from the ER .

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Characterization of ER-Containing Autophagosomes (ERAs) during the UPR(A) Images of representative DTT-treated wild-type cells that contain ERAs. Nuclei and cytoplasm are indicated as N and C, respectively.(B) Enlargement of representative images of ERAs from different cells. The bottom right image is likely to show a section through a cup-shaped ERA. Note that there are no connections between the stacked cisternae and the envelope.(C) High magnification of the ERA double membrane envelope.(D) Some ERAs are found attached to or are in close proximity to ER tubules/sheets (indicated by the arrow). Note that the section in (A) includes two such junctions.(E) High-pressure freezing/freeze substitution image of an ERA linked to an ER tubule/sheet. The osmium/lead staining used in this technique visualizes ribosomes and demonstrates that the outer ERA envelope membrane, but not the stacked internal cisternae, are tightly studded with ribosomes, indicating that they originate from ER membranes.(F) High-pressure freezing/freeze substitution image of an ER-ERA junction using an improved protocol to visualize membranes.(G) Using the same technique as in (F), we visualized the internal membrane content of an ERA. Note that both portions of the internal membranes and of the sequestering double membrane envelope contain bound ribosomes, and hence are likely derived from the ER.


Fluorescence Visualization of an ER Marker after UPR Induction ( A ) Cells treated with the UPR - inducing drug DTT ( + DTT ) or with no drug were visualized using a fusion protein between the translocon component Sec61 and the red - fluorescent protein " cherry . " Top panels show untreated cells , and bottom panels show representative UPR - induced cells . ( B ) Representative images showing UPR - induced cells that contain ERAs ( indicated by arrows ) .

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Fluorescence Visualization of an ER Marker after UPR Induction(A) Cells treated with the UPR-inducing drug DTT (+DTT) or with no drug were visualized using a fusion protein between the translocon component Sec61 and the red-fluorescent protein "cherry." Top panels show untreated cells, and bottom panels show representative UPR-induced cells.(B) Representative images showing UPR-induced cells that contain ERAs (indicated by arrows).


Immunogold Labeling of ERAs with an Antibody Directed against an ER Membrane Marker ( A ) Representative section of a cell immunolabeled against a myc - tagged Sec63 , an integral ER membrane protein . As a primary antibody , we used a rabbit polyclonal anti - myc and , as a secondary , we used 15 - nm gold particles - conjugated anti - rabbit antibody . Nucleus , nuclear envelope , ER , and ERA are indicated as N , NE , ER , and ERA , respectively . ( B ) High magnification of an electron micrograph of a section of ER . Quantification showed that there are 5 ± 2 gold particles per linear micrometer of ER . ( C ) High magnification of ERAs . To predict how many gold particles one should expect in a particular ERA , we first calculated and averaged the amount of ER ( expressed as length in linear micrometers ) present in an ERA ( similar to the ones shown in Figure 3B ) , and normalized the value for its area . These calculations determined that there are 20 . 8 ± 3 . 3 μm of ER per μm2 inside the ERAs . These values allowed us to predict how many gold particles would be expected over a section of an ERA if it were packed with ER membranes . Two representative ERAs are shown . The ERA shown in the middle picture should hold 2 . 4 μm of ER inside and , therefore , should have 12 gold particles . We counted 12 gold particles . The ERA on the right could contain 2 . 7 μm of ER and should contain14 gold particles ; we counted 16 gold particles . The image on the right shows a representative view of a nucleoplasmic region . ( D ) Quantification of gold - labeling density per area . To assess the signal - to - noise ratio of our immunogold - labeling procedure , we assessed background labeling by counting the number of gold particles over an areas of nucleoplasm ( N ) and over ERAs , and normalized the counts to the respective areas .

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Immunogold Labeling of ERAs with an Antibody Directed against an ER Membrane Marker(A) Representative section of a cell immunolabeled against a myc-tagged Sec63, an integral ER membrane protein. As a primary antibody, we used a rabbit polyclonal anti-myc and, as a secondary, we used 15-nm gold particles-conjugated anti-rabbit antibody. Nucleus, nuclear envelope, ER, and ERA are indicated as N, NE, ER, and ERA, respectively.(B) High magnification of an electron micrograph of a section of ER. Quantification showed that there are 5 ± 2 gold particles per linear micrometer of ER.(C) High magnification of ERAs. To predict how many gold particles one should expect in a particular ERA, we first calculated and averaged the amount of ER (expressed as length in linear micrometers) present in an ERA (similar to the ones shown in Figure 3B), and normalized the value for its area. These calculations determined that there are 20.8 ± 3.3 μm of ER per μm2 inside the ERAs. These values allowed us to predict how many gold particles would be expected over a section of an ERA if it were packed with ER membranes. Two representative ERAs are shown. The ERA shown in the middle picture should hold 2.4 μm of ER inside and, therefore, should have 12 gold particles. We counted 12 gold particles. The ERA on the right could contain 2.7 μm of ER and should contain14 gold particles; we counted 16 gold particles. The image on the right shows a representative view of a nucleoplasmic region.(D) Quantification of gold-labeling density per area. To assess the signal-to-noise ratio of our immunogold-labeling procedure, we assessed background labeling by counting the number of gold particles over an areas of nucleoplasm (N) and over ERAs, and normalized the counts to the respective areas.


( A ) Wild - type cells transformed with a plasmid containing GFP - Atg8 were grown for 4 h in synthetic media with no drug , with UPR - inducing conditions ( + DTT and + TM ) , or under nitrogen starvation conditions ( N starv ) , and then harvested for protein preparation . Protein extracts were analyzed by Western blotting using antibodies against GFP ( top panel ) or Hac1 ( bottom panel ) . Total protein concentration was measured by BCA protein assay . Same concentration of protein was loaded in each lane , and transfer efficiency was checked by Ponceau staining . The identities of the different bands are indicated .

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(A) Wild-type cells transformed with a plasmid containing GFP-Atg8 were grown for 4 h in synthetic media with no drug, with UPR-inducing conditions (+DTT and +TM), or under nitrogen starvation conditions (N starv), and then harvested for protein preparation. Protein extracts were analyzed by Western blotting using antibodies against GFP (top panel) or Hac1 (bottom panel). Total protein concentration was measured by BCA protein assay. Same concentration of protein was loaded in each lane, and transfer efficiency was checked by Ponceau staining. The identities of the different bands are indicated.


( B ) Wild - type cells expressing GFP - Atg8 grown under the conditions described above were visualized by fluorescence microscopy .

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(B) Wild-type cells expressing GFP-Atg8 grown under the conditions described above were visualized by fluorescence microscopy.


( C ) GFP - Atg8 was detected in extracts from untreated hac1Δ cells or cells expressing HAC1i ( + DOC ) by Western blotting using antibodies against GFP .

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(C) GFP-Atg8 was detected in extracts from untreated hac1Δ cells or cells expressing HAC1i (+DOC) by Western blotting using antibodies against GFP.


( D ) Western blot using antibodies against GFP of extracts from hac1Δ , ire1Δ , or vps4Δ pep4Δ cells expressing GFP - Atg8 . Mutant cells were grown under regular conditions , UPR - inducing conditions ( + DTT ) , or nitrogen starvation conditions ( N starv ) .

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(D) Western blot using antibodies against GFP of extracts from hac1Δ, ire1Δ, or vps4Δ pep4Δ cells expressing GFP-Atg8. Mutant cells were grown under regular conditions, UPR-inducing conditions (+DTT), or nitrogen starvation conditions (N starv).


Localization of GFP - Atg8 during UPR InductionSome of the DTT - treated cells shown in Figure 4B expressing GFP - Atg8 and Sec61 - cherry ( as an ER marker ) were visualized using fluorescence microscopy . GFP - Atg8 localizes in close proximity to the ERAs detected by the ER marker .

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Localization of GFP-Atg8 during UPR InductionSome of the DTT-treated cells shown in Figure 4B expressing GFP-Atg8 and Sec61-cherry (as an ER marker) were visualized using fluorescence microscopy. GFP-Atg8 localizes in close proximity to the ERAs detected by the ER marker.


Atg8 and Other ATG Genes Are Necessary during UPR InductionSerial dilutions for wild - type , hac1Δ , atg1Δ , atg8Δ , atg9Δ , atg16Δ , and atg20Δ deletion cells and vps4Δ pep4Δ double deletion cells were grown on rich - media plates with no drug ( YPD ) or with different concentrations of tunicamycin ( TM ; 0 . 2 or 1 . 0 μg / ml ) . atg19Δ gave an identical result to the other autophagy genes shown here ( unpublished data ) .

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Atg8 and Other ATG Genes Are Necessary during UPR InductionSerial dilutions for wild-type, hac1Δ, atg1Δ, atg8Δ, atg9Δ, atg16Δ, and atg20Δ deletion cells and vps4Δ pep4Δ double deletion cells were grown on rich-media plates with no drug (YPD) or with different concentrations of tunicamycin (TM; 0.2 or 1.0 μg/ml). atg19Δ gave an identical result to the other autophagy genes shown here (unpublished data).


( A ) The bar graph represents top 10 biological pathways upregulated in gene ontology ( GO ) based Reactome pathway analysis using a set of genes induced ( 1 . 5 fold , p < 0 . 05 ( Wald Test ) , 3 biological replicates ) in RNA - seq analysis in IRGM shRNA knockdown HT29 cells compared to control shRNA cells . Heatmaps were generated for sentinel interferon - regulated genes ( three biological replicates ) using ' ComplexHeatmap ' library using ' R ' Bioconductor package where the gene expression matrix was transformed into z - score . The heat map was generated from the common genes present in the three GO terms indicated by the three black lines . The numbers on the bars indicate the p - value of that particular GO term .

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(A) The bar graph represents top 10 biological pathways upregulated in gene ontology (GO) based Reactome pathway analysis using a set of genes induced (1.5 fold, p<0.05 (Wald Test), 3 biological replicates) in RNA-seq analysis in IRGM shRNA knockdown HT29 cells compared to control shRNA cells. Heatmaps were generated for sentinel interferon-regulated genes (three biological replicates) using 'ComplexHeatmap' library using 'R' Bioconductor package where the gene expression matrix was transformed into z-score. The heat map was generated from the common genes present in the three GO terms indicated by the three black lines. The numbers on the bars indicate the p-value of that particular GO term.


( D ) The qRT - PCR validation of RNA - seq data in control and IRGM KD HT29 cells .

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(D) The qRT-PCR validation of RNA- seq data in control and IRGM KD HT29 cells.


( E ) The bar graph represents the top pathways upregulated in GO - based Reactome pathway analysis using set of genes induced ( 1 . 5 fold , p < 0 . 05 ( Wald Test ) , 3 mice each group ) in the brain of Irgm1 - / - mice compared to Irgm1 + / + wild type mice . Heatmaps were generated for sentinel interferon - regulated genes ( three biological replicates ) using ' ComplexHeatmap ' library using ' R ' Bioconductor package where the gene expression matrix was transformed into z - score . The heat map was generated from the common genes present in the three GO terms indicated by the three black lines . The numbers on the bars indicate the p - value of that particular GO term .

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(E) The bar graph represents the top pathways upregulated in GO-based Reactome pathway analysis using set of genes induced (1.5 fold, p<0.05 (Wald Test), 3 mice each group) in the brain of Irgm1-/- mice compared to Irgm1+/+ wild type mice. Heatmaps were generated for sentinel interferon-regulated genes (three biological replicates) using 'ComplexHeatmap' library using 'R' Bioconductor package where the gene expression matrix was transformed into z-score. The heat map was generated from the common genes present in the three GO terms indicated by the three black lines. The numbers on the bars indicate the p-value of that particular GO term.


( H ) The qRT - PCR validation of RNA - seq data in Irgm1 + / + and Irgm1 - / - mouse brain .

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(H) The qRT-PCR validation of RNA- seq data in Irgm1+/+ and Irgm1-/- mouse brain.


( A ) Heat map of nucleic acid sensor proteins upregulated in IRGM KD HT29 cells and Irgm1 - / - mouse brain and BMDMs .

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(A) Heat map of nucleic acid sensor proteins upregulated in IRGM KD HT29 cells and Irgm1-/- mouse brain and BMDMs.


Western blot analysis to determine levels of nucleic acid sensor proteins with lysates of ( B ) HT29 control ( henceforth IRGM + / + ) and single allele CRISPR knockout IRGM cells ( henceforth IRGM + / - ) ,

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Western blot analysis to determine levels of nucleic acid sensor proteins with lysates of (B) HT29 control (henceforth IRGM+/+) and single allele CRISPR knockout IRGM cells (henceforth IRGM+/-),


Western blot analysis to determine levels of nucleic acid sensor proteins with lysates of ( C ) HT29 cells stably expressing control shRNA or IRGM shRNA ,

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Western blot analysis to determine levels of nucleic acid sensor proteins with lysates of (C) HT29 cells stably expressing control shRNA or IRGM shRNA,


Western blot analysis to determine levels of nucleic acid sensor proteins with lysates of ( D ) Irgm1 + / + and Irgm1 - / - mouse brain ( n = 3 mice ) .

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Western blot analysis to determine levels of nucleic acid sensor proteins with lysates of (D) Irgm1+/+ and Irgm1-/- mouse brain (n=3 mice).


( E ) Western blot analysis to determine levels of adaptor proteins in control and IRGM + / - HT29 cells .

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(E) Western blot analysis to determine levels of adaptor proteins in control and IRGM+/- HT29 cells.


( F ) SDD - AGE followed by western blot analysis with a mitochondrial fraction from control and IRGM siRNA knockdown THP1 cells . Western blot analysis with cytoplasmic fraction was also performed .

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(F) SDD-AGE followed by western blot analysis with a mitochondrial fraction from control and IRGM siRNA knockdown THP1 cells. Western blot analysis with cytoplasmic fraction was also performed.


Western blot analysis performed with lysates of ( G ) control and stable IRGM shRNA knockdown HT29 cells ( H ) IRGM + / + and IRGM + / - HT29

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Western blot analysis performed with lysates of (G) control and stable IRGM shRNA knockdown HT29 cells (H) IRGM+/+ and IRGM+/- HT29 cells


Western blot analysis performed with lysates of ( I ) Irgm1 + / + and Irgm1 - / - mouse brain to determine levels of TBK1 protein .

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Western blot analysis performed with lysates of (I) Irgm1+/+ and Irgm1-/- mouse brain to determine levels of TBK1 protein.


Western blot analysis performed with lysates of ( J ) IRGM + / + and IRGM + / - HT29

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Western blot analysis performed with lysates of (J) IRGM+/+ and IRGM+/- HT29 cells


Western blot analysis performed with lysates of ( K - L ) Irgm1 + / + and Irgm1 - / - mouse brain to determine levels of IRF proteins .

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Western blot analysis performed with lysates of (K-L) Irgm1+/+ and Irgm1-/- mouse brain to determine levels of IRF proteins.


Western blot analysis performed with lysates of ( M ) IRGM + / + and IRGM + / - HT29

Western blot analysis performed with lysates of (M) IRGM+/+ and IRGM+/- HT29 cells


Western blot analysis performed with lysates of ( N ) Irgm1 + / + and Irgm1 - / - mouse brain to determine levels of STAT proteins ( n = 3 mice ) .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O B-ORGANISM B-TISSUE O O O O O O O O O O O O O

Western blot analysis performed with lysates of (N) Irgm1+/+ and Irgm1-/- mouse brain to determine levels of STAT proteins (n=3 mice).


Western blot analysis performed with lysates of ( O ) IRGM + / + and IRGM + / - HT29 cells . 2 biological replicates are shown .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O B-CELL_LINE O O O O O O O O

Western blot analysis performed with lysates of (O) IRGM+/+ and IRGM+/- HT29 cells. 2 biological replicates are shown.


Western blot analysis performed with lysates of ( P ) Irgm1 + / + and Irgm1 - / - mouse brain to determine levels of ISG proteins ( n = 2 mice ) .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O B-ORGANISM B-TISSUE O O O O O O O O O O B-ORGANISM O O

Western blot analysis performed with lysates of (P) Irgm1+/+ and Irgm1-/- mouse brain to determine levels of ISG proteins (n=2 mice).


( Q ) Representative confocal image of immunofluorescence assay performed with IRGM + / + and IRGM + / - HT29 cells stained with MX1 antibodies . Scale bar , 10 µm .

O O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O B-GENEPROD O O O O B-GENEPROD O O O B-CELL_LINE O B-EXP_ASSAY O B-GENEPROD O O O O O O O O

(Q) Representative confocal image of immunofluorescence assay performed with IRGM+/+ and IRGM+/- HT29 cells stained with MX1 antibodies. Scale bar, 10 µm.


( A ) Immunoprecipitation ( IP ) analysis of the interaction between endogenous IRGM and endogenous RIG - I , cGAS , MDA5 , TLR3 , TLR4 , TLR7 , NLRC4 , and AIM2 in THP - 1 cell lysates . IgG ( H ) , IgG heavy chain .

O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O B-EXP_ASSAY O O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O

(A) Immunoprecipitation (IP) analysis of the interaction between endogenous IRGM and endogenous RIG-I, cGAS, MDA5, TLR3, TLR4, TLR7, NLRC4, and AIM2 in THP-1 cell lysates. IgG (H), IgG heavy chain.


( B - D ) Co - immunoprecipitation ( Co - IP ) analysis of the interaction between ( B ) GFP - IRGM and Flag - RIG - I , ( C ) GFP - IRGM , and Flag - cGAS , ( D ) GFP - IRGM and Flag - TLR3 in HEK293T cell lysates . L . E , Long exposure of input blot . IgG ( L ) , IgG Light chain ; IgG ( H ) , IgG heavy chain .

O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-CELL_LINE O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O

(B-D) Co-immunoprecipitation (Co-IP) analysis of the interaction between (B) GFP-IRGM and Flag-RIG-I, (C) GFP-IRGM, and Flag-cGAS, (D) GFP-IRGM and Flag-TLR3 in HEK293T cell lysates. L.E, Long exposure of input blot. IgG (L), IgG Light chain; IgG (H), IgG heavy chain.


( E ) GST pull - down assay of in - vitro translated and radiolabelled myc - tagged cGAS , RIG - I , and TLR3 with GST or GST - tagged IRGM .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY B-GENEPROD O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O

(E) GST pull-down assay of in-vitro translated and radiolabelled myc-tagged cGAS, RIG-I, and TLR3 with GST or GST-tagged IRGM.


( F ) Western blot analysis with the lysates of HT - 29 cells stably transfected with control or Flag - IRGM plasmid and probed with the indicated antibodies .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O

(F) Western blot analysis with the lysates of HT-29 cells stably transfected with control or Flag-IRGM plasmid and probed with the indicated antibodies.


( G - I ) Western blot analysis with lysates of HEK293T cells transiently expressing GFP or GFP - IRGM and ( G ) Flag - RIG - I or ( H ) Flag - cGAS or ( I ) or Flag - TLR3 .

O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O

(G-I) Western blot analysis with lysates of HEK293T cells transiently expressing GFP or GFP-IRGM and (G) Flag-RIG-I or (H) Flag-cGAS or (I) or Flag-TLR3.


( J - K ) The qRT - PCR analysis with RNA isolated from ( J ) control and Flag - IRGM stable HT - 29 cells ( K ) THP - 1 cells transiently transfected with control or Flag - IRGM plasmid .

O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O B-GENEPROD O B-GENEPROD O O

(J-K) The qRT-PCR analysis with RNA isolated from (J) control and Flag-IRGM stable HT-29 cells (K) THP-1 cells transiently transfected with control or Flag-IRGM plasmid.


Luciferase reporter assays in HEK293T cells transiently transfected with ISRE reporter plasmid and other plasmids as indicated .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O O O O O O O O O O O O

Luciferase reporter assays in HEK293T cells transiently transfected with ISRE reporter plasmid and other plasmids as indicated.


( O ) Western blotting analysis with lysates of control and untreated or Bafilomycin A1 ( 300 nM ; 4 h and 8 h ) treated Flag - IRGM expressing HT29 stable cell lines .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-CELL_LINE O O O O

(O) Western blotting analysis with lysates of control and untreated or Bafilomycin A1 (300 nM; 4 h and 8 h) treated Flag-IRGM expressing HT29 stable cell lines.


( P ) Western blotting analysis with lysates of control and untreated or chloroquine ( 50 µM ; 4 h and 8 h ) treated Flag - IRGM expressing HT29 stable cell lines .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-CELL_LINE O O O O

(P) Western blotting analysis with lysates of control and untreated or chloroquine (50 µM; 4 h and 8 h) treated Flag-IRGM expressing HT29 stable cell lines.


( Q ) The qRT - PCR analysis with RNA isolated from untreated or Bafilomycin A1 ( 300 nM ; 4 h , 8 h , and 12 h ) treated control or Flag - IRGM expressing HT - 29 stable cells as indicated .

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O

(Q) The qRT-PCR analysis with RNA isolated from untreated or Bafilomycin A1 (300 nM; 4 h, 8 h, and 12 h) treated control or Flag-IRGM expressing HT-29 stable cells as indicated.


( A ) The cell lysates of control or BECLIN1 siRNA transfected THP - 1 cells expressing GFP or GFP - IRGM were subjected to immunoblotting with indicated antibodies .

O O O O O O O O O B-GENEPROD O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY O O O O

(A) The cell lysates of control or BECLIN1 siRNA transfected THP-1 cells expressing GFP or GFP-IRGM were subjected to immunoblotting with indicated antibodies.


( B ) The cell lysates of control or ATG5 siRNA transfected THP - 1 cells expressing GFP or GFP - IRGM were subjected to immunoblotting with indicated antibodies .

(B) The cell lysates of control or ATG5 siRNA transfected THP-1 cells expressing GFP or GFP-IRGM were subjected to immunoblotting with indicated antibodies.


( C - D ) qRT - PCR analysis with RNA isolated from control or BECLIN1 siRNA transfected THP - 1 cells expressing 3X - Flag epitope or Flag - IRGM as indicated .

O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O

(C-D) qRT-PCR analysis with RNA isolated from control or BECLIN1 siRNA transfected THP-1 cells expressing 3X-Flag epitope or Flag-IRGM as indicated.


qRT - PCR analysis with RNA isolated from control or ATG5 siRNA transfected THP - 1 cells expressing 3X - Flag epitope or Flag - IRGM as indicated .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O

qRT-PCR analysis with RNA isolated from control or ATG5 siRNA transfected THP-1 cells expressing 3X-Flag epitope or Flag-IRGM as indicated.


( G - H ) qRT - PCR analysis with RNA isolated from control or p62 siRNA or BECLIN1 siRNA transfected HT29 cells expressing 3X - Flag epitope or Flag - IRGM as indicated .

O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD O O B-GENEPROD O O B-CELL_LINE O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O

(G-H) qRT-PCR analysis with RNA isolated from control or p62 siRNA or BECLIN1 siRNA transfected HT29 cells expressing 3X-Flag epitope or Flag-IRGM as indicated.


Co - IP analysis of the interaction between GFP IRGM with ( I ) Flag - p62 or Flag - NDP52 or Flag - NBR1 in HEK293T cell lysates .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-CELL_LINE O O O

Co-IP analysis of the interaction between GFP IRGM with (I) Flag-p62 or Flag-NDP52 or Flag-NBR1 in HEK293T cell lysates.


Co - IP analysis of the interaction between GFP IRGM with ( J ) Flag - Optineurin or Flag - TAX1BP1 in HEK293T cell lysates .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-CELL_LINE O O O

Co-IP analysis of the interaction between GFP IRGM with (J) Flag-Optineurin or Flag-TAX1BP1 in HEK293T cell lysates.


( K - M ) Co - IP analysis of the interaction between GFP IRGM with ( K ) Flag - cGAS , ( L ) Flag - RIG - I , ( M ) Flag - TLR3 in control siRNA or p62 siRNA transfected HEK293T cell lysates .

O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O B-CELL_LINE O O O

(K-M) Co-IP analysis of the interaction between GFP IRGM with (K) Flag-cGAS, (L) Flag-RIG-I, (M) Flag-TLR3 in control siRNA or p62 siRNA transfected HEK293T cell lysates.


( N ) The cell lysates of control or p62 siRNA transfected THP - 1 cells expressing GFP or GFP - IRGM ( as indicate ) were subjected to immunoblotting with indicated antibodies .

O O O O O O O O O B-GENEPROD O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O B-EXP_ASSAY O O O O

(N) The cell lysates of control or p62 siRNA transfected THP-1 cells expressing GFP or GFP- IRGM (as indicate) were subjected to immunoblotting with indicated antibodies.


( O ) The qRT - PCR analysis with RNA isolated from control or p62 transfected THP - 1 cells expressing 3X - Flag epitope or Flag - IRGM as indicated .

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O

(O) The qRT-PCR analysis with RNA isolated from control or p62 transfected THP-1 cells expressing 3X-Flag epitope or Flag-IRGM as indicated.


( P - R ) Co - IP analysis of the interaction between HA - p62 and ( P ) Flag - RIG - I , ( Q ) Flag - TLR3 , ( R ) Flag - cGAS in the absence and presence of GFP or GFP - IRGM in HEK293T cell lysates .

O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-CELL_LINE O O O

(P-R) Co-IP analysis of the interaction between HA-p62 and (P) Flag-RIG-I, (Q) Flag-TLR3, (R) Flag-cGAS in the absence and presence of GFP or GFP-IRGM in HEK293T cell lysates.


( A ) Representative confocal images of Irgm1 + / + and Irgm1 - / - mouse BMDMs processed for IF analysis with Tom20 antibody . Scale bar , 5 µm . Zoom panels are digital magnifications .

O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O B-ORGANISM B-CELL_TYPE O O B-EXP_ASSAY O O B-GENEPROD O O O O O O O O O O O O O O

(A) Representative confocal images of Irgm1+/+ and Irgm1-/- mouse BMDMs processed for IF analysis with Tom20 antibody. Scale bar, 5 µm. Zoom panels are digital magnifications.


( B ) The graph depicts the percentage of cells with swollen / rounded mitochondria in Irgm1 + / + and Irgm1 - / - mouse BMDMs .

O O O O O O O O O O O O O O B-SUBCELLULAR O B-GENEPROD O O O O B-GENEPROD O O O B-ORGANISM B-CELL_TYPE O

(B) The graph depicts the percentage of cells with swollen/rounded mitochondria in Irgm1+/+ and Irgm1-/- mouse BMDMs.


( C ) Representative confocal ( STED ) images of Irgm1 + / + and Irgm1 - / - mouse BMDMs processed for IF analysis with Tom20 and Parkin antibodies . Line profile : Co - localization analysis using line intensity profiles .

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O B-ORGANISM B-CELL_TYPE O O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O

(C) Representative confocal (STED) images of Irgm1+/+ and Irgm1-/- mouse BMDMs processed for IF analysis with Tom20 and Parkin antibodies. Line profile: Co-localization analysis using line intensity profiles.


( D ) Representative confocal ( STED ) images of Irgm1 + / + and Irgm1 - / - mouse BMDMs processed for IF analysis with Tom20 and Pink1 antibodies . Line profile : Co - localization analysis using line intensity profiles .

(D) Representative confocal (STED) images of Irgm1+/+ and Irgm1-/- mouse BMDMs processed for IF analysis with Tom20 and Pink1 antibodies. Line profile: Co-localization analysis using line intensity profiles.


( E ) Representative confocal images of control siRNA and IRGM siRNA transfected THP - 1 cells processed for IF analysis with Ubiquitin ( green ) and TOM20 ( red ) antibodies . Scale bar , 10 µm .

O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-EXP_ASSAY O O B-SMALL_MOLECULE O O O O B-GENEPROD O O O O O O O O O O O

(E) Representative confocal images of control siRNA and IRGM siRNA transfected THP-1 cells processed for IF analysis with Ubiquitin (green) and TOM20 (red) antibodies. Scale bar, 10 µm.


( F ) Western blot analysis of mitochondrial fraction of control siRNA and IRGM siRNA transfected THP - 1 cells , probed with indicated antibodies .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR O O O O O B-GENEPROD O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O

(F) Western blot analysis of mitochondrial fraction of control siRNA and IRGM siRNA transfected THP-1 cells, probed with indicated antibodies.


( G ) Western blot analysis of mitochondrial fraction of Irgm1 + / + and Irgm1 - / - mouse BMDM cells , probed with indicated antibodies ( n = 2 mice ) .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR O O B-GENEPROD O O O O B-GENEPROD O O O B-ORGANISM B-CELL_TYPE O O O O O O O O O O B-ORGANISM O O

(G) Western blot analysis of mitochondrial fraction of Irgm1+/+ and Irgm1-/- mouse BMDM cells, probed with indicated antibodies (n=2 mice).


( H ) Western blot analysis of mitochondrial fraction of Irgm1 + / + and Irgm1 - / - mouse BMDM cells untreated or treated with Bafilomycin A1 ( 300 nM , 3 h ) , probed with indicated antibodies .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR O O B-GENEPROD O O O O B-GENEPROD O O O B-ORGANISM B-CELL_TYPE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O

(H) Western blot analysis of mitochondrial fraction of Irgm1+/+ and Irgm1-/- mouse BMDM cells untreated or treated with Bafilomycin A1 (300 nM, 3 h), probed with indicated antibodies.


( I ) Western blot analysis of mitochondrial fraction of control siRNA and IRGM siRNA transfected THP - 1 cells untreated or treated with Bafilomycin A1 ( 300 nM , 3 h ) , probed with indicated antibodies .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR O O O O O B-GENEPROD O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O

(I) Western blot analysis of mitochondrial fraction of control siRNA and IRGM siRNA transfected THP-1 cells untreated or treated with Bafilomycin A1 (300 nM, 3 h), probed with indicated antibodies.


( J ) Seahorse XF Cell Mito stress test for analysis of mitochondrial function in Irgm1 + / + and Irgm1 - / - mouse BMDM cells . The experiments were performed using the XF24 extracellular flux analyzer , and the flow chart showed the measurement of oxygen consumption rate ( OCR )

O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-SUBCELLULAR O O B-GENEPROD O O O O B-GENEPROD O O O B-ORGANISM B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O

(J) Seahorse XF Cell Mito stress test for analysis of mitochondrial function in Irgm1+/+ and Irgm1-/- mouse BMDM cells. The experiments were performed using the XF24 extracellular flux analyzer, and the flow chart showed the measurement of oxygen consumption rate (OCR)


( K ) Representative confocal images of control and IRGM siRNA transfected THP - 1 cells stained with CMXRos red dye .

(K) Representative confocal images of control and IRGM siRNA transfected THP-1 cells stained with CMXRos red dye.


( L ) Flow cytometry analysis of control siRNA and IRGM siRNA transfected THP - 1 cells stained with CMXRos red dye ( 10 nM , 30 mins ) . ( M ) Graph depicts the normalized mean fluorescent intensity of control and IRGM knockdown THP - 1 cells stained with CMXRos .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-EXP_ASSAY O B-SMALL_MOLECULE O

(L) Flow cytometry analysis of control siRNA and IRGM siRNA transfected THP-1 cells stained with CMXRos red dye (10 nM, 30 mins). (M) Graph depicts the normalized mean fluorescent intensity of control and IRGM knockdown THP-1 cells stained with CMXRos.


( N ) Representative dot plot showing flow cytometry analysis of control and IRGM siRNA knockdown THP - 1 cells stained with JC - 1 dye ( 2 µM , 30 mins ) . At low mitochondrial membrane potential , JC - 1 is predominantly a monomer that yields green fluorescence , whereas at mitochondrial membrane potential the dye aggregates producing a red to orange colored emission .

O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O

(N) Representative dot plot showing flow cytometry analysis of control and IRGM siRNA knockdown THP-1 cells stained with JC-1 dye (2 µM, 30 mins). At low mitochondrial membrane potential, JC-1 is predominantly a monomer that yields green fluorescence, whereas at mitochondrial membrane potential the dye aggregates producing a red to orange colored emission.


( P ) Representative flow cytometry analysis of control and IRGM siRNA transfected THP - 1 cells stained with MitoSox red dye ( 1µM , 20 min ) . The percentage of control and IRGM knockdown cells with increased red fluorescence ( mitochondrial ROS generation ) is depicted .

O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O B-GENEPROD O O O O O O O B-SUBCELLULAR B-SMALL_MOLECULE O O O O O

(P) Representative flow cytometry analysis of control and IRGM siRNA transfected THP-1 cells stained with MitoSox red dye (1µM, 20 min). The percentage of control and IRGM knockdown cells with increased red fluorescence (mitochondrial ROS generation) is depicted.


( Q ) Representative flow cytometry analysis of control and IRGM siRNA transfected THP - 1 cells stained with CellRox - green and CellRox - orange dye ( 1µM , 30 min ) .

O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-EXP_ASSAY O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O O

(Q) Representative flow cytometry analysis of control and IRGM siRNA transfected THP-1 cells stained with CellRox-green and CellRox-orange dye (1µM, 30 min).


( A ) Representative confocal images of Irgm1 + / + and Irgm1 - / - mouse BMDMs processed for IF analysis with Tom20 ( red ) and dsDNA ( green ) antibody .

O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O B-ORGANISM B-CELL_TYPE O O B-EXP_ASSAY O O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O O

(A) Representative confocal images of Irgm1+/+ and Irgm1-/- mouse BMDMs processed for IF analysis with Tom20 (red) and dsDNA (green) antibody.


( B ) Representative STED microscopy images of control THP - 1 cells processed for IF analysis with cGAS ( green ) , dsDNA ( red ) , and Lamin B1 ( cyan ) antibodies . ( C ) The graph depicts the percentage of cells with micronuclei in control siRNA and IRGM siRNA knockdown THP - 1 cells .

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-EXP_ASSAY O O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O B-GENEPROD O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O

(B) Representative STED microscopy images of control THP-1 cells processed for IF analysis with cGAS (green), dsDNA (red), and Lamin B1 (cyan) antibodies. (C) The graph depicts the percentage of cells with micronuclei in control siRNA and IRGM siRNA knockdown THP-1 cells.


( D - E ) Representative STED microscopy images of Irgm1 - / - mouse BMDMs ( D ) transfected with mCherry - cGAS and immunostained with dsDNA ( green ) antibody . The white line depicts the periphery of the cells . Scale bar , 3 µm . ( E ) The graph depicts the percentage of cells with micronuclei in Irgm1 + / + and Irgm1 - / - mouse BMDMs .

O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O B-ORGANISM B-CELL_TYPE O O O O O B-GENEPROD O B-GENEPROD O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O 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-SUBCELLULAR O B-GENEPROD O O O O B-GENEPROD O O O B-ORGANISM B-CELL_TYPE O

(D-E) Representative STED microscopy images of Irgm1-/- mouse BMDMs (D) transfected with mCherry-cGAS and immunostained with dsDNA (green) antibody. The white line depicts the periphery of the cells. Scale bar, 3 µm. (E) The graph depicts the percentage of cells with micronuclei in Irgm1+/+ and Irgm1-/- mouse BMDMs.


( F ) The qRT - PCR analysis with cytosolic DNA ( minus mitochondria ) isolated from Irgm1 + / + and Irgm1 - / - mouse BMDMs .

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(F) The qRT-PCR analysis with cytosolic DNA (minus mitochondria) isolated from Irgm1+/+ and Irgm1-/- mouse BMDMs.


( G ) The qRT - PCR analysis with cytosolic DNA ( minus mitochondria ) isolated from control and IRGM siRNA transfected HT - 29 cells .

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(G) The qRT-PCR analysis with cytosolic DNA (minus mitochondria) isolated from control and IRGM siRNA transfected HT-29 cells.


( H ) The qRT - PCR analysis with cytosolic DNA ( minus mitochondria ) isolated from control or IRGM shRNA or EtBr treated IRGM shRNA HT - 29 ( Rho0 ) cells .

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(H) The qRT-PCR analysis with cytosolic DNA (minus mitochondria) isolated from control or IRGM shRNA or EtBr treated IRGM shRNA HT-29 (Rho0) cells.


( I ) The qRT - PCR analysis with total RNA isolated from control or IRGM shRNA or EtBr treated IRGM shRNA HT - 29 ( Rho0 ) cells .

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(I) The qRT-PCR analysis with total RNA isolated from control or IRGM shRNA or EtBr treated IRGM shRNA HT-29 (Rho0) cells.


( J ) The qRT - PCR analysis with cytosolic DNA ( minus mitochondria ) isolated from Irgm1 + / + and Irgm1 - / - mouse BMDMs transfected with DNase I ( 15 μg , 6hr ) .

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(J) The qRT-PCR analysis with cytosolic DNA (minus mitochondria) isolated from Irgm1+/+ and Irgm1-/- mouse BMDMs transfected with DNase I (15 μg, 6hr).


( K ) The qRT - PCR analysis with RNA isolated from control and IRGM siRNA knockdown THP - 1 cells electroporated with DNase I ( 5 μg , 1hr ) as indicated .

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(K) The qRT-PCR analysis with RNA isolated from control and IRGM siRNA knockdown THP-1 cells electroporated with DNase I (5 μg, 1hr) as indicated.


( L - M ) Representative confocal images of Irgm1 + / + and Irgm1 - / - mouse BMDMs processed for IF analysis with ( L ) dsRNA ( green ) and Tom20 ( red ) antibodies . ( M ) The graph depicts the percentage of Irgm1 + / + and Irgm1 - / - mouse BMDMs with dsRNA aggregates .

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(L-M) Representative confocal images of Irgm1+/+ and Irgm1-/- mouse BMDMs processed for IF analysis with (L) dsRNA (green) and Tom20 (red) antibodies. (M) The graph depicts the percentage of Irgm1+/+ and Irgm1-/- mouse BMDMs with dsRNA aggregates.


( N ) Representative confocal images of Irgm1 - / - mouse BMDMs processed for IF analysis with RIG - I ( red ) and dsRNA ( green ) antibodies . Line profile : Co - localization analysis using line intensity profiles .

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(N) Representative confocal images of Irgm1-/- mouse BMDMs processed for IF analysis with RIG-I (red) and dsRNA (green) antibodies. Line profile: Co-localization analysis using line intensity profiles.


Representative confocal images of Irgm1 - / - mouse BMDMs processed for IF analysis with ( O ) Mda5 ( red ) and dsRNA ( green ) antibodies Co - localization analysis using line intensity profiles .

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Representative confocal images of Irgm1-/- mouse BMDMs processed for IF analysis with (O) Mda5 (red) and dsRNA (green) antibodies Co-localization analysis using line intensity profiles.


Representative confocal images of Irgm1 - / - mouse BMDMs processed for IF analysis with ( P ) Mda5 ( red ) , dsRNA ( green ) and Tia1 ( blue ) antibodies ( Scale bar , 3 µm ) . Line profile : Co - localization analysis using line intensity profiles .

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Representative confocal images of Irgm1-/- mouse BMDMs processed for IF analysis with (P) Mda5 (red), dsRNA (green) and Tia1 (blue) antibodies (Scale bar, 3 µm). Line profile: Co-localization analysis using line intensity profiles.


( A ) qRT - PCR analysis with total RNA isolated from Irgm1 + / + and Irgm1 - / - mouse BMDMs transfected with control siRNA or cGAS siRNA or RIG - I siRNA or cGAS siRNA and RIG - I siRNA as indicated .

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(A) qRT-PCR analysis with total RNA isolated from Irgm1+/+ and Irgm1-/- mouse BMDMs transfected with control siRNA or cGAS siRNA or RIG-I siRNA or cGAS siRNA and RIG-I siRNA as indicated.


( B ) qRT - PCR analysis with total RNA isolated from HT - 29 cells transfected with control siRNA or IRGM siRNA or doubly transfected with IRGM siRNA and cGAS siRNA or IRGM siRNA and RIG - I siRNA or transfected with three siRNAs as indicated .

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(B) qRT-PCR analysis with total RNA isolated from HT-29 cells transfected with control siRNA or IRGM siRNA or doubly transfected with IRGM siRNA and cGAS siRNA or IRGM siRNA and RIG-I siRNA or transfected with three siRNAs as indicated.


( C ) qRT - PCR analysis with total RNA isolated from HT - 29 cells transfected with control siRNA or IRGM siRNA or doubly transfected with IRGM siRNA and STING siRNA or IRGM siRNA and MAVS siRNA or transfected with all the three siRNAs as indicated .

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(C) qRT-PCR analysis with total RNA isolated from HT-29 cells transfected with control siRNA or IRGM siRNA or doubly transfected with IRGM siRNA and STING siRNA or IRGM siRNA and MAVS siRNA or transfected with all the three siRNAs as indicated.


( D ) qRT - PCR analysis with total RNA isolated from HT - 29 cells transfected with control siRNA or IRGM siRNA or doubly transfected with IRGM siRNA and MDA5 siRNA as indicated .

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(D) qRT-PCR analysis with total RNA isolated from HT-29 cells transfected with control siRNA or IRGM siRNA or doubly transfected with IRGM siRNA and MDA5 siRNA as indicated.


( E ) qRT - PCR analysis with total RNA isolated from HT - 29 cells transfected with control siRNA or IRGM siRNA or doubly transfected with IRGM siRNA and IRF3 siRNA as indicated .

(E) qRT-PCR analysis with total RNA isolated from HT-29 cells transfected with control siRNA or IRGM siRNA or doubly transfected with IRGM siRNA and IRF3 siRNA as indicated.


( F ) qRT - PCR analysis with total RNA isolated from HT - 29 cells transfected with control siRNA or IRGM siRNA or doubly transfected with IRGM siRNA and STAT1 siRNA or IRGM siRNA and STAT2 siRNA as indicated .

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(F) qRT-PCR analysis with total RNA isolated from HT-29 cells transfected with control siRNA or IRGM siRNA or doubly transfected with IRGM siRNA and STAT1 siRNA or IRGM siRNA and STAT2 siRNA as indicated.


Immunofluorescence microscopy analysis reveals highly similar and mostly nuclear localization of the SMARCB1 wild type and the R377C mutant , visualized by anti - HA staining ( green ) . Phalloidin and DAPI staining was used to visualize the actin cytoskeleton and the nucleus , respectively . A novel MS - microscopy approach was used to further define the molecular context of the proteins . This analysis identified possible chromosomal , endosomal and membrane localization of the proteins . The possible endosomal localization is in agreement with the anti - HA immunofluorescence microscopy results . ( key : the scale bar for immunofluorescence images is 10 µm , and the color gradient on the MS - microscopy indicates the localization scores calculated by the MS - microscopy tool .

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Immunofluorescence microscopy analysis reveals highly similar and mostly nuclear localization of the SMARCB1 wild type and the R377C mutant, visualized by anti-HA staining (green). Phalloidin and DAPI staining was used to visualize the actin cytoskeleton and the nucleus, respectively. A novel MS-microscopy approach was used to further define the molecular context of the proteins. This analysis identified possible chromosomal, endosomal and membrane localization of the proteins. The possible endosomal localization is in agreement with the anti-HA immunofluorescence microscopy results. (key: the scale bar for immunofluorescence images is 10 µm, and the color gradient on the MS-microscopy indicates the localization scores calculated by the MS-microscopy tool.


The cell proliferation assay in HCT116 CRC cells shows growth advantage conferred by the SMARCB1 R377C mutation compared with the wild type SMARCB1 or the vector control . The error bars designate the standard deviation ( SD )

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The cell proliferation assay in HCT116 CRC cells shows growth advantage conferred by the SMARCB1 R377C mutation compared with the wild type SMARCB1 or the vector control. The error bars designate the standard deviation (SD)


Immunofluorescence microscopy analysis of the STK38L wild type and the R105W mutant display no clear difference in localization . Both the wild type and the R105W mutant display nuclear , cytoplasmic and plasma membrane localization . However , the MS - microscopy analysis suggests a more prominent endosomal localization with the R105W mutant . ( key : the scale bar for the immunofluorescence images is 10 µm , and the color gradient on the MS - microscopy indicates the localization scores calculated by the MS - Microscopy tool .

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Immunofluorescence microscopy analysis of the STK38L wild type and the R105W mutant display no clear difference in localization. Both the wild type and the R105W mutant display nuclear, cytoplasmic and plasma membrane localization. However, the MS-microscopy analysis suggests a more prominent endosomal localization with the R105W mutant. (key: the scale bar for the immunofluorescence images is 10 µm, and the color gradient on the MS-microscopy indicates the localization scores calculated by the MS-Microscopy tool.


The cell proliferation assay in HCT116 CRC cells shows growth advantage conferred by the expression of the wild type STK38L , whereas the R105W mutant only slightly increased the number of colonies . The error bars designate the standard deviation ( SD )

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The cell proliferation assay in HCT116 CRC cells shows growth advantage conferred by the expression of the wild type STK38L, whereas the R105W mutant only slightly increased the number of colonies. The error bars designate the standard deviation (SD)


Examples of CT scan images of several metastases ( M032R1 , M032R2 , and M032R5 ) .

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Examples of CT scan images of several metastases (M032R1, M032R2, and M032R5).


Volumetric measurements based on the PET and CT scans could be generated from M032 , M032R1 , and M032R5 . Graph illustrates that the metastases R1 and R5 initially expanded before the start of the treatment , but regressed upon vemurafenib treatment . M032R5 showed progressive disease after 4 months , whereas M032R1 still displayed stable disease . M032 was excised and no recurrence was observed .

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Volumetric measurements based on the PET and CT scans could be generated from M032, M032R1, and M032R5. Graph illustrates that the metastases R1 and R5 initially expanded before the start of the treatment, but regressed upon vemurafenib treatment. M032R5 showed progressive disease after 4 months, whereas M032R1 still displayed stable disease. M032 was excised and no recurrence was observed.


Hematoxylin - eosin ( H & amp ; amp ; E ) and p - ERK stainings on FFPE material of all metastases showed that all vemurafenib - resistant tumors had reactivation of the MAPK pathway . Scale bar represents 100

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Hematoxylin-eosin (H&amp;E) and p-ERK stainings on FFPE material of all metastases showed that all vemurafenib-resistant tumors had reactivation of the MAPK pathway. Scale bar represents 100 μm


qPCR was performed on gDNA retrieved from each of the metastases , using primers for BRAF and CRAF and normalized on LINE levels . Bars represent the mean of three replicates , error bars indicate standard deviation . The results confirmed that BRAF was amplified in M032R1 , M032R2 , and M032R5 .

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qPCR was performed on gDNA retrieved from each of the metastases, using primers for BRAF and CRAF and normalized on LINE levels. Bars represent the mean of three replicates, error bars indicate standard deviation. The results confirmed that BRAF was amplified in M032R1, M032R2, and M032R5.


Staining for BRAFV600E with a mutant epitope - specific antibody confirmed the upregulation of BRAFV600E in R1 , R2 , and R5 . Scale bar represents 100 μm .

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Staining for BRAFV600E with a mutant epitope-specific antibody confirmed the upregulation of BRAFV600E in R1, R2, and R5. Scale bar represents 100 μm.


Immunoblotting for MAPK pathway components confirmed reactivation of p - ERK and p - MEK in all resistant metastases .

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Immunoblotting for MAPK pathway components confirmed reactivation of p-ERK and p-MEK in all resistant metastases.


Immunoblotting for M032 patient ' s samples using an antibody directed against either the mutant BRAFV600E epitope or an N - terminal region of BRAF revealed the expression of a shorter variant of BRAFV600E in M032R3 , of approximately 80 kDa . This 80 - kDa band could not be detected with the N - terminus - recognizing antibody .

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Immunoblotting for M032 patient's samples using an antibody directed against either the mutant BRAFV600E epitope or an N-terminal region of BRAF revealed the expression of a shorter variant of BRAFV600E in M032R3, of approximately 80 kDa. This 80-kDa band could not be detected with the N-terminus-recognizing antibody.


Validation of T55delinsRT was done by PCR using insertion - specific primers . As a control , primers amplifying exon 2 of MEK1WT were used .

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Validation of T55delinsRT was done by PCR using insertion-specific primers. As a control, primers amplifying exon 2 of MEK1WT were used.


Transfection of pQXCIP - GFP , MEK1 - WT , or MEK1 - T55delinsRT into HEK293T cells showed that this mutation induces p - ERK and p - RSK .

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Transfection of pQXCIP-GFP, MEK1-WT, or MEK1-T55delinsRT into HEK293T cells showed that this mutation induces p-ERK and p-RSK.


Validation of expression of pQXCIP - GFP , MEK1WT , or MEK1T55delinsRT in A375 melanoma cells by immunoblotting .

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Validation of expression of pQXCIP-GFP, MEK1WT, or MEK1T55delinsRT in A375 melanoma cells by immunoblotting.


Dose - response curves for A375 melanoma cells expressing GFP , MEK1WT , or MEK1T55delinsRT with indicated doses of BRAF inhibitor vemurafenib , ERK inhibitor SCH772984 , or MEK inhibitor trametinib . Error bars indicate standard deviation .

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Dose-response curves for A375 melanoma cells expressing GFP, MEK1WT, or MEK1T55delinsRT with indicated doses of BRAF inhibitor vemurafenib, ERK inhibitor SCH772984, or MEK inhibitor trametinib. Error bars indicate standard deviation.


Colony formation assays with A375 melanoma cells , infected with GFP , MEK1WT , or MEK1T55delinsRT - encoding lentivirus , and treated with indicated doses and inhibitors .

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Colony formation assays with A375 melanoma cells, infected with GFP, MEK1WT, or MEK1T55delinsRT-encoding lentivirus, and treated with indicated doses and inhibitors.


Treatment of A375 MEK1T55delinsRT melanoma cells with DMSO ( − ) , 250 nM dabrafenib ( D ) , 10 nM trametinib ( T ) , or a combination ( D + T ) .

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Treatment of A375 MEK1T55delinsRT melanoma cells with DMSO (−), 250 nM dabrafenib (D), 10 nM trametinib (T), or a combination (D+T).


A375 melanoma cells expressing GFP , MEK1WT , or MEK1T55delinsRT were injected into immune - deficient mice ( n = 8 per group ) , and after the tumor size of ˜ 100 mm3 was reached , mice were treated with 30 mg / kg dabrafenib or vehicle . Graphs represent fold change in tumor volume normalized on the tumor volume on the day of the start of the treatment . Error bars indicate standard error of the mean .

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A375 melanoma cells expressing GFP, MEK1WT, or MEK1T55delinsRT were injected into immune-deficient mice (n = 8 per group), and after the tumor size of ˜100 mm3 was reached, mice were treated with 30 mg/kg dabrafenib or vehicle. Graphs represent fold change in tumor volume normalized on the tumor volume on the day of the start of the treatment. Error bars indicate standard error of the mean.


Average tumor weight of all groups at the end of the experiment described in ( A ) . Error bars indicate standard deviation . Unpaired t - test was used to calculate statistical significance . P - values are indicated in the graph .

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Average tumor weight of all groups at the end of the experiment described in (A). Error bars indicate standard deviation. Unpaired t-test was used to calculate statistical significance. P-values are indicated in the graph.


Immunoblotting for MAPK pathway components performed on tumors of all groups .

Immunoblotting for MAPK pathway components performed on tumors of all groups.


Immunohistochemistry for Sirius Red ( staining for collagen ) and H & amp ; amp ; E on tumors from all groups , to identify grade of demarcation and local invasion . Scale bar represents 500 ( upper row ) and 100 ( lower row ) μm .

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Immunohistochemistry for Sirius Red (staining for collagen) and H&amp;E on tumors from all groups, to identify grade of demarcation and local invasion. Scale bar represents 500 (upper row) and 100 (lower row) μm.


gDNA was isolated from another fragment of the tumor resection , and the PCR with the insertion - specific primers was repeated . Primers amplifying exon 2 of MEK1WT were used as an input control . Source data are available online for this figure .

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gDNA was isolated from another fragment of the tumor resection, and the PCR with the insertion-specific primers was repeated. Primers amplifying exon 2 of MEK1WT were used as an input control.Source data are available online for this figure.


Staining for hematoxylin - eosin ( H & amp ; amp ; E ) and p - ERK on the PDX samples M032 . X1 , M032R1 . X1 , M032R2 . X1 , M032R4 . X1 , and M032R5 . X1 . Stainings showed that p - ERK is higher in the PDX derived from the resistant metastases . Scale bar represents 100 μm .

B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O O O O O 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-GENEPROD O O O O O O O O O B-TISSUE O O O O O O O

Staining for hematoxylin-eosin (H&amp;E) and p-ERK on the PDX samples M032.X1, M032R1.X1, M032R2.X1, M032R4.X1, and M032R5.X1. Stainings showed that p-ERK is higher in the PDX derived from the resistant metastases. Scale bar represents 100 μm.


Immunoblotting for components of the MAPK pathway confirmed reactivation of p - ERK in the PDX derived from the vemurafenib - resistant metastases , although the p - ERK signal is heterogeneous in the pre - treatment PDX .

B-EXP_ASSAY O O O O O O O O O O O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O B-TISSUE O O O O O B-GENEPROD O O O O O O O O O O

Immunoblotting for components of the MAPK pathway confirmed reactivation of p-ERK in the PDX derived from the vemurafenib-resistant metastases, although the p-ERK signal is heterogeneous in the pre-treatment PDX.


Validation of the BRAF amplification was performed by qPCR on gDNA derived from the PDX , using primers for BRAF and CRAF and normalized on LINE expression . Bars represent the mean of three replicates , error bars indicate standard deviation . The results confirmed that BRAF was amplified in PDX derived from M032R2 and M032R5 , but not from M032R1 .

O O O B-GENEPROD O O O O B-EXP_ASSAY O O O O O O O O O O B-GENEPROD O B-GENEPROD 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-GENEPROD O O O O O O O O O O O O O O O

Validation of the BRAF amplification was performed by qPCR on gDNA derived from the PDX, using primers for BRAF and CRAF and normalized on LINE expression. Bars represent the mean of three replicates, error bars indicate standard deviation. The results confirmed that BRAF was amplified in PDX derived from M032R2 and M032R5, but not from M032R1.