C Expression ( cells ) and secretion ( media ) of VgrG1 from V . natriegens containing an arabinose - inducible vp1407 in the chromosomal dns locus ( VnatReg ) and harboring an empty plasmid ( pEmpty ) or plasmids containing VpT6SS1 that lacks vp1407 ( pT6SS1Ind ) or both vp1407 and hcp1 ( pT6SS1Ind / ∆ hcp1 ; T6SS - ) . Samples were grown in media containing 3 % NaCl and either supplemented ( + ) or not ( - ) with 0 . 1 % arabinose ( Ara ) at 30 ° C . Loading control ( LC ) is shown for total protein lysates .

C Expression (cells) and secretion (media) of VgrG1 from V. natriegens containing an arabinose-inducible vp1407 in the chromosomal dns locus (VnatReg) and harboring an empty plasmid (pEmpty) or plasmids containing VpT6SS1 that lacks vp1407 (pT6SS1Ind) or both vp1407 and hcp1 (pT6SS1Ind/∆hcp1; T6SS-). Samples were grown in media containing 3% NaCl and either supplemented (+) or not (-) with 0.1% arabinose (Ara) at 30 °C. Loading control (LC) is shown for total protein lysates.


E Viability counts of V . natriegens prey before ( 0 h ) and after ( 4 h ) co - incubation with VnatReg attackers carrying the indicated plasmids on solid media plates , as described in C . Data information : data are shown as the mean ± SD , n = 3 technical replicates ; statistical significance between samples at the 4 h timepoint by an unpaired , two - tailed Student ' s t - test is denoted above and is color coded to match the relevant samples . A significant difference was considered as P < 0 . 05 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Viability counts of V. natriegens prey before (0 h) and after (4 h) co-incubation with VnatReg attackers carrying the indicated plasmids on solid media plates, as described in C. Data information: data are shown as the mean ± SD, n = 3 technical replicates; statistical significance between samples at the 4 h timepoint by an unpaired, two-tailed Student's t-test is denoted above and is color coded to match the relevant samples. A significant difference was considered as P < 0.05.


A , B Viability counts of V . natriegens prey before ( 0 h ) and after ( 4 or 24 h ) co - incubation with VnatReg attackers harboring the indicated plasmids , on media containing 3 % NaCl and 0 . 1 % arabinose ( A ) , or on media containing 0 . 1 % arabinose at 28 ° C ( B ) . Data information : Data are shown as the mean ± SD , n = 3 technical replicates . Statistical significance between samples at the 4 or 24 h timepoints by an unpaired , two - tailed Student ' s t - test is denoted above and is color coded to match the relevant samples . A significant difference was considered as P < 0 . 05 .

O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, B Viability counts of V. natriegens prey before (0 h) and after (4 or 24 h) co-incubation with VnatReg attackers harboring the indicated plasmids, on media containing 3% NaCl and 0.1% arabinose (A), or on media containing 0.1% arabinose at 28 °C (B). Data information: Data are shown as the mean ± SD, n = 3 technical replicates. Statistical significance between samples at the 4 or 24 h timepoints by an unpaired, two-tailed Student's t-test is denoted above and is color coded to match the relevant samples. A significant difference was considered as P < 0.05.


C - F Viability counts of the indicated prey bacteria before ( 0 h ) and after ( 4 h ) co - incubation with VnatReg attackers harboring the indicated plasmids , on media containing 3 % NaCl and 0 . 1 % arabinose at 28 ° C . Data information : Data are shown as the mean ± SD , n = 3 technical replicates . Statistical significance between samples at the 4 or 24 h timepoints by an unpaired , two - tailed Student ' s t - test is denoted above and is color coded to match the relevant samples . A significant difference was considered as P < 0 . 05 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-F Viability counts of the indicated prey bacteria before (0 h) and after (4 h) co-incubation with VnatReg attackers harboring the indicated plasmids, on media containing 3% NaCl and 0.1% arabinose at 28 °C. Data information: Data are shown as the mean ± SD, n = 3 technical replicates. Statistical significance between samples at the 4 or 24 h timepoints by an unpaired, two-tailed Student's t-test is denoted above and is color coded to match the relevant samples. A significant difference was considered as P < 0.05.


Viability counts of V . natriegens ( cyan ) , V . vulnificus ( purple ) , A . jandaei ∆ tssB ( brown ) , E . coli ( green ) , and S . enterica ( gray ) prey before ( 0 h ) and after ( 4 h ) co - incubation with VnatReg attackers ( yellow ) harboring the indicated plasmids , on media containing 3 % NaCl at 28 ° C . The left panels show prey survival when competing alone ( single prey ) against the attacker at a 4 : 1 ( attacker : prey ) ratio ; the right panels show the results of a competition experiment in which all prey were mixed together with the attacker at a 10 : 1 : 1 : 1 : 1 : 1 ( attacker : prey ) ratio . Data are shown as the mean ± SD , n = 3 technical replicates . Statistical significance between samples at the 4 h timepoint by an unpaired , two - tailed Student ' s t - test is denoted above and is color coded to match the relevant samples . A significant difference was considered as P < 0 . 05 .

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

Viability counts of V. natriegens (cyan), V. vulnificus (purple), A. jandaei ∆tssB (brown), E. coli (green), and S. enterica (gray) prey before (0 h) and after (4 h) co-incubation with VnatReg attackers (yellow) harboring the indicated plasmids, on media containing 3% NaCl at 28 °C. The left panels show prey survival when competing alone (single prey) against the attacker at a 4:1 (attacker:prey) ratio; the right panels show the results of a competition experiment in which all prey were mixed together with the attacker at a 10:1:1:1:1:1 (attacker:prey) ratio. Data are shown as the mean ± SD, n = 3 technical replicates. Statistical significance between samples at the 4 h timepoint by an unpaired, two-tailed Student's t-test is denoted above and is color coded to match the relevant samples. A significant difference was considered as P < 0.05.


A - D ( A , B , D ) Viability counts of V . parahaemolyticus RIMD 2210633 ∆ hcp1 ( A ) , V . alginolyticus 12G01 ∆ hcp1 / ∆ hcp2 ( B ) , and V . natriegens ( D ) prey bacteria before ( 0 h ) and after ( 4 h ) co - incubation with VnatReg attackers harboring the indicated plasmids , on media containing 3 % NaCl and 0 . 1 % arabinose at 28 ° C . Data are shown as the mean ± SD , n = 3 technical replicates . Statistical significance between samples at the 4 h timepoint by an unpaired , two - tailed Student ' s t - test is denoted above . A significant difference was considered as P < 0 . 05 . ( C ) Illustration summarizing the competitions shown in ( A , B ) . Effector and immunity modules expressed by VnatReg carrying pT6SSEffectorless ( yellow ) are colored to match the bacterium from which they were derived .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-D (A, B, D) Viability counts of V. parahaemolyticus RIMD 2210633 ∆hcp1 (A), V. alginolyticus 12G01 ∆hcp1/∆hcp2 (B), and V. natriegens (D) prey bacteria before (0 h) and after (4 h) co-incubation with VnatReg attackers harboring the indicated plasmids, on media containing 3% NaCl and 0.1% arabinose at 28 °C. Data are shown as the mean ± SD, n = 3 technical replicates. Statistical significance between samples at the 4 h timepoint by an unpaired, two-tailed Student's t-test is denoted above. A significant difference was considered as P < 0.05. ( C) Illustration summarizing the competitions shown in (A, B). Effector and immunity modules expressed by VnatReg carrying pT6SSEffectorless (yellow) are colored to match the bacterium from which they were derived.


BACE1 or APP was immunoprecipitated from mouse brains and blotted with E4 - PHA lectin ( lower ) or anti - BACE1 or APP antibodies ( upper ) . hAPP indicates the APP23 transgenic mouse model for AD .

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

BACE1 or APP was immunoprecipitated from mouse brains and blotted with E4-PHA lectin (lower) or anti-BACE1 or APP antibodies (upper). hAPP indicates the APP23 transgenic mouse model for AD.


Proteins from mouse brain membrane fractions were treated with or without PNGase F and then immunoblotted for BACE1 or for syntaxin 6 ( loading control ) .

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

Proteins from mouse brain membrane fractions were treated with or without PNGase F and then immunoblotted for BACE1 or for syntaxin 6 (loading control).


LC - MS base peak chromatogram of desialo - alditol N - glycans derived from mouse brain BACE1 . To simplify the results , N - glycans were chemically desialylated before LC - MS analysis . BACE1 - specific glycans , judged by comparison with N - glycan structures from anti - BACE1 IgG ( shown in Supplementary Fig S2C ) , are highlighted by red squares . Asterisks indicate glycans demonstrated by MS / MS analysis to contain a bisecting GlcNAc structure . Numbers in parentheses indicate the charge state .

O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_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

LC-MS base peak chromatogram of desialo-alditol N-glycans derived from mouse brain BACE1. To simplify the results, N-glycans were chemically desialylated before LC-MS analysis. BACE1-specific glycans, judged by comparison with N-glycan structures from anti-BACE1 IgG (shown in Supplementary Fig S2C), are highlighted by red squares. Asterisks indicate glycans demonstrated by MS/MS analysis to contain a bisecting GlcNAc structure. Numbers in parentheses indicate the charge state.


BACE1 from temporal lobe membrane fractions of NC , eAD , or AD patients was immunoprecipitated and blotted with E4 - PHA ( upper ) or anti - BACE1 ( lower ) . The signal intensity of E4 - PHA relative to that of BACE1 was calculated ( n = 10 ) . The graph shows means ± SEM ( * P < 0 . 05 ; one - way ANOVA with post hoc Tukey - Kramer test . P = 0 . 014 for NC versus eAD , P = 0 . 028 for NC versus AD ) .

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

BACE1 from temporal lobe membrane fractions of NC, eAD, or AD patients was immunoprecipitated and blotted with E4-PHA (upper) or anti-BACE1 (lower). The signal intensity of E4-PHA relative to that of BACE1 was calculated (n = 10). The graph shows means ± SEM (*P < 0.05; one-way ANOVA with post hoc Tukey-Kramer test. P = 0.014 for NC versus eAD, P = 0.028 for NC versus AD).


APP metabolites from 3 - month - old mouse brain membrane ( A ) or soluble ( B ) fractions were immunoblotted . The signal intensity was quantified ( n = 4 - 5 ) . All graphs show means ± SEM ( * P < 0 . 05 ; Student ' s t - test for ( A ) βCTF and ( B ) sAPPα , and Mann - Whitney U - test for the others . P = 0 . 386 for APP , P = 0 . 602 for αCTF , P = 0 . 045 for βCTF , P = 0 . 218 for sAPPα , P = 0 . 022 for sAPPβ ) .

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

APP metabolites from 3-month-old mouse brain membrane (A) or soluble (B) fractions were immunoblotted. The signal intensity was quantified (n = 4-5). All graphs show means ± SEM (*P < 0.05; Student's t-test for (A) βCTF and (B) sAPPα, and Mann-Whitney U-test for the others. P = 0.386 for APP, P = 0.602 for αCTF, P = 0.045 for βCTF, P = 0.218 for sAPPα, P = 0.022 for sAPPβ).


Amounts of Aβ40 or Aβ42 in the TBS - soluble or Gu - HCl - extractable fraction from ( A ) 3 - month - old or ( B ) 12 - month - old hAPP / Mgat3 + / + , hAPP / Mgat3 + / − , or hAPP / Mgat3 − / − brains ( n = 5 ) . P = 0 . 015 for TBS Aβ40 , P = 0 . 034 for TBS Aβ42 , P = 0 . 001 and 0 . 012 for Gn - HCl Aβ40 , P = 0 . 024 for Gn - HCl Aβ42 in ( A ) , P = 0 . 040 for Gn - HCl Aβ40 , P = 0 . 047 for Gn - HCl Aβ42 in ( B ) .

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

Amounts of Aβ40 or Aβ42 in the TBS-soluble or Gu-HCl-extractable fraction from (A) 3-month-old or (B) 12-month-old hAPP/Mgat3+/+, hAPP/Mgat3+/−, or hAPP/Mgat3−/− brains (n = 5). P = 0.015 for TBS Aβ40, P = 0.034 for TBS Aβ42, P = 0.001 and 0.012 for Gn-HCl Aβ40, P = 0.024 for Gn-HCl Aβ42 in (A), P = 0.040 for Gn-HCl Aβ40, P = 0.047 for Gn-HCl Aβ42 in (B).


Amounts of Aβ40 or Aβ42 in the TBS - soluble or Gu - HCl - extractable fraction from 3 - to 4 - month - old Mgat3 + / + or Mgat3 − / − brains ( n = 7 - 8 ) . An outlier value was rejected by the Smirnov - Grubbs ' test ( P < 0 . 05 ) . P = 0 . 045 for Gn - HCl Aβ40 .

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

Amounts of Aβ40 or Aβ42 in the TBS-soluble or Gu-HCl-extractable fraction from 3- to 4-month-old Mgat3+/+ or Mgat3−/− brains (n = 7-8). An outlier value was rejected by the Smirnov-Grubbs' test (P < 0.05). P = 0.045 for Gn-HCl Aβ40.


Immunostaining of Aβ plaques in 12 - month - old mouse brain ( left ) . Scale bar , 300 μm . The number of FSB - stained Aβ plaques in brain sections prepared from 12 - month - old male mice was quantified ( right ) ( n = 6 ) . P = 0 . 004 for hippocampus , P = 0 . 0003 for cortex .

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

Immunostaining of Aβ plaques in 12-month-old mouse brain (left). Scale bar, 300 μm. The number of FSB-stained Aβ plaques in brain sections prepared from 12-month-old male mice was quantified (right) (n = 6). P = 0.004 for hippocampus, P = 0.0003 for cortex.


The Y - maze test was performed using 12 - month - old male Mgat3 + / + , Mgat3 − / − , hAPP / Mgat3 + / + , or hAPP / Mgat3 − / − mice ( n = 8 - 10 ) . * * P = 0 . 004 for Mgat3 + / + versus hAPP / Mgat3 + / + , * P = 0 . 022 for hAPP / Mgat3 + / + versus hAPP / Mgat3 − / − .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Y-maze test was performed using 12-month-old male Mgat3+/+, Mgat3−/−, hAPP/Mgat3+/+, or hAPP/Mgat3−/− mice (n = 8-10). **P = 0.004 for Mgat3+/+ versus hAPP/Mgat3+/+, *P = 0.022 for hAPP/Mgat3+/+ versus hAPP/Mgat3−/−.


BACE1 from 2 - week - old mouse brains was immunoprecipitated and immunoblotted ( left ) . Immunoprecipitated BACE1 activity was measured in vitro ( right , n = 3 ) . The values of Bace1 − / − samples were subtracted as a background . N . S . , P = 0 . 101 .

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

BACE1 from 2-week-old mouse brains was immunoprecipitated and immunoblotted (left). Immunoprecipitated BACE1 activity was measured in vitro (right, n = 3). The values of Bace1−/− samples were subtracted as a background. N.S., P = 0.101.


MEF homogenates were fractionated by sucrose density centrifugation and immunoblotted for BACE1 , APP , rab5 , or rab7 . EE , early endosome ; LE , late endosome .

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

MEF homogenates were fractionated by sucrose density centrifugation and immunoblotted for BACE1, APP, rab5, or rab7. EE, early endosome; LE, late endosome.


Brain homogenates were fractionated by sucrose density centrifugation and immunoblotted for BACE1 , rab5 , or rab9 . EE , early endosome ; LE , late endosome . Signal intensities were quantified and are shown in the right graphs .

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

Brain homogenates were fractionated by sucrose density centrifugation and immunoblotted for BACE1, rab5, or rab9. EE, early endosome; LE, late endosome. Signal intensities were quantified and are shown in the right graphs.


Immunostaining of 12 - month - old mouse cerebral cortex for BACE1 and APP . A typical image in the vicinity of plaque - forming area is shown for hAPP / Mgat3 + / + brain . The area of co - localization was quantified using random images of cerebral cortex ( n = 10 ) . * P = 0 . 015 .

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

Immunostaining of 12-month-old mouse cerebral cortex for BACE1 and APP. A typical image in the vicinity of plaque-forming area is shown for hAPP/Mgat3+/+ brain. The area of co-localization was quantified using random images of cerebral cortex (n = 10). *P = 0.015.


Immunoblot of BACE1 or GAPDH ( loading control ) from immortalized MEFs treated with a proteasome inhibitor ( MG132 ) or a lysosome inhibitor ( chloroquine ; CQ ) .

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 B-CONTROLLED_VAR O B-CONTROLLED_VAR O O

Immunoblot of BACE1 or GAPDH (loading control) from immortalized MEFs treated with a proteasome inhibitor (MG132) or a lysosome inhibitor (chloroquine; CQ).


Immunostaining of 12 - month - old mouse cerebral cortex for BACE1 ( F ) , or nicastrin ( G ) , and Lamp1 . LE , late endosome . Scale bar , 10 μm . The area in which co - localized staining was observed was quantified as a percentage of the total BACE1 - positive area ( right , n = 10 ) . * * P = 0 . 007 .

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

Immunostaining of 12-month-old mouse cerebral cortex for BACE1 (F), or nicastrin (G), and Lamp1. LE, late endosome. Scale bar, 10 μm. The area in which co-localized staining was observed was quantified as a percentage of the total BACE1-positive area (right, n = 10). **P = 0.007.


Immunostaining of 12 - month - old brain sections ( cerebral cortex ) with anti - BACE1 . Scale bar , 100 μm .

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

Immunostaining of 12-month-old brain sections (cerebral cortex) with anti-BACE1. Scale bar, 100 μm.


Proteins from the membrane fractions of 12 - month - old mouse brains ( n = 4 - 5 ) were immunoblotted for BACE1 , and BACE1 intensity was quantified . * P = 0 . 044 .

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

Proteins from the membrane fractions of 12-month-old mouse brains (n = 4-5) were immunoblotted for BACE1, and BACE1 intensity was quantified. *P = 0.044.


Lysates from MEFs were immunoblotted for APP , BACE1 , or GAPDH ( loading control ) , and BACE1 intensity was quantified ( n = 4 ) .

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

Lysates from MEFs were immunoblotted for APP, BACE1, or GAPDH (loading control), and BACE1 intensity was quantified (n = 4).


Amount of Aβ40 in the culture medium of immortalized MEFs ( n = 3 ) . * P = 0 . 045 .

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

Amount of Aβ40 in the culture medium of immortalized MEFs (n = 3). *P = 0.045.


MEFs were transfected with control siRNA or GGA3 - siRNA and then immunoblotted for BACE1 , GGA3 , or GAPDH ( loading control ) ( n = 6 ) .

MEFs were transfected with control siRNA or GGA3-siRNA and then immunoblotted for BACE1, GGA3, or GAPDH (loading control) (n = 6).


Membrane fractions from 3 - week - old Mgat3 + / + , Mgat3 − / − , and Bace1 − / − mice were immunoblotted for CHL1 ( upper ) , contactin - 2 ( middle ) , or syntaxin 6 ( lower ) ( n = 4 - 5 ) . The graphs show means ± SEM ( * * P < 0 . 01 ; two - way ANOVA with post hoc Tukey - Kramer test , P = 0 . 006 for Mgat3 + / + versus Bace1 − / − , P = 0 . 008 for Mgat3 − / − versus Bace1 − / − in CHL1 / syn6 , P = 0 . 001 for Mgat3 + / + versus Bace1 − / − , P = 0 . 002 for Mgat3 − / − versus Bace1 − / − in Contactin - 2 / syn6 ) .

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

Membrane fractions from 3-week-old Mgat3+/+, Mgat3−/−, and Bace1−/− mice were immunoblotted for CHL1 (upper), contactin-2 (middle), or syntaxin 6 (lower) (n = 4-5). The graphs show means ± SEM (**P < 0.01; two-way ANOVA with post hoc Tukey-Kramer test, P = 0.006 for Mgat3+/+ versus Bace1−/−, P = 0.008 for Mgat3−/− versus Bace1−/− in CHL1/syn6, P = 0.001 for Mgat3+/+ versus Bace1−/−, P = 0.002 for Mgat3−/− versus Bace1−/− in Contactin-2/syn6).


Number of pups surviving at 4 weeks following a cross between heterozygous male and female mice .

Number of pups surviving at 4 weeks following a cross between heterozygous male and female mice.


( B ) YTS KIR2DL1 cells were transfected with either N . S siRNA ( blue curve ) or with SHP - 1 and Cbls siRNA ( red curve ) . After 48 hours , cells were loaded with calcium - sensitive Fluo - 3 - AM and analyzed for basal intracellular calcium levels for 1 min . The NK cells were then mixed with 721 . 221 Cw4 target cells at 37 ° C and monitoring of calcium levels was continued . The data shown are representative of three independent experiments .

O O O O O O O O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O

(B) YTS KIR2DL1 cells were transfected with either N.S siRNA (blue curve) or with SHP-1 and Cbls siRNA (red curve). After 48 hours, cells were loaded with calcium-sensitive Fluo-3-AM and analyzed for basal intracellular calcium levels for 1 min. The NK cells were then mixed with 721.221 Cw4 target cells at 37°C and monitoring of calcium levels was continued. The data shown are representative of three independent experiments.


( C ) YTS KIR2DL1 cells were incubated with either 721 . 221 Cw4 or Cw7 target cells for 2 hours and analyzed by flow cytometry to determine the expression of CD107a . Expression of CD107a was measured by mean fluorescence Intensity ( MFI ) . Relative expression of CD107a MFI was normalized to the mock - transfected sample following Cw4 incubation . Data are means ± SEM of five independent experiments . ( n = 5 ) . P values were calculated by One - sample t - tests and One way ANOVA following Tukey ' s post hoc analysis are indicated by asterisks * P < 0 . 05 , * * * * P < 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) YTS KIR2DL1 cells were incubated with either 721.221 Cw4 or Cw7 target cells for 2 hours and analyzed by flow cytometry to determine the expression of CD107a. Expression of CD107a was measured by mean fluorescence Intensity (MFI). Relative expression of CD107a MFI was normalized to the mock-transfected sample following Cw4 incubation. Data are means ± SEM of five independent experiments.(n=5). P values were calculated by One-sample t-tests and One way ANOVA following Tukey's post hoc analysis are indicated by asterisks *P < 0.05, ****P<0.0001 .


( E ) Killing assay . YTS KIR2DL1 cells were mock - transfected or transfected with N . S siRNA or SHP - 1 and Cbls siRNA . After 48 hours , cells were incubated with [ 35S ] Met - loaded 721 . 221 Cw4 or Cw7 target cells . After 5 hours of co - culture , the supernatant was collected , and the radioactive signal was measured . Data are means ± SEM of three independent experiments . P values were calculated by One - way ANOVA following Tukey ' s post hoc analysis and are indicated by asterisks * P < 0 . 05 , * * P < 0 . 01 .

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(E) Killing assay. YTS KIR2DL1 cells were mock-transfected or transfected with N.S siRNA or SHP-1 and Cbls siRNA. After 48 hours, cells were incubated with [35S] Met-loaded 721.221 Cw4 or Cw7 target cells. After 5 hours of co-culture, the supernatant was collected, and the radioactive signal was measured. Data are means ± SEM of three independent experiments. P values were calculated by One-way ANOVA following Tukey's post hoc analysis and are indicated by asterisks *P < 0.05, **P < 0.01 .


( B ) Characterization of the diameter and zeta potential ( Z - potential ) with the addition of each layer . Graph below presenting NP size distribution .

O 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-MEASURED_VAR O O O

(B) Characterization of the diameter and zeta potential (Z-potential) with the addition of each layer. Graph below presenting NP size distribution.


( A ) NK92 - NKp46low , NK92 - NKp46high , YTS KIR2DL1 , K562 and 721 . 221 HLA - Cw4 cells were stained with anti - NKp46 monoclonal antibody followed by staining with Alexa568 - Fluor goat anti - mouse IgG secondary antibody . Cells were then analyzed using flow cytometry . ( B ) NK92 - NKp46low , NK92 - NKp46 + , YTS KIR2DL1 , K562 , and 721 . 221 HLA - Cw4 cells were incubated with 50 µg rhodamine - labeled NPs and analyzed using flow cytometry to confirm the internalization of the coated NPs in a NKp46 - specific manner .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O

(A) NK92-NKp46low, NK92-NKp46high, YTS KIR2DL1, K562 and 721.221 HLA-Cw4 cells were stained with anti-NKp46 monoclonal antibody followed by staining with Alexa568-Fluor goat anti-mouse IgG secondary antibody. Cells were then analyzed using flow cytometry. (B) NK92-NKp46low, NK92-NKp46+, YTS KIR2DL1, K562, and 721.221 HLA-Cw4 cells were incubated with 50 µg rhodamine-labeled NPs and analyzed using flow cytometry to confirm the internalization of the coated NPs in a NKp46-specific manner.


( C - F ) ( C ) NK92 - NKp46low , ( D ) NK92 - NKp46high , ( E ) YTS KIR2DL1 and ( F ) K562 cells were incubated with 50 µg rhodamine - labeled NPs and analyzed using confocal microscopy . Upper panel shows wide field images . Lower panel shows single - cell images . The data shown are representative of three independent experiments .

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(C-F) (C) NK92-NKp46low, (D) NK92-NKp46high, (E) YTS KIR2DL1 and (F) K562 cells were incubated with 50 µg rhodamine-labeled NPs and analyzed using confocal microscopy. Upper panel shows wide field images. Lower panel shows single-cell images. The data shown are representative of three independent experiments.


( A ) YTS KIR2DL1 and primary NK cells were incubated with NPs loaded with Cbl - b , c - Cbl and SHP - 1 siRNA , or NPs loaded with 1500 pmol N . S siRNA . Following 48 hours of incubation , cells were lysed , and the membrane was blotted with anti - Cbl - b , anti - c - Cbl or anti - SHP - 1 antibodies . GAPDH served as loading control . Densitometric analysis of the bands was performed using ImageJ and normalized to the GAPDH densitometry values . Analysis by ImageJ densitometry is summarized in the graph below . Data are means ± SEM of four independent experiments . ( n = 4 ) . P values were calculated by One - sample t - tests and are indicated by asterisks . * P < 0 . 05 , * * P < 0 . 01 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) YTS KIR2DL1 and primary NK cells were incubated with NPs loaded with Cbl-b, c-Cbl and SHP-1 siRNA, or NPs loaded with 1500 pmol N.S siRNA. Following 48 hours of incubation, cells were lysed, and the membrane was blotted with anti-Cbl-b, anti-c-Cbl or anti-SHP-1 antibodies. GAPDH served as loading control. Densitometric analysis of the bands was performed using ImageJ and normalized to the GAPDH densitometry values. Analysis by ImageJ densitometry is summarized in the graph below. Data are means ± SEM of four independent experiments. (n=4). P values were calculated by One-sample t-tests and are indicated by asterisks. *P < 0.05, **P < 0.01.


( B ) YTS KIR2DL1 cells were incubated with SHP - 1 and Cbls siRNAs - loaded NPs ( red curve ) , with NS siRNA loaded NPs ( green curve ) or with empty NPs ( blue curve ) . After 48 hours , cells were loaded with calcium - sensitive Fluo - 3 - AM and analyzed for basal intracellular calcium levels for 1 min . The NK cells were then mixed with 721 . 221 Cw4 target cells at 37 ° C , and monitoring of calcium levels was continued . The data shown are representative of three independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O

(B) YTS KIR2DL1 cells were incubated with SHP-1 and Cbls siRNAs-loaded NPs (red curve), with NS siRNA loaded NPs (green curve) or with empty NPs (blue curve). After 48 hours, cells were loaded with calcium-sensitive Fluo-3-AM and analyzed for basal intracellular calcium levels for 1 min. The NK cells were then mixed with 721.221 Cw4 target cells at 37°C, and monitoring of calcium levels was continued. The data shown are representative of three independent experiments.


( D ) YTS KIR2DL1 cells were treated with SHP - 1 and Cbls siRNAs - loaded NPs or N . S siRNA - loaded NPs . After 48 hours , cells were incubated with either 721 . 221 Cw4 or Cw7 target cells , the supernatant was then collected , and granzyme B levels were evaluated using ELISA sandwich assay . The levels of granzyme B were quantified based on a standard curve . Data are means ± SEM of five independent experiments . ( n = 5 ) . P values were calculated by One way ANOVA following Tukey ' s post hoc analysis , and are indicated by asterisks . * * P < 0 . 01 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) YTS KIR2DL1 cells were treated with SHP-1 and Cbls siRNAs-loaded NPs or N.S siRNA-loaded NPs. After 48 hours, cells were incubated with either 721.221 Cw4 or Cw7 target cells, the supernatant was then collected, and granzyme B levels were evaluated using ELISA sandwich assay. The levels of granzyme B were quantified based on a standard curve. Data are means ± SEM of five independent experiments. (n=5). P values were calculated by One way ANOVA following Tukey's post hoc analysis, and are indicated by asterisks. **P < 0.01.


( F ) pNK cells were pretreated with either SHP - 1 and Cbls siRNA - loaded NPs or N . S siRNA - loaded NPs . After 48 hours , cells were incubated with either 721 . 221 Cw4 or Cw7 target cells for 2 hours and analyzed by flow cytometry to determine the expression of CD107a . Expression of CD107a was quantitated by mean fluorescence intensity ( MFI ) . The data represent five independent experiments ( n = 5 ) Data are shown as mean ± SEM . ) ( n = 5 ) . P values were calculated by One - sample t - tests and One way ANOVA following Tukey ' s post hoc analysis , and are indicated by asterisks . * P ≤ 0 . 05 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) pNK cells were pretreated with either SHP-1 and Cbls siRNA-loaded NPs or N.S siRNA-loaded NPs. After 48 hours, cells were incubated with either 721.221 Cw4 or Cw7 target cells for 2 hours and analyzed by flow cytometry to determine the expression of CD107a. Expression of CD107a was quantitated by mean fluorescence intensity (MFI). The data represent five independent experiments (n=5) Data are shown as mean ± SEM.) (n=5). P values were calculated by One-sample t-tests and One way ANOVA following Tukey's post hoc analysis, and are indicated by asterisks. *P≤0.05.


( A ) YTS KIR2DL1 cells were incubated with SHP - 1 and Cbls siRNA - loaded NPs ( red curve ) or empty NPs ( purple curve ) . After 48 hours , cells were loaded with Fluo - 3 - AM and analyzed for intracellular calcium levels for 20 min at 37 ° C . YTS KIR2DL1 cells that were treated with PBS served as a negative control ( green curve ) . NK cells that were mixed with 721 . 221 Cw7 target cells served as a positive control ( blue curve ) . Calcium levels were analyzed by spectrofluorometry . ( n = 3 )

O O O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_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 B-MEASURED_VAR O O O O O O O O O O O

(A) YTS KIR2DL1 cells were incubated with SHP-1 and Cbls siRNA-loaded NPs (red curve) or empty NPs (purple curve). After 48 hours, cells were loaded with Fluo-3-AM and analyzed for intracellular calcium levels for 20 min at 37°C. YTS KIR2DL1 cells that were treated with PBS served as a negative control (green curve). NK cells that were mixed with 721.221 Cw7 target cells served as a positive control (blue curve). Calcium levels were analyzed by spectrofluorometry. (n=3)


( B ) Freshly isolated PBMCs were treated with SHP - 1 and Cbls siRNA - loaded NPs , empty NPs , PBS as negative control or PHA as a positive control . Following incubation , supernatant from each sample was collected and the levels of human cytokines TNF - α , IL - 6 , IL - 10 and IFN - γ were determined using ELISA . The data represent four independent experiments ( n = 4 ) . P values were calculated by One way ANOVA following Tukey ' s post hoc analysis , and are indicated by asterisks . * P ≤ 0 . 008 . Data are shown as mean ± SEM .

(B) Freshly isolated PBMCs were treated with SHP-1 and Cbls siRNA-loaded NPs, empty NPs, PBS as negative control or PHA as a positive control. Following incubation, supernatant from each sample was collected and the levels of human cytokines TNF-α, IL-6, IL-10 and IFN-γ were determined using ELISA. The data represent four independent experiments (n=4). P values were calculated by One way ANOVA following Tukey's post hoc analysis, and are indicated by asterisks. *P≤0.008. Data are shown as mean ± SEM.


( C ) Average growth rates of tumors from the first treatment to the end point from same experimental groups of mice as indicated in B . P value was calculated by two - tailed Student ' s t - test and is indicated within the graph P = 0 . 001 . Data are shown as mean ± SEM .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Average growth rates of tumors from the first treatment to the end point from same experimental groups of mice as indicated in B. P value was calculated by two-tailed Student's t-test and is indicated within the graph P =0.001. Data are shown as mean ± SEM.


( E ) Following 24 hours after the last treatment , extracted cells from tumors of either SHP - 1 and Cbls or N . S siRNA loaded NKp46 - NPs treated mice were analyzed by flow cytometry . Expression of CD107a was measured by mean fluorescence Intensity ( MFI ) . Relative expression of CD107a MFI was normalized to the sample treated with N . S siRNA loaded NPs . The data represent three independent experiments . Data are shown as mean ± SEMn = 3 ; P value was calculated by one sample t - test and is indicated within the graph P = 0 . 009 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Following 24 hours after the last treatment, extracted cells from tumors of either SHP-1 and Cbls or N.S siRNA loaded NKp46-NPs treated mice were analyzed by flow cytometry. Expression of CD107a was measured by mean fluorescence Intensity (MFI). Relative expression of CD107a MFI was normalized to the sample treated with N.S siRNA loaded NPs. The data represent three independent experiments. Data are shown as mean ± SEMn=3; P value was calculated by one sample t-test and is indicated within the graph P =0.009.


( F ) Survival analysis in mice treated with NK cells ' gene silenced for SHP - 1 and Cbls using siRNA - loaded NPs ( n = 18 ) or N . S siRNA - loaded NPs ( n = 18 ) as demonstrated by Kaplan - Meier survival curve . P values were calculated using the log - rank test and is indicated within the graph P = 0 . 00009 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Survival analysis in mice treated with NK cells' gene silenced for SHP-1 and Cbls using siRNA-loaded NPs (n=18) or N.S siRNA-loaded NPs (n=18) as demonstrated by Kaplan-Meier survival curve. P values were calculated using the log-rank test and is indicated within the graph P =0.00009.


Wild - type and LGP2 - KO HEK293 cells in 24 - well plates were transfected with 200 ng / ml of short poly I : C IFN - β and IP - 10 mRNA expression was determined by RT - qPCR . The data represent the mean ± SD ( n = 3 , t - test , * p < 0 . 05 ) . LGP2 KO2 and KO3 are independently isolated LGP2 KO clones . Data information : All data are representative at least two independent experiments , and " n " represents the number of samples ( biological replicates ) . Error bars represent standard deviation from the mean .

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

Wild-type and LGP2-KO HEK293 cells in 24-well plates were transfected with 200 ng/ml of short poly I:C IFN-β and IP-10 mRNA expression was determined by RT-qPCR. The data represent the mean ± SD (n = 3, t-test, *p < 0.05). LGP2 KO2 and KO3 are independently isolated LGP2 KO clones. Data information: All data are representative at least two independent experiments, and "n" represents the number of samples (biological replicates). Error bars represent standard deviation from the mean.


Wild - type and LGP2 - KO HEK293 cells in 24 - well plates were infected with SeV . IFN - β and IP - 10 mRNA expression was determined by RT - qPCR . The data represent the mean ± SD ( n = 3 , t - test , * p < 0 . 05 ) . Data information : All data are representative at least two independent experiments , and " n " represents the number of samples ( biological replicates ) . Error bars represent standard deviation from the mean .

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

Wild-type and LGP2-KO HEK293 cells in 24-well plates were infected with SeV. IFN-β and IP-10 mRNA expression was determined by RT-qPCR. The data represent the mean ± SD (n = 3, t-test, *p < 0.05). Data information: All data are representative at least two independent experiments, and "n" represents the number of samples (biological replicates). Error bars represent standard deviation from the mean.


( F ) HEK293 cells in 24 - well plates were transfected with LGP2 ( 0 . 1 , 0 . 3 , or 0 . 6 μg / well ) and RIG - I ( 0 . 1 μg / well ) expression vectors as indicated . 24 h after transfection , cells were stimulated with 200 ng of short poly I : C for 4 hr . Total RNAs were extracted , and the expression of IFN - β mRNA was determined by RT - qPCR and normalized to GAPDH ( n = 3 , t - test , * p < 0 . 05 ) . Data information : All data are representative at least two independent experiments , and " n " represents the number of samples ( biological replicates ) . Error bars represent standard deviation from the mean .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HEK293 cells in 24-well plates were transfected with LGP2 (0.1, 0.3, or 0.6 μg/well) and RIG-I (0.1 μg/well) expression vectors as indicated. 24 h after transfection, cells were stimulated with 200 ng of short poly I:C for 4 hr. Total RNAs were extracted, and the expression of IFN-β mRNA was determined by RT-qPCR and normalized to GAPDH (n = 3, t-test, *p < 0.05). Data information: All data are representative at least two independent experiments, and "n" represents the number of samples (biological replicates). Error bars represent standard deviation from the mean.


( G ) HEK293FT cells were transfected with FLAG - tagged LGP2 and HA - tagged ubiquitin expression vectors . Whole cell extracts ( WCE ) were prepared 24 h after transfection . Immunoprecipitation was performed with anti - FLAG antibody ( IP : FLAG ) and the proteins were detected by western blot analysis with the indicated 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

(G) HEK293FT cells were transfected with FLAG-tagged LGP2 and HA-tagged ubiquitin expression vectors. Whole cell extracts (WCE) were prepared 24 h after transfection. Immunoprecipitation was performed with anti-FLAG antibody (IP: FLAG) and the proteins were detected by western blot analysis with the indicated antibodies.


( H ) FLAG - tagged LGP2 was transfected into HEK293 cells . Cells were then infected with SeV at MOI = 10 for 24 h . Immunoprecipitation was performed with anti - FLAG antibody , and the proteins were detected with the indicated 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

(H) FLAG-tagged LGP2 was transfected into HEK293 cells. Cells were then infected with SeV at MOI = 10 for 24 h. Immunoprecipitation was performed with anti-FLAG antibody, and the proteins were detected with the indicated antibodies.


A ) HEK293 cells were transfected with FLAG - RIG - I or FLAG - LGP2 expressing vector . Cells were then infected with SeV , and whole cell extracts were collected at indicated time points . Immunoprecipitation was performed with anti - FLAG antibody . The proteins were subjected to SDS - PAGE and detected by western blotting .

A) HEK293 cells were transfected with FLAG-RIG-I or FLAG-LGP2 expressing vector. Cells were then infected with SeV, and whole cell extracts were collected at indicated time points. Immunoprecipitation was performed with anti-FLAG antibody. The proteins were subjected to SDS-PAGE and detected by western blotting.


B ) A549 cells transfected with HA - tagged ubiquitin were stimulated with 200 ng / ml of short poly I : C for 6 h . PLA was performed with anti - HA , anti - RIG - I , and anti - LGP2 antibodies . The number of PLA signals were counted ( t - test , * p < 0 . 05 , n = 20 ) . Each dot represents the number of PLA signals of each cell . Data information : Scale bars represent 10 μm . All data are representative at least two independent experiments , and " n " represents the number of samples ( biological replicates ) . Error bars represent standard deviation from the mean .

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 I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 transfected with HA-tagged ubiquitin were stimulated with 200 ng/ml of short poly I:C for 6 h. PLA was performed with anti-HA, anti-RIG-I, and anti-LGP2 antibodies. The number of PLA signals were counted (t-test, *p < 0.05, n = 20). Each dot represents the number of PLA signals of each cell. Data information: Scale bars represent 10 μm. All data are representative at least two independent experiments, and "n" represents the number of samples (biological replicates). Error bars represent standard deviation from the mean.


C ) HEK293 cells were transfected with empty and LGP2 expression vectors for 24 hr . PLA was performed with anti - LGP2 and anti - K63 - Ub antibodies . The number of PLA signals were counted ( t - test , * p < 0 . 05 , n = 33 ) . Each dot represents the number of PLA signals of each cell . Data information : Scale bars represent 10 μm . All data are representative at least two independent experiments , and " n " represents the number of samples ( biological replicates ) . Error bars represent standard deviation from the mean .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HEK293 cells were transfected with empty and LGP2 expression vectors for 24 hr. PLA was performed with anti-LGP2 and anti-K63-Ub antibodies. The number of PLA signals were counted (t-test, *p < 0.05, n = 33). Each dot represents the number of PLA signals of each cell. Data information: Scale bars represent 10 μm. All data are representative at least two independent experiments, and "n" represents the number of samples (biological replicates). Error bars represent standard deviation from the mean.


D - F ) HEK293 cells were transfected with 200 ng / ml of short poly I : C . Cells were fixed at the indicated time points , and PLA was performed with anti - RIG - I , anti - LGP2 , and anti - K63 - Ub antibodies ( D ) . The number of PLA signals were counted ( E , F ) ( t - test , * p < 0 . 05 , n = 30 ) . Each dot represents the number of PLA signals of each cell . Data information : Scale bars represent 10 μm . All data are representative at least two independent experiments , and " n " represents the number of samples ( biological replicates ) . Error bars represent standard deviation from the mean .

O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-F) HEK293 cells were transfected with 200 ng/ml of short poly I:C. Cells were fixed at the indicated time points, and PLA was performed with anti-RIG-I, anti-LGP2, and anti-K63-Ub antibodies (D). The number of PLA signals were counted (E, F) (t-test, *p < 0.05, n = 30). Each dot represents the number of PLA signals of each cell. Data information: Scale bars represent 10 μm. All data are representative at least two independent experiments, and "n" represents the number of samples (biological replicates). Error bars represent standard deviation from the mean.


A ) HEK293FT cells were transfected with FLAG - tagged LGP2 and HA - tagged Riplet expression vectors . 24 h after transfection , cells were lysed , and WCE were prepared . Immunoprecipitation was performed with anti - FLAG antibody , and the proteins were detected by western blotting with the indicated 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

A) HEK293FT cells were transfected with FLAG-tagged LGP2 and HA-tagged Riplet expression vectors. 24 h after transfection, cells were lysed, and WCE were prepared. Immunoprecipitation was performed with anti-FLAG antibody, and the proteins were detected by western blotting with the indicated antibodies.


B ) HEK293FT cells were transfected with FLAG - tagged LGP2 expression vector . 24 h after transfection , immunoprecipitation was performed with anti - FLAG antibody and control IgG . Immunoprecipitates were subjected to SDS - PAGE , and the proteins were detected by western blotting with indicated 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

B) HEK293FT cells were transfected with FLAG-tagged LGP2 expression vector. 24 h after transfection, immunoprecipitation was performed with anti-FLAG antibody and control IgG. Immunoprecipitates were subjected to SDS-PAGE, and the proteins were detected by western blotting with indicated antibodies.


C ) HEK293FT cells were transfected with FLAG - tagged LGP2 , Riplet , and HA - tagged ubiquitin expression vectors , and immunoprecipitation was performed with anti - FLAG antibody .

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

C) HEK293FT cells were transfected with FLAG-tagged LGP2, Riplet, and HA-tagged ubiquitin expression vectors, and immunoprecipitation was performed with anti-FLAG antibody.


D ) HEK293FT cells were transfected with FLAG - tagged LGP2 , Riplet , and myc - tagged K63 - only - Ub expression vectors . 24 h after transfection , immunoprecipitation was performed with anti - FLAG antibody .

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

D) HEK293FT cells were transfected with FLAG-tagged LGP2, Riplet, and myc-tagged K63-only-Ub expression vectors. 24 h after transfection, immunoprecipitation was performed with anti-FLAG antibody.


E ) WT and Riplet KO HEK293 cells were transfected with FLAG - tagged LGP2 and myc - tagged K63 - only - Ub expression vectors , and immunoprecipitation was performed with anti - FLAG antibody .

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E) WT and Riplet KO HEK293 cells were transfected with FLAG-tagged LGP2 and myc-tagged K63-only-Ub expression vectors, and immunoprecipitation was performed with anti-FLAG antibody.


( F , G ) WT and Riplet KO HEK293 cells were transfected with 200 ng / ml of short poly I : C for 6 h , and PLA was performed with anti - LGP2 and anti - K63 - Ub antibodies . The number of PLA signals were counted ( t - test , * p < 0 . 05 , n = 100 ) . Each dot represents the number of PLA signals in each cell . Scale bars represent 10 μm . Data information : All data are representative at least two independent experiments , and " n " represents the number of samples ( biological replicates ) . Error bars represent standard deviation from the mean .

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 I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, G) WT and Riplet KO HEK293 cells were transfected with 200 ng/ml of short poly I:C for 6 h, and PLA was performed with anti-LGP2 and anti-K63-Ub antibodies. The number of PLA signals were counted (t-test, *p < 0.05, n = 100). Each dot represents the number of PLA signals in each cell. Scale bars represent 10 μm. Data information: All data are representative at least two independent experiments, and "n" represents the number of samples (biological replicates). Error bars represent standard deviation from the mean.


( A ) Liquid chromatography - tandem mass spectrometry ( LC - MS / MS ) of LGP2 in Riplet - expressing cells . LC - MS / MS data was quantified using LFQ method . The chromatographic profiles for each ubiquitination site are shown in the right panels . Schematic representation of ubiquitination sites of RIG - I and LGP2 are shown in the upper panel .

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(A) Liquid chromatography-tandem mass spectrometry (LC-MS/MS) of LGP2 in Riplet-expressing cells. LC-MS/MS data was quantified using LFQ method. The chromatographic profiles for each ubiquitination site are shown in the right panels. Schematic representation of ubiquitination sites of RIG-I and LGP2 are shown in the upper panel.


( B ) FLAG - tagged LGP2 fragments and HA - tagged Riplet expression vectors were transfected into HEK293FT cells . 24 h after transfection WCE were prepared . immunoprecipitation was performed with anti - FLAG antibody . Immunoprecipitates were subjected to SDS - PAGE , and the proteins were detected by western blotting with indicated antibodies . The data is a representative of two independent experiments .

(B) FLAG-tagged LGP2 fragments and HA-tagged Riplet expression vectors were transfected into HEK293FT cells. 24 h after transfection WCE were prepared. immunoprecipitation was performed with anti-FLAG antibody. Immunoprecipitates were subjected to SDS-PAGE, and the proteins were detected by western blotting with indicated antibodies. The data is a representative of two independent experiments.


( C ) FLAG - tagged LGP2 and FLAG - tagged LGP2 - 4KR expression vectors were transfected into HEK293FT cells . 24 h after transfection , WCE were prepared . Immunoprecipitation was performed with anti - FLAG antibody . The data is a representative of two independent experiments .

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(C) FLAG-tagged LGP2 and FLAG-tagged LGP2-4KR expression vectors were transfected into HEK293FT cells. 24 h after transfection, WCE were prepared. Immunoprecipitation was performed with anti-FLAG antibody. The data is a representative of two independent experiments.


( A ) RIG - I , WT LGP2 , and KR mutants of LGP2 were transfected into HEK293 cells together with p125luc reporter plasmids . Luciferase activities were determined 24 h after transfection . The data represent the mean ± SD ( n = 3 , t - test , * p < 0 . 05 ) . WCEs were subjected to SDS - PAGE , and the proteins were detected by western blotting ( lower panels ) . Data information : All data are representative at least two independent experiments , and " n " represents the number of samples ( biological replicates ) . Error bars represent standard deviation from the mean .

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(A) RIG-I, WT LGP2, and KR mutants of LGP2 were transfected into HEK293 cells together with p125luc reporter plasmids. Luciferase activities were determined 24 h after transfection. The data represent the mean ± SD (n = 3, t-test, *p < 0.05). WCEs were subjected to SDS-PAGE, and the proteins were detected by western blotting (lower panels). Data information: All data are representative at least two independent experiments, and "n" represents the number of samples (biological replicates). Error bars represent standard deviation from the mean.


( B ) HE293 cells stably expressing GFP ( control ) , LGP2 , or LGP2 - 4KR were transfected with p125luc reporter plasmids and then stimulated with indicated amounts of short poly I : C . WCEs were prepared 24 h after transfection and stimulation , and luciferase activity was determined . The proteins were subjected to SDS - PAGE and detected by western blotting with the indicated Abs . The data represent the mean ± SD ( n = 3 , two - way ANOVA , * p < 0 . 05 ) . Data information : All data are representative at least two independent experiments , and " n " represents the number of samples ( biological replicates ) . Error bars represent standard deviation from the mean .

O O O O O O O O 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 I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HE293 cells stably expressing GFP (control), LGP2, or LGP2-4KR were transfected with p125luc reporter plasmids and then stimulated with indicated amounts of short poly I:C. WCEs were prepared 24 h after transfection and stimulation, and luciferase activity was determined. The proteins were subjected to SDS-PAGE and detected by western blotting with the indicated Abs. The data represent the mean ± SD (n = 3, two-way ANOVA, *p < 0.05). Data information: All data are representative at least two independent experiments, and "n" represents the number of samples (biological replicates). Error bars represent standard deviation from the mean.


HEK293 cells stably expressing GFP , LGP2 , or LGP2 - 4KR were transfected with 200 ng / ml of short poly I : C ( C - E ) , Total RNA was isolated at the indicated time points . The expression of IFN - β , IP - 10 , and Ccl5 mRNA was determined by RT - qPCR and normalized to GAPDH . The data represent the mean ± SD ( n = 3 , two - way ANOVA , * p < 0 . 05 ) . Data information : All data are representative at least two independent experiments , and " n " represents the number of samples ( biological replicates ) . Error bars represent standard deviation from the mean .

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HEK293 cells stably expressing GFP, LGP2, or LGP2-4KR were transfected with 200 ng/ml of short poly I:C (C-E), Total RNA was isolated at the indicated time points. The expression of IFN-β, IP-10, and Ccl5 mRNA was determined by RT-qPCR and normalized to GAPDH. The data represent the mean ± SD (n = 3, two-way ANOVA, *p < 0.05). Data information: All data are representative at least two independent experiments, and "n" represents the number of samples (biological replicates). Error bars represent standard deviation from the mean.


HEK293 cells stably expressing LGP2 , or LGP2 - 4KR were infected with SeV at MOI = 5 ( F - H ) , Total RNA was isolated at the indicated time points . The expression of IFN - β , IP - 10 , and Ccl5 mRNA was determined by RT - qPCR and normalized to GAPDH . The data represent the mean ± SD ( n = 3 , two - way ANOVA , * p < 0 . 05 ) . Data information : All data are representative at least two independent experiments , and " n " represents the number of samples ( biological replicates ) . Error bars represent standard deviation from the mean .

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HEK293 cells stably expressing LGP2, or LGP2-4KR were infected with SeV at MOI = 5 (F-H), Total RNA was isolated at the indicated time points. The expression of IFN-β, IP-10, and Ccl5 mRNA was determined by RT-qPCR and normalized to GAPDH. The data represent the mean ± SD (n = 3, two-way ANOVA, *p < 0.05). Data information: All data are representative at least two independent experiments, and "n" represents the number of samples (biological replicates). Error bars represent standard deviation from the mean.


HEK293 cells stably expressing LGP2 , or LGP2 - 4KR were infected with influenza A virus ( Flu ) at MOI = 10 ( I , J ) . Total RNA was isolated at the indicated time points . The expression of IFN - β , IP - 10 mRNA was determined by RT - qPCR and normalized to GAPDH . The data represent the mean ± SD ( n = 3 , two - way ANOVA , * p < 0 . 05 Data information : All data are representative at least two independent experiments , and " n " represents the number of samples ( biological replicates ) . Error bars represent standard deviation from the mean .

HEK293 cells stably expressing LGP2, or LGP2-4KR were infected with influenza A virus (Flu) at MOI =10 (I, J). Total RNA was isolated at the indicated time points. The expression of IFN-β, IP-10 mRNA was determined by RT-qPCR and normalized to GAPDH. The data represent the mean ± SD (n = 3, two-way ANOVA, *p < 0.05 Data information: All data are representative at least two independent experiments, and "n" represents the number of samples (biological replicates). Error bars represent standard deviation from the mean.


( K , L ) HEK293 cell clones stably expressing LGP2 or LGP2 - 4KR at lower levels were transfected with short poly I : C , and the expression of mRNAs were determined by RT - qPCR . The data represent the mean ± SD ( n = 3 , two - way ANOVA , * p < 0 . 05 ) . Data information : All data are representative at least two independent experiments , and " n " represents the number of samples ( biological replicates ) . Error bars represent standard deviation from the mean .

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 I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, L) HEK293 cell clones stably expressing LGP2 or LGP2-4KR at lower levels were transfected with short poly I:C, and the expression of mRNAs were determined by RT-qPCR. The data represent the mean ± SD (n = 3, two-way ANOVA, *p < 0.05). Data information: All data are representative at least two independent experiments, and "n" represents the number of samples (biological replicates). Error bars represent standard deviation from the mean.


A ) Microarray analysis was performed as described in materials and methods . A heatmap of the microarray data of LGP2 and LGP2 - 4KR stably expressing cells stimulated with short poly I : C for 12 h .

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A) Microarray analysis was performed as described in materials and methods. A heatmap of the microarray data of LGP2 and LGP2-4KR stably expressing cells stimulated with short poly I:C for 12 h.


B , LGP2 and LGP2 - 4KR stably expressing HEK293 cells were stimulated with 200 ng / ml of short poly I : C , and the expression of the genes at the indicated time points were determined by RT - qPCR and normalized to GAPDH ( n = 3 , two - way ANOVA , * p < 0 . 05 ) . Data information : All data are representative at least two independent experiments , and " n " represents the number of samples ( biological replicates ) . Error bars represent standard deviation from the mean .

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 I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, LGP2 and LGP2-4KR stably expressing HEK293 cells were stimulated with 200 ng/ml of short poly I:C, and the expression of the genes at the indicated time points were determined by RT-qPCR and normalized to GAPDH (n = 3, two-way ANOVA, *p < 0.05). Data information: All data are representative at least two independent experiments, and "n" represents the number of samples (biological replicates). Error bars represent standard deviation from the mean.


C ) LGP2 and LGP2 - 4KR stably expressing HEK293 cells were stimulated with 200 ng / ml of short poly I : C , and the expression of the genes at the indicated time points were determined by RT - qPCR and normalized to GAPDH ( n = 3 , two - way ANOVA , * p < 0 . 05 ) . Data information : All data are representative at least two independent experiments , and " n " represents the number of samples ( biological replicates ) . Error bars represent standard deviation from the mean .

C) LGP2 and LGP2-4KR stably expressing HEK293 cells were stimulated with 200 ng/ml of short poly I:C, and the expression of the genes at the indicated time points were determined by RT-qPCR and normalized to GAPDH (n = 3, two-way ANOVA, *p < 0.05). Data information: All data are representative at least two independent experiments, and "n" represents the number of samples (biological replicates). Error bars represent standard deviation from the mean.


LGP2 and LGP2 - 4KR stably expressing A549 cells were infected with influenza A virus ( Flu ) at MOI = 10 , and the expression of the genes at the indicated time points were determined by RT - qPCR and normalized to GAPDH ( n = 3 , two - way ANOVA , * p < 0 . 05 ) . Data information : All data are representative at least two independent experiments , and " n " represents the number of samples ( biological replicates ) . Error bars represent standard deviation from the mean .

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

LGP2 and LGP2-4KR stably expressing A549 cells were infected with influenza A virus (Flu) at MOI = 10, and the expression of the genes at the indicated time points were determined by RT-qPCR and normalized to GAPDH (n = 3, two-way ANOVA, *p < 0.05). Data information: All data are representative at least two independent experiments, and "n" represents the number of samples (biological replicates). Error bars represent standard deviation from the mean.


H , I ) WT and indicated mutant HEK293 cells were stimulated with 200 ng / ml of short poly I : C for 8 h . WCEs were prepared and subjected to SDS - PAGE . The proteins were detected with indicated antibodies .

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H, I) WT and indicated mutant HEK293 cells were stimulated with 200 ng/ml of short poly I:C for 8 h. WCEs were prepared and subjected to SDS-PAGE. The proteins were detected with indicated antibodies.


J , K ) LGP2 and LGP2 - 4KR expressing vectors were transected into HeLa cells with Riplet expressing vector . 18 hr after transfection , cells were fixed , and then the PLA signals of LGP2 and endogenous MAVS were detected . Peroxisome was visualized using CellLight Peroxisome - GFP reagent . Colocalization of GFP ( peroxisome ) and the PLA signals ( red ) were observed by confocal microscopy . Scale bars represent 10 μm ( J ) . The colocalization area was normalized to the area of the PLA signals ( n = 25 , t - test , * p < 0 . 05 ) ( K ) . Data information : All data are representative at least two independent experiments , and " n " represents the number of samples ( biological replicates ) . Error bars represent standard deviation from the mean .

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J, K) LGP2 and LGP2-4KR expressing vectors were transected into HeLa cells with Riplet expressing vector. 18 hr after transfection, cells were fixed, and then the PLA signals of LGP2 and endogenous MAVS were detected. Peroxisome was visualized using CellLight Peroxisome-GFP reagent. Colocalization of GFP (peroxisome) and the PLA signals (red) were observed by confocal microscopy. Scale bars represent 10 μm (J). The colocalization area was normalized to the area of the PLA signals (n = 25, t-test, *p < 0.05) (K). Data information: All data are representative at least two independent experiments, and "n" represents the number of samples (biological replicates). Error bars represent standard deviation from the mean.


WT , and Riplet KO HEK293 cells were stimulated with 200 ng / ml of short poly I : C . The expression of SP100 , PML , and ANKRD1 mRNA was determined by RT - qPCR and normalized to GAPDH ( n = 3 , two - way ANOVA , * p < 0 . 05 ) . Data information : All data are representative at least two independent experiments , and " n " represents the number of samples ( biological replicates ) . Error bars represent standard deviation from the mean .

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

WT, and Riplet KO HEK293 cells were stimulated with 200 ng/ml of short poly I:C. The expression of SP100, PML, and ANKRD1 mRNA was determined by RT-qPCR and normalized to GAPDH (n = 3, two-way ANOVA, *p <0.05). Data information: All data are representative at least two independent experiments, and "n" represents the number of samples (biological replicates). Error bars represent standard deviation from the mean.


B ) WT , LGP2 KO , HEK293 cells were stimulated with 200 ng / ml of short poly I : C . The expression of SP100 , PML , and ANKRD1 mRNA was determined by RT - qPCR and normalized to GAPDH ( n = 3 , two - way ANOVA , * p < 0 . 05 ) . Data information : All data are representative at least two independent experiments , and " n " represents the number of samples ( biological replicates ) . Error bars represent standard deviation from the mean .

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B) WT, LGP2 KO, HEK293 cells were stimulated with 200 ng/ml of short poly I:C. The expression of SP100, PML, and ANKRD1 mRNA was determined by RT-qPCR and normalized to GAPDH (n = 3, two-way ANOVA, *p <0.05). Data information: All data are representative at least two independent experiments, and "n" represents the number of samples (biological replicates). Error bars represent standard deviation from the mean.


C , WT and Riplet KO A549 ( C ) cells were stimulated with 200 ng / ml of short poly I : C . The expression of SP100 was determined by RT - qPCR ( n = 3 , two - way ANOVA , * p < 005 ) . Data information : All data are representative at least two independent experiments , and " n " represents the number of samples ( biological replicates ) . Error bars represent standard deviation from the mean .

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C, WT and Riplet KO A549 (C) cells were stimulated with 200 ng/ml of short poly I:C. The expression of SP100 was determined by RT-qPCR (n = 3, two-way ANOVA, *p < 005). Data information: All data are representative at least two independent experiments, and "n" represents the number of samples (biological replicates). Error bars represent standard deviation from the mean.


D ) WT and Riplet KO MEF ( D ) cells were stimulated with 200 ng / ml of short poly I : C . The expression of SP100 was determined by RT - qPCR ( n = 3 , two - way ANOVA , * p < 005 ) . Data information : All data are representative at least two independent experiments , and " n " represents the number of samples ( biological replicates ) . Error bars represent standard deviation from the mean .

D) WT and Riplet KO MEF (D) cells were stimulated with 200 ng/ml of short poly I:C. The expression of SP100 was determined by RT-qPCR (n = 3, two-way ANOVA, *p < 005). Data information: All data are representative at least two independent experiments, and "n" represents the number of samples (biological replicates). Error bars represent standard deviation from the mean.


E ) WT and Riplet KO BMM were infected with influenza A virus ( Flu ) at MOI = 10 . PML and SP100 mRNA expression was determined by RT - qPCR ( n = 3 , two - way ANOVA , * p < 0 . 05 ) . Data information : All data are representative at least two independent experiments , and " n " represents the number of samples ( biological replicates ) . Error bars represent standard deviation from the mean .

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E) WT and Riplet KO BMM were infected with influenza A virus (Flu) at MOI = 10. PML and SP100 mRNA expression was determined by RT-qPCR (n = 3, two-way ANOVA, *p < 0.05). Data information: All data are representative at least two independent experiments, and "n" represents the number of samples (biological replicates). Error bars represent standard deviation from the mean.


F ) WT and Riplet KO A549 cells were infected with influenza A virus infection at MOI = 10 . WCE was prepared at the indicated time points and subjected to SDS - PAGE . Proteins were detected by western blotting with the indicated antibody .

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F) WT and Riplet KO A549 cells were infected with influenza A virus infection at MOI = 10. WCE was prepared at the indicated time points and subjected to SDS-PAGE. Proteins were detected by western blotting with the indicated antibody.


G , LGP2 or LGP2 - 4KR stably expressing WT or Riplet KO HEK293 cells were infected with SeV ( G ) IFN - β mRNA levels at indicated time points were determined by RT - qPCR . ( n = 3 , two - way ANOVA , * p < 0 . 05 , ns : not significant ( p > 0 . 05 ) ) . Right graph in panel G is an enlargement of Y - axis of the left graph ( dotted line ) . Data information : All data are representative at least two independent experiments , and " n " represents the number of samples ( biological replicates ) . Error bars represent standard deviation from the mean .

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G, LGP2 or LGP2-4KR stably expressing WT or Riplet KO HEK293 cells were infected with SeV (G) IFN-β mRNA levels at indicated time points were determined by RT-qPCR. (n = 3, two-way ANOVA, *p < 0.05, ns: not significant (p > 0.05)). Right graph in panel G is an enlargement of Y-axis of the left graph (dotted line). Data information: All data are representative at least two independent experiments, and "n" represents the number of samples (biological replicates). Error bars represent standard deviation from the mean.


H ) LGP2 or LGP2 - 4KR stably expressing WT or Riplet KO HEK293 cells were stimulated with 200 ng / ml of short poly I : C ( H ) . IFN - β mRNA levels at indicated time points were determined by RT - qPCR . ( n = 3 , two - way ANOVA , * p < 0 . 05 , ns : not significant ( p > 0 . 05 ) ) . Data information : All data are representative at least two independent experiments , and " n " represents the number of samples ( biological replicates ) . Error bars represent standard deviation from the mean .

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H) LGP2 or LGP2-4KR stably expressing WT or Riplet KO HEK293 cells were stimulated with 200 ng/ml of short poly I:C (H). IFN-β mRNA levels at indicated time points were determined by RT-qPCR. (n = 3, two-way ANOVA, *p < 0.05, ns: not significant (p > 0.05)). Data information: All data are representative at least two independent experiments, and "n" represents the number of samples (biological replicates). Error bars represent standard deviation from the mean.


C TIMM50S levels in primary and immortalized skin fibroblast from control ( C1 - 4 ) and mutant TIMM50 ( Pt ) . SDHA and ETHE1 are shown as loading controls .

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C TIMM50S levels in primary and immortalized skin fibroblast from control (C1-4) and mutant TIMM50 (Pt). SDHA and ETHE1 are shown as loading controls.


D qPCR quantification of TIMM50 transcript levels in control and TIMM50 mutant fibroblasts . Data are shown as mean ± SD , n = 4 .

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D qPCR quantification of TIMM50 transcript levels in control and TIMM50 mutant fibroblasts. Data are shown as mean ± SD, n = 4.


A Western - blot of whole cell extracts for different components of the protein import machinery in control and TIMM50 mutant fibroblasts grown in either glucose or galactose containing medium .

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A Western-blot of whole cell extracts for different components of the protein import machinery in control and TIMM50 mutant fibroblasts grown in either glucose or galactose containing medium.


B Western - blot of whole cell extracts for different components of the protein import machinery in control and TIMM50 mutant fibroblasts , transduced with the empty vector ( EV ) or with wild - type TIMM50 grown in either glucose or galactose containing medium .

B Western-blot of whole cell extracts for different components of the protein import machinery in control and TIMM50 mutant fibroblasts, transduced with the empty vector (EV) or with wild-type TIMM50 grown in either glucose or galactose containing medium.


C Mitochondrial membrane potential in fibroblasts grown in glucose or galactose using JC - 1 staining in untreated or FCCP treated conditions . Data are shown as mean ± SD , n = 4 biological replicates ; ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 , ∗ * ∗ p < 0 . 001 .

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C Mitochondrial membrane potential in fibroblasts grown in glucose or galactose using JC-1 staining in untreated or FCCP treated conditions. Data are shown as mean ± SD, n = 4 biological replicates; ∗p < 0.05, ∗∗p < 0.01, ∗*∗p < 0.001.


D TMRM live staining of control and TIMM50 mutant fibroblasts , transduced with the empty vector ( EV ) or with wild - type TIMM50 grown in either glucose or galactose containing medium . Scale bar corresponds to 10 μm .

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D TMRM live staining of control and TIMM50 mutant fibroblasts, transduced with the empty vector (EV) or with wild-type TIMM50 grown in either glucose or galactose containing medium. Scale bar corresponds to 10 μm.


A In organello import of the precursors of TFAM and AAC1 into isolated mitochondria from control and TIMM50 mutant fibroblasts in the absence or presence of the uncoupler FCCP . The radiolabeled precursors were incubated with isolated mitochondria for the indicated times followed by trypsin treatment only in the case of AAC1 . Coomassie Blue ( CB ) staining was used as loading control and the radiolabeled protein ( TNT ) as reference for quantification . The position of the precursor ( empty arrowheads ) and the mature protein ( filled arrowhead ) are also shown . Western blot for TIMM50 is shown as cell line control . B Quantification of TFAM and AAC1 import from control and TIMM50 mutant fibroblasts expressed as fraction of the input radiolabeled precursor converted into mature protein and normalized to Coomassie Blue signal . Data are shown as mean ± SD , n = 3 biological replicates ; ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 , ∗ * ∗ p < 0 . 001 grouping both controls together .

O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 In organello import of the precursors of TFAM and AAC1 into isolated mitochondria from control and TIMM50 mutant fibroblasts in the absence or presence of the uncoupler FCCP. The radiolabeled precursors were incubated with isolated mitochondria for the indicated times followed by trypsin treatment only in the case of AAC1. Coomassie Blue (CB) staining was used as loading control and the radiolabeled protein (TNT) as reference for quantification. The position of the precursor (empty arrowheads) and the mature protein (filled arrowhead) are also shown. Western blot for TIMM50 is shown as cell line control. B Quantification of TFAM and AAC1 import from control and TIMM50 mutant fibroblasts expressed as fraction of the input radiolabeled precursor converted into mature protein and normalized to Coomassie Blue signal. Data are shown as mean ± SD, n = 3 biological replicates; ∗p < 0.05, ∗∗p < 0.01, ∗*∗p < 0.001 grouping both controls together.


C In organello import of the precursors of TFAM and AAC1 into isolated mitochondria from control and TIMM50 mutant fibroblasts transduced with the empty vector ( EV ) or with wild - type TIMM50 in the absence or presence of the uncoupler FCCP The position of the precursor ( empty arrowheads ) and the mature protein ( filled arrowhead ) are also shown . D Quantification of TFAM and AAC1 import from control and TIMM50 mutant fibroblasts transduced with the empty vector ( EV ) or with wild - type TIMM50 expressed as fraction of the input radiolabeled precursor converted into mature protein and normalized to Coomassie Blue signal . Data are shown as mean ± SD , n = 3 biological replicates ; ∗ p < 0 . 05 .

O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 In organello import of the precursors of TFAM and AAC1 into isolated mitochondria from control and TIMM50 mutant fibroblasts transduced with the empty vector (EV) or with wild-type TIMM50 in the absence or presence of the uncoupler FCCP The position of the precursor (empty arrowheads) and the mature protein (filled arrowhead) are also shown. D Quantification of TFAM and AAC1 import from control and TIMM50 mutant fibroblasts transduced with the empty vector (EV) or with wild-type TIMM50 expressed as fraction of the input radiolabeled precursor converted into mature protein and normalized to Coomassie Blue signal. Data are shown as mean ± SD, n = 3 biological replicates; ∗p < 0.05.


Western blot for representatives of all five respiratory complexes in control and TIMM50 mutant fibroblasts grown in glucose or galactose . Two biological replicates are shown for each cell line and condition . Significant changes in protein levels in TIMM50 mutant fibroblasts compared to controls have been boxed in red .

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

Western blot for representatives of all five respiratory complexes in control and TIMM50 mutant fibroblasts grown in glucose or galactose. Two biological replicates are shown for each cell line and condition. Significant changes in protein levels in TIMM50 mutant fibroblasts compared to controls have been boxed in red.


B Western blot for representatives of all five respiratory complexes in control and TIMM50 mutant fibroblasts , transduced with the empty vector ( EV ) or with wild - type TIMM50 grown in glucose or galactose . Two biological replicates were obtained for each cell line and condition . Significant changes in protein levels in TIMM50 mutant fibroblasts complemented with TIMM50 compared to EV have been boxed in red .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Western blot for representatives of all five respiratory complexes in control and TIMM50 mutant fibroblasts, transduced with the empty vector (EV) or with wild-type TIMM50 grown in glucose or galactose. Two biological replicates were obtained for each cell line and condition. Significant changes in protein levels in TIMM50 mutant fibroblasts complemented with TIMM50 compared to EV have been boxed in red.


Oxygen consumption measurements in control and TIMM50 mutant fibroblasts grown in glucose or galactose . Values of basal and maximal respiration along with ATP - production dependent , proton leak respiration and spare capacity are shown . Data are shown as mean ± SD , n = 4 biological replicates ; ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 , ∗ * ∗ p < 0 . 001 grouping both controls together .

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

Oxygen consumption measurements in control and TIMM50 mutant fibroblasts grown in glucose or galactose. Values of basal and maximal respiration along with ATP-production dependent, proton leak respiration and spare capacity are shown. Data are shown as mean ± SD, n = 4 biological replicates; ∗p < 0.05, ∗∗p < 0.01, ∗*∗p < 0.001 grouping both controls together.


D Oxygen consumption measurements in control and TIMM50 mutant fibroblasts , transduced with the empty vector ( EV ) or with wild - type TIMM50 grown in glucose or galactose . Values of basal and maximal respiration along with ATP - production dependent , proton leak respiration and spare capacity are shown . Data are shown as mean ± SD , n = 4 biological replicates ; ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 , ∗ * ∗ p < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Oxygen consumption measurements in control and TIMM50 mutant fibroblasts, transduced with the empty vector (EV) or with wild-type TIMM50 grown in glucose or galactose. Values of basal and maximal respiration along with ATP-production dependent, proton leak respiration and spare capacity are shown. Data are shown as mean ± SD, n = 4 biological replicates; ∗p < 0.05, ∗∗p < 0.01, ∗*∗p < 0.001.


A ROS production as measured by intracellular oxidation of the cell - permeant probe DCF - DA in control and TIMM50 mutant fibroblasts grown in glucose or galactose . Measurements were made in untreated cells and after treatment with 250μM hydrogen peroxide for 30 min . Data are shown as mean ± SD , n = 3 biological replicates ; ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 , ∗ * ∗ p < 0 . 001 grouping both controls together .

O B-MEASURED_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 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

A ROS production as measured by intracellular oxidation of the cell-permeant probe DCF-DA in control and TIMM50 mutant fibroblasts grown in glucose or galactose. Measurements were made in untreated cells and after treatment with 250μM hydrogen peroxide for 30 min. Data are shown as mean ± SD, n = 3 biological replicates; ∗p < 0.05, ∗∗p < 0.01, ∗*∗p < 0.001 grouping both controls together.


B Steady state levels of ROS related proteins SOD2 and ACO2 in control and TIMM50 mutant fibroblasts growing in either glucose or galactose were assessed by Western blot using GAPDH as internal control . C Quantification of the steady state levels of SOD2 and ACO2 in control and TIMM50 mutant fibroblasts growing in either glucose or galactose . Data are shown as mean ± SD , n = 4 biological replicates ; ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 grouping both controls together .

B Steady state levels of ROS related proteins SOD2 and ACO2 in control and TIMM50 mutant fibroblasts growing in either glucose or galactose were assessed by Western blot using GAPDH as internal control. C Quantification of the steady state levels of SOD2 and ACO2 in control and TIMM50 mutant fibroblasts growing in either glucose or galactose. Data are shown as mean ± SD, n = 4 biological replicates; ∗p < 0.05, ∗∗p < 0.01 grouping both controls together.


D Steady state levels of mitophagy related proteins VDAC1 , p62 and LC3 - II in control and TIMM50 mutant fibroblasts growing in either glucose or galactose were assessed by Western blot using GAPDH as internal control . E Quantification of the steady state levels of VDAC1 , p62 and LC3 - II in control and TIMM50 mutant fibroblasts growing in either glucose or galactose . Data are shown as mean ± SD , n = 4 biological replicates ; ∗ ∗ p < 0 . 01 , ∗ * ∗ p < 0 . 001 grouping both controls together .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Steady state levels of mitophagy related proteins VDAC1, p62 and LC3-II in control and TIMM50 mutant fibroblasts growing in either glucose or galactose were assessed by Western blot using GAPDH as internal control. E Quantification of the steady state levels of VDAC1, p62 and LC3-II in control and TIMM50 mutant fibroblasts growing in either glucose or galactose. Data are shown as mean ± SD, n = 4 biological replicates; ∗∗p < 0.01, ∗*∗p < 0.001 grouping both controls together.


A Cell size in control and TIMM50 mutant fibroblasts , transduced with the empty vector ( EV ) or with wild - type TIMM50 grown in glucose or galactose . Data are shown as mean ± SD , n = 5 biological replicates ; ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 , ∗ * ∗ p < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Cell size in control and TIMM50 mutant fibroblasts, transduced with the empty vector (EV) or with wild-type TIMM50 grown in glucose or galactose. Data are shown as mean ± SD, n = 5 biological replicates; ∗p < 0.05, ∗∗p < 0.01, ∗*∗p < 0.001.


B Growth curves of control and TIMM50 mutant fibroblasts , transduced with the empty vector ( EV ) or with wild - type TIMM50 grown in glucose or galactose . Cell growth was monitored continuously by the Incucyte live cell imager ( Essen Bioscience ) . One of the three independent experiments carried out is presented . Data are shown as mean of three technical replicates ± SD .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Growth curves of control and TIMM50 mutant fibroblasts, transduced with the empty vector (EV) or with wild-type TIMM50 grown in glucose or galactose. Cell growth was monitored continuously by the Incucyte live cell imager (Essen Bioscience). One of the three independent experiments carried out is presented. Data are shown as mean of three technical replicates ± SD.


C Plot of Annexin V intensity against propidium iodine signal in control and TIMM50 mutant fibroblasts , transduced with the empty vector ( EV ) or with wild - type TIMM50 grown in glucose or galactose . Cells in the late stage of apoptosis are boxed in the top right corner .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Plot of Annexin V intensity against propidium iodine signal in control and TIMM50 mutant fibroblasts, transduced with the empty vector (EV) or with wild-type TIMM50 grown in glucose or galactose. Cells in the late stage of apoptosis are boxed in the top right corner.


D Quantification of the number of late apoptotic cells in control and TIMM50 mutant fibroblasts , transduced with the empty vector ( EV ) or with wild - type TIMM50 grown in glucose or galactose . Data are shown as mean ± SD , n = 5 or 4 biological replicates in the case of transduced cells ; ∗ * ∗ p < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Quantification of the number of late apoptotic cells in control and TIMM50 mutant fibroblasts, transduced with the empty vector (EV) or with wild-type TIMM50 grown in glucose or galactose. Data are shown as mean ± SD, n = 5 or 4 biological replicates in the case of transduced cells; ∗*∗p < 0.001.


A Western - blot of whole cell extracts for TIMM50 and GAPDH in control and TIMM50 , RNASEH1 and ISCU mutant fibroblasts grown in either glucose or galactose containing medium .

A Western-blot of whole cell extracts for TIMM50 and GAPDH in control and TIMM50, RNASEH1 and ISCU mutant fibroblasts grown in either glucose or galactose containing medium.


B Mitochondrial membrane potential in control and TIMM50 , RNASEH1 and ISCU mutant fibroblasts grown in glucose or galactose using JC - 1 staining in untreated or FCCP treated conditions . Data are shown as mean ± SD , n = 4 biological replicates ; ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 , ∗ * ∗ p < 0 . 001 .

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

B Mitochondrial membrane potential in control and TIMM50, RNASEH1 and ISCU mutant fibroblasts grown in glucose or galactose using JC-1 staining in untreated or FCCP treated conditions. Data are shown as mean ± SD, n = 4 biological replicates; ∗p < 0.05, ∗∗p < 0.01, ∗*∗p < 0.001.


C Western blot for representatives of all five respiratory complexes in control and TIMM50 , RNASEH1 and ISCU mutant fibroblasts grown in glucose or galactose . GAPDH is from the same blot as panel A . Quantification based on two biological replicates for RNASEH1 and ISCU mutant fibroblasts Significant changes in protein levels in RNASEH1 and ISCU mutant fibroblasts compared to controls have been boxed in red .

C Western blot for representatives of all five respiratory complexes in control and TIMM50, RNASEH1 and ISCU mutant fibroblasts grown in glucose or galactose. GAPDH is from the same blot as panel A. Quantification based on two biological replicates for RNASEH1 and ISCU mutant fibroblasts Significant changes in protein levels in RNASEH1 and ISCU mutant fibroblasts compared to controls have been boxed in red.


D Oxygen consumption measurements in control and TIMM50 , RNASEH1 and ISCU mutant fibroblasts grown in glucose or galactose . Values of basal and maximal respiration along with ATP - production dependent , proton leak respiration and spare capacity are shown . Data are shown as mean ± SD , n = 4 biological replicates ; ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 , ∗ * ∗ p < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Oxygen consumption measurements in control and TIMM50, RNASEH1 and ISCU mutant fibroblasts grown in glucose or galactose. Values of basal and maximal respiration along with ATP-production dependent, proton leak respiration and spare capacity are shown. Data are shown as mean ± SD, n = 4 biological replicates; ∗p < 0.05, ∗∗p < 0.01, ∗*∗p < 0.001.


E Steady state levels of ROS related proteins SOD2 and ACO2 and mitophagy related proteins VDAC1 , p62 and LC3 in control and TIMM50 , RNASEH1 and ISCU mutant fibroblasts growing in either glucose or galactose were assessed by Western blot using GAPDH as internal control . GAPDH is from the same blot as panel A .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Steady state levels of ROS related proteins SOD2 and ACO2 and mitophagy related proteins VDAC1, p62 and LC3 in control and TIMM50, RNASEH1 and ISCU mutant fibroblasts growing in either glucose or galactose were assessed by Western blot using GAPDH as internal control. GAPDH is from the same blot as panel A.


F Quantification of the number of late apoptotic cells in control and TIMM50 , RNASEH1 and ISCU mutant fibroblasts grown in glucose or galactose . Data are shown as mean ± SD , n = 4 or more biological replicates ; ∗ * ∗ p < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Quantification of the number of late apoptotic cells in control and TIMM50, RNASEH1 and ISCU mutant fibroblasts grown in glucose or galactose. Data are shown as mean ± SD, n = 4 or more biological replicates; ∗*∗p < 0.001.


D . Selected 2D class averages obtained by cryo - electron microscopy of the yeast Smc5 / 6 hexamer . Representative classes with dimers of Smc5 / 6 hexamers are displayed ( left images ) . Emerging details are indicated schematically ( right panel ) .

O O O O O O O O O O O O O O O O O O O O O O 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. Selected 2D class averages obtained by cryo-electron microscopy of the yeast Smc5/6 hexamer. Representative classes with dimers of Smc5/6 hexamers are displayed (left images). Emerging details are indicated schematically (right panel).