D , E Seahorse analysis of basal and maximal ECAR ( D ) or OCR ( E ) using wildtype or Peli1 - KO OT - I CD8 T cells that were activated for 16 h with plate - bound anti - CD3 ( 1 μg / ml ) plus anti - CD28 ( 1 μg / ml ) in the presence of solvent control ( DMSO ) , rapamycin ( Rapa , 10 nM ) , or Torin 1 ( 100 nM ) . Data are presented as a representative plot ( left ) and summary graphs ( right ) .

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

D,E Seahorse analysis of basal and maximal ECAR (D) or OCR (E) using wildtype or Peli1-KO OT-I CD8 T cells that were activated for 16 h with plate-bound anti-CD3 (1 μg/ml) plus anti-CD28 (1 μg/ml) in the presence of solvent control (DMSO), rapamycin (Rapa, 10 nM), or Torin 1 (100 nM). Data are presented as a representative plot (left) and summary graphs (right).


F , G Immunoblot analysis of the indicated phosphorylated ( p - ) and total proteins in whole - cell lysates of wildtype or Peli1 - KO OT - I CD8 T cells that were either not treated ( - ) or stimulated ( + ) for 1 h ( F ) or 24 h ( G ) with anti - CD3 ( 1 μg / ml ) and anti - CD28 ( 1 μg / ml ) in the presence ( + ) or absence ( - ) of rapamycin ( 10 nM ) and Torin 1 ( 100 nM ) .

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

F,G Immunoblot analysis of the indicated phosphorylated (p-) and total proteins in whole-cell lysates of wildtype or Peli1-KO OT-I CD8 T cells that were either not treated (-) or stimulated (+) for 1 h (F) or 24 h (G) with anti-CD3 (1 μg/ml) and anti-CD28 (1 μg/ml) in the presence (+) or absence (-) of rapamycin (10 nM) and Torin 1 (100 nM).


H qRT - PCR analysis of Ifng gene expression in naïve wildtype or Peli1 - KO OT - I CD8 T cells that were either not treated ( - ) or stimulated for 6 h ( + ) with anti - CD3 plus anti - CD28 in the presence ( + ) or absence ( - ) or rapamycin .

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

H qRT-PCR analysis of Ifng gene expression in naïve wildtype or Peli1-KO OT-I CD8 T cells that were either not treated (-) or stimulated for 6 h (+) with anti-CD3 plus anti-CD28 in the presence (+) or absence (-) or rapamycin.


I - K Schematic of experimental design ( I ) , tumor growth curve ( J ) , and summary of day 15 tumor weight ( K ) of B6 . SJL mice inoculated with B16 - OVA melanoma cells ( 2 x 105 ) and adoptively transferred on day 7 with in vitro activated and rapamycin - or DMSO - treated wildtype or Peli1 - KO OT - I CD8 T cells ( 4 x 105 ) .

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

I-K Schematic of experimental design (I), tumor growth curve (J), and summary of day 15 tumor weight (K) of B6.SJL mice inoculated with B16-OVA melanoma cells (2 x 105) and adoptively transferred on day 7 with in vitro activated and rapamycin- or DMSO-treated wildtype or Peli1-KO OT-I CD8 T cells (4 x 105).


Immunoblot analysis of the indicated phosphorylated ( p - ) and total proteins in whole - cell lysates of wildtype ( WT ) or Peli1 - KO ( KO ) OT - I CD8 T cells that were activated for 16 h with plate - coated anti - CD3 ( 1 μg / ml ) plus anti - CD28 , starved for 6 h in serum - free RPMI medium , and then treated for indicated time periods with FBS ( 10 % ) ( 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Immunoblot analysis of the indicated phosphorylated (p-) and total proteins in whole-cell lysates of wildtype (WT) or Peli1-KO (KO) OT-I CD8 T cells that were activated for 16 h with plate-coated anti-CD3 (1 μg/ml) plus anti-CD28, starved for 6 h in serum-free RPMI medium, and then treated for indicated time periods with FBS (10%) (A)


Immunoblot analysis of the indicated phosphorylated ( p - ) and total proteins in whole - cell lysates of wildtype ( WT ) or Peli1 - KO ( KO ) OT - I CD8 T cells that were activated for 16 h with plate - coated anti - CD3 ( 1 μg / ml ) plus anti - CD28 , starved for 6 h in serum - free RPMI medium , and then treated for indicated time periods with insulin ( 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 B-CONTROLLED_VAR O O O O

Immunoblot analysis of the indicated phosphorylated (p-) and total proteins in whole-cell lysates of wildtype (WT) or Peli1-KO (KO) OT-I CD8 T cells that were activated for 16 h with plate-coated anti-CD3 (1 μg/ml) plus anti-CD28, starved for 6 h in serum-free RPMI medium, and then treated for indicated time periods with insulin (B).


Immunoblot analysis of the indicated phosphorylated ( p - ) and total proteins in whole - cell lysates of wildtype ( WT ) or Peli1 - KO ( KO ) MEFs that were starved for 16 h in serum - free DMEM medium and then treated for the indicated time periods with FBS ( C )

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

Immunoblot analysis of the indicated phosphorylated (p-) and total proteins in whole-cell lysates of wildtype (WT) or Peli1-KO (KO) MEFs that were starved for 16 h in serum-free DMEM medium and then treated for the indicated time periods with FBS (C)


Immunoblot analysis of the indicated phosphorylated ( p - ) and total proteins in whole - cell lysates of wildtype ( WT ) or Peli1 - KO ( KO ) MEFs that were starved for 16 h in serum - free DMEM medium and then treated for the indicated time periods with insulin ( D )

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

Immunoblot analysis of the indicated phosphorylated (p-) and total proteins in whole-cell lysates of wildtype (WT) or Peli1-KO (KO) MEFs that were starved for 16 h in serum-free DMEM medium and then treated for the indicated time periods with insulin (D)


Immunoblot analysis of the indicated phosphorylated ( p - ) and total proteins in whole - cell lysates of wildtype ( WT ) or Peli1 - KO ( KO ) MEFs that were starved for 16 h in serum - free DMEM medium and then treated for the indicated time periods with EGF ( E ) .

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

Immunoblot analysis of the indicated phosphorylated (p-) and total proteins in whole-cell lysates of wildtype (WT) or Peli1-KO (KO) MEFs that were starved for 16 h in serum-free DMEM medium and then treated for the indicated time periods with EGF (E).


A Immunoblot analysis of the indicated proteins in whole - cell lysates of wildtype ( WT ) and Peli1 - KO ( KO ) CD8 T cells that were either not treated ( NT ) or activated for 16 h with plate - bound anti - CD3 and anti - CD28 . Data are presented as a representative blot ( left panel ) and a summary graph of quantified TSC1 ( middle panel ) and TSC2 ( right panel ) protein bands relative to the level of α - Tubulin .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Immunoblot analysis of the indicated proteins in whole-cell lysates of wildtype (WT) and Peli1-KO (KO) CD8 T cells that were either not treated (NT) or activated for 16 h with plate-bound anti-CD3 and anti-CD28. Data are presented as a representative blot (left panel) and a summary graph of quantified TSC1 (middle panel) and TSC2 (right panel) protein bands relative to the level of α-Tubulin.


Immunoblot analysis of the indicated proteins in whole - cell lysates of wildtype and Peli1 - KO CD8 T cells that were activated for 1 h with anti - CD3 and anti - CD28 and then treated with cycloheximide ( CHX ) for the indicated time periods . Data are presented as a representative blot ( 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 B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O

Immunoblot analysis of the indicated proteins in whole-cell lysates of wildtype and Peli1-KO CD8 T cells that were activated for 1 h with anti-CD3 and anti-CD28 and then treated with cycloheximide (CHX) for the indicated time periods. Data are presented as a representative blot (B)


a summary graph of quantified TSC1 and TSC2 protein bands presented as percentage of the level in untreated ( 0 min ) cells ( C ) .

O O O 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 summary graph of quantified TSC1 and TSC2 protein bands presented as percentage of the level in untreated (0 min) cells (C).


D Immunoblot analysis of TSC1 and TSC2 in wildtype or Peli1 - KO CD8 T cells stimulated with anti - CD3 plus anti - CD28 for 1 h and then incubated with ( + ) or without ( - ) cycloheximide ( CHX ) and / or MG132 for 2 h . Data are presented as a representative blot ( left panel ) and a summary graph of quantified TSC1 ( middle panel ) and TSC2 ( right panel ) protein bands relative to the level of α - Tubulin .

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

D Immunoblot analysis of TSC1 and TSC2 in wildtype or Peli1-KO CD8 T cells stimulated with anti-CD3 plus anti-CD28 for 1 h and then incubated with (+) or without (-) cycloheximide (CHX) and/or MG132 for 2 h. Data are presented as a representative blot (left panel) and a summary graph of quantified TSC1 (middle panel) and TSC2 (right panel) protein bands relative to the level of α-Tubulin.


Ubiquitination analysis of immunoprecipitated TSC2 ( E ) from activated ( anti - CD3 plus anti - CD28 for 2 h ) wildtype or Peli1 - KO CD8 T cells by immunobloting using anti - ubiquitin Abs detecting total ( Ub ) , K48 ( Ub - K48 ) , or K63 ( Ub - K63 ) polyubiquitin chains .

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

Ubiquitination analysis of immunoprecipitated TSC2 (E) from activated (anti-CD3 plus anti-CD28 for 2 h) wildtype or Peli1-KO CD8 T cells by immunobloting using anti-ubiquitin Abs detecting total (Ub), K48 (Ub-K48), or K63 (Ub-K63) polyubiquitin chains.


Ubiquitination analysis of immunoprecipitated TSC1 ( F ) from activated ( anti - CD3 plus anti - CD28 for 2 h ) wildtype or Peli1 - KO CD8 T cells by immunobloting using anti - ubiquitin Abs detecting total ( Ub ) , K48 ( Ub - K48 ) , or K63 ( Ub - K63 ) polyubiquitin chains .

Ubiquitination analysis of immunoprecipitated TSC1 (F) from activated (anti-CD3 plus anti-CD28 for 2 h) wildtype or Peli1-KO CD8 T cells by immunobloting using anti-ubiquitin Abs detecting total (Ub), K48 (Ub-K48), or K63 (Ub-K63) polyubiquitin chains.


Analysis of ubiquitin conjugation to TSC1 ( G , H ) in 293T cells transiently transfected ( for 48 h ) with the indicated expression vectors .

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

Analysis of ubiquitin conjugation to TSC1 (G, H) in 293T cells transiently transfected (for 48 h) with the indicated expression vectors.


Analysis of ubiquitin conjugation to TSC1 TSC2 ( I , J ) in 293T cells transiently transfected ( for 48 h ) with the indicated expression vectors .

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

Analysis of ubiquitin conjugation to TSC1 TSC2 (I, J) in 293T cells transiently transfected (for 48 h) with the indicated expression vectors.


K Co - immunoprecipitaton analysis of TSC1 - TSC2 interaction in 293T cells transiently transfected ( for 48 h ) with the indicated expression vectors .

O O 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 Co-immunoprecipitaton analysis of TSC1-TSC2 interaction in 293T cells transiently transfected (for 48 h) with the indicated expression vectors.


L Co - immunoprecipitation analysis of endogenous TSC1 - TSC2 interaction in wildtype and Peli1 - KO CD8 T cells that were either not treated ( NT ) or stimulated for 2 h with anti - CD3 plus anti - CD28 . The right panel is a summary graph of TSC2 / TSC1 ratio based on quantification of the blot from anti - TSC1 IP ( the right four lanes of the top two panels ) .

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

L Co-immunoprecipitation analysis of endogenous TSC1-TSC2 interaction in wildtype and Peli1-KO CD8 T cells that were either not treated (NT) or stimulated for 2 h with anti-CD3 plus anti-CD28. The right panel is a summary graph of TSC2/TSC1 ratio based on quantification of the blot from anti-TSC1 IP (the right four lanes of the top two panels).


A Analysis of ubiquitin conjugation to wildtype ( WT ) TSC1 and the indicated TSC1 mutants in 293T cells transiently transfected Flag - tagged TSC1 and its mutants along with ( + ) or without ( - ) the indicated expression vectors .

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

A Analysis of ubiquitin conjugation to wildtype (WT) TSC1 and the indicated TSC1 mutants in 293T cells transiently transfected Flag-tagged TSC1 and its mutants along with (+) or without (-) the indicated expression vectors.


B Immunoblot analysis of the indicated phosphorylated ( p - ) and total proteins in lysates of TSC1 - KO MEFs transfected with ( + ) or without ( - ) TSC1 wildtype or K30A expression vectors . The cells were serum starved and then left either untreated ( NT ) or stimulated with 10 % FBS for 30 mins . Data are presented as a representative blot ( left panel ) and a summary graph of quantified TSC2 ( right panel ) protein bands relative to the level of β - Actin .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Immunoblot analysis of the indicated phosphorylated (p-) and total proteins in lysates of TSC1-KO MEFs transfected with (+) or without (-) TSC1 wildtype or K30A expression vectors. The cells were serum starved and then left either untreated (NT) or stimulated with 10% FBS for 30 mins. Data are presented as a representative blot (left panel) and a summary graph of quantified TSC2 (right panel) protein bands relative to the level of β-Actin.


C Analysis of TSC2 ubiquitination in 293 cells transfected ( for 48 h ) with ( + ) or without ( - ) the indicated expression vectors .

O O O O 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 Analysis of TSC2 ubiquitination in 293 cells transfected (for 48 h) with (+) or without (-) the indicated expression vectors.


D Co - immunoprecipitation analysis of the interaction between endogenous TSC2 and exogenous TSC1 wildtype and K30A in TSC1 - KO MEFs transfected ( for 48 h ) with ( + ) or without ( - ) the indicated expression vectors . KO MEF cells were transfected with indicated expression plasmids . The right panel is a summary graph of TSC2 / TSC1 ratio based on quantification of the last four lanes of the anti - Flag IP blot in left 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 O O O O O O O O O O O O O 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 Co-immunoprecipitation analysis of the interaction between endogenous TSC2 and exogenous TSC1 wildtype and K30A in TSC1-KO MEFs transfected (for 48 h) with (+) or without (-) the indicated expression vectors. KO MEF cells were transfected with indicated expression plasmids. The right panel is a summary graph of TSC2/TSC1 ratio based on quantification of the last four lanes of the anti-Flag IP blot in left panel.


E Analysis of ubiquitin conjugation to endogenous TSC2 in TSC1 - KO MEFs transfected ( for 48 h ) with ( + ) or without ( - ) TSC1 WT or K30A and treated with MG132 for 2 h before lysis .

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 B-CONTROLLED_VAR O O O O O O

E Analysis of ubiquitin conjugation to endogenous TSC2 in TSC1-KO MEFs transfected (for 48 h) with (+) or without (-) TSC1 WT or K30A and treated with MG132 for 2 h before lysis.


F Immunoblot analysis of endogenous TSC2 in TSC1 - KO MEFs transfected ( for 48 h ) with Myc - TSC1 wildtype or K30A and subsequently treated with cycloheximide ( 10 μg / ml ) for the indicated time periods before lysis . The right panel is a summary graph of quantified TSC2 WT and K30A protein bands presented as percentages of the level in the untreated ( 0 min ) lane .

O O O O 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

F Immunoblot analysis of endogenous TSC2 in TSC1-KO MEFs transfected (for 48 h) with Myc-TSC1 wildtype or K30A and subsequently treated with cycloheximide (10 μg/ml) for the indicated time periods before lysis. The right panel is a summary graph of quantified TSC2 WT and K30A protein bands presented as percentages of the level in the untreated (0 min) lane.


( A ) The time courses of normalized FRET ratio ( Mean ± SEM ) of calcium biosensor before and after shockwave stimulation in HBSS Ca2 + medium with ( yellow line , n = 6 , N = 3 ) or without the Piezo1 expression ( black line , n = 6 , N = 3 ) . ( B ) The representative FRET / ECFP ratio images of calcium biosensor in Piezo1 - expressed HEK cells before ( top left ) and after ( top right ) LIS stimulation . Phase image with laser initiating point ( left ) and fluorescent image indicating Piezo1 expression ( right ) were also shown on the bottom panels ( Scale bar , 40 μm ) . Color scale bars in the figures are to show the FRET / ECFP ratios , with cold and hot colors representing low and high ratios , respectively . ( C ) The time course of normalized FRET ratio ( Mean ± SEM ) of calcium biosensor in the Piezo1 - expressing HEK cells before and after multiple shockwave stimulations in HBSS Ca2 + medium ( n = 6 , N = 3 ) .

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

(A) The time courses of normalized FRET ratio (Mean ± SEM) of calcium biosensor before and after shockwave stimulation in HBSS Ca2+ medium with (yellow line, n = 6, N = 3) or without the Piezo1 expression (black line, n = 6, N = 3). (B) The representative FRET/ECFP ratio images of calcium biosensor in Piezo1-expressed HEK cells before (top left) and after (top right) LIS stimulation. Phase image with laser initiating point (left) and fluorescent image indicating Piezo1 expression (right) were also shown on the bottom panels (Scale bar, 40 μm). Color scale bars in the figures are to show the FRET/ECFP ratios, with cold and hot colors representing low and high ratios, respectively. (C) The time course of normalized FRET ratio (Mean ± SEM) of calcium biosensor in the Piezo1-expressing HEK cells before and after multiple shockwave stimulations in HBSS Ca2+ medium (n = 6, N = 3).


( A ) The representative ECFP / FRET ratio images of FAK biosensor in Piezo1 overexpressed HEK cells before ( left ) and after ( right ) shockwave stimulation ( Scale bar , 40 μm ) . Color scale bars in the figures are to show the ECFP / FRET ratios , with cold and hot colors representing low and high ratios , respectively . ( B ) The time courses of normalized ECFP / FRET ratio ( Mean ± SEM ) of FAK FRET biosensor before and after shockwave stimulation in HBSS Ca2 + media with ( green line , n = 16 , N = 3 ) or without Piezo1 ( black line , n = 10 , N = 3 ) . ( C ) The percentage change of FRET ratio of the FAK FRET biosensor in HEK cells with or without Piezo1 after shockwave stimulation in HBSS Ca2 + medium . * P < 0 . 05 from student two - tailed t - test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) The representative ECFP/FRET ratio images of FAK biosensor in Piezo1 overexpressed HEK cells before (left) and after (right) shockwave stimulation (Scale bar, 40 μm). Color scale bars in the figures are to show the ECFP/FRET ratios, with cold and hot colors representing low and high ratios, respectively. (B) The time courses of normalized ECFP/FRET ratio (Mean ± SEM) of FAK FRET biosensor before and after shockwave stimulation in HBSS Ca2+ media with (green line, n = 16, N = 3) or without Piezo1 (black line, n = 10, N = 3). (C) The percentage change of FRET ratio of the FAK FRET biosensor in HEK cells with or without Piezo1 after shockwave stimulation in HBSS Ca2+ medium. *P < 0.05 from student two-tailed t-test.


( A ) The representative ECFP / FRET ratio images of FAK biosensor in Piezo1 expressing HEK cells before and after 25 μM Yoda1 stimulation ( Scale bar , 10 μm ) . Color scale bars in the figures indicate ECFP / FRET ratios , with cold and hot colors representing low and high ratios , respectively . ( B ) The time courses of normalized FRET ratio ( Mean ± SEM ) of FAK FRET biosensor before and after Yoda1 stimulation with ( pink line , n = 10 , N = 3 ) or without Piezo1 ( black line , n = 11 , N = 3 ) . The grey line represents the DMSO control group ( n = 8 , 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 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 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

(A) The representative ECFP/FRET ratio images of FAK biosensor in Piezo1 expressing HEK cells before and after 25 μM Yoda1 stimulation (Scale bar, 10 μm). Color scale bars in the figures indicate ECFP/FRET ratios, with cold and hot colors representing low and high ratios, respectively. (B) The time courses of normalized FRET ratio (Mean ± SEM) of FAK FRET biosensor before and after Yoda1 stimulation with (pink line, n = 10, N = 3) or without Piezo1 (black line, n = 11, N = 3). The grey line represents the DMSO control group (n = 8, N = 3).


( C ) The representative FRET / ECFP ratio images of the calcium biosensor in the Piezo1 - expressing HEK cells before and after 25 μM Yoda1 stimulation ( Scale bar , 10 μm ) . Color scale bars in the figures indicate FRET / ECFP ratios , with cold and hot colors representing low and high ratios , respectively . ( D ) The time courses of normalized FRET ratio ( Mean ± SEM ) of calcium biosensor in the Piezo1 - expressing HEK cells ( purple line , n = 9 , N = 3 ) before and after 25 μM Yoda1 stimulation in culture medium .

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 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 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-CONTROLLED_VAR O O O O O

(C) The representative FRET/ECFP ratio images of the calcium biosensor in the Piezo1-expressing HEK cells before and after 25 μM Yoda1 stimulation (Scale bar, 10 μm). Color scale bars in the figures indicate FRET/ECFP ratios, with cold and hot colors representing low and high ratios, respectively. (D) The time courses of normalized FRET ratio (Mean ± SEM) of calcium biosensor in the Piezo1-expressing HEK cells (purple line, n = 9, N = 3) before and after 25 μM Yoda1 stimulation in culture medium.


( A ) & ( B ) , The time courses ( Mean ± SEM ) ( A ) and representative images ( Scale bar , 10 μm ) of FRET ratio of SHP2 biosensor ( Y542F ) before and after 25 μM or 0 . 5 μM Yoda1 stimulation in cells with or without Piezo1 overexpression ( red bar , n = 5 , N = 3 ; blue bar , n = 5 , N = 3 ; purple bar , n = 11 , N = 3 ; green bar , n = 5 , 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 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

(A)&(B), The time courses (Mean ± SEM) (A) and representative images (Scale bar, 10 μm) of FRET ratio of SHP2 biosensor (Y542F) before and after 25 μM or 0.5 μM Yoda1 stimulation in cells with or without Piezo1 overexpression (red bar, n = 5, N=3; blue bar, n = 5, N=3; purple bar, n = 11, N=3; green bar, n = 5, N=3).


Survival rates of control and Cdc50a cKO mice after oil or 4 - OHT injection ( n = 5 for each group , P * * * = 0 . 0003 ) .

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

Survival rates of control and Cdc50a cKO mice after oil or 4-OHT injection (n = 5 for each group, P*** = 0.0003).


Bar graphs showing audiogenic seizure scores following auditory stimuli ( 5 , 10 , 15 , 20 , 40 kHz or white noise ( WN ) at 70 , 80 , 90 , or 100 dB ) in control ( E ) and Cdc50a cKO ( F ) mice ( n = 8 for each group ) .

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

Bar graphs showing audiogenic seizure scores following auditory stimuli (5, 10, 15, 20, 40 kHz or white noise (WN) at 70, 80, 90, or 100 dB) in control (E) and Cdc50a cKO (F) mice (n = 8 for each group).


Representative confocal z - stack images showing cFos ( magenta ) - positive neurons from control and Cdc50a cKO mice ( CTX : cortex , HP : hippocampus , and IC : inferior colliculus ) . Scale bar , 20 μm . Bar graphs showing the relative area of cFos - positive cells in the brains of control and Cdc50a cKO mice ( n = 5 for each group , * * P = 0 . 0030 , n . s . , not significant ) .

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

Representative confocal z-stack images showing cFos (magenta)-positive neurons from control and Cdc50a cKO mice (CTX: cortex, HP: hippocampus, and IC: inferior colliculus). Scale bar, 20 μm. Bar graphs showing the relative area of cFos-positive cells in the brains of control and Cdc50a cKO mice (n = 5 for each group, **P = 0.0030, n.s., not significant).


Schematic diagram of the experiment . Representative confocal z - stack images of PSD95 ( green ) - and VGLUT1 ( red ) - positive excitatory ( A ) and Gephyrin ( green ) - and VGAT ( magenta ) - positive inhibitory ( B ) synapses in the CTX , HP , and IC ( CTX : cortex , HP : hippocampus , and IC : inferior colliculus ) of control and Cdc50a cKO mice . The dashed line represents where the high magnified insert was taken from . Scale bar , 20 μm .

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

Schematic diagram of the experiment. Representative confocal z-stack images of PSD95 (green)- and VGLUT1 (red)-positive excitatory (A) and Gephyrin (green)- and VGAT (magenta)-positive inhibitory (B) synapses in the CTX, HP, and IC (CTX: cortex, HP: hippocampus, and IC: inferior colliculus) of control and Cdc50a cKO mice. The dashed line represents where the high magnified insert was taken from. Scale bar, 20 μm.


Representative current clamp traces from the IC of control and Cdc50a cKO mice ; + 100 pA ( upper ) and + 400 pA ( lower ) currents were injected into control mice ( left ) and Cdc50a cKO mice ( right ) for 500 ms .

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

Representative current clamp traces from the IC of control and Cdc50a cKO mice; +100 pA (upper) and +400 pA (lower) currents were injected into control mice (left) and Cdc50a cKO mice (right) for 500 ms.


The bar graphs show the relative percentages of neurons showing PS exposure only in the soma ( left ) and overall PS exposure ( right ) ( n = 30 - 40 cells for each group ) in control and Cdc50a cKO mice .

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

The bar graphs show the relative percentages of neurons showing PS exposure only in the soma (left) and overall PS exposure (right) (n = 30-40 cells for each group) in control and Cdc50a cKO mice.


Schematic diagram of imaging of the neuronal somas and proximal and distal dendrites of neurons in the CA1 region of the HP . Representative confocal z - stack images showing Gephyrin - positive inhibitory post - synapses in the somas ( red ) and proximal ( green ) and distal dendrites ( cyan ) of neurons in the CA1 region of the HP in control and Cdc50a cKO mice . Scale bar , 20 μm . Bar graphs showing the number of Gephyrin - positive inhibitory post - synapses in the somas and proximal and distal dendrites of neurons in the CA1 of the HP in control and Cdc50a cKO mice ( n = 4 for each group , P * * * = 0 . 0002 , 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 O O O O O O O O O O O O O O O O O

Schematic diagram of imaging of the neuronal somas and proximal and distal dendrites of neurons in the CA1 region of the HP. Representative confocal z-stack images showing Gephyrin-positive inhibitory post-synapses in the somas (red) and proximal (green) and distal dendrites (cyan) of neurons in the CA1 region of the HP in control and Cdc50a cKO mice. Scale bar, 20 μm. Bar graphs showing the number of Gephyrin-positive inhibitory post-synapses in the somas and proximal and distal dendrites of neurons in the CA1 of the HP in control and Cdc50a cKO mice (n = 4 for each group, P*** = 0.0002, P**** < 0.0001).


Representative confocal z - stack and colocalization images of Gephyrin ( green ) , IBA1 ( blue ) , and CD68 ( red ) in the IC of control and Cdc50a cKO mice . Scale bar , 20 μm . Bar graphs showing the relative colocalization area of Gephyrin and CD68 in the IC of control and Cdc50a cKO mice ( n = 3 for each group , * * P = 0 . 0024 ) .

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

Representative confocal z-stack and colocalization images of Gephyrin (green), IBA1 (blue), and CD68 (red) in the IC of control and Cdc50a cKO mice. Scale bar, 20 μm. Bar graphs showing the relative colocalization area of Gephyrin and CD68 in the IC of control and Cdc50a cKO mice (n = 3 for each group, **P = 0.0024).


Representative confocal z - stack images of microglia ( IBA1 , green , upper ) and inhibitory synapses ( Gephyrin : green , VGAT : red , lower ) in the IC of control ; DMSO , control ; PLX3397 , Cdc50a cKO ; DMSO and Cdc50a cKO ; PLX3397 mice . The dashed line represents where the high magnified insert was taken from . Scale bar , 20 μm . Bar graphs showing the relative area of IBA1 - positive cells in the IC of control ; DMSO , control ; PLX3397 , Cdc50a cKO ; DMSO and Cdc50a cKO ; PLX3397 mice ( n = 3 for each group , control ; DMSO versus control ; PLX3397 , * P = 0 . 0428 , control ; DMSO versus Cdc50a cKO ; DMSO , * * * P = 0 . 0002 , Cdc50a cKO ; DMSO versus Cdc50a cKO ; PLX3397 , * * * P = 0 . 0005 ) . Bar graphs showing the number of inhibitory ( Gephyrin - and VGAT - positive ) synapses in the IC of control ; DMSO , control ; PLX3397 , Cdc50a cKO ; DMSO and Cdc50a cKO ; PLX3397 mice ( n = 3 for each group , 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 B-CONTROLLED_VAR 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 B-CONTROLLED_VAR 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 B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O 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 B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O

Representative confocal z-stack images of microglia (IBA1, green, upper) and inhibitory synapses (Gephyrin: green, VGAT: red, lower) in the IC of control;DMSO, control;PLX3397, Cdc50a cKO;DMSO and Cdc50a cKO;PLX3397 mice. The dashed line represents where the high magnified insert was taken from. Scale bar, 20 μm. Bar graphs showing the relative area of IBA1-positive cells in the IC of control;DMSO, control;PLX3397, Cdc50a cKO;DMSO and Cdc50a cKO;PLX3397 mice (n = 3 for each group, control;DMSO versus control;PLX3397, *P = 0.0428, control;DMSO versus Cdc50a cKO;DMSO, ***P = 0.0002, Cdc50a cKO;DMSO versus Cdc50a cKO;PLX3397, ***P = 0.0005). Bar graphs showing the number of inhibitory (Gephyrin- and VGAT-positive) synapses in the IC of control;DMSO, control;PLX3397, Cdc50a cKO;DMSO and Cdc50a cKO;PLX3397 mice (n = 3 for each group, P**** < 0.0001).


Representative confocal z - stack images of Gephyrin ( green ) - and VGAT ( red ) - positive inhibitory synapses in the IC ( inferior colliculus ) of control ; C3 + / + , control ; C3 - / - , Cdc50a cKO ; C3 + / + and Cdc50a cKO ; C3 - / - mice . The dashed line represents where the high magnified insert was taken from . Scale bar , 10 μm .

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

Representative confocal z-stack images of Gephyrin (green)- and VGAT (red)-positive inhibitory synapses in the IC (inferior colliculus) of control;C3+/+, control;C3-/-, Cdc50a cKO;C3+/+ and Cdc50a cKO;C3-/- mice. The dashed line represents where the high magnified insert was taken from. Scale bar, 10 μm.


Representative confocal z - stack images of Gephyrin ( green ) - and VGAT ( red ) - positive inhibitory synapses in the IC of control ; Mertk + / + , control ; Mertk - / - , Cdc50a cKO ; Mertk + / + and Cdc50a cKO ; Mertk - / - mice . The dashed line represents where the high magnified insert was taken from . Scale bar , 20 μm .

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

Representative confocal z-stack images of Gephyrin (green)- and VGAT (red)-positive inhibitory synapses in the IC of control;Mertk+/+, control;Mertk-/-, Cdc50a cKO;Mertk+/+ and Cdc50a cKO;Mertk-/- mice. The dashed line represents where the high magnified insert was taken from. Scale bar, 20 μm.


Schematic diagram of the experiment in which PS exposure was measured in the brains of 1 - month - and 3 - month - old wild - type mice by injecting AAV9 - GFAP - secA5 - mCherry in the neonatal stage ( P0 ) . Representative confocal z - stack colocalization images of Gephyrin ( green ) , VGAT ( blue ) , and secA5 ( red ) in the HP ( hippocampus ) of 1 - month - and 3 - month - old wild - type mice . The white circles indicate secA5 - positive inhibitory synapses . Scale bar , 10 μm . Bar graphs showing the expression of secA5 ( B ) and IBA1 ( C ) in inhibitory post - synapses ( Gephyrin - positive , green ) and pre - synapses ( VGAT - positive , blue ) in the HP of 1 - month - and 3 - month - old wild - type mice ( n = 4 for each group , * * * P = 0 . 0009 , * P = 0 . 0248 , n . s . , not significant ) .

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

Schematic diagram of the experiment in which PS exposure was measured in the brains of 1-month- and 3-month-old wild-type mice by injecting AAV9-GFAP-secA5-mCherry in the neonatal stage (P0). Representative confocal z-stack colocalization images of Gephyrin (green), VGAT (blue), and secA5 (red) in the HP (hippocampus) of 1-month- and 3-month-old wild-type mice. The white circles indicate secA5-positive inhibitory synapses. Scale bar, 10 μm. Bar graphs showing the expression of secA5 (B) and IBA1 (C) in inhibitory post-synapses (Gephyrin-positive, green) and pre-synapses (VGAT-positive, blue) in the HP of 1-month- and 3-month-old wild-type mice (n = 4 for each group, ***P = 0.0009, *P = 0.0248, n.s., not significant).


Schematic diagram of the experiment in which Annexin V was injected intravenously ( every two days ) to block PS in the brains of 1 - month - and 3 - month - old wild - type mice . Representative confocal z - stack images of Gephyrin ( green ) - and VGAT ( red ) - positive inhibitory synapses in the HP of 1 - month - old wild - type mice . The white circles indicate inhibitory synapses . Scale bar , 10 μm . Bar graphs showing the number of inhibitory ( Gephyrin - and VGAT - positive ) synapses in the HP of 1 - month - old mice after PBS or Annexin V injection ( n = 4 for each group , * * * P = 0 . 0013 , * * P = 0 . 0090 ) .

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

Schematic diagram of the experiment in which Annexin V was injected intravenously (every two days) to block PS in the brains of 1-month- and 3-month-old wild-type mice. Representative confocal z-stack images of Gephyrin (green)- and VGAT (red)-positive inhibitory synapses in the HP of 1-month-old wild-type mice. The white circles indicate inhibitory synapses. Scale bar, 10 μm. Bar graphs showing the number of inhibitory (Gephyrin- and VGAT-positive) synapses in the HP of 1-month-old mice after PBS or Annexin V injection (n = 4 for each group, ***P = 0.0013, **P = 0.0090).


Schematic diagram of the experiment in which microglial Mertk KO ( CX3CR1 - CreERT2 ; Mertkfl / fl ) and control ( Mertkfl / fl ) mice were generated . Both 1 - month - old microglial Mertk KO mice and 1 - month - old control mice were injected with 4 - OHT for 5 consecutive days . Representative confocal z - stack images of Gephyrin ( green ) - and VGAT ( magenta ) - positive inhibitory synapses in the HP of Mertkfl / fl and CX3CR1 - CreERT2 ; Mertkfl / fl mice . The white circles indicate inhibitory synapses . Scale bar , 10 μm . Bar graphs showing the number of inhibitory ( Gephyrin - and VGAT - positive ) synapses in the HP of Mertkfl / fl and CX3CR1 - CreERT2 ; Mertkfl / fl mice ( n = 4 for each group , * * P = 0 . 0027 , * P = 0 . 0062 ) . Representative confocal z - stack colocalization images of Gephyrin ( green ) , VGAT ( blue ) and secA5 ( red ) in the HP of Mertkfl / fl and CX3CR1 - CreERT2 ; Mertkfl / fl mice . The white circles indicate secA5 - positive inhibitory synapses . Scale bar , 10 μm . Bar graphs showing the relative expression of secA5 in inhibitory synapses in the HP of Mertkfl / fl and CX3CR1 - CreERT2 ; Mertkfl / fl mice ( n = 4 for each group , * P = 0 . 0015 ) .

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

Schematic diagram of the experiment in which microglial Mertk KO (CX3CR1-CreERT2;Mertkfl/fl) and control (Mertkfl/fl) mice were generated. Both 1-month-old microglial Mertk KO mice and 1-month-old control mice were injected with 4-OHT for 5 consecutive days. Representative confocal z-stack images of Gephyrin (green)- and VGAT (magenta)-positive inhibitory synapses in the HP of Mertkfl/fl and CX3CR1-CreERT2;Mertkfl/fl mice. The white circles indicate inhibitory synapses. Scale bar, 10 μm. Bar graphs showing the number of inhibitory (Gephyrin- and VGAT-positive) synapses in the HP of Mertkfl/fl and CX3CR1-CreERT2;Mertkfl/fl mice (n = 4 for each group, **P = 0.0027, *P = 0.0062). Representative confocal z-stack colocalization images of Gephyrin (green), VGAT (blue) and secA5 (red) in the HP of Mertkfl/fl and CX3CR1-CreERT2;Mertkfl/fl mice. The white circles indicate secA5-positive inhibitory synapses. Scale bar, 10 μm. Bar graphs showing the relative expression of secA5 in inhibitory synapses in the HP of Mertkfl/fl and CX3CR1-CreERT2;Mertkfl/fl mice (n = 4 for each group, *P = 0.0015).


B Sequencing chromatograms depicting the identified 22 nucleotide deletion ( c . 346 _ * 7del ) identified in Subject 1 ( S1 ) , the heterozygous state of the deletion in the father , and the wild - type NDUFC2 sequence in Control DNA .

O O O O O O O O O O O O O O O O O O O O O O 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 Sequencing chromatograms depicting the identified 22 nucleotide deletion (c.346_*7del) identified in Subject 1 (S1), the heterozygous state of the deletion in the father, and the wild-type NDUFC2 sequence in Control DNA.


C Sequencing chromatograms depicting the identified missense variant ( c . 173A > T ; p . ( His58Leu ) ) in Subject 3 ( S3 ) and the wild - type NDUFC2 sequence in Control DNA .

O O O O O O O O O O O O O O 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 Sequencing chromatograms depicting the identified missense variant (c.173A>T; p.(His58Leu)) in Subject 3 (S3) and the wild-type NDUFC2 sequence in Control DNA.


A NDUFC2 mRNA transcript levels in fibroblast cell lines of controls ( white ) , Subject 1 ( grey ) and Subject 3 ( black ) . Quantification of NDUFC2 mRNA transcript levels ( normalised to ACTB ) showed diminished transcript levels in Subject 1 while transcript levels were unaffected in Subject 3 . Bars correspond to standard deviations of two independent experiments , performed in duplicate and analysed by Student ' s t - test ; * 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

A NDUFC2 mRNA transcript levels in fibroblast cell lines of controls (white), Subject 1 (grey) and Subject 3 (black). Quantification of NDUFC2 mRNA transcript levels (normalised to ACTB) showed diminished transcript levels in Subject 1 while transcript levels were unaffected in Subject 3. Bars correspond to standard deviations of two independent experiments, performed in duplicate and analysed by Student's t-test; *p<0.05.


B Assessment of respiratory chain enzyme activities , normalised to citrate synthase ( CS ) , in fibroblast cell lines from controls ( white ) , Subject 1 ( grey ) and Subject 3 ( black ) . Mean residual enzyme activities of control fibroblasts ( n = 8 ) are set at 100 % and error bars represent 1 standard deviation of control activities . Asterisks ( * ) denote enzyme activity below control range .

B Assessment of respiratory chain enzyme activities, normalised to citrate synthase (CS), in fibroblast cell lines from controls (white), Subject 1 (grey) and Subject 3 (black). Mean residual enzyme activities of control fibroblasts (n = 8) are set at 100% and error bars represent 1 standard deviation of control activities. Asterisks (*) denote enzyme activity below control range.


C Assessment of respiratory chain enzyme activities , normalised to citrate synthase ( CS ) , in skeletal muscle from controls ( white ) and Subject 3 ( black ) . Mean residual enzyme activities of control skeletal muscle ( n > 100 samples ) are set at 100 % and bars are 1 standard deviation of control activities . Asterisks ( * ) denote enzyme activity below control range .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Assessment of respiratory chain enzyme activities, normalised to citrate synthase (CS), in skeletal muscle from controls (white) and Subject 3 (black). Mean residual enzyme activities of control skeletal muscle (n>100 samples) are set at 100% and bars are 1 standard deviation of control activities. Asterisks (*) denote enzyme activity below control range.


D Respiration of Subject 3 and Subject 1 derived fibroblasts ( black and grey , respectively ) compared to control fibroblasts ( white ) using high - resolution respirometry . ROX - corrected analysis of basal respiration , Oligomycin inhibited LEAK respiration and maximum uncoupled respiration ( ETS ) . Both subject fibroblasts exhibited a severe mitochondrial respiration defect as measured by oxygen consumption . Data are mean ± SD from at least 3 experiments and analysed by Student ' s t - test . * * 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 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

D Respiration of Subject 3 and Subject 1 derived fibroblasts (black and grey, respectively) compared to control fibroblasts (white) using high-resolution respirometry. ROX-corrected analysis of basal respiration, Oligomycin inhibited LEAK respiration and maximum uncoupled respiration (ETS). Both subject fibroblasts exhibited a severe mitochondrial respiration defect as measured by oxygen consumption. Data are mean ± SD from at least 3 experiments and analysed by Student's t-test. **p<0.01, ***p<0.001.


A Western blot analysis of various structural subunits of complex I in fibroblasts from Subjects 1 and 3 ( S1 and S3 respectively ) with three age - matched controls ( C1 , C2 , and C3 ) .

O O O O O O O O O O O O O O O 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 Western blot analysis of various structural subunits of complex I in fibroblasts from Subjects 1 and 3 (S1 and S3 respectively) with three age-matched controls (C1, C2, and C3).


B BN - PAGE analysis of OXPHOS complex assembly in enriched mitochondria from subject and control fibroblasts solubilised using DDM . Immunoblotting was performed using antibodies to structural subunits from each OXPHOS complex ( CI [ NDUFB8 ] , CII [ SDHA ] , CIII [ UQCRC2 ] , CIV [ MT - CO1 ] , and CV [ ATP5A ] ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 BN-PAGE analysis of OXPHOS complex assembly in enriched mitochondria from subject and control fibroblasts solubilised using DDM. Immunoblotting was performed using antibodies to structural subunits from each OXPHOS complex (CI [NDUFB8], CII [SDHA], CIII [UQCRC2], CIV [MT-CO1], and CV [ATP5A]).


C Western blot analysis of whole cell lysates from controls ( C1 and C2 ) and subject cell lines ( S1 and S3 ) that were either untransduced ( - ) or transduced with the lentiviral vector ( pLVX ) containing wild - type NDUFC2 ( + ) . Transduced cell lines were either uninduced or induced with 100 or 200 ng / ml doxycycline ( for 48 and 96 hours respectively ) where indicated .

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C Western blot analysis of whole cell lysates from controls (C1 and C2) and subject cell lines (S1 and S3) that were either untransduced (-) or transduced with the lentiviral vector (pLVX) containing wild-type NDUFC2 (+). Transduced cell lines were either uninduced or induced with 100 or 200 ng/ml doxycycline (for 48 and 96 hours respectively) where indicated.


D BN - PAGE analysis of OXPHOS complex assembly in enriched mitochondria isolated from controls ( C1 and C2 ) and subject cell lines that were either untransduced ( - ) or transduced with the lentiviral vector ( pLVX ) containing wild - type NDUFC2 ( + ) . Transduced cell lines were either uninduced or induced with 200 ng / ml doxycycline for 72 hours .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 BN-PAGE analysis of OXPHOS complex assembly in enriched mitochondria isolated from controls (C1 and C2) and subject cell lines that were either untransduced (-) or transduced with the lentiviral vector (pLVX) containing wild-type NDUFC2 (+). Transduced cell lines were either uninduced or induced with 200 ng/ml doxycycline for 72 hours.


A Complexome profiling identified assembly intermediates in subject cells with NDUFC2 variants . Complexes from mitochondrial membranes of Control , Subject 1 and Subject 3 fibroblasts were separated by BN - PAGE ( Fig EV2 ) and analysed by complexome profiling . Sample preparation , mass spectrometry , data processing and raw data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository ( Vizcaíno et al . , 2013 ) with the dataset identifier < PXD014936 > . Assignment of complexes : III , complex III ; IV , complex IV ; III / IV , supercomplex containing dimer of complex III and 1 - 2 copies of complex IV ; S , supercomplex containing complex I , dimer of complex III and 1 - 4 copies of complex IV . Assembly intermediates are highlighted in dashed boxes indicating stalled complex I assembly . In subject complexomes , various stalled complex I intermediates are assembled : ND4 module ( blue dashed box ) ; Q - TIMMDC1 - NDUFA13 complex I intermediate ( orange dashed box ) ; and Q - TIMMDC1 - NDUFA13 - MCIA ( green dashed box ) . In control complexome , the Q module ( orange dashed box ) is assembled without TIMMDC1 or NDUFA13 . Complex I modules are denoted according to Stroud and colleagues ( Stroud et al . , 2016 ) .

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A Complexome profiling identified assembly intermediates in subject cells with NDUFC2 variants. Complexes from mitochondrial membranes of Control, Subject 1 and Subject 3 fibroblasts were separated by BN-PAGE (Fig EV2) and analysed by complexome profiling. Sample preparation, mass spectrometry, data processing and raw data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository (Vizcaíno et al., 2013) with the dataset identifier <PXD014936>. Assignment of complexes: III, complex III; IV, complex IV; III/IV, supercomplex containing dimer of complex III and 1-2 copies of complex IV; S, supercomplex containing complex I, dimer of complex III and 1-4 copies of complex IV. Assembly intermediates are highlighted in dashed boxes indicating stalled complex I assembly. In subject complexomes, various stalled complex I intermediates are assembled: ND4 module (blue dashed box); Q-TIMMDC1-NDUFA13 complex I intermediate (orange dashed box); and Q-TIMMDC1-NDUFA13-MCIA (green dashed box). In control complexome, the Q module (orange dashed box) is assembled without TIMMDC1 or NDUFA13. Complex I modules are denoted according to Stroud and colleagues (Stroud et al., 2016).


B Abundance data for all the subunits of complex I were averaged to plot an abundance profile of complex I . Heatmap ( upper panel ) and profile plot ( lower panel ) compare complex I between control ( black ) , Subject 1 ( red ) and Subject 3 ( blue ) . C Profile plot of complex III ( average of all subunits ) . Black arrows indicate shifted complex III to the position of the individual complex and small supercomplex ( III / IV ) in Subjects 1 and 3 . D Profile plot of complex IV ( average of all subunits ) indicate less complex IV at the position of the supercomplexes for Subject 1 and Subject 3 , but higher amounts of individual complex IV and small supercomplex ( III / IV , black arrow ) .

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B Abundance data for all the subunits of complex I were averaged to plot an abundance profile of complex I. Heatmap (upper panel) and profile plot (lower panel) compare complex I between control (black), Subject 1 (red) and Subject 3 (blue). C Profile plot of complex III (average of all subunits). Black arrows indicate shifted complex III to the position of the individual complex and small supercomplex (III/IV) in Subjects 1 and 3. D Profile plot of complex IV (average of all subunits) indicate less complex IV at the position of the supercomplexes for Subject 1 and Subject 3, but higher amounts of individual complex IV and small supercomplex (III/IV, black arrow).


E Details of Q module - containing assembly intermediates of Subject 3 . Intensity based absolute quantification ( iBAQ ) values of assembly factors , single subunit and averaged Q - module subunits were plotted to show abundance profiles . The cartoon ( lower panel ) indicate assembled intermediate complexes relative to the plot .

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E Details of Q module-containing assembly intermediates of Subject 3. Intensity based absolute quantification (iBAQ) values of assembly factors, single subunit and averaged Q-module subunits were plotted to show abundance profiles. The cartoon (lower panel) indicate assembled intermediate complexes relative to the plot.


F Details about ND4 module assembly intermediate of Subject 3 . Abundance profile of averaged iBAQ values of ND4 subunits and assembly factors show accumulation of a complex containing the ND4 module , FOXRED1 and TMEM70 .

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F Details about ND4 module assembly intermediate of Subject 3. Abundance profile of averaged iBAQ values of ND4 subunits and assembly factors show accumulation of a complex containing the ND4 module, FOXRED1 and TMEM70.


The intestine - specific RNAi against irx - 1 reduces the Smurf phenotype at day 8 of adulthood . Mean ± SD . Scale bar : 100 μm . Unpaired t - test , two - tailed . More than 30 worms analysed in each of the three biological replicates .

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The intestine-specific RNAi against irx-1 reduces the Smurf phenotype at day 8 of adulthood. Mean ± SD. Scale bar: 100 μm. Unpaired t-test, two-tailed. More than 30 worms analysed in each of the three biological replicates.


, C The intestine - specific RNAi against irx - 1 reduces the Smurf phenotype at day 8 of adulthood . Mean ± SD . Scale bar : 100 μm . Unpaired t - test , two - tailed . More than 30 worms analysed in each of the three biological replicates .

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, C The intestine-specific RNAi against irx-1 reduces the Smurf phenotype at day 8 of adulthood. Mean ± SD. Scale bar: 100 μm. Unpaired t-test, two-tailed. More than 30 worms analysed in each of the three biological replicates.


B , C The BWM - specific RNAi against myrf - 2 improves sarcomere in the worms at day 8 of adulthood . Arrowheads and arrows respectively denote the gaps of myofilaments and the disorganised myofilaments with GFP aggregations . Scale Bar : 10 μm . Mean ± SD . Unpaired t - test , two - tailed . n > = 50 .

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B, C The BWM-specific RNAi against myrf-2 improves sarcomere in the worms at day 8 of adulthood. Arrowheads and arrows respectively denote the gaps of myofilaments and the disorganised myofilaments with GFP aggregations. Scale Bar: 10 μm. Mean ± SD. Unpaired t-test, two-tailed. n>=50.


D , E Suppressing myrf - 2 in BWM increases worms ' motility at day 5 of adulthood . The tracks were from worms ' movement in 30 sec ( D ) . The speed of worm movement was measured by the ratio of track length versus body length ( E ) . Mean ± SD . n > = 120 . Unpaired t - test , two - tailed .

D, E Suppressing myrf-2 in BWM increases worms' motility at day 5 of adulthood. The tracks were from worms' movement in 30 sec (D). The speed of worm movement was measured by the ratio of track length versus body length (E). Mean ± SD. n>=120. Unpaired t-test, two-tailed.


B . Relative quantification of all four histone proteins in each tissue replicate ( order of appearance per replicate : H2A , H2B , H3 . 3 and H4 ) .

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B. Relative quantification of all four histone proteins in each tissue replicate (order of appearance per replicate: H2A, H2B, H3.3 and H4).


C . Immunohistochemistry images from the Human Protein Atlas ( www . proteinatlas . org ) for protein ANXA1 with nuclear staining ( blue ) for three selected tissues .

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C. Immunohistochemistry images from the Human Protein Atlas (www.proteinatlas.org) for protein ANXA1 with nuclear staining (blue) for three selected tissues.


D . Calibration curves for two of the four histone peptides , with decreasing amount of QPrEST standard spiked into a U2OS cell lysate .

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D. Calibration curves for two of the four histone peptides, with decreasing amount of QPrEST standard spiked into a U2OS cell lysate.


A . Absolute copy number of protein in kidney tissue and human embryonal kidney cells ( HEK293 ) , liver tissue and liver cancer cell line ( HepG2 ) , lung tissue and lung cancer cell line ( A549 ) , and breast tissue and breast cancer cell line ( MCF7 ) . The order of proteins are the same in the tissue and corresponding cell line and the proteins have been order according to the abundance in the respective tissue .

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A. Absolute copy number of protein in kidney tissue and human embryonal kidney cells (HEK293), liver tissue and liver cancer cell line (HepG2), lung tissue and lung cancer cell line (A549), and breast tissue and breast cancer cell line (MCF7). The order of proteins are the same in the tissue and corresponding cell line and the proteins have been order according to the abundance in the respective tissue.


B . The direct correlation between RNA ( TPM ) and protein abundances ( copy number ) for all quantified genes in the same tissues and cell lines . Spearman ( ρ ) and Pearson ( r ) correlation between the two values across the quantified genes are shown . The other seven tissues and five cell lines are shown in Figure EV4 .

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B. The direct correlation between RNA (TPM) and protein abundances (copy number) for all quantified genes in the same tissues and cell lines. Spearman (ρ) and Pearson (r) correlation between the two values across the quantified genes are shown. The other seven tissues and five cell lines are shown in Figure EV4.


Subcellular localization by immunofluorescence staining and immunohistochemistry staining in tissue sections by three different antibodies ( SELENBP1 , HPA011731 ; STOM , HPA010961 ; ASS1 , HPA020896 ) . Microtubule - and nuclear probes are visualized in red and blue respectively . Antibody staining is shown in green . RNA to protein ratio across nine cell lines and 11 tissues with Spearman rho , Pearson r and R2 for each gene . All other genes can be found in Figure EV1 .

Subcellular localization by immunofluorescence staining and immunohistochemistry staining in tissue sections by three different antibodies (SELENBP1, HPA011731; STOM, HPA010961; ASS1, HPA020896). Microtubule- and nuclear probes are visualized in red and blue respectively. Antibody staining is shown in green. RNA to protein ratio across nine cell lines and 11 tissues with Spearman rho, Pearson r and R2 for each gene. All other genes can be found in Figure EV1.


Four to six - week - old C57BL / 6 mice were injected i . p . with 104 - 107 PFU of WNV - poly ( A ) , 104 PFU of WT WNV or PBS ( negative control ) in a volume of 200 μL . Survival ( n = 5 ) in infected mice were monitored daily Data information : Log - rank test was

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Four to six-week-old C57BL/6 mice were injected i.p. with 104-107 PFU of WNV-poly(A), 104 PFU of WT WNV or PBS (negative control) in a volume of 200 μL. Survival (n=5) in infected mice were monitored daily Data information: Log-rank test was used


Four to six - week - old C57BL / 6 mice were injected i . p . with 104 - 107 PFU of WNV - poly ( A ) , 104 PFU of WT WNV or PBS ( negative control ) in a volume of 200 μL . Histological analysis of mouse brain . Scale bars represent 100 μm and 50 μm for left and right panels , respectively . Severe vacuolization ( black arrows ) and inflammation ( yellow arrow ) in brain small vessels as well as degenerative changes in neurons with cytoplasmic rarefaction ( blue arrow ) and necrosis with nuclear compaction ( red arrow ) were indicated . Scale bars represent 100 μm and 50 μm for left and right panels ,

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Four to six-week-old C57BL/6 mice were injected i.p. with 104-107 PFU of WNV-poly(A), 104 PFU of WT WNV or PBS (negative control) in a volume of 200 μL. Histological analysis of mouse brain. Scale bars represent 100 μm and 50 μm for left and right panels, respectively. Severe vacuolization (black arrows) and inflammation (yellow arrow) in brain small vessels as well as degenerative changes in neurons with cytoplasmic rarefaction (blue arrow) and necrosis with nuclear compaction (red arrow) were indicated. Scale bars represent 100 μm and 50 μm for left and right panels, respectively


E Viral load and replication kinetics in different tissues of C57BL / 6 mice . Six - week - old female C57BL / 6 mice were inoculated subcutaneously with 106 PFU of WT WNV or WNV - poly ( A ) in the left rear footpad , and tissues were harvested from two mice at the indicated times post - inoculation . Viral loads in brain , spleen , left lymph node , right lymph node and liver were measured by plaque assay and expressed as PFU per gram of tissue . n = 3 , values are mean ± sd , the dotted line corresponds to the limit of detection ( LOD ) .

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E Viral load and replication kinetics in different tissues of C57BL/6 mice. Six-week-old female C57BL/6 mice were inoculated subcutaneously with 106 PFU of WT WNV or WNV-poly(A) in the left rear footpad, and tissues were harvested from two mice at the indicated times post-inoculation. Viral loads in brain, spleen, left lymph node, right lymph node and liver were measured by plaque assay and expressed as PFU per gram of tissue. n=3, values are mean ± sd, the dotted line corresponds to the limit of detection (LOD).


B - C Total anti - WNV IgG antibody ( B ) and neutralizing antibody titers ( C ) in mouse serum at the indicated days post immunization . ( For B and C , the values represent mean ± sd in each group and the horizontal dotted line represents the limit of detection ) Data information : For statistical analysis , one - way ANOVA analysis were used * p < 0 . 5 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , ns represents not

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B-C Total anti-WNV IgG antibody (B) and neutralizing antibody titers (C) in mouse serum at the indicated days post immunization. (For B and C, the values represent mean ± sd in each group and the horizontal dotted line represents the limit of detection) Data information: For statistical analysis, one-way ANOVA analysis were used *p<0.5, **p<0.01, *** p<0.001,****p< 0.0001, ns represents not significant


D - E Survival ( D ) and body - weight changes were monitored daily after challenge . ( The values represent mean ± sd in each group ) ( E ) . Data information : For statistical analysis , two - way ANOVA analysis were used Log - rank test was performed * p < 0 . 5 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , ns represents not

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D-E Survival (D) and body-weight changes were monitored daily after challenge. (The values represent mean ± sd in each group) (E). Data information: For statistical analysis, two-way ANOVA analysis were used Log-rank test was performed *p<0.5, **p<0.01, *** p<0.001,****p< 0.0001, ns represents not significant


G - H T - cell responses in vaccinated mice were measured through ELISpot assay . Two groups of C57BL / 6 mice ( n = 4 / group ) were i . p . inoculated with 107 of WNV - poly ( A ) or equal volume of PBS . ELISpot assay specific for IFN - γ in splenocytes was performed at 7 days post - immunization . Representative images were shown in ( G ) and the numbers of antibody secreting cells ( ASCs ) were counted ( The values represent mean ± sd in each group , each symbol represents a mouse ) ( H ) . Data information : Mann - Whitney test was used for H . * p < 0 . 5 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , ns represents not

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G-H T-cell responses in vaccinated mice were measured through ELISpot assay. Two groups of C57BL/6 mice (n = 4 / group) were i.p. inoculated with 107 of WNV-poly(A) or equal volume of PBS. ELISpot assay specific for IFN-γ in splenocytes was performed at 7 days post-immunization. Representative images were shown in (G) and the numbers of antibody secreting cells (ASCs) were counted (The values represent mean ± sd in each group, each symbol represents a mouse) (H). Data information: Mann-Whitney test was used for H. *p<0.5, **p<0.01, *** p<0.001,****p< 0.0001, ns represents not significant


C Comparison of growth kinetics between WT WNV and WNV - poly ( A ) viruses in MEF cells . MEF cells were infected with either WT WNV or WNV - poly ( A ) virus at an MOI of 0 . 01 . Values indicate mean ± sd from two replicates of one representative experiment . Data information : Three independent assays were performed for above experiments with similar results and data of one representative data was shown . Scale bars represent 50 μm .

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C Comparison of growth kinetics between WT WNV and WNV-poly(A) viruses in MEF cells. MEF cells were infected with either WT WNV or WNV-poly(A) virus at an MOI of 0.01. Values indicate mean ± sd from two replicates of one representative experiment. Data information: Three independent assays were performed for above experiments with similar results and data of one representative data was shown. Scale bars represent 50 μm.


D Survival rate of IFNAR - / - mice infected with different dosages of WNV - poly ( A ) ( n = 5 ) . Data information : Three independent assays were performed for above experiments with similar results and data of one representative data was shown . Scale bars represent 50 μm .

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D Survival rate of IFNAR-/- mice infected with different dosages of WNV-poly(A) (n=5). Data information: Three independent assays were performed for above experiments with similar results and data of one representative data was shown. Scale bars represent 50 μm.


E Cell morphology changes of MEF cells infected with WT WNV and WNV - poly ( A ) . Data information : Three independent assays were performed for above experiments with similar results and data of one representative data was shown . Scale bars represent 50 μm .

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E Cell morphology changes of MEF cells infected with WT WNV and WNV-poly(A). Data information: Three independent assays were performed for above experiments with similar results and data of one representative data was shown. Scale bars represent 50 μm.


Virulence and efficacy of the passaged WNV - poly ( A ) ( WNV - poly ( A ) - P50 ) in mice ( n = 5 ) . ( E ) Survival of mice infected with 107 PFU of WNV - poly ( A ) - P50 . Data information : For statistical analysis , one - way ANOVA analysis and Log - rank test were used * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , ns represents not significant .

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Virulence and efficacy of the passaged WNV-poly(A) (WNV-poly(A)-P50) in mice (n=5). (E) Survival of mice infected with 107 PFU of WNV-poly(A)-P50. Data information: For statistical analysis, one-way ANOVA analysis and Log-rank test were used **p<0.01, *** p<0.001,****p< 0.0001, ns represents not significant.


Virulence and efficacy of the passaged WNV - poly ( A ) ( WNV - poly ( A ) - P50 ) in mice ( n = 5 ) . ( F ) Antibody titers of mice infected with WNV - poly ( A ) - P50 at different days post immunization . ( For the values represent mean ± sd in each group ( n = 5 ) and the horizontal dotted line represents the limit of detection ) . Data information : For statistical analysis , Mann - Whitney test were used * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , ns represents not significant .

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Virulence and efficacy of the passaged WNV-poly(A) (WNV-poly(A)-P50) in mice (n=5). (F) Antibody titers of mice infected with WNV-poly(A)-P50 at different days post immunization. (For the values represent mean ± sd in each group (n=5) and the horizontal dotted line represents the limit of detection). Data information: For statistical analysis, Mann-Whitney test were used **p<0.01, *** p<0.001,****p< 0.0001, ns represents not significant.


( I ) Survival of mice after WT WNV challenge . Data information : For statistical analysis , one - way ANOVA analysis and Log - rank test were used * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , ns represents not significant .

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(I) Survival of mice after WT WNV challenge. Data information: For statistical analysis, one-way ANOVA analysis and Log-rank test were used **p<0.01, *** p<0.001,****p< 0.0001, ns represents not significant.


A A549 and U937 cells were infected with each IAV at MOI 1 or 5 for 24 h , and lysates were analyzed by Western blotting using polyclonal anti - PB1 - F2 antibody .

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A A549 and U937 cells were infected with each IAV at MOI 1 or 5 for 24 h, and lysates were analyzed by Western blotting using polyclonal anti-PB1-F2 antibody.


B A549 cells were transfected with a plasmid encoding Flag - tagged PB1 - F2 derived from the PR8 or 1918 strain and treated with the proteasome inhibitor MG132 ( 25 μM ) or vehicle for 6 h before harvesting . PB1 - F2 transcripts were analyzed by semi - quantitative RT - PCR ( top panel ) , and The PB1 - F2 protein was analyzed by Western blotting with monoclonal anti - Flag antibody .

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B A549 cells were transfected with a plasmid encoding Flag-tagged PB1-F2 derived from the PR8 or 1918 strain and treated with the proteasome inhibitor MG132 (25 μM) or vehicle for 6 h before harvesting. PB1-F2 transcripts were analyzed by semi-quantitative RT-PCR (top panel), and The PB1-F2 protein was analyzed by Western blotting with monoclonal anti-Flag antibody.


C Representative immunofluorescence images showing expression of PB1 - F2 ( red ) . A549 cells were transfected with plasmids encoding indicated PB1 - F2 . At 18 h after transfection , cells were treated with MG132 for 6 h and subjected to immunofluorescence analysis . Magnification , × 100 ; scale bar , 100 μm .

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C Representative immunofluorescence images showing expression of PB1-F2 (red). A549 cells were transfected with plasmids encoding indicated PB1-F2. At 18 h after transfection, cells were treated with MG132 for 6 h and subjected to immunofluorescence analysis. Magnification, ×100; scale bar, 100 μm.


D Each HA - tagged PB1 - F2 was transfected into 293T cells with or without Flag - ubiquitin . At 18 h after transfection , cells were treated with MG132 for 6 h . The lysates were immunoprecipitated with anti - HA antibody and analyzed by Western blotting with anti - Flag antibody . PR8 , A / Puerto Rico / 8 / 34 ( H1N1 ) ; 1918 , A / Brevig Mission / 1 / 1918 ( H1N1 ) .

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D Each HA-tagged PB1-F2 was transfected into 293T cells with or without Flag-ubiquitin. At 18 h after transfection, cells were treated with MG132 for 6 h. The lysates were immunoprecipitated with anti-HA antibody and analyzed by Western blotting with anti-Flag antibody. PR8, A/Puerto Rico/8/34 (H1N1); 1918, A/Brevig Mission/1/1918 (H1N1).


B - D A549 cells were transfected with the indicated plasmids encoding Flag - tagged PB1 - F2 derived from the PR8 or 1918 strain and chimeric PB1 - F2 mutants . At 18 h after transfection , cells were treated with or without MG132 for 6 h . The expression of PB1 - F2 and control genes was detected by semi - quantitative RT - PCR ( upper panel ) or Western blotting ( lower panel ) . Results shown are representative of three independent experiments .

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B-D A549 cells were transfected with the indicated plasmids encoding Flag-tagged PB1-F2 derived from the PR8 or 1918 strain and chimeric PB1-F2 mutants. At 18 h after transfection, cells were treated with or without MG132 for 6 h. The expression of PB1-F2 and control genes was detected by semi-quantitative RT-PCR (upper panel) or Western blotting (lower panel). Results shown are representative of three independent experiments.


E Representative immunofluorescence images of PB1 - F2 expression in A549 cells . Cells transfected with the Flag - tagged PB1 - F2 plasmid were stained with anti - Flag ( red ) antibody and analyzed by immunofluorescence assay . Magnification , × 400 ; scale bar , 50 μm .

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E Representative immunofluorescence images of PB1-F2 expression in A549 cells. Cells transfected with the Flag-tagged PB1-F2 plasmid were stained with anti-Flag (red) antibody and analyzed by immunofluorescence assay. Magnification, ×400; scale bar, 50 μm.


A Relative activity of NF - kB luciferase reporter was measured in A549 cells transfected with plasmids encoding PB1 - F2 . The mRNA levels of pro - inflammatory cytokines were determined by semi - quantitative RT - PCR . All data are shown as mean ( ± SEM ) from at least three independent experiments ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ) .

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A Relative activity of NF-kB luciferase reporter was measured in A549 cells transfected with plasmids encoding PB1-F2. The mRNA levels of pro-inflammatory cytokines were determined by semi-quantitative RT-PCR. All data are shown as mean (±SEM) from at least three independent experiments (*p < 0.05, **p <0.01, ***p <0.001).


Cells were transfected with PB1 - F2 - expressing plasmids . Relative IFNβ mRNA levels were determined by semi - quantitative RT - PCR ( top ) and qPCR ( bottom ) . All data are shown as mean ( ± SEM ) from at least three independent experiments ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ) .

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Cells were transfected with PB1-F2-expressing plasmids. Relative IFNβ mRNA levels were determined by semi-quantitative RT-PCR (top) and qPCR (bottom). All data are shown as mean (±SEM) from at least three independent experiments (*p < 0.05, **p <0.01, ***p <0.001).


C U937 cells were co - transfected with the IFNβ luciferase reporter plasmid and the PB1 - F2 or NS1 expressing plasmid and treated with polyI : C for 12 h before harvesting . IFNβ promoter activity was determined by luciferase reporter assay . All data are shown as mean ( ± SEM ) from at least three independent experiments ( * 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 B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 U937 cells were co-transfected with the IFNβ luciferase reporter plasmid and the PB1-F2 or NS1 expressing plasmid and treated with polyI:C for 12 h before harvesting. IFNβ promoter activity was determined by luciferase reporter assay. All data are shown as mean (±SEM) from at least three independent experiments (*p < 0.05, **p <0.01, ***p <0.001).


Cells were infected for 24 h with each virus at 1 MOI . The mRNA level of IFNβ was determined by semi - quantitative RT - PCR , and PB1 - F2 protein expression was assessed by Western blotting . All data are shown as mean ( ± SEM ) from at least three independent experiments ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ) .

Cells were infected for 24 h with each virus at 1 MOI. The mRNA level of IFNβ was determined by semi-quantitative RT-PCR, and PB1-F2 protein expression was assessed by Western blotting. All data are shown as mean (±SEM) from at least three independent experiments (*p < 0.05, **p <0.01, ***p <0.001).


E Cells were transfected with each plasmid encoding PB1 - F2 . 18 h after transfection , cells were treated with MG132 for 6 h and the level of IFNβ mRNA was determined by semi - quantitative RT - PCR ( left ) and qPCR ( right ) . PB1 - F2 protein expression was assessed by Western blotting . All data are shown as mean ( ± SEM ) from at least three independent experiments ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ) .

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E Cells were transfected with each plasmid encoding PB1-F2. 18 h after transfection, cells were treated with MG132 for 6 h and the level of IFNβ mRNA was determined by semi-quantitative RT-PCR (left) and qPCR (right). PB1-F2 protein expression was assessed by Western blotting. All data are shown as mean (±SEM) from at least three independent experiments (*p < 0.05, **p <0.01, ***p <0.001).


F A549 cells were transfected with PB1 - F2 clones mutated at positions 68 and 69 ; 24 h after transfection , the IFNβ mRNA level and PB1 - F2 protein expression were determined by semi - quantitative RT - PCR and Western blotting , respectively ( left ) . The relative expression level of IFNβ mRNA was also analyzed by qPCR ( right ) . All data are shown as mean ( ± SEM ) from at least three independent experiments ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ) .

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F A549 cells were transfected with PB1-F2 clones mutated at positions 68 and 69; 24 h after transfection, the IFNβ mRNA level and PB1-F2 protein expression were determined by semi-quantitative RT-PCR and Western blotting, respectively (left). The relative expression level of IFNβ mRNA was also analyzed by qPCR (right). All data are shown as mean (±SEM) from at least three independent experiments (*p < 0.05, **p <0.01, ***p <0.001).


A Mice ( n = 5 or 6 per group ) were infected with IAV with indicated pfu . Survival was monitored daily for 2 weeks .

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A Mice (n = 5 or 6 per group) were infected with IAV with indicated pfu. Survival was monitored daily for 2 weeks.


B Mice ( n = 6 - 9 per group ) were infected with each IAV as indicated . Body weight was recorded daily after infection for 2 weeks .

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B Mice (n = 6-9 per group) were infected with each IAV as indicated. Body weight was recorded daily after infection for 2 weeks.


C Mice ( n = 5 or 6 per group ) were infected with viruses ( 1000 pfu ) carrying PR8 PB1 - F2 with the I68T , L69P , or I68T + L69P mutation . Body weight changes and survival rate were monitored daily . Detailed analyses on the virulence necessitated the monitoring of body weight below the standard level ( 80 % ) of mortality .

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C Mice (n = 5 or 6 per group) were infected with viruses (1000 pfu) carrying PR8 PB1-F2 with the I68T, L69P, or I68T+L69P mutation. Body weight changes and survival rate were monitored daily. Detailed analyses on the virulence necessitated the monitoring of body weight below the standard level (80%) of mortality.


Three cohorts of mice ( 5 or 6 per group ) were infected with 500 pfu of one of the three IAVs . Viral protein expression in mouse lung was determined by Western blotting 2 days after infection .

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Three cohorts of mice (5 or 6 per group) were infected with 500 pfu of one of the three IAVs. Viral protein expression in mouse lung was determined by Western blotting 2 days after infection.


E Mice ( 6 per group ) were infected with 500 pfu of one of the three IAVs . Viral titration by plaque assay was performed at 2 days post - infection using lung homogenates .

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E Mice (6 per group) were infected with 500 pfu of one of the three IAVs. Viral titration by plaque assay was performed at 2 days post-infection using lung homogenates.


F Mice ( 5 - 9 per group ) were infected with 500 pfu of each virus . At 2 days post - infection , IFNβ mRNA levels were determined by qPCR .

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F Mice (5-9 per group) were infected with 500 pfu of each virus. At 2 days post-infection, IFNβ mRNA levels were determined by qPCR.


G Mice ( 5 - 9 per group ) were infected with 1 × 105 pfu of each virus . At 2 days post - infection , IFNβ protein levels in lung homogenates were measured by ELISA .

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G Mice (5-9 per group) were infected with 1 × 105 pfu of each virus. At 2 days post-infection, IFNβ protein levels in lung homogenates were measured by ELISA.


A SDS - PAGE analysis of proteins immunoprecipitated from A549 cell lysates using HA - antibody . Bands were stained by Coomassie Blue . Results were confirmed in three independent experiments , and representative data are shown .

A SDS-PAGE analysis of proteins immunoprecipitated from A549 cell lysates using HA-antibody. Bands were stained by Coomassie Blue. Results were confirmed in three independent experiments, and representative data are shown.


E Analysis of interaction between DDX3 and 1918 PB1 - F2 . Cells were transfected with indicated plasmids ; 18 h later , cells were treated with MG132 for 6 h . Cell lysates were immunoprecipitated with anti - HA antibody . Proteins were detected by immunoblotting with the indicated antibodies ( left panel ) . IAV - infected cell lysates were immunoprecipitated with anti - PB1 - F2 antibody and immunoblotted with the indicated antibodies ( middle panel ) . Purified recombinant 1918 PB1 - F2 protein was incubated with recombinant LysRS or LysRS - DDX3 , and the interaction was analyzed by immunoblotting ( right panel ) . Results were confirmed in three independent experiments , and representative data are shown .

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E Analysis of interaction between DDX3 and 1918 PB1-F2. Cells were transfected with indicated plasmids; 18 h later, cells were treated with MG132 for 6 h. Cell lysates were immunoprecipitated with anti-HA antibody. Proteins were detected by immunoblotting with the indicated antibodies (left panel). IAV-infected cell lysates were immunoprecipitated with anti-PB1-F2 antibody and immunoblotted with the indicated antibodies (middle panel). Purified recombinant 1918 PB1-F2 protein was incubated with recombinant LysRS or LysRS-DDX3, and the interaction was analyzed by immunoblotting (right panel). Results were confirmed in three independent experiments, and representative data are shown.


F Analysis of interaction between DDX3 and PB1 - F2 mutants . A549 cells were transfected with the indicated clones ; 18 h after transfection , cells were treated with MG132 for 6 h and analyzed by immunoprecipitation . Results were confirmed in three independent experiments , and representative data are shown .

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F Analysis of interaction between DDX3 and PB1-F2 mutants. A549 cells were transfected with the indicated clones; 18 h after transfection, cells were treated with MG132 for 6 h and analyzed by immunoprecipitation. Results were confirmed in three independent experiments, and representative data are shown.


G A549 cells were treated with IFNβ for the indicated time periods and subjected to Western blotting ( top ) . A549 cells were infected with indicated IAV . At 24 h post - infection , cell lysates were analyzed by Western blotting ( bottom ) . Results were confirmed in three independent experiments , and representative data are shown .

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G A549 cells were treated with IFNβ for the indicated time periods and subjected to Western blotting (top). A549 cells were infected with indicated IAV. At 24 h post-infection, cell lysates were analyzed by Western blotting (bottom). Results were confirmed in three independent experiments, and representative data are shown.


H Representative fluorescence images showing expression level of GFP - DDX3 ( green ) . A549 cells were co - transfected with plasmids encoding the GEP - DDX3 and Flag - tagged PR8 or 1918 PB1 - F2 . At 18 h after transfection , cells were treated with MG132 for 6 h and subjected to fluorescence analysis . Results were confirmed in three independent experiments , and representative data are shown .

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H Representative fluorescence images showing expression level of GFP-DDX3 (green). A549 cells were co-transfected with plasmids encoding the GEP-DDX3 and Flag-tagged PR8 or 1918 PB1-F2. At 18 h after transfection, cells were treated with MG132 for 6 h and subjected to fluorescence analysis. Results were confirmed in three independent experiments, and representative data are shown.


I Mice were infected with 1 × 105 pfu of each virus , and lung homogenates were examined for endogenous Ddx3 expression . Results were confirmed in three independent experiments , and representative data are shown .

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I Mice were infected with 1×105 pfu of each virus, and lung homogenates were examined for endogenous Ddx3 expression. Results were confirmed in three independent experiments, and representative data are shown.


J A549 cells were transfected with the indicated plasmids or treated with poly I : C . Cytoplasmic and nuclear fractions were separated and subjected to Western blotting . Results were confirmed in three independent experiments , and representative data are shown .

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J A549 cells were transfected with the indicated plasmids or treated with poly I:C. Cytoplasmic and nuclear fractions were separated and subjected to Western blotting. Results were confirmed in three independent experiments, and representative data are shown.


K A549 cells were transfected with the indicated plasmids , and IFNβ mRNA levels were determined by semi - quantitative RT - PCR ( top ) and qPCR ( middle ) . IFNβ promoter activity was determined by luciferase reporter assay ( bottom ) . Results were confirmed in three independent experiments , and representative data are shown .

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K A549 cells were transfected with the indicated plasmids, and IFNβ mRNA levels were determined by semi-quantitative RT-PCR (top) and qPCR (middle). IFNβ promoter activity was determined by luciferase reporter assay (bottom). Results were confirmed in three independent experiments, and representative data are shown.


Groups of five mice were intranasally infected with IAV ( 1918 ) , and recombinant DDX3 protein was administered intranasally . Survival was monitored daily for 2 weeks .

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Groups of five mice were intranasally infected with IAV (1918), and recombinant DDX3 protein was administered intranasally. Survival was monitored daily for 2 weeks.


Groups of five mice were intranasally infected with IAV ( 1918 ) , and recombinant DDX3 protein was administered intranasally . Two days post - infection , virus titers in lung homogenates were determined by plaque assay . A p value of < 0 . 05 was considered significant ( * * p < 0 . 01 ) .

Groups of five mice were intranasally infected with IAV (1918), and recombinant DDX3 protein was administered intranasally. Two days post-infection, virus titers in lung homogenates were determined by plaque assay. A p value of <0.05 was considered significant (** p < 0.01).


Groups of five mice were intranasally infected with IAV ( 1918 ) , and recombinant DDX3 protein was administered intranasally . Two days post - infection , the expression level of IFNβ in the lungs was analyzed by semi - quantitative RT - PCR ( left panel ) and qPCR ( right panel ) . A p value of < 0 . 05 was considered significant ( * * p < 0 . 01 ) .

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Groups of five mice were intranasally infected with IAV (1918), and recombinant DDX3 protein was administered intranasally. Two days post-infection, the expression level of IFNβ in the lungs was analyzed by semi-quantitative RT-PCR (left panel) and qPCR (right panel). A p value of <0.05 was considered significant (** p < 0.01).


( A - B ' ' ) Nr2f1 ( red ) and Pax6 ( green , retinal progenitors ) immunofluorescences ( IF ) on sagittal sections of E10 . 5 and E12 . 5 mouse optic vesicles . Note the high - ventral to low - dorsal Nr2f1 gradient in the presumptive neural retina ( pNR ) , and high Nr2f1 expression in both the presumptive dorsal and ventral optic stalk ( pvOS and pdOS ) . Cr : crystal lens ; Di , distal ; Pr , proximal ; presumptive retinal pigmented epithelium ( pRPE ) . Scale bars : 50µm for mouse

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(A-B'') Nr2f1 (red) and Pax6 (green, retinal progenitors) immunofluorescences (IF) on sagittal sections of E10.5 and E12.5 mouse optic vesicles. Note the high-ventral to low-dorsal Nr2f1 gradient in the presumptive neural retina (pNR), and high Nr2f1 expression in both the presumptive dorsal and ventral optic stalk (pvOS and pdOS). Cr: crystal lens; Di, distal; Pr, proximal; presumptive retinal pigmented epithelium (pRPE). Scale bars: 50µm for mouse sections


( C - D " ) IF on E13 . 5 mouse eye sagittal sections with Nr2f1 ( red ) and Sox2 ( green , progenitors in C , C ' ) or Brn3a ( green , RGCs in D , D ' ) showing Nr2f1 expression in NR progenitors and post - mitotic RGCs ( insets in C ' - D " ) . Scale bars : 50µm for mouse

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(C-D") IF on E13.5 mouse eye sagittal sections with Nr2f1 (red) and Sox2 (green, progenitors in C,C') or Brn3a (green, RGCs in D,D') showing Nr2f1 expression in NR progenitors and post-mitotic RGCs (insets in C'-D"). Scale bars: 50µm for mouse sections


( E - F " ) Nr2f1 ( red ) and Brn3a ( green ) IF on E18 . 5 and P7 mouse eyes depicting Nr2f1 expression in virtually all RGCs in the ganglion cell layer ( GCL ) . Scale bars : 50µm for mouse

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(E-F") Nr2f1 (red) and Brn3a (green) IF on E18.5 and P7 mouse eyes depicting Nr2f1 expression in virtually all RGCs in the ganglion cell layer (GCL). Scale bars: 50µm for mouse sections


( G , G ' ) Nr2f1 ( red ) and Sox2 ( green ) IF on P7 mouse retina illustrating low Nr2f1 expression in the inner nuclear layer ( INL ) and progenitors ( NPs ) , and high levels in the GCL and ciliary marginal zone ( CMZ ) . No expression in the outer nuclear layer ( ONL ) . Scale bars : 50µm for mouse

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(G,G') Nr2f1 (red) and Sox2 (green) IF on P7 mouse retina illustrating low Nr2f1 expression in the inner nuclear layer (INL) and progenitors (NPs), and high levels in the GCL and ciliary marginal zone (CMZ). No expression in the outer nuclear layer (ONL). Scale bars: 50µm for mouse sections