( C ) RNF26 RNF5 competed with each other on MITA polyubiquitination . 293 ( 5 × 106 ) were transfected with MITA ( 5 µg ) , Flag - Ub - K48O HA - Ub - K11O ( 1 µg each ) together with indicated amount of RNF26 RNF5 . Twenty - four hours after transfection , lysates were subjected to IP under denatured conditions with anti - MITA immunoprecipitates were analyzed by immunoblots with anti - Flag ( upper panel ) , anti - HA ( middle panel ) or anti - MITA ( lower panel ) . whole lysates were analyzed by immunoblots with antibodies against indicated .

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(C) RNF26 and RNF5 competed with each other on MITA polyubiquitination. The 293 cells (5×106) were transfected with MITA (5 µg), Flag-Ub-K48O and HA-Ub-K11O (1 µg each) together with indicated amount of RNF26 and RNF5. Twenty-four hours after transfection, cell lysates were subjected to IP under denatured conditions with anti-MITA and the immunoprecipitates were analyzed by immunoblots with anti-Flag (upper panel), anti-HA (middle panel) or anti-MITA (lower panel). The whole cell lysates were analyzed by immunoblots with antibodies against the indicated proteins.


( D ) Effects of RNF26 knockdown on - triggered MITA degradation . THP - 1 - RNF26 - RNAi or control ( 1 × 106 ) were infected with SeV or HSV - 1 for indicated time points or left uninfected . were lysed whole lysates were analyzed by immunoblots with antibodies against indicated ( upper immunoblots ) . relative of MITA in reference to β - actin were analyzed by Quantity One program data shown are mean ± S . D . of three independent experiments ( lower histographs ) .

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(D) Effects of RNF26 knockdown on virus-triggered MITA degradation. The THP-1-RNF26-RNAi or control cells (1×106) were infected with SeV or HSV-1 for the indicated time points or left uninfected. Cells were lysed and whole cell lysates were analyzed by immunoblots with antibodies against the indicated proteins (upper immunoblots). The relative protein levels of MITA in reference to β-actin were analyzed by Quantity One program and data shown are mean ± S.D. of three independent experiments (lower histographs).


( A ) Effects of RNF26 knockdown on SeV - triggered activation of IFN - β promoter . 293 ( 2 × 105 ) were transfected with IFN - β promoter reporter ( 0 . 1 µg ) indicated RNAi plasmid ( 0 . 5 µg each ) . Thirty hours after transfection , were infected with SeV for 12 hours or left uninfected before reporter assays were performed .

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(A) Effects of RNF26 knockdown on SeV-triggered activation of the IFN-β promoter. The 293 cells (2×105) were transfected with the IFN-β promoter reporter (0.1 µg) and the indicated RNAi plasmid (0.5 µg each). Thirty hours after transfection, cells were infected with SeV for 12 hours or left uninfected before reporter assays were performed.


( B C ) Effects of RNF26 knockdown on - triggered induction of IFN - β in THP - 1 . THP - 1 - RNF26 - RNAi or control ( 1 × 106 ) were infected with SeV or HSV - 1 for indicated time points or left uninfected followed by quantitative real - time PCR ( B )

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(B and C) Effects of RNF26 knockdown on virus-triggered induction of IFN-β in THP-1 cells. The THP-1-RNF26-RNAi or control cells (1×106) were infected with SeV or HSV-1 for the indicated time points or left uninfected followed by quantitative real-time PCR (B)


( B C ) Effects of RNF26 knockdown on - triggered induction of IFN - β in THP - 1 . THP - 1 - RNF26 - RNAi or control ( 1 × 106 ) were infected with SeV or HSV - 1 for indicated time points or left uninfected followed by quantitative real - time PCR ( B ) or ELISA (

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(B and C) Effects of RNF26 knockdown on virus-triggered induction of IFN-β in THP-1 cells. The THP-1-RNF26-RNAi or control cells (1×106) were infected with SeV or HSV-1 for the indicated time points or left uninfected followed by quantitative real-time PCR (B) or ELISA analysis (C


( D ) Effects of RNF26 knockdown on - triggered induction of IFNB1 gene in THP - 1 . THP - 1 - RNF26 - RNAi or control ( 1 × 106 ) were infected with VSV , EMCV or ECTV for indicated time points or left uninfected before quantitative real - time PCR was performed as in ( B ) .

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(D) Effects of RNF26 knockdown on virus-triggered induction of IFNB1 gene in THP-1 cells. The THP-1-RNF26-RNAi or control cells (1×106) were infected with VSV, EMCV or ECTV for the indicated time points or left uninfected before quantitative real-time PCR analysis was performed as in (B).


( E F ) Effects of RNF26 knockdown on - triggered induction of TNFα in THP - 1 . THP - 1 - RNF26 - RNAi or control ( 1 × 106 ) were infected with SeV or HSV - 1 for indicated time points or left uninfected followed by quantitative real - time PCR ( E ) ELISA ( F ) .

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(E and F) Effects of RNF26 knockdown on virus-triggered induction of TNFα in THP-1 cells. The THP-1-RNF26-RNAi or control cells (1×106) were infected with SeV or HSV-1 for the indicated time points or left uninfected followed by quantitative real-time PCR (E) and ELISA analysis (F).


( G ) Effects of RNF26 knockdown on - triggered phosphorylation of TBK1 , IRF3 IκBα . THP - 1 - RNF26 - RNAi or control ( 1 × 106 ) were infected with SeV or HSV - 1 for indicated time points or left uninfected , whole lysates were analyzed by immunoblots with anti - p - TBK1 , anti - TBK1 , anti - p - IRF3 , anti - IRF3 , anti - p - IκBα , anti - IκBα , anti - RNF26 or anti - β - actin as indicated . experiments were repeated for at least three times with similar results . bar graphs show mean ± S . D . ( n = 3 ) of a representative experiment performed in triplicate .

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(G) Effects of RNF26 knockdown on virus-triggered phosphorylation of TBK1, IRF3 and IκBα. The THP-1-RNF26-RNAi or control cells (1×106) were infected with SeV or HSV-1 for the indicated time points or left uninfected, whole cell lysates were analyzed by immunoblots with anti-p-TBK1, anti-TBK1, anti-p-IRF3, anti-IRF3, anti-p-IκBα, anti-IκBα, anti-RNF26 or anti-β-actin as indicated. All experiments were repeated for at least three times with similar results. The bar graphs show mean ± S.D. (n = 3) of a representative experiment performed in triplicate.


A ) Effects of RNF26 knockdown on IRF3 level . In left panel , whole lysates of THP - 1 - RNF26 - RNAi or control ( 1 × 106 ) were analyzed by immunoblots with indicated antibodies . In right panel , ( 1 × 106 ) were subjected to quantitative real - time PCR .

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A) Effects of RNF26 knockdown on IRF3 level. In the left panel, whole cell lysates of THP-1-RNF26-RNAi or control cells (1×106) were analyzed by immunoblots with the indicated antibodies. In the right panel, cells (1×106) were subjected to quantitative real-time PCR analysis.


B ) Effects of RNF26 on IRF3 stability . 293 ( 1 × 106 ) were transfected with HA - IRF3 ( 0 . 5 µg ) HA - β - actin ( 0 . 1 µg ) together with RNF26 or its mutant ( 0 . 3 µg each ) . Twenty - four hours later , whole lysates were analyzed by immunoblots with anti - HA or anti - RNF26 .

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B) Effects of RNF26 on IRF3 stability. The 293 cells (1×106) were transfected with HA-IRF3 (0.5 µg) and HA-β-actin (0.1 µg) together with RNF26 or its mutant (0.3 µg each). Twenty-four hours later, whole cell lysates were analyzed by immunoblots with anti-HA or anti-RNF26.


( C ) Effects of RNF26 its mutant on SeV - triggered activation of IFN - β promoter . 293 ( 2 × 105 ) were transfected with IFN - β promoter reporter ( 0 . 1 µg ) RNF26 or its mutant ( 0 . 1 µg each ) . Twenty hours after transfection , were infected with SeV for 12 hours or left uninfected before reporter assays were performed .

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(C) Effects of RNF26 and its mutant on SeV-triggered activation of the IFN-β promoter. The 293 cells (2×105) were transfected with the IFN-β promoter reporter (0.1 µg) and RNF26 or its mutant (0.1 µg each). Twenty hours after transfection, cells were infected with SeV for 12 hours or left uninfected before reporter assays were performed.


( D ) Effects of RNF26 on IRF3 ubiquitination . 293 ( 5 × 106 ) were transfected with Flag - IRF3 ( 5 µg ) RNF26 ( 1 µg ) together with HA - Ub or its mutants ( 1 µg each ) . Eighteen hours after transfection , 3 - MA ( 500 ng / mL ) was added into culture medium for 4 hours to protect IRF3 from autophagosomal degradation during experiment . lysates were subjected to IP under denatured conditions with anti - Flag immunoprecipitates were analyzed by immunoblots with anti - HA ( upper panels ) or anti - Flag ( lower panels ) . whole lysates were analyzed by immunoblots with anti - Flag or anti - RNF26 as indicated .

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(D) Effects of RNF26 on IRF3 ubiquitination. The 293 cells (5×106) were transfected with Flag-IRF3 (5 µg) and RNF26 (1 µg) together with HA-Ub or its mutants (1 µg each). Eighteen hours after transfection, 3-MA (500 ng/mL) was added into culture medium for 4 hours to protect IRF3 from autophagosomal degradation during the experiment. The cell lysates were subjected to IP under denatured conditions with anti-Flag and the immunoprecipitates were analyzed by immunoblots with anti-HA (upper panels) or anti-Flag (lower panels). The whole cell lysates were analyzed by immunoblots with anti-Flag or anti-RNF26 as indicated.


( E ) Effects of inhibitors on RNF26 - mediated destabilization of IRF3 . 293 ( 1 × 106 ) were transfected with indicated plasmids as described in ( B ) . Eighteen hours after transfection , 3 - MA ( 500 ng / mL ) , NH4Cl ( 25 mM ) or MG132 ( 100 µM ) was added into culture medium for four hours . Whole lysates were analyzed by immunoblots with anti - HA or anti - RNF26 .

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(E) Effects of inhibitors on RNF26-mediated destabilization of IRF3. The 293 cells (1×106) were transfected with the indicated plasmids as described in (B). Eighteen hours after transfection, 3-MA (500 ng/mL), NH4Cl (25 mM) or MG132 (100 µM) was added into culture medium for four hours. Whole cell lysates were analyzed by immunoblots with anti-HA or anti-RNF26.


( F ) Effects of ATG12 knockdown on RNF26 - mediated destabilization of IRF3 . 293 - ATG12 - RNAi or control ( 1 × 106 ) were transfected with indicated plasmids . Twenty - four hours later , whole lysates were analyzed by immunoblots with anti - HA , anti - RNF26 or anti - ATG12 as indicated . experiments were repeated for at least three times with similar results . bar graphs show mean ± S . D . ( n = 3 ) of a representative experiment performed in triplicate .

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(F) Effects of ATG12 knockdown on RNF26-mediated destabilization of IRF3. The 293-ATG12-RNAi or control cells (1×106) were transfected with the indicated plasmids. Twenty-four hours later, whole cell lysates were analyzed by immunoblots with anti-HA, anti-RNF26 or anti-ATG12 as indicated. All experiments were repeated for at least three times with similar results. The bar graphs show mean ± S.D. (n = 3) of a representative experiment performed in triplicate.


( A , B ) Expression of UVRAG under stress . HL60 HCT116 were treated with doxorubicin ( 1 μg / ml ) , cisplatin ( 10 μm ) UV irradiation ( 5 min after 50 mJ / cm2 ) for indicated time , then apoptosis , autophagy of UVRAG was assayed . Scale bar , 10 μm .

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(A,B) Expression of UVRAG under cell stress. HL60 and HCT116 cells were treated with doxorubicin (1 μg/ml), cisplatin (10 μm) and UV irradiation (5 min after 50 mJ/cm2) for the indicated time, and then apoptosis, autophagy and fluorescence intensity of UVRAG was assayed. Scale bar, 10 μm.


( C ) In parallel , total extracts were used for western blot .

(C) In parallel, the total protein extracts were used for western blot analysis.


( D , E ) Pharmacological inhibition ( for example , 3 ‐ methyladenine ( 3 ‐ MA ) , 10 mM ) or genetic deletion ( for example , ATG5 − / − ) of autophagy pathway decreases mRNA expression of UVRAG increases apoptosis at 12 h in HL60 treated with doxorubicin ( Doxo ) UV irradiation ( n = 3 , * P0 . 05 ) . AU , arbitrary units ; C ‐ PARP , poly ‐ ADP ribose polymerase ; LC3 , chain 3 ; mRNA , messenger ; PI , propidium iodide ; UV , ultraviolet ; UVRAG , ultraviolet irradiation resistance ‐ associated gene .

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(D,E) Pharmacological inhibition (for example, 3‐methyladenine (3‐MA), 10 mM) or genetic deletion (for example, ATG5−/−) of the autophagy pathway decreases the protein and mRNA expression of UVRAG and increases apoptosis at 12 h in HL60 cells treated with doxorubicin (Doxo) and UV irradiation (n=3, *P0.05). AU, arbitrary units; C‐PARP, cleaved poly‐ADP ribose polymerase; LC3, light chain 3; mRNA, messenger RNA; PI, propidium iodide; UV, ultraviolet; UVRAG, ultraviolet irradiation resistance‐associated gene.


( A - C ) After transfection with UVRAG shRNA or control shRNA for 48 h , HL60 ( A ) , HCT116 ( B ) HeLa ( C ) were treated with doxorubicin ( Doxo , 1 μg / ml ) , cisplatin ( Cis , 10 μm ) UV irradiation ( 5 min after 50 mJ / cm2 ) for 12 h or starvation ( HBSS ) for 3 h , then apoptosis autophagy were assayed ( n = 3 , * P0 . 05 ) .

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(A-C) After transfection with UVRAG shRNA or control shRNA for 48 h, HL60 (A), HCT116 (B) and HeLa (C) cells were treated with doxorubicin (Doxo, 1 μg/ml), cisplatin (Cis, 10 μm) and UV irradiation (5 min after 50 mJ/cm2) for 12 h or starvation (HBSS) for 3 h, and then apoptosis and autophagy were assayed (n=3, *P0.05).


( D ) of LC3 processing by autophagy in presence or absence of lysosomal protease inhibitors pepstatin A ( 10 μg / ml ) E64D ( 10 μg / ml ) after doxorubicin ( 1 μg / ml ) treatment for 12 h . * P0 . 05 compared with control shRNA group .

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(D) Analysis of LC3 processing by autophagy in the presence or absence of lysosomal protease inhibitors pepstatin A (10 μg/ml) and E64D (10 μg/ml) after doxorubicin (1 μg/ml) treatment for 12 h. *P0.05 compared with control shRNA group.


( E ) After transfection with UVRAG shRNA or control shRNA for 48 h , ATG5 + / + ATG5 − / − murine embryonic fibroblasts ( MEFs ) were treated with doxorubicin ( 1 μg / ml ) , with or without Z ‐ VAD ‐ FMK ( 20 μM ) for 12 h , then apoptosis was assayed ( n = 3 , * P0 . 05 ) .

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(E) After transfection with UVRAG shRNA or control shRNA for 48 h, ATG5+/+ and ATG5−/− murine embryonic fibroblasts (MEFs) were treated with doxorubicin (1 μg/ml), with or without Z‐VAD‐FMK (20 μM) for 12 h, and then apoptosis was assayed (n=3, *P0.05).


( F ) Nude mice were inoculated with 4 × 106 HL60 tumour following transfection of control ( shControl ) or UVRAG ( shUVRAG ) ‐ specific shRNA treated with doxorubicin ( 10 mg / kg ) beginning at day 7 . Tumours were measured twice weekly , volumes were calculated for 21 days ( n = 10 , * P0 . 05 ; shControl + Doxo compared with shUVRAG + Doxo ) .

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(F) Nude mice were inoculated with 4 × 106 HL60 tumour cells following transfection of control (shControl) or UVRAG (shUVRAG)‐specific shRNA and treated with doxorubicin (10 mg/kg) beginning at day 7. Tumours were measured twice weekly, and volumes were calculated for 21 days (n=10, *P0.05; shControl+Doxo compared with shUVRAG+Doxo).


( G ) On day 21 in experiments described in ( F ) , apoptosis UVRAG expression in tumour samples were assayed by TUNEL or western blotting , respectively ( n = 10 , * P0 . 05 ) . BAX , BCL2 ‐ associated X ; HBSS , Hank ' s Buffered Salt Solution ; LC3 , chain 3 ; shRNA , short ‐ hairpin ; TUNEL , terminal deoxytransferase uridine triphosphate nick end labelling ; UV , ultraviolet ; UVRAG , ultraviolet irradiation resistance associated gene .

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(G) On day 21 in the experiments described in (F), apoptosis and UVRAG expression in tumour samples were assayed by TUNEL or western blotting, respectively (n=10, *P0.05). BAX, BCL2‐associated X protein; HBSS, Hank's Buffered Salt Solution; LC3, light chain 3; shRNA, short‐hairpin RNA; TUNEL, terminal deoxytransferase uridine triphosphate nick end labelling; UV, ultraviolet; UVRAG, ultraviolet irradiation resistance associated gene.


( A ) Control shRNA or UVRAG shRNA were transfected with BAX plasmid , apoptosis was assayed ( n = 3 , * P0 . 05 ) .

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(A) Control shRNA or UVRAG shRNA cells were transfected with BAX plasmid, and apoptosis was assayed (n=3, *P0.05).


( B ) In parallel , mitochondrial potential , cytochrome c release from mitochondria , caspase ‐ 3 ‐ 9 activation in HL60 were analysed ( n = 3 , * P0 . 05 ) .

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(B) In parallel, the mitochondrial membrane potential, cytochrome c release from mitochondria, and caspase‐3 and ‐9 activation in HL60 cells were analysed (n=3, *P0.05).


( C ) After transfection with UVRAG vector or control vector for 48 h , were transected or treated with BAX ( 1 μg ) , BAD ( 1 μg ) , BID ( 1 μg ) , FAS antibody ( 10 μg / ml ) or TRAIL ( 100 ng / ml ) for 24 h , then apoptosis was analysed ( n = 3 , * P0 . 05 ) .

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(C) After transfection with UVRAG vector or control vector for 48 h, cells were transected or treated with BAX (1 μg), BAD (1 μg), BID (1 μg), FAS antibody (10 μg/ml) or TRAIL (100 ng/ml) for 24 h, then cell apoptosis was analysed (n=3, *P0.05).


( D ) Knockdown of UVRAG in BAX − / − HCT116 restored sensitivity to UV treatment ‐ induced apoptosis ( n = 3 , * P0 . 05 ) . AU , arbitrary units ; BAX , BCL2 ‐ associated X ; shRNA , short ‐ hairpin ; UV , ultraviolet ; UVRAG , ultraviolet irradiation resistance associated gene .

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(D) Knockdown of UVRAG in BAX−/− HCT116 cells restored the sensitivity to UV treatment‐induced apoptosis (n=3, *P0.05). AU, arbitrary units; BAX, BCL2‐associated X protein; shRNA, short‐hairpin RNA; UV, ultraviolet; UVRAG, ultraviolet irradiation resistance associated gene.


( A , B ) HL60 HCT116 were treated with UV irradiation for 24 h then assayed for expression as indicated by co ‐ immunoprecipitation ( RIPA or CHAPS buffer ) or western blotting .

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(A,B) HL60 and HCT116 cells were treated with UV irradiation for 24 h and then assayed for protein expression levels as indicated by co‐immunoprecipitation (RIPA or CHAPS buffer) or western blotting.


( C ) in pellet after GST pull ‐ down assays in HCT116 lysate were analysed by SDS - PAGE immunodetected with UVRAG or GST antibodies .

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(C) Proteins in the pellet after GST pull‐down assays in HCT116 cell lysate were analysed by SDS-PAGE and immunodetected with UVRAG or GST antibodies.


( D ) UVRAG C2 domain deletion terminates BAX binding . HCT116 were transfected with Flag ‐ UVRAG or its mutants ( ΔC2 , ΔCCD ) , then assayed for expression as indicated by IP or western blot .

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(D) UVRAG C2 domain deletion terminates BAX binding. HCT116 cells were transfected with Flag‐UVRAG or its mutants (ΔC2, ΔCCD), and then assayed for protein expression levels as indicated by IP or western blot.


( E ) Apoptosis was assayed in indicated HCT116 after treatment with doxorubicin ( Doxo , 1 μg / ml ) , UV irradiation ( 5 min after 50 mJ / cm2 ) or transfected with BAX ( 1 μg ) for 24 h . BAX , BCL2 ‐ associated X ; CCD , coiled ‐ coil domain ; CHAPS , 3 ‐ [ ( 3 ‐ cholamidopropyl ) ‐ dimethylammonio ] ‐ 1 ‐ propane sulphonate ; GST , glutathione ‐ S ‐ transferase ; IB , immunoblotting ; IgG , immunoglobulin G ; IP , immunoprecipitation ; RIPA , radioimmunoprecipitation ; SDS - PAGE , SDS - polyacrylamide gel electrophoresis ; UV , ultraviolet ; UVRAG , ultraviolet irradiation resistance associated gene .

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(E) Apoptosis was assayed in indicated HCT116 cells after treatment with doxorubicin (Doxo, 1 μg/ml), UV irradiation (5 min after 50 mJ/cm2) or transfected with BAX (1 μg) for 24 h. BAX, BCL2‐associated X protein; CCD, coiled‐coil domain; CHAPS, 3‐[(3‐cholamidopropyl)‐dimethylammonio]‐1‐propane sulphonate; GST, glutathione‐S‐transferase; IB, immunoblotting; IgG, immunoglobulin G; IP, immunoprecipitation; RIPA, radioimmunoprecipitation assay; SDS-PAGE, SDS-polyacrylamide gel electrophoresis; UV, ultraviolet; UVRAG, ultraviolet irradiation resistance associated gene.


UVRAG inhibits mitochondrial translocation of BAX . After transfection with indicated shRNA or cDNA for 48 h , HL60 were treated with doxorubicin ( Doxo ) UV irradiation for 12 h , then BAX in cytosol ( Cyt ) mitochondria ( Mit ) was assayed by western blotting ( A - C ) activation of BAX ( D - F ) was assayed by western blotting after IP using BAX monoclonal antibody ( clone 6A7 ) * P0 . 05 . AU , arbitrary units ; BAX , BCL2 ‐ associated X ; CHAPS , 3 ‐ [ ( 3 ‐ cholamidopropyl ) ‐ dimethylammonio ] ‐ 1 ‐ propane sulphonate buffer ; Cyt c , cytochrome c ; IP , immunoprecipitation ; shRNA , short ‐ hairpin ; UV , ultraviolet ; UVRAG , ultraviolet irradiation resistance associated gene .

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

UVRAG inhibits mitochondrial translocation of BAX. After transfection with indicated shRNA or cDNA for 48 h, HL60 cells were treated with doxorubicin (Doxo) and UV irradiation for 12 h, and then BAX in cytosol (Cyt) and mitochondria (Mit) was assayed by western blotting (A-C) and activation of BAX (D-F) was assayed by western blotting after IP using BAX monoclonal antibody (clone 6A7) *P0.05. AU, arbitrary units; BAX, BCL2‐associated X protein; CHAPS, 3‐[(3‐cholamidopropyl)‐dimethylammonio]‐1‐propane sulphonate buffer; Cyt c, cytochrome c; IP, immunoprecipitation; shRNA, short‐hairpin RNA; UV, ultraviolet; UVRAG, ultraviolet irradiation resistance associated gene.


A . Average ribosome occupancy their respective codon bias - derived occupancy scores across CDS of transcript ( in 25 bins ) . Ribosome occupancy for 16 , 423 transcripts their respective codon bias - derived occupancy were firstly binned into 25 bins mean occupancy was calculated for each bin . B . Cumulative distribution plots showing distributions of correlations between ribosome occupancy codon bias - derived occupancy . Correlations obtained from ribosome occupancy scrambled codon bias - derived occupancy served as control . A Kolmogorov - Smirnov was performed between codon bias - derived occupancy control group . Data information : error bars represent 95 % confidence intervals .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Average ribosome occupancy and their respective codon bias-derived occupancy scores across the CDS of transcript (in 25 bins). Ribosome occupancy for 16,423 transcripts and their respective codon bias-derived occupancy were firstly binned into 25 bins and the mean occupancy was calculated for each bin. B. Cumulative distribution plots showing the distributions of correlations between ribosome occupancy and codon bias-derived occupancy. Correlations obtained from the ribosome occupancy and scrambled codon bias-derived occupancy served as the control. A Kolmogorov-Smirnov test was performed between the codon bias-derived occupancy and the control group. Data information: error bars represent the 95% confidence intervals.


C . Comparison of average transcript translation efficiencies ( TEs ) across their respective GC3 - content ranges . Number of transcripts within each gene optimality range is indicated above their respective points . Data information : error bars represent 95 % confidence intervals .

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C. Comparison of average transcript translation efficiencies (TEs) across their respective GC3-content ranges. Number of transcripts within each gene optimality range is indicated above their respective points. Data information: error bars represent the 95% confidence intervals.


A . HEK293 Tet - off experiments showing degradation of REL - OPT REL - WT transcripts ( left ) as well as IL6 - OPT , IL6 - WT IL6 - DE transcripts ( right ) , post - doxycycline addition . Data information : data is representative of 3 independent experiments each with 3 replicates . data represents mean ± SD for 3 replicates . A two - way ANOVA with Holm - Sidak multiple comparisons was performed . P - values are denoted as follows : p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) p < 0 . 001 ( * * * ) . half - lives of respective transcripts are indicated in brackets .

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

A. HEK293 Tet-off experiments showing the degradation of REL-OPT and REL-WT transcripts (left) as well as IL6-OPT, IL6-WT and IL6-DE transcripts (right), post-doxycycline addition. Data information: data is representative of 3 independent experiments each with 3 replicates. The data represents the mean ± SD for 3 replicates. A two-way ANOVA with Holm-Sidak multiple comparisons was performed. P-values are denoted as follows: p < 0.05 (*), p<0.01 (**) and p<0.001 (***). The half-lives of the respective transcripts are indicated in brackets.


B . Representative immunoblot of FLAG - tagged REL - OPT REL - WT in HEK293T transfected with either empty plasmids , plasmids bearing REL - OPT or REL - WT . immunoblot is representative of 3 independent experiments . ACTB is shown as loading controls .

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

B. Representative immunoblot of FLAG-tagged REL-OPT and REL-WT in HEK293T cells transfected with either empty plasmids, plasmids bearing REL-OPT or REL-WT. The immunoblot is representative of 3 independent experiments. ACTB is shown as the loading controls.


C . ELISA of secreted IL6 concentrations of IL6 - OPT , IL6 - WT IL6 - DE from HEK293T transfected with plasmids bearing IL6 - OPT , IL6 - WT IL6 - DE . Data information : In ( C ) , data is representative of 3 independent experiments each with 3 replicates . data represents mean ± SD for 3 replicates . A one - way ANOVA with Tukey ' s multiple comparisons was performed between samples where , p < 0 . 01 ( * * ) p < 0 . 001 ( * * * ) .

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

C. ELISA of secreted IL6 concentrations of IL6-OPT, IL6-WT and IL6-DE from HEK293T cells transfected with plasmids bearing IL6-OPT, IL6-WT and IL6-DE. Data information: In (C), the data is representative of 3 independent experiments each with 3 replicates. The data represents the mean ± SD for 3 replicates. A one-way ANOVA with Tukey's multiple comparisons was performed between samples where, p<0.01 (**) and p<0.001 (***).


D . Fold changes of REL - OPT REL - WT transcript ( top ) relative to their abundances from fraction 1 as detected by qPCR across polysome fractions ( below ) . Data represents mean ± SD for 3 biological replicates . Data information data is representative of 3 independent experiments each with 3 replicates . data represents mean ± SD for 3 replicates . A two - way ANOVA with Holm - Sidak multiple comparisons was performed . P - values are denoted as follows : p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) p < 0 . 001 ( * * * ) . half - lives of respective transcripts are indicated in brackets .

O O O O O B-CONTROLLED_VAR 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

D. Fold changes of REL-OPT and REL-WT transcript levels (top) relative to their abundances from fraction 1 as detected by qPCR across polysome fractions (below). Data represents the mean ± SD for 3 biological replicates. Data information data is representative of 3 independent experiments each with 3 replicates. The data represents the mean ± SD for 3 replicates. A two-way ANOVA with Holm-Sidak multiple comparisons was performed. P-values are denoted as follows: p < 0.05 (*), p<0.01 (**) and p<0.001 (***). The half-lives of the respective transcripts are indicated in brackets.


HEK293 Tet - off experiments showing degradation of REL - OPT REL - WT transcripts under vehicle ( DMSO ) - cycloheximide ( CHX ) - treatment , post - doxycycline addition . data is representative of 3 independent experiments each with 3 replicates . data represents mean ± SD for 3 replicates . A two - way ANOVA with Holm - Sidak multiple comparisons was performed . P - values are denoted as follows : p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) p < 0 . 001 ( * * * ) .

O O O O O O O O B-CONTROLLED_VAR 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

HEK293 Tet-off experiments showing the degradation of REL-OPT and REL-WT transcripts under vehicle (DMSO)- and cycloheximide (CHX)-treatment, post-doxycycline addition. data is representative of 3 independent experiments each with 3 replicates. The data represents the mean ± SD for 3 replicates. A two-way ANOVA with Holm-Sidak multiple comparisons was performed. P-values are denoted as follows: p < 0.05 (*), p<0.01 (**) and p<0.001 (***).


HEK293 Tet - off experiments showing degradation of IL6 - OPT , IL6 - WT IL6 - DE transcripts ( B ) , under vehicle ( DMSO ) - cycloheximide ( CHX ) - treatment , post - doxycycline addition . data is representative of 3 independent experiments each with 3 replicates . data represents mean ± SD for 3 replicates . A two - way ANOVA with Holm - Sidak multiple comparisons was performed . P - values are denoted as follows : p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) p < 0 . 001 ( * * * ) .

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

HEK293 Tet-off experiments showing the degradation of IL6-OPT, IL6-WT and IL6-DE transcripts (B), under vehicle (DMSO)- and cycloheximide (CHX)-treatment, post-doxycycline addition. data is representative of 3 independent experiments each with 3 replicates. The data represents the mean ± SD for 3 replicates. A two-way ANOVA with Holm-Sidak multiple comparisons was performed. P-values are denoted as follows: p < 0.05 (*), p<0.01 (**) and p<0.001 (***).


HEK293 Tet - off experiments showing degradation of REL - OPT REL - WT transcripts ( C ) under vehicle ( PBS ) - anisomycin ( ANI ) - treatment , post - doxycycline addition . data is representative of 3 independent experiments each with 3 replicates . data represents mean ± SD for 3 replicates . A two - way ANOVA with Holm - Sidak multiple comparisons was performed . P - values are denoted as follows : p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) p < 0 . 001 ( * * * ) .

O O O O O O O O B-CONTROLLED_VAR 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

HEK293 Tet-off experiments showing the degradation of REL-OPT and REL-WT transcripts (C) under vehicle (PBS)- and anisomycin (ANI)-treatment, post-doxycycline addition. data is representative of 3 independent experiments each with 3 replicates. The data represents the mean ± SD for 3 replicates. A two-way ANOVA with Holm-Sidak multiple comparisons was performed. P-values are denoted as follows: p < 0.05 (*), p<0.01 (**) and p<0.001 (***).


HEK293 Tet - off experiments showing degradation of IL6 - OPT , IL6 - WT IL6 - DE transcripts ( D ) , under vehicle ( PBS ) - anisomycin ( ANI ) - treatment , post - doxycycline addition . data is representative of 3 independent experiments each with 3 replicates . data represents mean ± SD for 3 replicates . A two - way ANOVA with Holm - Sidak multiple comparisons was performed . P - values are denoted as follows : p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) p < 0 . 001 ( * * * ) .

HEK293 Tet-off experiments showing the degradation of IL6-OPT, IL6-WT and IL6-DE transcripts (D), under vehicle (PBS)- and anisomycin (ANI)-treatment, post-doxycycline addition. data is representative of 3 independent experiments each with 3 replicates. The data represents the mean ± SD for 3 replicates. A two-way ANOVA with Holm-Sidak multiple comparisons was performed. P-values are denoted as follows: p < 0.05 (*), p<0.01 (**) and p<0.001 (***).


HEK293 Tet - off experiments showing degradation of REL - OPT , REL - OPT ( + 1 Frameshift ) REL - WT transcripts ( E ) post - doxycycline addition . data is representative of 3 independent experiments each with 3 replicates . data represents mean ± SD for 3 replicates . A two - way ANOVA with Holm - Sidak multiple comparisons was performed . P - values are denoted as follows : p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) p < 0 . 001 ( * * * ) .

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

HEK293 Tet-off experiments showing the degradation of REL-OPT, REL-OPT (+1 Frameshift) and REL-WT transcripts (E) post-doxycycline addition. data is representative of 3 independent experiments each with 3 replicates. The data represents the mean ± SD for 3 replicates. A two-way ANOVA with Holm-Sidak multiple comparisons was performed. P-values are denoted as follows: p < 0.05 (*), p<0.01 (**) and p<0.001 (***).


HEK293 Tet - off experiments showing degradation of IL6 - OPT , IL6 - WT , IL6 - DE IL6 - OPT ( + 1 Frameshift ) transcripts ( F ) post - doxycycline addition . data is representative of 3 independent experiments each with 3 replicates . data represents mean ± SD for 3 replicates . A two - way ANOVA with Holm - Sidak multiple comparisons was performed . P - values are denoted as follows : p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) p < 0 . 001 ( * * * ) .

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

HEK293 Tet-off experiments showing the degradation of IL6-OPT, IL6-WT, IL6-DE and IL6-OPT (+1 Frameshift) transcripts (F) post-doxycycline addition. data is representative of 3 independent experiments each with 3 replicates. The data represents the mean ± SD for 3 replicates. A two-way ANOVA with Holm-Sidak multiple comparisons was performed. P-values are denoted as follows: p < 0.05 (*), p<0.01 (**) and p<0.001 (***).


Volcano plots showing enrichment of RBPs which bind to REL - WT relative to REL - OPT transcripts ( A ) Data information : vertical dotted lines indicate a 1 . 5 - fold enrichment while horizontal dashed lines indicate p - value cut - off of 0 . 05 . Points shaded in blue indicate RBPs which have a differential fold change of more than 1 . 5 p < 0 . 05 .

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

Volcano plots showing the enrichment of RBPs which bind to REL-WT relative to REL-OPT transcripts (A) Data information: vertical dotted lines indicate a 1.5-fold enrichment while horizontal dashed lines indicate the p-value cut-off of 0.05. Points shaded in blue indicate RBPs which have a differential fold change of more than 1.5 and p<0.05.


Volcano plots showing enrichment of RBPs which bind to , IL6 - DE relative to IL6 - WT transcripts ( B ) Data information : vertical dotted lines indicate a 1 . 5 - fold enrichment while horizontal dashed lines indicate p - value cut - off of 0 . 05 . Points shaded in blue indicate RBPs which have a differential fold change of more than 1 . 5 p < 0 . 05 .

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

Volcano plots showing the enrichment of RBPs which bind to , IL6-DE relative to IL6-WT transcripts (B) Data information: vertical dotted lines indicate a 1.5-fold enrichment while horizontal dashed lines indicate the p-value cut-off of 0.05. Points shaded in blue indicate RBPs which have a differential fold change of more than 1.5 and p<0.05.


Volcano plots showing enrichment of RBPs which bind to IL6 - WT relative to IL6 - OPT transcripts ( C ) . Data information : , vertical dotted lines indicate a 1 . 5 - fold enrichment while horizontal dashed lines indicate p - value cut - off of 0 . 05 . Points shaded in blue indicate RBPs which have a differential fold change of more than 1 . 5 p < 0 . 05 .

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

Volcano plots showing the enrichment of RBPs which bind to IL6-WT relative to IL6-OPT transcripts (C). Data information: , vertical dotted lines indicate a 1.5-fold enrichment while horizontal dashed lines indicate the p-value cut-off of 0.05. Points shaded in blue indicate RBPs which have a differential fold change of more than 1.5 and p<0.05.


A . Cumulative distribution plots showing difference in distribution of transcript optimality between upregulated downregulated transcripts in K562 subject to ILF2 CRISPR interference targeting ILF2 . Transcript quantities are indicated in figure legend . Data information : Wilcoxon signed rank were performed on upregulated downregulated groups against control group . P - values are denoted ( right ) .

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 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

A. Cumulative distribution plots showing the difference in distribution of transcript optimality between upregulated and downregulated transcripts in K562 cells subject to ILF2 CRISPR interference targeting ILF2. Transcript quantities are indicated in the figure legend. Data information: Wilcoxon signed rank tests were performed on the upregulated and downregulated groups against the control group. P-values are denoted (right).


B , C . HEK293 Tet - off experiments showing degradation of REL - OPT ( B ) REL - WT ( C ) transcripts with ILF2 ILF3 siRNA Control ( CTR ) siRNA treatment , post - doxycycline addition . Data information : data is representative of 3 independent experiments in which data represents mean ± SD for 3 biological replicates . A two - way ANOVA with Holm - Sidak multiple comparisons was performed . P - values are denoted as follows : p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) .

O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, C. HEK293 Tet-off experiments showing the degradation of REL-OPT (B) and REL-WT (C) transcripts with ILF2 and ILF3 siRNA and Control (CTR) siRNA treatment, post-doxycycline addition. Data information: data is representative of 3 independent experiments in which the data represents the mean ± SD for 3 biological replicates. A two-way ANOVA with Holm-Sidak multiple comparisons was performed. P-values are denoted as follows: p < 0.05 (*), p<0.01 (**).


D . Representative immunoblot of FLAG - tagged REL - OPT REL - WT expressed in HEK293T under ILF2 ILF3 siRNA treatment . immunoblot is representative of 3 independent experiments . ACTB is shown as loading controls .

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D. Representative immunoblot of FLAG-tagged REL-OPT and REL-WT expressed in HEK293T cells under ILF2 and ILF3 siRNA treatment. The immunoblot is representative of 3 independent experiments. ACTB is shown as loading controls.


E . Representative immunoblot of FLAG - tagged REL - OPT REL - WT in HEK293T co - expressed with two different isoforms of ILF2 . immunoblot is representative of 3 independent experiments . ACTB is shown as loading controls .

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E. Representative immunoblot of FLAG-tagged REL-OPT and REL-WT in HEK293T cells co-expressed with two different isoforms of ILF2. The immunoblot is representative of 3 independent experiments. ACTB is shown as loading controls.


A . KD efficiency of three different Syt11 shRNAs in DRG neurons . Cultured DRG neurons were infected with control or shRNA - expressing lentiviruses at DIV 1 , Western blotting for Syt11 , Syt1 , Syt4 , synaptobrevin 2 ( Syb2 ) , SNAP25 , complexin 1 / 2 ( Cpx1 , Cpx2 ) , clathrin , adaptor 2 ( AP - 2 ) , β - actin were performed at DIV 6 - 7 . N = 4 independent experiments .

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A. KD efficiency of three different Syt11 shRNAs in DRG neurons. Cultured DRG neurons were infected with control or shRNA-expressing lentiviruses at DIV 1, and Western blotting for Syt11, Syt1, Syt4, synaptobrevin 2 (Syb2), SNAP25, complexin 1/2 (Cpx1, Cpx2), clathrin, adaptor protein 2 (AP-2), and β-actin were performed at DIV 6-7. N = 4 independent experiments.


B , C . Representative Cm traces induced by 200 - ms depolarization ( arrows ) ib DRG neurons . DRG neurons were transfected with plasmids expressing shSyt11 - 2 ( Syt11 KD , KD ) or scrambled shRNA ( Sc ) Cm recording was performed 5 days after transfection . Endo - 5s represents Cm decay 5 s after stimulation . Insets show Ca2 + currents recorded in same neurons .

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B, C. Representative Cm traces induced by 200-ms pulse depolarization (arrows) ib DRG neurons. DRG neurons were transfected with plasmids expressing shSyt11-2 (Syt11 KD, KD) or scrambled shRNA (Sc) and Cm recording was performed 5 days after transfection. Endo-5s represents the Cm decay 5 s after stimulation. Insets show Ca2+ currents recorded in the same neurons.


D . Representative Cm overshoot recorded from an Syt11 KD neuron . About 45 % of KD neurons showed excessive retrieval after a 200 - ms depolarizing .

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D. Representative Cm overshoot recorded from an Syt11 KD neuron. About 45% of the KD neurons showed excessive membrane retrieval after a 200-ms depolarizing pulse.


E . Averaged Cm traces recorded from Control ( Ctrl ) , Sc , KD ( from three shRNAs , n = 39 for shSyt11 - 2 , n = 7 for shSyt11 - 1 , n = 14 for shSyt11 - 3 ) , rescued ( with shSyt11 - 2 - resistant form of Syt11 ) DRG neurons .

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E. Averaged Cm traces recorded from Control (Ctrl), Sc, KD (from all three shRNAs, n = 39 for shSyt11-2, n = 7 for shSyt11-1, n = 14 for shSyt11-3), and rescued (with the shSyt11-2-resistant form of Syt11) DRG neurons.


F - H . Statistics of Cm jumps , Endo - 5s , Ca2 + current recorded from DRGneurons as in ( B - E ) . Data were collected from 4 ( Sc , Rescue ) , 8 ( Ctrl ) , 16 ( KD ) independent experiments .

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F-H. Statistics of Cm jumps, Endo-5s, and Ca2+ current recorded from DRGneurons as in (B-E). Data were collected from 4 (Sc, Rescue), 8 (Ctrl), and 16 (KD) independent experiments.


I . Normalized Cm changes induced by two pulses of 200 - ms depolarization ( arrows ) at a 1 - s interval . Data were normalized to capacitance jump induced by first depolarization .

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I. Normalized Cm changes induced by two pulses of 200-ms depolarization (arrows) at a 1-s interval. Data were normalized to the capacitance jump induced by the first depolarization.


J . Paired - ratios of ΔCm with different interstimulus intervals as in ( I ) . DRG neurons were stimulated with two 200 - ms pulses at different intervals as indicated , paired - was calculated by normalizing ΔCm in response to second stimulation to that induced by first one .

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J. Paired-pulse ratios of ΔCm with different interstimulus intervals as in (I). DRG neurons were stimulated with two 200-ms pulses at different intervals as indicated, and the paired-pulse ratio was calculated by normalizing the ΔCm in response to the second stimulation to that induced by the first one.


A , B . FM 1 - 43 uptake into DRG neurons stimulated by 100 mM K + for 30 s . right - shifted cumulative frequency in ( B ) indicates increased uptake level in KD neurons . Scale bars , 5 μm .

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A, B. FM 1-43 uptake into DRG neurons stimulated by 100 mM K+ for 30 s. The right-shifted cumulative frequency in (B) indicates the increased uptake level in KD neurons. Scale bars, 5 μm.


C . of KCl - evoked FM 1 - 43 uptake with different stimulation times .

C. Quantification of KCl-evoked FM 1-43 uptake with different stimulation times.


D . Representative showing increased constitutive FM 1 - 43 uptake ( for 5 min ) in KD neurons . Scale bars , 5 μm .

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D. Representative micrographs showing the increased constitutive FM 1-43 uptake (for 5 min) in KD neurons. Scale bars, 5 μm.


E . of constitutive FM 1 - 43 uptake with different loading times .

E. Quantification of constitutive FM 1-43 uptake with different loading times.


F . Basal [ Ca2 + ] i in DRG neurons measured with Fura - 2 .

F. Basal [Ca2+]i in DRG neurons measured with Fura-2.


G . FM1 - 43 uptake by Syt11 KD ( RFP - positive ) control ( RFP - negative ) hippocampal neurons stimulated by 100 mM K + for 2 min . Quantitative data are shown in lower - right panel . Scale bars , 20 μm .

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G. FM1-43 uptake by Syt11 KD (RFP-positive) and control (RFP-negative) hippocampal neurons stimulated by 100 mM K+ for 2 min. Quantitative data are shown in the lower-right panel. Scale bars, 20 μm.


H . Representative showing preloaded FM4 - 64 in presynaptic boutons of hippocampal neurons before after 800 stimuli at 40 Hz . Scale bars , 5 μm .

H. Representative images showing the preloaded FM4-64 fluorescence in presynaptic boutons of hippocampal neurons before and after 800 stimuli at 40 Hz. Scale bars, 5 μm.


I . Time - course of FM4 - 64 unloading from control , Syt11 KD , rescued nerve terminals within same field of view ( 6 coverslips from at least 3 biological repeats each ) .

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I. Time-course of FM4-64 unloading from control, Syt11 KD, and rescued nerve terminals within the same field of view (6 coverslips from at least 3 biological repeats each).


A . Averaged ΔCm traces induced by 200 - ms depolarization in presence absence of dynasore . Endocytic inhibition by dynasore ( Dyna ) was estimated by comparison of Cm traces recorded during 1 - 3 min ( KD ) 5 - 10 min ( KD + Dyna Ctrl + Dyna ) after whole - dialysis .

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A. Averaged ΔCm traces induced by 200-ms depolarization in the presence and absence of dynasore. Endocytic inhibition by dynasore (Dyna) was estimated by comparison of Cm traces recorded during 1-3 min (KD) and 5-10 min (KD + Dyna and Ctrl + Dyna) after whole-cell dialysis.


B , C . Endo - 5s Cm jumps recorded as in ( A ) .

B, C. Endo-5s and Cm jumps recorded as in (A).


D . Averaged ΔCm traces induced by 200 - ms depolarization in presence absence of dynole - 34 - 2TM ( Dynole ) . Cm traces endocytic rate were measured as in ( A ) except that 10 μM dynole - 34 - 2TM with 0 . 1 % DMSO was dialyzed into patched .

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D. Averaged ΔCm traces induced by 200-ms depolarization in the presence and absence of dynole-34-2TM (Dynole). Cm traces and endocytic rate were measured as in (A) except that 10 μM dynole-34-2TM with 0.1% DMSO was dialyzed into patched cells.


E , F . Endo - 5s Cm jumps as in ( D ) .

E, F. Endo-5s and Cm jumps as in (D).


G , H . Averaged ∆ Cm traces Endo - 5s recorded from KD neurons under control conditions ( 0 . 1 % DMSO ) .

G, H. Averaged ∆Cm traces and Endo-5s recorded from KD neurons under control conditions (0.1 % DMSO).


I . KCl - evoked FM 1 - 43 uptake in DRG neurons after 10 - min pre - incubation with 0 . 1 % DMSO ( control ) or with 100 μM dynasore .

I. KCl-evoked FM 1-43 uptake in DRG neurons after 10-min pre-incubation with 0.1% DMSO (control) or with 100 μM dynasore.


A . DRG neurons expressing Myc - Syt11 were immunostained for Myc - Syt11 , dynamin ( Dyn ) , AP - 2 , clathrin ( CL ) , TGN46 , synaptobrevin 2 ( Syb2 ) , LAMP1 . For dynamin , box along plasma was straightened , enlarged , is shown in upper right panels with arrows indicating co - localized . An enlarged inset of inside of ( box ) is shown in lower panel . For localization of transferrin receptor , EGFP - transferrin receptor ( TfR ) was expressed in DRG neurons . Scale bars , 10 μm .

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A. DRG neurons expressing Myc-Syt11 were immunostained for Myc-Syt11, dynamin (Dyn), AP-2, clathrin (CL), TGN46, synaptobrevin 2 (Syb2), and LAMP1. For dynamin, the box along the plasma membrane was straightened, enlarged, and is shown in the upper right panels with arrows indicating co-localized puncta. An enlarged inset of the inside of the cell (box) is shown in the lower panel. For localization of the transferrin receptor, EGFP-transferrin receptor (TfR) was expressed in DRG neurons. Scale bars, 10 μm.


B . of co - localization of Myc - Syt11 with dynamin , AP - 2 , clathrin , TGN46 , synaptobrevin 2 , transferrin receptor , LAMP1 . N = 3 independent experiments .

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B. Analysis of co-localization of Myc-Syt11 with dynamin, AP-2, clathrin, TGN46, synaptobrevin 2, transferrin receptor, and LAMP1. N = 3 independent experiments.


A . Transferrin ( Tf , red ) uptake in Syt11 KD ( GFP - positive ) control neurons . Quantitative data are on right . Scale bars , 20 μm .

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A. Transferrin (Tf, red) uptake in Syt11 KD (GFP-positive) and control neurons. Quantitative data are on the right. Scale bars, 20 μm.


B . Tf uptake in Syt11 over - expressing control neurons . Quantitative data are on right . Scale bars , 20 μm .

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B. Tf uptake in Syt11 over-expressing and control neurons. Quantitative data are on the right. Scale bars, 20 μm.


C . Normalized ∆ Cm induced by 200 - ms depolarization of a Syt11 KD neuron in presence of 100 μM MDC was fitted to a double - exponential decay function ( solid black red , fitted curves ) .

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C. Normalized ∆Cm induced by 200-ms depolarization of a Syt11 KD neuron in the presence of 100 μM MDC was fitted to a double-exponential decay function (solid black and red, fitted curves).


D . Fast slow time - constants of endocytosis in Syt11 KD neurons recorded as in ( C ) .

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D. Fast and slow time-constants of endocytosis in Syt11 KD neurons recorded as in (C).


E . KCl - evoked FM 1 - 43 uptake in DRG neurons after 1 - h pre - incubation with 0 . 1 % DMSO or 100 μM MDC in bath solution .

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E. KCl-evoked FM 1-43 uptake in DRG neurons after 1-h pre-incubation with 0.1% DMSO or 100 μM MDC in the bath solution.


A , B . Cm traces with bulk endocytosis ( arrowheads ) in control ( A ) KD ( B ) neurons induced by 200 - ms depolarization . Expanded bulk events are shown on right .

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A, B. Cm traces with bulk endocytosis (arrowheads) in control (A) and KD (B) neurons induced by 200-ms depolarization. Expanded bulk events are shown on the right.


C , D . Frequency distribution bulk endocytic events / within 16 s after stimulation .

C, D. Frequency distribution and bulk endocytic events/cell within 16 s after stimulation.


E , F . duration of Cm decay during bulk endocytosis .

E, F. Size and duration of Cm decay during bulk endocytosis.


G , H . Representative z - projected statistics showing large dextran uptake in Syt11 KD control DRG neurons . Neurons were loaded with 50 μM tetramethylrhodamine - dextran ( 40 kD ) by 2 - min exposure to 100 mM KCl . Scale bars , 10 μm .

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G, H. Representative z-projected fluorescence images and statistics showing the large dextran uptake in Syt11 KD and control DRG neurons. Neurons were loaded with 50 μM tetramethylrhodamine-dextran (40 kD) by 2-min exposure to 100 mM KCl. Scale bars, 10 μm.


A . Representative electron showing a small / clear vesicle ( SV , arrow ) , dense - vesicles ( DCVs , arrowheads ) a clathrin - coated pit ( hatched arrow ) in DRG neurons .

A. Representative electron micrographs showing a small/clear vesicle (SV, arrow), dense-core vesicles (DCVs, arrowheads) and a clathrin-coated pit (hatched arrow) in DRG neurons.


B , C . Diameter distributions of SVs DCVs ( statistics from 6 control 9 KD ) in DRG neurons . red green lines are curves fitted to a Gaussian function .

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B, C. Diameter distributions of SVs and DCVs (statistics from 6 control cells and 9 KD cells) in DRG neurons. The red and green lines are curves fitted to a Gaussian function.


D , E . Diameters of SVs DCVs in Syt11 KD control neurons with ( 100K , statistics from 6 control 9 KD ) or without 100 mM K + stimulation ( SE , statistics from 6 for each group ) .

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D, E. Diameters of SVs and DCVs in Syt11 KD and control neurons with (100K, statistics from 6 control cells and 9 KD cells) or without 100 mM K+ stimulation (SE, statistics from 6 cells for each group).


A . Representative electron of HRP uptake from standard ( SE ) or 100 mM K + ( 100K ) bath solution . Scale bar , 2 μm .

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A. Representative electron micrographs of HRP uptake from standard (SE) or 100 mM K+ (100K) bath solution. Scale bar, 2 μm.


B - D . HRP - labeled small vesicles ( filled arrows ) endosomes ( arrowheads ) in DRG neurons . Quantitative data are shown in ( C ) ( D ) . Scale bar , 200 nm .

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B-D. HRP-labeled small vesicles (filled arrows) and endosomes (arrowheads) in DRG neurons. Quantitative data are shown in (C) and (D). Scale bar, 200 nm.


E . Representative docked vesicles ( arrows ) in a DRG neuron . Quantitative data are on right . Scale bar , 200 nm .

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E. Representative docked vesicles (arrows) in a DRG neuron. Quantitative data are on the right. Scale bar, 200 nm.


F - H . Clathrin - coated pits ( CCPs , hatched arrows ) bulk - endocytosis - related structures of invaginating ( open arrowhead ) in DRGneurons . Quantitative data are shown in ( G ) ( H ) . Scale bars , 200 nm .

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F-H. Clathrin-coated pits (CCPs, hatched arrows) and bulk-endocytosis-related structures of invaginating membrane (open arrowhead) in DRGneurons. Quantitative data are shown in (G) and (H). Scale bars, 200 nm.


I , J . Cumulative frequencies of diameter distributions of small vesicles endosomes ( statistics from 6 control 9 KD ) . Kolmogorov - Smirnov , P = 0 . 304 for ( I ) P = 0 . 553 for ( J ) .

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I, J. Cumulative frequencies of diameter distributions of small vesicles and endosomes (statistics from 6 control cells and 9 KD cells). Kolmogorov-Smirnov test, P = 0.304 for (I) and P = 0.553 for (J).


Figure 9 . Functional domains of Syt11 in endocytosisA . Diagram of mutant forms of Syt11 used for rescue experiments . B - D . Statistics of FM4 - 64 ( 5 min ) , Tf , large dextran ( 40 - KD ) uptake into KD neurons with or without rescue by indicated forms of Syt11 . data are presented as mean ± s . e . m . of 3 independent experiments . One - way ANOVA , values labeled with different letters ( a , b , c ) are significantly different from each other , * * * P < 0 . 001 . Student ' s t - for comparison between KD control in ( B - D ) , * * P < 0 . 01 , * * * P < 0 . 001 .

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Figure 9. Functional domains of Syt11 in endocytosisA. Diagram of the mutant forms of Syt11 used for rescue experiments.B-D. Statistics of FM4-64 (5 min), Tf, and large dextran (40-KD) uptake into KD neurons with or without rescue by indicated forms of Syt11.All data are presented as mean ± s.e.m. of 3 independent experiments. One-way ANOVA, values labeled with different letters (a, b, and c) are significantly different from each other, ***P < 0.001. Student's t-test for the comparison between KD and control cells in (B-D), **P < 0.01, ***P < 0.001.


SUD binds to Paip1 Paip1M with same binding . n . s . : not significant .

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SUD binds to Paip1 and Paip1M with the same binding intensity. n. s.: not significant.


Mac2 subdomain is crucial for interacting with Paip1M .

The Mac2 subdomain is crucial for interacting with Paip1M.


Δ16 - Mac2 cannot bind Paip1M , Mac1 − 2 binds to Paip1M in split - YFP . Scale bars represent 50 µM .

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Δ16-Mac2 cannot bind Paip1M, Mac1−2 binds to Paip1M in the split-YFP assay. Scale bars represent 50 µM.


SUD PABP do not compete with each other for interacting with Paip1 . ( A ) Excess expression of SUD - RFP does not impair split - YFP signal between PABP Paip1 ( lower ) , compared to control ( upper ) . ( B ) Co - expression of excess RFP - PABP does not result in an impaired interaction between Paip1 SUD either . RFP : red . Scale bars represent 50 µM .

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SUD and PABP do not compete with each other for interacting with Paip1. (A) Excess expression of SUD-RFP does not impair the split-YFP signal between PABP and Paip1 (lower images), compared to the control (upper images). (B) Co-expression of excess RFP-PABP does not result in an impaired interaction between Paip1 and SUD either. RFP: red fluorescent protein. Scale bars represent 50 µM.


GFP - trap - co - immunoprecipitation ( CoIP ) demonstrating a formed by PABP , Paip1 , SUD . GFP : green .

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GFP-trap-based co-immunoprecipitation (CoIP) assay demonstrating a complex formed by PABP, Paip1, and SUD. GFP: green fluorescent protein.


Left image : SUD generally stimulates translation level detected by luciferase - pcDNA3 reporter ( n = 6 ) . Middle image : SUD does not increase amounts of total synthesis ( host viral ) in replicon - transfected . Right image : SUD augments viral synthesis ( n = 8 ) . HEK - 293 growing in 12 - well plates were transfected with indicated plasmids replicon . Twenty - four hours post - transfection , were harvested for Renilla luciferase activity measurement ( left right image ) . For ribopuromycylation , 24 hours post - transfection were pulsed with 3 µM puromycin for 1 hour at 37 ° C before harvesting for western blot ( middle image ) .

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Left image: SUD generally stimulates protein translation level detected by the luciferase-pcDNA3 reporter (n=6). Middle image: SUD does not increase the amounts of total protein synthesis (host and viral proteins) in the replicon-transfected cells. Right image: SUD augments viral protein synthesis (n=8). HEK-293 cells growing in 12-well plates were transfected with the indicated plasmids and replicon DNA. Twenty-four hours post-transfection, cells were harvested for Renilla luciferase activity measurement (left and right image). For the ribopuromycylation assay, 24 hours post-transfection cells were pulsed with 3 µM puromycin for 1 hour at 37°C before harvesting for western blot analysis (middle image).


SARS - CoV - 2 SUD ( SARS2 - SUD ) interacts with Paip1 in HEK - 293 as demonstrated by split - YFP .

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SARS-CoV-2 SUD (SARS2-SUD) interacts with Paip1 in HEK-293 cells as demonstrated by the split-YFP assay.


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SARS2-Mac2 binds to Paip1. SARS2-Mac2 but not Δ16-SARS2-Mac2 interacts with Paip1M.


MERS - CoV SUD - MC ( Mac3 − DPUP ) or XMC ( Mac1 − Mac3 − DPUP ) do not bind to Paip1 . Scale bars represent 50 µM .

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MERS-CoV SUD-MC (Mac3−DPUP) or XMC (Mac1−Mac3−DPUP) do not bind to Paip1. Scale bars represent 50 µM.


( A ) Venn diagram of STK38 partners identified in both starvation suspension conditions by proximity biotinylation coupled to mass spectrometry identification ( B ) representation of common STK38 interactors identified in both starvation suspension conditions according to their dynamic of association with STK38 ( with a minimal fold increase of 1 . 38 ± 0 . 06 ) . Unsupervised hierarchical clustering was generated on their association fold using Pearson correlation . ( C ) Representation of 10 most component enriched terms of two clusters ( assessed by using Gene Ontology ( GO )

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(A) Venn diagram of STK38 partners identified in both starvation and suspension conditions by proximity biotinylation assay coupled to mass spectrometry identification (B) Heatmap representation of common STK38 interactors identified in both starvation and suspension conditions according to their dynamic of association with STK38 (with a minimal fold increase of 1.38 ± 0.06). Unsupervised hierarchical clustering was generated based on their association fold using Pearson correlation. (C) Representation of the 10 most cellular component enriched terms of the two clusters (assessed by using the Gene Ontology (GO) database


( A ) STK38 interacts with XPO1 . HekRasV12 were transiently transfected with myc - STK38 ( wt ) together with either Flag - XPO1 ( wt ) plasmid , Flag - control ( ctrl = Sirt3 ) plasmid or without . 24h later , were incubated with Okadaic Acid ( OA ) , ( final = 1µM ) for 1 hour or with DMSO . Flag fusions were pulled - down co - immunoprecipited were analyzed by western blotting ( WB ) . Upper panel displays whole lysates ( WCL ) lower panel represents immunoprecipitated .

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(A) STK38 interacts with XPO1. HekRasV12 cells were transiently transfected with myc-STK38(wt) together with either Flag-XPO1(wt) plasmid, Flag-control (ctrl = Sirt3) plasmid or without DNA. 24h later, cells were incubated with Okadaic Acid (OA), (final concentration = 1µM) for 1 hour or with DMSO. Flag fusions were pulled-down and co-immunoprecipited proteins were analyzed by western blotting (WB). Upper panel displays whole cell lysates (WCL) and lower panel represents immunoprecipitated proteins.


STK38 accumulates in cytoplasm upon nutrient starvation in a XPO1 - dependant manner . ( B ) HeLa were transfected with myc - STK38 ( wt ) plasmid . next day , were incubated with DMEM or EBSS in presence of XPO1 inhibitors KPT - 185 or KPT - 330 as indicated ( final = 1 µM ) or DMSO for 4 hours . were then fixed stained for myc - tag . Representative are shown scale bars are 40 µm .

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STK38 accumulates in the cytoplasm upon nutrient starvation in a XPO1-dependant manner. (B) HeLa cells were transfected with myc-STK38(wt) plasmid. The next day, cells were incubated with DMEM or EBSS in the presence of XPO1 inhibitors KPT-185 or KPT-330 as indicated (final concentration = 1 µM) or DMSO for 4 hours. Cells were then fixed and stained for myc-tag. Representative images are shown and scale bars are 40 µm.


STK38 accumulates in cytoplasm upon nutrient starvation in a XPO1 - dependant manner . ( C ) of myc - STK38 ( wt ) / ( n > 30 from 3 independent experiments , mean ± ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , Mann - Whitney ) .

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STK38 accumulates in the cytoplasm upon nutrient starvation in a XPO1-dependant manner. (C) Quantification of myc-STK38(wt) nuclear/cytoplasmic staining (n > 30 cells from 3 independent experiments, mean ± SEM; *p<0.05, **p<0.01, ***p<0.001, Mann-Whitney test).


XPO1 activity is required for nutrient starvation - induced autophagy . ( D ) Immunoblotting with indicated antibodies of whole lysates of its graphical representation ( n = 3 independent experiments , mean ± ; * * * p < 0 . 001 , ns , not significant , Student ' s t ) . As expected , p62 degradation in starvation conditions was inhibited in presence of XPO1 inhibitors .

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XPO1 activity is required for nutrient starvation-induced autophagy. (D) Immunoblotting with indicated antibodies of whole cell lysates of cells and its graphical representation (n = 3 independent experiments, mean ± SEM; ***p<0.001, ns, not significant, Student's t test). As expected, p62 degradation in starvation conditions was inhibited in presence of XPO1 inhibitors.


XPO1 activity is required for nutrient starvation - induced autophagy . ( E ) HeLa stably expressing GFP - LC3 - RFP - LC3ΔG reporter autophagic probe [ 26 ] were incubated with DMEM or EBSS in presence of XPO1 inhibitors KPT - 185 or KPT - 330 ( final = 1 µM ) or DMSO for 4 hours . GFP - LC3 ( degraded upon autophagy ) RFP - LC3ΔG ( non - degraded upon autophagy ) signals were recorded by FACS then shown as a recapitulating overall LC3 level ( n = 3 independent experiments , mean ± ; * p < 0 . 05 , * * p < 0 . 01 , ns , not significant , Mann - Whitney ) . Here again , incubation with XPO1 inhibitors significantly impaired autophagy

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XPO1 activity is required for nutrient starvation-induced autophagy. (E) HeLa cells stably expressing the GFP-LC3-RFP-LC3ΔG reporter autophagic probe [26] were incubated with DMEM or EBSS in the presence of XPO1 inhibitors KPT-185 or KPT-330 (final concentration = 1 µM) or DMSO for 4 hours. The GFP-LC3 (degraded upon autophagy) and RFP-LC3ΔG (non-degraded upon autophagy) signals were recorded by FACS analysis and then shown as a ratio recapitulating overall LC3 level (n=3 independent experiments, mean ± SEM; *p<0.05, **p<0.01, ns, not significant, Mann-Whitney test). Here again, incubation with XPO1 inhibitors significantly impaired the autophagy process


STK38 kinase activity is required sufficient for its accumulation upon nutrient starvation . ( A ) HeLa silenced for endogenous STK38 were transfected with myc - STK38 ( wt ) expressing plasmid , myc - STK38 ( K118R ) ( STK38 kinase - dead version ) plasmid or with HA - STK38 ( PIF ) ( STK38 constitutively active version ) plasmid . 24 hours later , were incubated with DMEM or EBSS for 4 hours , fixed stained for myc - tag or HA - tag . Representative are shown scale bars are 40 µm .

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STK38 kinase activity is required and sufficient for its cytoplasmic accumulation upon nutrient starvation. (A) HeLa cells silenced for endogenous STK38 were transfected with myc-STK38(wt) expressing plasmid, myc-STK38(K118R) (STK38 kinase-dead version) plasmid or with HA-STK38(PIF) (STK38 constitutively active version) plasmid. 24 hours later, cells were incubated with DMEM or EBSS for 4 hours, fixed and stained for myc-tag or HA-tag. Representative images are shown and scale bars are 40 µm.


STK38 kinase activity is required sufficient for its accumulation upon nutrient starvation . ( B ) Graphical representation of tag - STK38 variants / ( n > 30 from 3 independent experiments , mean ± ; * * p < 0 . 01 , * * * p < 0 . 001 , ns , not significant , Mann - Whitney ) .

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STK38 kinase activity is required and sufficient for its cytoplasmic accumulation upon nutrient starvation. (B) Graphical representation of tag-STK38 variants nuclear staining/cytoplasmic staining (n > 30 cells from 3 independent experiments, mean ± SEM; **p<0.01, ***p<0.001, ns, not significant, Mann-Whitney test).


STK38 kinase activity is required sufficient for nutrient - starvation induced autophagy . ( C ) HeLa stably expressing GFP - LC3 - RFP - LC3ΔG autophagic probe [ 26 ] were transiently transfected with siRNA targeting 3 ' UTR region of endogenous STK38 ( or with non - targeting siRNA ( siNT ) ) . next day , were transiently transfected with indicated STK38 mutants expressing plasmids 24 hours after plasmid transfection , were incubated with DMEM or EBSS for 4 hours . GFP - LC3 ( degraded upon autophagy ) RFP - LC3ΔG ( non - degraded upon autophagy ) signals were recorded by FACS then shown as a recapitulating overall LC3 level ( n = 4 independent experiments , mean ± ; * p < 0 . 05 , * * * p < 0 . 001 , ns , not significant , Mann - Whitney ) . depleting STK38 prevents autophagy to take place . This effect was partially reversed by expressing wt ORF as well as constitutively active STK38 whereas kinase - dead version failed to reproduce endogenous STK38 effect . Expression of constitutively active STK38 is sufficient to induce a substantial change in autophagic flux upon nutrient rich

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

STK38 kinase activity is required and sufficient for nutrient-starvation induced autophagy. (C) HeLa cells stably expressing the GFP-LC3-RFP-LC3ΔG autophagic probe [26] were transiently transfected with siRNA targeting the 3'UTR region of endogenous STK38 (or with non-targeting siRNA (siNT)). The next day, cells were transiently transfected with the indicated STK38 mutants expressing plasmids 24 hours after plasmid transfection, cells were incubated with DMEM or EBSS for 4 hours. The GFP-LC3 (degraded upon autophagy) and RFP-LC3ΔG (non-degraded upon autophagy) signals were recorded by FACS analysis and then shown as a ratio recapitulating overall LC3 level (n=4 independent experiments, mean ± SEM; *p<0.05, ***p<0.001, ns, not significant, Mann-Whitney test). depleting STK38 prevents autophagy to take place. This effect was partially reversed by expressing the wt ORF as well as constitutively active STK38 whereas kinase-dead version failed to reproduce endogenous STK38 effect. Expression of constitutively active STK38 is sufficient to induce a substantial change in the autophagic flux upon nutrient rich conditions


STK38 kinase activity is required sufficient for nutrient - starvation induced autophagy . ( D ) HeLa were transiently transfected with siRNA targeting 3 ' UTR region of endogenous STK38 ( or with non - targeting siRNA ( siNT ) ) then transiently transfected with indicated STK38 mutants expressing plasmids 48h after . next day , were incubated with DMEM or EBSS for 4 hours overall p62 level was quantified for autophagy estimation ( n = 3 independent experiments , mean ± ; * * * p < 0 . 001 , ns , not significant , Student ' s t ) .

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STK38 kinase activity is required and sufficient for nutrient-starvation induced autophagy. (D) HeLa cells were transiently transfected with siRNA targeting the 3'UTR region of endogenous STK38 (or with non-targeting siRNA (siNT)) and then transiently transfected with the indicated STK38 mutants expressing plasmids 48h after. The next day, the cells were incubated with DMEM or EBSS for 4 hours and overall p62 level was quantified for autophagy estimation (n = 3 independent experiments, mean ± SEM; ***p<0.001, ns, not significant, Student's t test).


( A - B ) STK38 is required for XPO1 _ S1055 phosphorylation . ( A ) HeLa were transiently transfected with indicated siRNA subjected to Flag - XPO1 ( wt ) transient transfection following day . 48 hours later , were incubated with Okadaic Acid ( OA , final = 1 µM ) for 1 hour or with DMSO . Immunoblotting was performed on whole lysates with indicated antibodies . ( B ) Graphical representation of phospho S1055 - XPO1 signal on total XPO1 ( n = 3 independent experiments , mean ± ; * p < 0 . 05 , ns , not significant , Mann - Whitney ) . As expected , XPO1 is phosphorylated on its Ser1055 upon OA treatment but not when STK38 is

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(A-B) STK38 is required for XPO1_S1055 phosphorylation. (A) HeLa cells were transiently transfected with the indicated siRNA and subjected to Flag-XPO1(wt) transient transfection the following day. 48 hours later, cells were incubated with Okadaic Acid (OA, final concentration = 1 µM) for 1 hour or with DMSO. Immunoblotting was performed on whole cell lysates with indicated antibodies. (B) Graphical representation of the phospho S1055-XPO1 signal on total XPO1 (n = 3 independent experiments, mean ± SEM; *p<0.05, ns, not significant, Mann-Whitney test). As expected, XPO1 is phosphorylated on its Ser1055 upon OA treatment but not when STK38 is silenced


( C ) STK38 kinase activity is required for XPO1 _ S1055 phosphorylation . HekRasV12 silenced ( or not ) for endogenous STK38 were transfected with Flag - XPO1 ( wt ) expression plasmid in addition to myc - STK38 ( wt ) or myc - STK38 ( K118R ) ( STK38 kinase - dead version ) plasmids . next day , were treated with 1 µM OA or with vehicle ( DMSO ) for 1 hour . Flag fusions were immunoprecipited pulled - down were analyzed by western blotting . Upper panel displays immunoprecipited lower panel represents whole lysates .

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(C) STK38 kinase activity is required for XPO1_S1055 phosphorylation. HekRasV12 cells silenced (or not) for endogenous STK38 were transfected with Flag-XPO1(wt) expression plasmid in addition to myc-STK38(wt) or myc-STK38(K118R) (STK38 kinase-dead version) plasmids. The next day, cells were treated with 1 µM OA or with vehicle (DMSO) for 1 hour. Flag fusions were immunoprecipited and pulled-down proteins were analyzed by western blotting. Upper panel displays immunoprecipited proteins and lower panel represents whole cell lysates.


( A ) Graphical representation of XPO1 . C528 to S acid mutation confers XPO1 resistance to both KPT - 185 KPT - 330 chemical inhibitors [ 24 ] . S1055 acid correspond to phosphorylation target of STK38 . Finally , ΔCter region is also highlighted corresponding to a XPO1 construct lacking 39 C - terminal residues ( B - C ) XPO1 _ S1055 phosphorylation is required sufficient for STK38 exit upon nutrient starvation . ( B ) HeLa were transiently transfected with myc - STK38 ( wt ) in addition to indicated Flag - XPO1 mutant plasmids . 24 hours later , were incubated with DMEM or EBSS for 4 hours both supplemented with KPT - 185 ( final = 1 µM ) in order to inhibit endogenous XPO1 activity . were then fixed stained for Flag myc tags . Only positives for both Flag - XPO1 myc - STK38 ( wt ) were captured . Representative are shown scale bars are 40 µm . ( C ) Graphical representation of myc - STK38 ( wt ) / ( n > 30 from 3 independent experiments , mean ± ; * * * p < 0 . 001 , ns , not significant , Mann - Whitney ) . Expression of wt XPO1 failed to induce localization of STK38 upon nutrient starvation in presence of KPT - 185 where C528S mutant recapitulates results described in Figure 2B - C . Expression of phosphonegative XPO1 ( S1055A ) failed to induces STK38 localization upon EBSS treatment while phosphomimetics XPO1 ( S1055D S1055E ) were sufficient to promote STK38 localization without autophagic stimuli . Finally , expression of XPO1 lacking in its 39 C - terminal residues mimicked phosphomimetics variants .

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(A) Graphical representation of XPO1 protein. C528 to S amino acid mutation confers XPO1 resistance to both KPT-185 and KPT-330 chemical inhibitors [24]. S1055 amino acid correspond to the phosphorylation target of the STK38. Finally, the ΔCter region is also highlighted corresponding to a XPO1 construct lacking the 39 C-terminal residues (B-C) XPO1_S1055 phosphorylation is required and sufficient for STK38 nuclear exit upon nutrient starvation. (B) HeLa cells were transiently transfected with myc-STK38(wt) in addition to indicated Flag-XPO1 mutant plasmids. 24 hours later, cells were incubated with DMEM or EBSS for 4 hours both supplemented with KPT-185 (final concentration = 1 µM) in order to inhibit endogenous XPO1 activity. Cells were then fixed and stained for Flag and myc tags. Only cells positives for both Flag-XPO1 and myc-STK38(wt) were captured. Representative images are shown and scale bars are 40 µm. (C) Graphical representation of myc-STK38(wt) nuclear staining/cytoplasmic staining (n > 30 cells from 3 independent experiments, mean ± SEM; ***p<0.001, ns, not significant, Mann-Whitney test). Expression of wt XPO1 failed to induce cytoplasmic localization of STK38 upon nutrient starvation in presence of KPT-185 where C528S mutant recapitulates results described in Figure 2B-C. Expression of phosphonegative XPO1 (S1055A) failed to induces STK38 cytoplasmic localization upon EBSS treatment while phosphomimetics XPO1 (S1055D and S1055E) were sufficient to promote STK38 cytoplasmic localization without autophagic stimuli. Finally, expression of XPO1 lacking in its 39 C-terminal residues mimicked phosphomimetics variants.


( D ) HAP1 carrying genomic XPO1 mutations of S1055 were subjected for IMDM ( nutrient rich medium ) or EBSS ( starvation ) incubation for 4 hours followed by western - blot for p62 level measurement . This result indicates that p62 degradation in starvation conditions was inhibited in XPO1 _ S1055A HAP1 whereas p62 degradation was potentiated in both phosphomimetics variant ( S1055D & S1055E ) ( n = 4 independent experiments , mean ± ; * p < 0 . 05 , ns , not significant , Mann - Whitney

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(D) HAP1 cells carrying genomic XPO1 mutations of S1055 were subjected for IMDM (nutrient rich medium) or EBSS (starvation) incubation for 4 hours followed by western-blot analysis for p62 level measurement. This result indicates that p62 degradation in starvation conditions was inhibited in XPO1_S1055A HAP1 cells whereas p62 degradation was potentiated in both phosphomimetics variant (S1055D & S1055E) (n = 4 independent experiments, mean ± SEM; *p<0.05, ns, not significant, Mann-Whitney test


( E ) Hek - HT - iRFP - LC3 were transiently transfected with siRNA targeting endogenous STK38 ( or with non - targeting siRNA ( siNT ) ) . Two days after , were transiently transfected with indicated Flag - XPO1 mutant plasmids . 24 hours later , were incubated with DMEM or EBSS for 4 hours both supplemented with KPT - 185 ( final = 1 µM ) in order to inhibit endogenous XPO1 activity with chloroquine ( final = 10 µM ) . were then fixed number of iRFP - LC3 dots per was recorded only in positive for Flag - XPO1 mutants As expected , silencing of endogenous STK38 prevented formation of LC3 dots upon starvation where introduction of phosphonegative XPO1 ( S1055A ) failed also to increase LC3 dots upon starvation . Interestingly , phosphomimetic XPO1 ( S1055D ) was sufficient to increase number of LC3 dots even in complete also STK38 - depleted conditions .

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(E) Hek-HT-iRFP-LC3 cells were transiently transfected with siRNA targeting endogenous STK38 (or with non-targeting siRNA (siNT)). Two days after, cells were transiently transfected with indicated Flag-XPO1 mutant plasmids. 24 hours later, cells were incubated with DMEM or EBSS for 4 hours both supplemented with KPT-185 (final concentration = 1 µM) in order to inhibit endogenous XPO1 activity and with chloroquine (final concentration = 10 µM). Cells were then fixed and the number of iRFP-LC3 dots per cell was recorded only in cells positive for Flag-XPO1 mutants As expected, silencing of endogenous STK38 prevented the formation of LC3 dots upon starvation where introduction of phosphonegative XPO1 (S1055A) failed also to increase LC3 dots upon starvation. Interestingly, phosphomimetic XPO1 (S1055D) was sufficient to increase the number of LC3 dots even in complete and also STK38-depleted conditions.


( A ) XPO1 STK38 are required for Beclin1 accumulation upon nutrient starvation - induced autophagy . HeLa were transiently transfected with indicated siRNA ( control KPT conditions = siNT ) . 72 hours later , were incubated with XPO1 inhibitors KPT - 185 or KPT - 330 as indicated ( final = 1 µM ) or with DMSO for other conditions for 2 hours prior incubation with DMEM or EBSS supplemented ( or not ) with inhibitors for 2 hours . were fixed stained for endogenous Beclin1 . Graphical representation of endogenous Beclin1 / ( n > 30 from 3 independent experiments , mean ± ; * p < 0 . 05 , * * * p < 0 . 001 , ns , not significant , Mann - Whitney ) . XPO1 activity inhibition by both KPT - 185 KPT - 330 as well as STK38 silencing failed to induce Beclin1 accumulation upon nutrient

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(A) XPO1 and STK38 are required for Beclin1 cytoplasmic accumulation upon nutrient starvation-induced autophagy. HeLa cells were transiently transfected with the indicated siRNA (control and KPT conditions = siNT). 72 hours later, cells were incubated with XPO1 inhibitors KPT-185 or KPT-330 as indicated (final concentration = 1 µM) or with DMSO for all other conditions for 2 hours prior incubation with DMEM or EBSS supplemented (or not) with inhibitors for 2 hours. Cells were fixed and stained for endogenous Beclin1. Graphical representation of endogenous Beclin1 nuclear staining/cytoplasmic staining (n > 30 cells from 3 independent experiments, mean ± SEM; *p<0.05, ***p<0.001, ns, not significant, Mann-Whitney test). XPO1 activity inhibition by both KPT-185 and KPT-330 as well as STK38 silencing failed to induce Beclin1 cytoplasmic accumulation upon nutrient starvation


( B ) XPO1 _ S1055 phosphorylation is required sufficient for Beclin1 exit upon nutrient starvation . Genome edited XPO1 mutant HAP1 were incubated with IMDM ( complete ) or EBSS ( starvation ) for 2 hours . were then fixed stained for endogenous Beclin1 . Graphical representation of endogenous Beclin1 / ( n > 30 from 3 independent experiments , mean ± ; * * * p < 0 . 001 , ns , not significant , Mann - Whitney ) . Here , phosphonegative XPO1 failed to induce Beclin1 accumulation upon nutrient starvation whereas phosphomimetics variant ( S1055D & S1055E ) potentiated Beclin1

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(B) XPO1_S1055 phosphorylation is required and sufficient for Beclin1 nuclear exit upon nutrient starvation. Genome edited XPO1 mutant HAP1 cells were incubated with IMDM (complete) or EBSS (starvation) for 2 hours. Cells were then fixed and stained for endogenous Beclin1. Graphical representation of endogenous Beclin1 nuclear staining/cytoplasmic staining (n > 30 cells from 3 independent experiments, mean ± SEM; ***p<0.001, ns, not significant, Mann-Whitney test). Here, phosphonegative XPO1 failed to induce Beclin1 cytoplasmic accumulation upon nutrient starvation whereas phosphomimetics variant (S1055D & S1055E) potentiated Beclin1 cytoplasmic accumulation


( C ) Genome edited XPO1 mutant HAP1 were transiently transfected with siRNA targeting endogenous STK38 ( siSTK38 # 206 or with non - targeting siRNA ( siNT ) ) . 72 hours later , were incubated with IMDM ( complete ) or EBSS ( starvation ) for 2 hours . were then fixed stained for endogenous Beclin1 . Graphical representation of endogenous Beclin1 / ( n = 30 from 2 independent experiments ) . Here , phosphomimetic XPO1 ( S1055D ) potentiated Beclin1 localization in both complete starvation condition even in absence of STK38 as compared to carrying wt

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(C) Genome edited XPO1 mutant HAP1 cells were transiently transfected with siRNA targeting endogenous STK38 (siSTK38#206 or with non-targeting siRNA (siNT)). 72 hours later, cells were incubated with IMDM (complete) or EBSS (starvation) for 2 hours. Cells were then fixed and stained for endogenous Beclin1. Graphical representation of endogenous Beclin1 nuclear staining/cytoplasmic staining (n = 30 cells from 2 independent experiments). Here, phosphomimetic XPO1 (S1055D) potentiated Beclin1 cytoplasmic localization in both complete and starvation condition even in the absence of STK38 as compared to cells carrying wt XPO1