C118 of caspase - 4 was required for the GPx8 - mediated inhibitory effect . THP - 1 cells stably expressing empty vector ( EV ) , FLAG - tagged caspase - 4 ( WT , C258S , C118S mutants ) , or GPx8 ( WT and C79S mutants ) were confirmed by immunoblotting ( B ) . Data information : All data shown are representative of at least 3 independent experiments .

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C118 of caspase-4 was required for the GPx8-mediated inhibitory effect. THP-1 cells stably expressing empty vector (EV), FLAG-tagged caspase-4 (WT, C258S, C118S mutants), or GPx8 (WT and C79S mutants) were confirmed by immunoblotting (B). Data information: All data shown are representative of at least 3 independent experiments.


C118 of caspase - 4 was required for the GPx8 - mediated inhibitory effect . ( C - D ) Casp4C118S completely abolished the inhibition of noncanonical inflammasome signaling in response to intracellular LPS . THP - 1 cells were differentiated , primed , and transfected with LPS . Activation of the noncanonical inflammasome by different quantities of cytoplasmic LPS is confirmed by the production of IL - 1β ( C ) and LDH ( D ) . In C and D , data are presented as the mean ± SD , n = 4 , technical repeats . * * * P < 0 . 001 ( Student ' s two ‐ tailed t ‐ test ) .

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C118 of caspase-4 was required for the GPx8-mediated inhibitory effect. (C-D) Casp4C118S completely abolished the inhibition of noncanonical inflammasome signaling in response to intracellular LPS. THP-1 cells were differentiated, primed, and transfected with LPS. Activation of the noncanonical inflammasome by different quantities of cytoplasmic LPS is confirmed by the production of IL-1β (C) and LDH (D). In C and D, data are presented as the mean ± SD, n=4, technical repeats. ***P < 0.001 (Student's two‐tailed t‐test).


A . The caspase - 4 / 11 and - 1 inhibitor , VX - 765 , inhibited noncanonical inflammasome activation . BMDMs isolated from mice were primed with LPS for 5 - 6 h , treated with 20 μM VX - 765 , and transfected with LPS for 12 h . Data information data are presented as the mean ± SD , n = 5 , technical repeats . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( Student ' s two ‐ tailed t ‐ test ) .

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A. The caspase-4/11 and -1 inhibitor, VX-765, inhibited noncanonical inflammasome activation. BMDMs isolated from mice were primed with LPS for 5-6 h, treated with 20 μM VX-765, and transfected with LPS for 12 h. Data information data are presented as the mean ± SD, n=5, technical repeats. *P < 0.05; **P < 0.01; ***P < 0.001 (Student's two‐tailed t‐test).


Mice were injected intraperitoneally with VX - 765 ( 20 mg / kg ) under colitis model and analyzed by a Kaplan - Meier survival plot ( B ) of mice with colitis induced by 4 % DSS for 6 days ( n = 10 - 11 per group ) . Data information : , data are presented as the mean ± SEM . NS : not significant ( P > 0 . 05 ) . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( Student ' s two ‐ tailed t ‐ test ) .

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Mice were injected intraperitoneally with VX-765 (20 mg/kg) under colitis model and analyzed by a Kaplan-Meier survival plot (B) of mice with colitis induced by 4% DSS for 6 days (n = 10-11 per group). Data information: , data are presented as the mean ± SEM. NS: not significant (P > 0.05). *P < 0.05; **P < 0.01; ***P < 0.001 (Student's two‐tailed t‐test).


Mice were injected intraperitoneally with VX - 765 ( 20 mg / kg ) under colitis model and the percentage of body weight ( C ) and clinical scores ( D ) of mice with colitis induced by 4 % DSS for 6 days ( n = 10 - 11 per group ) . data are presented as the mean ± SEM . NS : not significant ( P > 0 . 05 ) . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( Student ' s two ‐ tailed t ‐ test ) .

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Mice were injected intraperitoneally with VX-765 (20 mg/kg) under colitis model and the percentage of body weight (C) and clinical scores (D) of mice with colitis induced by 4% DSS for 6 days (n = 10-11 per group). data are presented as the mean ± SEM. NS: not significant (P > 0.05). *P < 0.05; **P < 0.01; ***P < 0.001 (Student's two‐tailed t‐test).


Mice were injected intraperitoneally with VX - 765 ( 20 mg / kg ) under colitis model Colon samples from mice treated with 4 % DSS for 5 days ( n = 6 - 10 per group ) . Full - length and cleavage forms of caspase - 11 , caspase - 1 , and GSDMD ( E ) in colon tissue lysates were analyzed by immunoblots on day 7 after DSS treatment . data are presented as the mean ± SEM . NS : not significant ( P > 0 . 05 ) . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( Student ' s 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 B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-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

Mice were injected intraperitoneally with VX-765 (20 mg/kg) under colitis model Colon samples from mice treated with 4% DSS for 5 days (n = 6-10 per group). Full-length and cleavage forms of caspase-11, caspase-1, and GSDMD (E) in colon tissue lysates were analyzed by immunoblots on day 7 after DSS treatment. data are presented as the mean ± SEM. NS: not significant (P > 0.05). *P < 0.05; **P < 0.01; ***P < 0.001 (Student's two‐tailed t‐test).


Mice were injected intraperitoneally with VX - 765 ( 20 mg / kg ) under colitis model Colon samples from mice treated with 4 % DSS for 5 days ( n = 6 - 10 per group ) . Production of IL - 1β and IL - 6 ( F ) in colon tissue lysates was assessed on day 14 after treatment . data are presented as the mean ± SEM . NS : not significant ( P > 0 . 05 ) . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( Student ' s 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 B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Mice were injected intraperitoneally with VX-765 (20 mg/kg) under colitis model Colon samples from mice treated with 4% DSS for 5 days (n = 6-10 per group). Production of IL-1β and IL-6 (F) in colon tissue lysates was assessed on day 14 after treatment. data are presented as the mean ± SEM. NS: not significant (P > 0.05). *P < 0.05; **P < 0.01; ***P < 0.001 (Student's two‐tailed t‐test).


A . GPx8 was predominantly expressed in macrophages of UC patients . Colon biopsies of non - inflamed regions from UC patients were stained with the pan - macrophage marker , CD68 , or the monocyte / macrophage activating marker , CD163 , combined with anti - GPx8 . Quantification of double - positive cells was calculated from 3 individual patients and is presented as the mean ± SEM of the percentage of double - positive cells . The total cell numbers are the sum of double - and single - positive cells .

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A. GPx8 was predominantly expressed in macrophages of UC patients. Colon biopsies of non-inflamed regions from UC patients were stained with the pan-macrophage marker, CD68, or the monocyte/macrophage activating marker, CD163, combined with anti-GPx8. Quantification of double-positive cells was calculated from 3 individual patients and is presented as the mean ± SEM of the percentage of double-positive cells. The total cell numbers are the sum of double- and single-positive cells.


B . Relative expression levels of GPx8 , GPx7 and caspase - 4 in non - inflamed biopsy specimens from healthy controls ( Ctrl ) ( n = 14 ) and UC patients ( n = 25 ) are demonstrated by immunoblots . C . Data are normalized to the mean of healthy controls and presented as the mean ± SEM . Mann Whitney test for GPx8 and Student ' s two ‐ tailed t ‐ test for GPx7 and caspase - 4 expression analysis . Data information : NS : not significant ( P > 0 . 05 ) . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 .

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B. Relative expression levels of GPx8, GPx7 and caspase-4 in non-inflamed biopsy specimens from healthy controls (Ctrl) (n = 14) and UC patients (n = 25) are demonstrated by immunoblots. C. Data are normalized to the mean of healthy controls and presented as the mean ± SEM. Mann Whitney test for GPx8 and Student's two‐tailed t‐test for GPx7 and caspase-4 expression analysis. Data information: NS: not significant (P > 0.05). *P < 0.05; **P < 0.01; ***P < 0.001.


D . Paired GPx8 and caspase - 4 expression from each individual are linked by lines and presented as the mean ± SEM . The p - value was analyzed by the Fisher ' s exact test . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 .

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D. Paired GPx8 and caspase-4 expression from each individual are linked by lines and presented as the mean ± SEM. The p-value was analyzed by the Fisher's exact test. *P < 0.05; **P < 0.01; ***P < 0.001.


( D ) Quantification of EGFP - LC3 puncta in MCF7 - EGFP - LC3 cells 56 h after transfection with the indicated siRNAs . When indicated , cells were exposed to 100 nM rapamycin for the last 3 h . RPTOR ( raptor ) and BECN1 ( beclin1 ) siRNAs served as positive and negative controls , respectively .

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(D) Quantification of EGFP-LC3 puncta in MCF7-EGFP-LC3 cells 56 h after transfection with the indicated siRNAs. When indicated, cells were exposed to 100 nM rapamycin for the last 3 h. RPTOR (raptor) and BECN1 (beclin1) siRNAs served as positive and negative controls, respectively.


( E ) Autophagic flux was measured as the ratio between luciferase activities in MCF7 - RLuc - LC3WT and MCF7 - RLuc - LC3G120A cells transfected with the indicated siRNAs 56 h earlier and left untreated or treated with 100 nM rapamycin for the last 3 h . Error bars are SDs for a representative ( n = 5 ) triplicate experiment with a minimum of 4 × 10 randomly chosen areas / sample analyzed ( D ) or three independent experiments ( F ) . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 , as compared with similarly treated control siRNA - transfected samples .

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(E) Autophagic flux was measured as the ratio between luciferase activities in MCF7-RLuc-LC3WT and MCF7-RLuc-LC3G120A cells transfected with the indicated siRNAs 56 h earlier and left untreated or treated with 100 nM rapamycin for the last 3 h. Error bars are SDs for a representative (n = 5) triplicate experiment with a minimum of 4 × 10 randomly chosen areas/sample analyzed (D) or three independent experiments (F). *, P < 0.05; **, P < 0.01; ***, P < 0.001, as compared with similarly treated control siRNA-transfected samples.


( A ) Representative immunoblots of indicated proteins from whole - cell lysates of MCF7 cells transfected with indicated siRNAs for 56 h and subjected to 0 - 40 min of amino acid starvation [ − AA ] ) .

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(A) Representative immunoblots of indicated proteins from whole-cell lysates of MCF7 cells transfected with indicated siRNAs for 56 h and subjected to 0-40 min of amino acid starvation [−AA]).


( B ) Representative immunoblots of the indicated proteins from whole - cell lysates of MCF7 - EGFP - LC3 cells stably infected with control or CIP2Ar ( siRNA - resistant CIP2A ) - encoding lentivirus , transfected with the indicated siRNAs for 53 h , and treated with 100 nM rapamycin ( Rapa ) for 0 - 20 min .

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(B) Representative immunoblots of the indicated proteins from whole-cell lysates of MCF7-EGFP-LC3 cells stably infected with control or CIP2Ar (siRNA-resistant CIP2A)-encoding lentivirus, transfected with the indicated siRNAs for 53 h, and treated with 100 nM rapamycin (Rapa) for 0-20 min.


( C ) MCF7 - EGFP - LC3 cells stably infected with control or CIP2Ar - encoding lentivirus were transfected with indicated siRNAs for 48 h and analyzed for EGFP - LC3 puncta formation .

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(C) MCF7-EGFP-LC3 cells stably infected with control or CIP2Ar-encoding lentivirus were transfected with indicated siRNAs for 48 h and analyzed for EGFP-LC3 puncta formation.


( D ) MCF7 - EGFP - LC3 cells transfected with control ( − ) and CIP2A siRNAs together with an empty vector ( − ) or MYC - S62A cDNA as indicated were analyzed 56 h later for EGFP - LC3 puncta ( left ) and expression of indicated proteins ( right ) .

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(D) MCF7-EGFP-LC3 cells transfected with control (−) and CIP2A siRNAs together with an empty vector (−) or MYC-S62A cDNA as indicated were analyzed 56 h later for EGFP-LC3 puncta (left) and expression of indicated proteins (right).


( E ) Representative immunoblots ( left ) of indicated proteins from whole - cell lysates of MCF7 cells treated with the control ( C ) , RPTOR ( RP ) , or CIP2A ( CIP ) siRNAs for 56 h and densitometric quantification of three independent experiments ( right ) . Error bars show SDs for three independent experiments . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 , compared with Student ' s t test as indicated ( C and D ) or with control siRNA - transfected samples ( E ) . Black lines indicate that intervening lanes have been spliced out .

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(E) Representative immunoblots (left) of indicated proteins from whole-cell lysates of MCF7 cells treated with the control (C), RPTOR (RP), or CIP2A (CIP) siRNAs for 56 h and densitometric quantification of three independent experiments (right). Error bars show SDs for three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001, compared with Student's t test as indicated (C and D) or with control siRNA-transfected samples (E). Black lines indicate that intervening lanes have been spliced out.


( A , top left ) Quantification of EGFP - LC3 puncta in MCF7 - EGFP - LC3 cells infected with control or CIP2A lentiviral shRNAs . Representative immunofluorescence images ( right ) and immunoblots ( bottom ) are shown . ( B ) Mean cell diameters of MCF7 - EGFP - LC3 cells infected as in A were measured with a cytometer ( NucleoCounter NC - 3000 ; Chemometec AS ) equipped with Via1 - Cassettes ( Chemometec AS ) .

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(A, top left) Quantification of EGFP-LC3 puncta in MCF7-EGFP-LC3 cells infected with control or CIP2A lentiviral shRNAs. Representative immunofluorescence images (right) and immunoblots (bottom) are shown. (B) Mean cell diameters of MCF7-EGFP-LC3 cells infected as in A were measured with a cytometer (NucleoCounter NC-3000; Chemometec AS) equipped with Via1-Cassettes (Chemometec AS).


( C ) MCF7 cells infected with shRNAs as in A were plated in equal density and left untreated or treated with 100 nM rapamycin ( Rapa ) for 72 h . The viable cells were either visualized after fixation and crystal violet staining ( left ; representative figure , n = 4 ) or trypsinized and counted with NucleoCounter NC - 3000 equipped with Via1 - Cassettes ( right ) .

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(C) MCF7 cells infected with shRNAs as in A were plated in equal density and left untreated or treated with 100 nM rapamycin (Rapa) for 72 h. The viable cells were either visualized after fixation and crystal violet staining (left; representative figure, n = 4) or trypsinized and counted with NucleoCounter NC-3000 equipped with Via1-Cassettes (right).


( D ) MCF7 cells infected with shRNAs as in A were left untreated or treated with 2 nM ConA , 2 µM MG132 , 5 µM siramesine ( Sira ) , or 100 µM etoposide ( Etop ) for 48 h , and the metabolic activity ( cell density ) was measured by the MTT assay ( left ) , and cell death was measured by the LDH assay ( right ) .

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(D) MCF7 cells infected with shRNAs as in A were left untreated or treated with 2 nM ConA, 2 µM MG132, 5 µM siramesine (Sira), or 100 µM etoposide (Etop) for 48 h, and the metabolic activity (cell density) was measured by the MTT assay (left), and cell death was measured by the LDH assay (right).


( E ) Light microscopy pictures of MCF10A cells at passage 8 after infection with control or CIP2A - encoding lentivirus .

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(E) Light microscopy pictures of MCF10A cells at passage 8 after infection with control or CIP2A-encoding lentivirus.


( F ) Representative immunoblots of the indicated proteins from whole - cell lysates of MCF10A ( left ) and HEK - 293 ( right ) cells infected as in E and treated with 100 nM rapamycin for the indicated times . Black lines indicate that intervening lanes have been spliced out .

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(F) Representative immunoblots of the indicated proteins from whole-cell lysates of MCF10A (left) and HEK-293 (right) cells infected as in E and treated with 100 nM rapamycin for the indicated times. Black lines indicate that intervening lanes have been spliced out.


CIP2A expression correlates with mTORC1 activity in primary human breast tumors . TMAs including 33 normal breast tissues and 210 primary breast carcinoma samples were analyzed by semiquantitative immunohistochemistry for the expression of the indicated proteins and mean scores ( arbitrary units [ a . u . ] ) for duplicate samples were averaged and plotted according to mean CIP2A expression ( left ) . The red lines are the regression lines , and the stippled red lines define the 95 % confidence intervals . Statistical analysis of data was performed using NCSS 2007 and Prism version 5 . Examples of the staining of breast cancer ( a ) and normal ( b ) cores indicated in the P - T389 - S6K1 / CIP2A plot are shown on the right . rsp , Spearman ' s correlation coefficient .

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CIP2A expression correlates with mTORC1 activity in primary human breast tumors. TMAs including 33 normal breast tissues and 210 primary breast carcinoma samples were analyzed by semiquantitative immunohistochemistry for the expression of the indicated proteins and mean scores (arbitrary units [a.u.]) for duplicate samples were averaged and plotted according to mean CIP2A expression (left). The red lines are the regression lines, and the stippled red lines define the 95% confidence intervals. Statistical analysis of data was performed using NCSS 2007 and Prism version 5. Examples of the staining of breast cancer (a) and normal (b) cores indicated in the P-T389-S6K1/CIP2A plot are shown on the right. rsp, Spearman's correlation coefficient.


( A and B ) In vitro translated full - length CIP2A was incubated with purified human PP2A complex and HA - Raptor ( A ) or HA - S6K1 ( B ) purified from transiently transfected MCF7 cells . Protein complexes were immunoprecipitated with anti - HA antibody and analyzed by immunoblotting as indicated . Nonspecific IgG served as a control . Note that human reticulocyte ( RC ) lysate used for in vitro translation of CIP2A contained detectable amounts of PP2Ac .

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(A and B) In vitro translated full-length CIP2A was incubated with purified human PP2A complex and HA-Raptor (A) or HA-S6K1 (B) purified from transiently transfected MCF7 cells. Protein complexes were immunoprecipitated with anti-HA antibody and analyzed by immunoblotting as indicated. Nonspecific IgG served as a control. Note that human reticulocyte (RC) lysate used for in vitro translation of CIP2A contained detectable amounts of PP2Ac.


( C ) Endogenous CIP2A protein complexes immunoprecipitated from MCF7 cells were analyzed by immunoblotting and PP2A activity assay . Immunoblots of corresponding cell lysates are shown on the right .

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(C) Endogenous CIP2A protein complexes immunoprecipitated from MCF7 cells were analyzed by immunoblotting and PP2A activity assay. Immunoblots of corresponding cell lysates are shown on the right.


( D ) Endogenous raptor protein complexes immunoprecipitated from MCF7 cells stably infected with control ( Ctr ) or CIP2A shRNA lentiviruses ( Fig . 3 A ) and starved for amino acids for 20 min when indicated were analyzed by immunoblotting and PP2A activity assay . Immunoblots of corresponding cell lysates are shown on the right .

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(D) Endogenous raptor protein complexes immunoprecipitated from MCF7 cells stably infected with control (Ctr) or CIP2A shRNA lentiviruses (Fig. 3 A) and starved for amino acids for 20 min when indicated were analyzed by immunoblotting and PP2A activity assay. Immunoblots of corresponding cell lysates are shown on the right.


( E ) Raptor protein complexes immunoprecipitated from MCF7 cells treated with control ( C ) or PPP2R1A ( R1A ) siRNAs for 56 h were analyzed by immunoblotting with the indicated antibodies . Immunoblots of corresponding cell lysates are shown on the left .

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(E) Raptor protein complexes immunoprecipitated from MCF7 cells treated with control (C) or PPP2R1A (R1A) siRNAs for 56 h were analyzed by immunoblotting with the indicated antibodies. Immunoblots of corresponding cell lysates are shown on the left.


( F ) HA - S6K1 protein complexes immunoprecipitated from MCF7 cells stably infected with control or CIP2A shRNA lentiviruses ( Fig . 3 A ) , transiently transfected with HA - RPS6K1 cDNA and starved for amino acids for 20 min when indicated , were analyzed by immunoblotting . Immunoblots of corresponding cell lysates are shown on the right .

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(F) HA-S6K1 protein complexes immunoprecipitated from MCF7 cells stably infected with control or CIP2A shRNA lentiviruses (Fig. 3 A), transiently transfected with HA-RPS6K1 cDNA and starved for amino acids for 20 min when indicated, were analyzed by immunoblotting. Immunoblots of corresponding cell lysates are shown on the right.


( G ) Enzymatic activity of a purified PP2A holoenzyme incubated with human reticulocyte lysate with or without in vitro translated full - length CIP2A . Error bars are SDs for four ( D ) independent experiments or a representative triplicate experiment ( C and G ) .

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(G) Enzymatic activity of a purified PP2A holoenzyme incubated with human reticulocyte lysate with or without in vitro translated full-length CIP2A. Error bars are SDs for four (D) independent experiments or a representative triplicate experiment (C and G).


( H ) Representative confocal microscopy images of untreated and amino acid - starved ( 4 h ) MCF7 cells stained for endogenous CIP2A and raptor . Black lines indicate that intervening lanes have been spliced out . − AA , amino acid starvation ; IP , immunoprecipitation ; a . u . , arbitrary unit . Bar , 10 µm .

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(H) Representative confocal microscopy images of untreated and amino acid-starved (4 h) MCF7 cells stained for endogenous CIP2A and raptor. Black lines indicate that intervening lanes have been spliced out. −AA, amino acid starvation; IP, immunoprecipitation; a.u., arbitrary unit. Bar, 10 µm.


( A ) Representative immunoblots of the indicated proteins from whole - cell lysates of MCF7 cells subjected to 100 nM rapamycin or amino acid starvation for 0 - 24 h .

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(A) Representative immunoblots of the indicated proteins from whole-cell lysates of MCF7 cells subjected to 100 nM rapamycin or amino acid starvation for 0-24 h.


( B ) qPCR analysis of CIP2A and MYC mRNA levels in MCF7 cells treated as in A . AA - starv , amino acid starvation ; a . u . , arbitrary unit . Error bars show SDs for three independent experiments .

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(B) qPCR analysis of CIP2A and MYC mRNA levels in MCF7 cells treated as in A. AA-starv, amino acid starvation; a.u., arbitrary unit. Error bars show SDs for three independent experiments.


( C ) Representative immunoblots of the indicated proteins from whole - cell lysates of MCF7 cells treated with ULK1 or SQSTM1 siRNAs for 54 h before the exposure to fresh medium ( 0 ) or amino acid starvation ( − AA ) for 12 h . Ctr , control .

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(C) Representative immunoblots of the indicated proteins from whole-cell lysates of MCF7 cells treated with ULK1 or SQSTM1 siRNAs for 54 h before the exposure to fresh medium (0) or amino acid starvation (−AA) for 12 h. Ctr, control.


( D ) Representative immunoblots of the indicated proteins from whole - cell lysates of MCF7 cells treated for 0 - 5 h with the indicated combinations of 100 µM cycloheximide ( CHX ) , 100 nM rapamycin , and 2 nM ConA .

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(D) Representative immunoblots of the indicated proteins from whole-cell lysates of MCF7 cells treated for 0-5 h with the indicated combinations of 100 µM cycloheximide (CHX), 100 nM rapamycin, and 2 nM ConA.


( E ) Representative confocal images of MCF7 ( left and middle ) and MCF7 - LC3 - EGFP ( right ) cells left untreated or treated with 100 nM rapamycin for 4 h or 100 nM rapamycin and 2 nM ConA for 8 h , fixed , and stained with antibodies against CIP2A . Nuclei in MCF - LC3 - EGFP cells were visualized with Hoechst . Green arrowheads indicate cells with many LC3 - positive puncta and low CIP2A levels , and yellow arrowheads show cells in which LC3 and CIP2A colocalize . Bars , 10 µm .

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(E) Representative confocal images of MCF7 (left and middle) and MCF7-LC3-EGFP (right) cells left untreated or treated with 100 nM rapamycin for 4 h or 100 nM rapamycin and 2 nM ConA for 8 h, fixed, and stained with antibodies against CIP2A. Nuclei in MCF-LC3-EGFP cells were visualized with Hoechst. Green arrowheads indicate cells with many LC3-positive puncta and low CIP2A levels, and yellow arrowheads show cells in which LC3 and CIP2A colocalize. Bars, 10 µm.


( F ) Representative immunoblots of an endogenous CIP2A protein complex immunoprecipitated from lysates of MCF7 cells left untreated or treated for 4 h with 100 nM rapamycin ( Rapa ) and 2 nM ConA as indicated . Mouse IgG served as a negative control .

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(F) Representative immunoblots of an endogenous CIP2A protein complex immunoprecipitated from lysates of MCF7 cells left untreated or treated for 4 h with 100 nM rapamycin (Rapa) and 2 nM ConA as indicated. Mouse IgG served as a negative control.


( G ) MCF7 cells were treated with 100 nM rapamycin , 2 nM ConA , and 2 µM MG132 for 24 h as indicated . Endogenous CIP2A was immunoprecipitated ( IP ) in stringent conditions ( no coimmunoprecipitation of p62 ) and analyzed by immunoblotting using antibodies against CIP2A , monoubiquitin , and polyubiquitin . Short ( < 100 kD ) , medium ( > 150 kD ) , and long ( 100 - 130 kD ) exposures are shown .

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(G) MCF7 cells were treated with 100 nM rapamycin, 2 nM ConA, and 2 µM MG132 for 24 h as indicated. Endogenous CIP2A was immunoprecipitated (IP) in stringent conditions (no coimmunoprecipitation of p62) and analyzed by immunoblotting using antibodies against CIP2A, monoubiquitin, and polyubiquitin. Short (<100 kD), medium (>150 kD), and long (100-130 kD) exposures are shown.


( H ) Proteins from MCF7 cell lysates were immunoprecipitated with the indicated antibodies in stringent conditions ( see G ) and analyzed by immunoblotting for CIP2A and monoubiquitin .

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(H) Proteins from MCF7 cell lysates were immunoprecipitated with the indicated antibodies in stringent conditions (see G) and analyzed by immunoblotting for CIP2A and monoubiquitin.


( I ) Lysates from MCF7 cells transfected with the indicated ubiquitin - HA constructs were subjected to anti - CIP2A immunopurification followed by immunoblotting with antibodies against CIP2A and HA .

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(I) Lysates from MCF7 cells transfected with the indicated ubiquitin-HA constructs were subjected to anti-CIP2A immunopurification followed by immunoblotting with antibodies against CIP2A and HA.


( J ) Lysates from MCF7 cells transfected with the CIP2A - FLAG construct ( Junttila et al . , 2007 ) and the indicated ubiquitin - HA constructs were subjected to anti - FLAG immunopurification in denaturing conditions with 0 . 1 % SDS followed by immunoblotting with the indicated antibodies . Black lines indicate that intervening lanes have been spliced out . Ub , ubiquitin ; WT , wild type .

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(J) Lysates from MCF7 cells transfected with the CIP2A-FLAG construct (Junttila et al., 2007) and the indicated ubiquitin-HA constructs were subjected to anti-FLAG immunopurification in denaturing conditions with 0.1% SDS followed by immunoblotting with the indicated antibodies. Black lines indicate that intervening lanes have been spliced out. Ub, ubiquitin; WT, wild type.


D Bax / BAK1 protein levels were detected by western blotting .

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D Bax/BAK1 protein levels were detected by western blotting.


G , Results of immunoblotting with immunoprecipitates ( IP ) or whole - cell lysates from HEK293T cells transfected with different constructs as indicated .

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G, Results of immunoblotting with immunoprecipitates (IP) or whole-cell lysates from HEK293T cells transfected with different constructs as indicated.


H Results of immunoblotting with immunoprecipitates ( IP ) or whole - cell lysates from HEK293T cells transfected with different constructs as indicated .

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H Results of immunoblotting with immunoprecipitates (IP) or whole-cell lysates from HEK293T cells transfected with different constructs as indicated.


I Immublotting results with IP or input ( whole - cell lysates ) from mouse midbrain , SH - SY5Y , HEK293T or HeLa cells as indicated . The α - ctrl represents that the magnetic beads used in IP were not pre - incubated with any antibody .

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I Immublotting results with IP or input (whole-cell lysates) from mouse midbrain, SH-SY5Y, HEK293T or HeLa cells as indicated. The α-ctrl represents that the magnetic beads used in IP were not pre-incubated with any antibody.


B Results of immunoblotting with immunoprecipitates ( IP ) or whole - cell lysates from cells transfected with Bcl - 2 and TMEM175 or its mutants TMEM175 - V145A , TMEM175 - R377V , as indicated . C Statistics of the relative TMEM175 protein levels normalized to GAPDH in vector , TMEM175 , TMEM175 - V145A or TMEM175 - R377V overexpressing HEK293T cells . Data information : The data are presented as the mean ± SEM . Statistical significance was calculated with two - sided student ' s t - tests ( C and is indicated with NS for not significant ( P > 0 . 05 ) , * * for p < 0 . 01 , and * * * for p < 0 . 001 . n value means the number of biological replicates made for each data point .

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B Results of immunoblotting with immunoprecipitates (IP) or whole-cell lysates from cells transfected with Bcl-2 and TMEM175 or its mutants TMEM175-V145A, TMEM175-R377V, as indicated. C Statistics of the relative TMEM175 protein levels normalized to GAPDH in vector, TMEM175, TMEM175-V145A or TMEM175-R377V overexpressing HEK293T cells. Data information: The data are presented as the mean ± SEM. Statistical significance was calculated with two-sided student's t-tests (C and is indicated with NS for not significant (P > 0.05), ** for p < 0.01, and *** for p < 0.001. n value means the number of biological replicates made for each data point.


K ( Upper ) schematic diagram showing the strategy of TMEM175 - KO in HEK293T cells . 92 bp in exon 4 and intron 4 were removed from the tmem175 locus . ( Lower ) TMEM175 levels in the WT or TMEM175 KO cells were detected by Western - blotting .

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K (Upper) schematic diagram showing the strategy of TMEM175-KO in HEK293T cells. 92 bp in exon 4 and intron 4 were removed from the tmem175 locus. (Lower) TMEM175 levels in the WT or TMEM175 KO cells were detected by Western-blotting.


L CM - H2DCFDA ( green ) indicating cellular ROS levels of WT or TMEM175 KO HEK293T cells pre - treatment with vehicle or 10 μM HA14 - 1 for 20 min . Scale bars = 10 μm . M Statistics of CM - H2DCFDA fluorescence intensity of ( L ) . The n values represent the cell numbers counted within the same experiment . Data information : The data are presented as the mean ± SEM . Statistical significance was calculated with two - sided student ' s t - tests M ) and is indicated with NS for not significant ( P > 0 . 05 ) , * for p < 0 . 05 , * * for p < 0 . 01 , and * * * for p < 0 . 001 . In panel M , n value means the number of technical replicates .

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L CM-H2DCFDA (green) indicating cellular ROS levels of WT or TMEM175 KO HEK293T cells pre-treatment with vehicle or 10 μM HA14-1 for 20 min. Scale bars = 10 μm. M Statistics of CM-H2DCFDA fluorescence intensity of (L). The n values represent the cell numbers counted within the same experiment. Data information: The data are presented as the mean ± SEM. Statistical significance was calculated with two-sided student's t-tests M) and is indicated with NS for not significant (P > 0.05), * for p < 0.05, ** for p < 0.01, and *** for p < 0.001. In panel M, n value means the number of technical replicates.


F Fluorescence of G3BP1 excited with 488 nm in control ( upper ) and TMEM175 - overexpressing ( lower ) cells . Scale bars = 10 μm . G Statistics of the numbers of puncta per cell ( left ) and the percentage of cells with puncta in ( F ) . We counted 547 cells in Vector group from 37 different fields of view and 332 cells in TMEM175 group from 76 different fields of view . n value represents the number of different fields of view randomly selected in the same imaging experiment . Data information : The data are presented as the mean ± SEM . Statistical significance was calculated with two - sided student ' s t - test , and is indicated with * * for p < 0 . 01 , and * * * for p < 0 . 001 . In panels G n value means the number of technical replicates .

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F Fluorescence of G3BP1 excited with 488 nm in control (upper) and TMEM175-overexpressing (lower) cells. Scale bars = 10 μm. G Statistics of the numbers of puncta per cell (left) and the percentage of cells with puncta in (F). We counted 547 cells in Vector group from 37 different fields of view and 332 cells in TMEM175 group from 76 different fields of view. n value represents the number of different fields of view randomly selected in the same imaging experiment. Data information: The data are presented as the mean ± SEM. Statistical significance was calculated with two-sided student's t-test, and is indicated with ** for p < 0.01, and *** for p < 0.001. In panels G n value means the number of technical replicates.


H Co - localization of mitochondrial indicator TOM20 - mCherry and lysosomal indicator LysoTracker in WT ( upper row ) or TMEM175 KO ( lower row ) HEK293T cells . Co - localization of mitochondrion and lysosome is observed as a yellow colour in the merged image ( marked by white arrows ) . Scale bars = 10 μm . I The number of merged puncta per cell in ( H ) . We counted 139 cells in WT group from 38 different fields of view and 148 cells in TMEM175 KO group from 42 different fields of view . n value represents the number of different fields of view randomly selected in the same imaging experiment . Data information : The data are presented as the mean ± SEM . Statistical significance was calculated with two - sided student ' s t - test , and is indicated with * * for p < 0 . 01 , and * * * for p < 0 . 001 . In panels I , n value means the number of technical replicates .

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H Co-localization of mitochondrial indicator TOM20-mCherry and lysosomal indicator LysoTracker in WT (upper row) or TMEM175 KO (lower row) HEK293T cells. Co-localization of mitochondrion and lysosome is observed as a yellow colour in the merged image (marked by white arrows). Scale bars = 10 μm. I The number of merged puncta per cell in (H). We counted 139 cells in WT group from 38 different fields of view and 148 cells in TMEM175 KO group from 42 different fields of view. n value represents the number of different fields of view randomly selected in the same imaging experiment. Data information: The data are presented as the mean ± SEM. Statistical significance was calculated with two-sided student's t-test, and is indicated with ** for p < 0.01, and *** for p < 0.001. In panels I, n value means the number of technical replicates.


A Representative immunoblots for TMEM175 expression levels in the midbrains of mice of different ages ( W : weeks ; M : months ) . B Statistics of the relative TMEM175 protein levels normalized to GAPDH in ( A ) . Data information : The data are presented as the mean ± SEM . Statistical significance was analyzed with two - sided student ' s t - tests , and is indicated with NS for not significant ( p > 0 . 05 ) , * for p < 0 . 05 , * * for p < 0 . 01 , and * * * for p < 0 . 001 . n value means the number of biological replicates made for each data point .

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A Representative immunoblots for TMEM175 expression levels in the midbrains of mice of different ages (W: weeks; M: months). B Statistics of the relative TMEM175 protein levels normalized to GAPDH in (A). Data information: The data are presented as the mean ± SEM. Statistical significance was analyzed with two-sided student's t-tests, and is indicated with NS for not significant (p > 0.05), * for p < 0.05, ** for p < 0.01, and *** for p < 0.001. n value means the number of biological replicates made for each data point.


D TMEM175 mRNA levels in the midbrains of WT or TMEM175 KO mice were detected by RT - qPCR using SYBR Green PCR Master Mix . Data information : The data are presented as the mean ± SEM . Statistical significance was analyzed with two - sided student ' s t - tests , and is indicated with NS for not significant ( p > 0 . 05 ) , * for p < 0 . 05 , * * for p < 0 . 01 , and * * * for p < 0 . 001 . n value means the number of biological replicates made for each data point .

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D TMEM175 mRNA levels in the midbrains of WT or TMEM175 KO mice were detected by RT-qPCR using SYBR Green PCR Master Mix. Data information: The data are presented as the mean ± SEM. Statistical significance was analyzed with two-sided student's t-tests, and is indicated with NS for not significant (p > 0.05), * for p < 0.05, ** for p < 0.01, and *** for p < 0.001. n value means the number of biological replicates made for each data point.


E The knockout of TMEM175 in mouse was verified by Western blot of brain tissues .

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E The knockout of TMEM175 in mouse was verified by Western blot of brain tissues.


F Representative TMEM175 basal lysosomal currents recorded in macrophages of WT or TMEM175 - KO mice . Ψ is lysosomal membrane potential ( deﬁned as Vcytosol - Vlumen ) . The currents were elicited with ramp voltage protocols ( - 100 to + 100 mV in 1 s , Vh = 0 mV ) . G Statistics of the current amplitudes of ( F ) . Data information : The data are presented as the mean ± SEM . Statistical significance was analyzed with two - sided student ' s t - tests , and is indicated with NS for not significant ( p > 0 . 05 ) , * for p < 0 . 05 , * * for p < 0 . 01 , and * * * for p < 0 . 001 . n value means the number of biological replicates made for each data point .

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F Representative TMEM175 basal lysosomal currents recorded in macrophages of WT or TMEM175-KO mice. Ψ is lysosomal membrane potential (deﬁned as Vcytosol - Vlumen). The currents were elicited with ramp voltage protocols (-100 to +100 mV in 1 s, Vh = 0 mV). G Statistics of the current amplitudes of (F). Data information: The data are presented as the mean ± SEM. Statistical significance was analyzed with two-sided student's t-tests, and is indicated with NS for not significant (p > 0.05), * for p < 0.05, ** for p < 0.01, and *** for p < 0.001. n value means the number of biological replicates made for each data point.


H , Quantification of rears in the cylinder ( H ) pre - and post - MPTP treatment . Data information : The data are presented as the mean ± SEM . Statistical significance was analyzed with two - sided student ' s t - tests , and is indicated with NS for not significant ( p > 0 . 05 ) , * for p < 0 . 05 , * * for p < 0 . 01 , and * * * for p < 0 . 001 . n value means the number of biological replicates made for each data point .

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H, Quantification of rears in the cylinder (H) pre- and post-MPTP treatment. Data information: The data are presented as the mean ± SEM. Statistical significance was analyzed with two-sided student's t-tests, and is indicated with NS for not significant (p > 0.05), * for p < 0.05, ** for p < 0.01, and *** for p < 0.001. n value means the number of biological replicates made for each data point.


I Quantification of the latency to fall from a wire hang apparatus ( I ) pre - and post - MPTP treatment . Data information : The data are presented as the mean ± SEM . Statistical significance was analyzed with two - sided student ' s t - tests , and is indicated with NS for not significant ( p > 0 . 05 ) , * for p < 0 . 05 , * * for p < 0 . 01 , and * * * for p < 0 . 001 . n value means the number of biological replicates made for each data point .

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I Quantification of the latency to fall from a wire hang apparatus (I) pre- and post-MPTP treatment. Data information: The data are presented as the mean ± SEM. Statistical significance was analyzed with two-sided student's t-tests, and is indicated with NS for not significant (p > 0.05), * for p < 0.05, ** for p < 0.01, and *** for p < 0.001. n value means the number of biological replicates made for each data point.


J Swing Duration CV ( CV % ) ( Left panel ) and Ataxia Coefficient in Gait tests 7 days post - MPTP . Data information : The data are presented as the mean ± SEM . Statistical significance was analyzed with two - sided student ' s t - tests , and is indicated with NS for not significant ( p > 0 . 05 ) , * for p < 0 . 05 , * * for p < 0 . 01 , and * * * for p < 0 . 001 . n value means the number of biological replicates made for each data point .

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J Swing Duration CV (CV%) (Left panel) and Ataxia Coefficient in Gait tests 7 days post-MPTP. Data information: The data are presented as the mean ± SEM. Statistical significance was analyzed with two-sided student's t-tests, and is indicated with NS for not significant (p > 0.05), * for p < 0.05, ** for p < 0.01, and *** for p < 0.001. n value means the number of biological replicates made for each data point.


K Latency to fall in rotarod tests 7 days post - MPTP . Data information : The data are presented as the mean ± SEM . Statistical significance was analyzed with two - sided student ' s t - tests , and is indicated with NS for not significant ( p > 0 . 05 ) , * for p < 0 . 05 , * * for p < 0 . 01 , and * * * for p < 0 . 001 . n value means the number of biological replicates made for each data point .

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K Latency to fall in rotarod tests 7 days post-MPTP. Data information: The data are presented as the mean ± SEM. Statistical significance was analyzed with two-sided student's t-tests, and is indicated with NS for not significant (p > 0.05), * for p < 0.05, ** for p < 0.01, and *** for p < 0.001. n value means the number of biological replicates made for each data point.


L Immunoblots of the TH levels post - MPTP treatment in WT and TMEM175 KO mouse midbrains . M Quantification of the relative TH levels normalized to GAPDH post - MPTP . Data information : The data are presented as the mean ± SEM . Statistical significance was analyzed with two - sided student ' s t - tests , and is indicated with NS for not significant ( p > 0 . 05 ) , * for p < 0 . 05 , * * for p < 0 . 01 , and * * * for p < 0 . 001 . n value means the number of biological replicates made for each data point .

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L Immunoblots of the TH levels post-MPTP treatment in WT and TMEM175 KO mouse midbrains. M Quantification of the relative TH levels normalized to GAPDH post-MPTP. Data information: The data are presented as the mean ± SEM. Statistical significance was analyzed with two-sided student's t-tests, and is indicated with NS for not significant (p > 0.05), * for p < 0.05, ** for p < 0.01, and *** for p < 0.001. n value means the number of biological replicates made for each data point.


N TH immunofluorescent labeling on substantia nigra slices of WT or TMEM175 - KO mice post - MPTP treatment . Scale bars = 100 μm . O Statistics of the TH - positive neurons in ( N ) . Data information : The data are presented as the mean ± SEM . Statistical significance was analyzed with two - sided student ' s t - tests , and is indicated with NS for not significant ( p > 0 . 05 ) , * for p < 0 . 05 , * * for p < 0 . 01 , and * * * for p < 0 . 001 . n value means the number of biological replicates made for each data point .

O B-MEASURED_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 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

N TH immunofluorescent labeling on substantia nigra slices of WT or TMEM175-KO mice post-MPTP treatment. Scale bars = 100 μm. O Statistics of the TH-positive neurons in (N). Data information: The data are presented as the mean ± SEM. Statistical significance was analyzed with two-sided student's t-tests, and is indicated with NS for not significant (p > 0.05), * for p < 0.05, ** for p < 0.01, and *** for p < 0.001. n value means the number of biological replicates made for each data point.


factor . a , Duplicate screens depicting HCoV - 229E infection ( 24 hours post - infection ) in Huh7 cells ectopically expressing ISGs .

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factor.a, Duplicate screens depicting HCoV-229E infection (24 hours post-infection) in Huh7 cells ectopically expressing ISGs.


b , HCoV - 229E infection of Huh7 cells expressing LY6E or control vector ( Empty ) . Blue : DAPI , green : HCoV - 229E N protein , red : TagRFP encoded in SCRPSY vector .

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b, HCoV-229E infection of Huh7 cells expressing LY6E or control vector (Empty). Blue: DAPI, green: HCoV-229E N protein, red: TagRFP encoded in SCRPSY vector.


c - h , Effect of LY6E expression on diverse coronaviruses . Stably transduced cells were infected with HCoV - 229E ( c ) , HCoV - OC43 ( d ) , MERS - CoV ( e ) , SARS - CoV ( f ) , SARS - CoV - 2 ( g ) , MHV - Gluc ( h ) . Viral replication readouts included : titer determination by plaque assay ( c ) , infectivity by nucleoprotein staining and quantitation by flow cytometry ( d ) , limiting dilution assay ( e , f , g ) , luciferase activity shown as relative light units ( RLU ) ( h , j , l ) .

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c-h, Effect of LY6E expression on diverse coronaviruses. Stably transduced cells were infected with HCoV-229E (c), HCoV-OC43 (d), MERS-CoV (e), SARS-CoV (f), SARS-CoV-2 (g), MHV-Gluc (h). Viral replication readouts included: titer determination by plaque assay (c), infectivity by nucleoprotein staining and quantitation by flow cytometry (d), limiting dilution assay (e,f,g), luciferase activity shown as relative light units (RLU) (h,j,l).


i , Western blot of WT A549 or two LY6E KO clones ( # 3 , # 4 ) .

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i, Western blot of WT A549 or two LY6E KO clones (#3, #4).


j , HCoV - 229E infection of WT or LY6E KO A549 .

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j, HCoV-229E infection of WT or LY6E KO A549.


k , Western blot of WT or LY6E KO A549 reconstituted with CRISPR - resistant LY6E ( CR LY6E ) or control vector ( Empty ) .

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k, Western blot of WT or LY6E KO A549 reconstituted with CRISPR-resistant LY6E (CR LY6E) or control vector (Empty).


l , HCoV - 229E infection of CR LY6E - reconstituted WT or LY6E KO A549 .

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l, HCoV-229E infection of CR LY6E-reconstituted WT or LY6E KO A549.


formation . a - d , VSV pseudoparticles ( PP ) harboring spike proteins from HCoV - 229E ( a ) , MERS - CoV ( b ) , SARS - CoV ( c ) , SARS - CoV - 2 ( d ) were inoculated on LY6E - or empty vector - expressing cells . Virus entry efficiency was quantified by measuring virus - encoded luciferase reporter activity .

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formation.a-d, VSV pseudoparticles (PP) harboring spike proteins from HCoV-229E (a), MERS-CoV (b), SARS-CoV (c), SARS-CoV-2 (d) were inoculated on LY6E- or empty vector-expressing cells. Virus entry efficiency was quantified by measuring virus-encoded luciferase reporter activity.


e , VSV pseudoparticles expressing VSV G protein on their surface and encoding CoV S protein and GFP ( VSV * ΔG ( CoV S ) ) were inoculated with LY6E - or empty vector - expressing Huh7 . Syncytia formation was analyzed by immunofluorescence microscopy . Blue : DAPI , green : GFP , red : TagRFP inserted in SCRPSY vector .

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e, VSV pseudoparticles expressing VSV G protein on their surface and encoding CoV S protein and GFP (VSV*ΔG(CoV S)) were inoculated with LY6E- or empty vector-expressing Huh7. Syncytia formation was analyzed by immunofluorescence microscopy. Blue: DAPI, green: GFP, red: TagRFP inserted in SCRPSY vector.


f , Quantification of VSV * ΔG ( CoV S ) induced syncytia depicted as percentage syncytia area . Three independent areas were analyzed per biological replicate ( circle , square , triangle ) .

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f, Quantification of VSV*ΔG(CoV S) induced syncytia depicted as percentage syncytia area. Three independent areas were analyzed per biological replicate (circle, square, triangle).


h , Cell - cell fusion was measured by co - incubating cells transfected with plasmids encoding CoV S proteins and a split - luciferase construct ( DSP1 - 7 ) together with CoV permissive LY6E - expressing or control cells transfected with the second fragment of split - luciferase ( DSP8 - 11 ) .

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h, Cell-cell fusion was measured by co-incubating cells transfected with plasmids encoding CoV S proteins and a split-luciferase construct (DSP1-7) together with CoV permissive LY6E-expressing or control cells transfected with the second fragment of split-luciferase (DSP8-11).


virus . a - b , Female Ly6efl / fl and Ly6eΔHSC mice were injected with PBS or MHV and monitored for survival ( a ) and weight loss ( b ) .

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virus.a-b, Female Ly6efl/fl and Ly6eΔHSC mice were injected with PBS or MHV and monitored for survival (a) and weight loss (b).


c - n , Mice were assessed at 3 ( c - h ) or 5 ( i - n ) days post - infection for serum ALT ( c , i ) , viral titers in liver ( d , j ) and spleen ( e , k ) , and liver necrosis and inflammation ( f - h , l - n ) .

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c-n, Mice were assessed at 3 (c-h) or 5 (i-n) days post-infection for serum ALT (c,i), viral titers in liver (d,j) and spleen (e,k), and liver necrosis and inflammation (f-h, l-n).


mice . a - e , Transcriptomic analysis from Ly6efl / fl ( WT ) and Ly6eΔHSC ( KO ) mice . a - b , Heat maps displaying significant changes ( mean RPKM > 0 . 5 ; fold change > 2 ; FDR ≤ 0 . 05 ) in liver ( a ) and spleen ( b ) . Dendrograms are normalized read data ( row z - score ) clustered with complete linkage method employing Spearman Rank correlation distance measurement .

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mice.a-e, Transcriptomic analysis from Ly6efl/fl (WT) and Ly6eΔHSC (KO) mice. a-b, Heat maps displaying significant changes (mean RPKM > 0.5; fold change > 2; FDR ≤ 0.05) in liver (a) and spleen (b). Dendrograms are normalized read data (row z-score) clustered with complete linkage method employing Spearman Rank correlation distance measurement.


d - e , Expression of select genes in liver ( d ) and spleen ( e ) .

d-e, Expression of select genes in liver (d) and spleen (e).


f - g , Immune cell counts from liver ( f ) and spleen ( g ) .

f-g, Immune cell counts from liver (f) and spleen (g).


h , Infection of cultured splenocytes . For a - e , n = 3 . For f - g , n = 8 MHV - injected , n = 4 PBS - injected from two pooled experiments .

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h, Infection of cultured splenocytes. For a-e, n=3. For f-g, n=8 MHV-injected, n=4 PBS-injected from two pooled experiments.


Schematic representation of the upstream region of myogenin and amplified regions by RT - PCR ( top ) . RT - PCR for the novel transcripts at the upstream region of myogenin in C2C12 myotubes ( bottom ) . The presence or absence of reverse transcriptase ( RT ) is shown by ( + ) or ( - ) , respectively . The templates ( cDNA or genomic DNA ) are indicated by C or G , respectively .

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Schematic representation of the upstream region of myogenin and amplified regions by RT-PCR (top). RT-PCR for the novel transcripts at the upstream region of myogenin in C2C12 myotubes (bottom). The presence or absence of reverse transcriptase (RT) is shown by (+) or (-), respectively. The templates (cDNA or genomic DNA) are indicated by C or G, respectively.


The primers used for RT - PCR ( top ) . Strand - specific RT - PCR for the novel transcripts at the upstream region of myogenin in C2C12 myotubes using total RNA ( middle and bottom ) and poly ( A ) + RNA ( bottom ) .

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The primers used for RT-PCR (top). Strand-specific RT-PCR for the novel transcripts at the upstream region of myogenin in C2C12 myotubes using total RNA (middle and bottom) and poly(A)+ RNA (bottom).


Relative expression of indicated RNAs in differentiating C2C12 cells . The presence or absence of RT is shown by ( + ) or ( - ) , respectively . PCR products were verified by sequencing . n = 3 , mean ± SD . The nuclear / cytoplasmic fractionation is confirmed by the expression of H2B and tubulin , respectively .

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Relative expression of indicated RNAs in differentiating C2C12 cells. The presence or absence of RT is shown by (+) or (-), respectively. PCR products were verified by sequencing. n = 3, mean ± SD. The nuclear/cytoplasmic fractionation is confirmed by the expression of H2B and tubulin, respectively.


Chromatin pellet extract ( CPE ) / soluble nuclear extract ( SNE ) ratio of SRA1 , Malat1 , and Myoparr in C2C12 myotubes on a log scale . n = 3 , mean ± SD .

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Chromatin pellet extract (CPE)/soluble nuclear extract (SNE) ratio of SRA1, Malat1, and Myoparr in C2C12 myotubes on a log scale. n = 3, mean ± SD.


Quantitative RT - PCR for myogenin ( A ) and Myoparr ( B ) during myogenesis of C2C12 cells , primary mouse myoblasts ( growth medium , GM ) , and mouse embryonic skeletal muscle ( S . K . ) . The x - axis shows days after differentiation induction or embryonic days . n = 3 , mean ± SD .

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Quantitative RT-PCR for myogenin (A) and Myoparr (B) during myogenesis of C2C12 cells, primary mouse myoblasts (growth medium, GM), and mouse embryonic skeletal muscle (S.K.). The x-axis shows days after differentiation induction or embryonic days. n = 3, mean ± SD.


qRT - PCR showing increased Myoparr expression by MyoD in C3H10T1 / 2 fibroblasts . n = 4 , mean ± SD . * * p < 0 . 01 ( unpaired two - tailed Welch ' s t - test ) .

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qRT-PCR showing increased Myoparr expression by MyoD in C3H10T1/2 fibroblasts. n = 4, mean ± SD. **p < 0.01 (unpaired two-tailed Welch's t-test).


Treatment of recombinant TGF - β for 24 h decreased Myoparr expression in differentiating C2C12 cells . n = 3 , mean ± SD . * p < 0 . 05 ( unpaired two - tailed Student ' s t - test ) .

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Treatment of recombinant TGF-β for 24 h decreased Myoparr expression in differentiating C2C12 cells. n = 3, mean ± SD. *p < 0.05 (unpaired two-tailed Student's t-test).


Relative luciferase activities of the indicated promoter in differentiating C2C12 cells by exogenous MyoD . n = 3 , mean ± SD . * * p < 0 . 01 . ( unpaired two - tailed Student ' s t - test ) .

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Relative luciferase activities of the indicated promoter in differentiating C2C12 cells by exogenous MyoD. n = 3, mean ± SD. **p < 0.01. (unpaired two-tailed Student's t-test).


Relative luciferase activities of the indicated promoter with / without E - box mutations in differentiating C2C12 cells . n = 3 , mean ± SD . * * p < 0 . 01 . ( unpaired two - tailed Student ' s t - test ) .

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Relative luciferase activities of the indicated promoter with/without E-box mutations in differentiating C2C12 cells. n = 3, mean ± SD. **p < 0.01. (unpaired two-tailed Student's t-test).


Relative luciferase activities of the indicated promoter with / without E - box mutations in differentiating C2C12 cells by TGF - β treatment . n = 3 , mean ± SD . * p < 0 . 05 . * * p < 0 . 01 . n . s . , not significant ( unpaired two - tailed Student ' s t - test ) .

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Relative luciferase activities of the indicated promoter with/without E-box mutations in differentiating C2C12 cells by TGF-β treatment. n = 3, mean ± SD. *p < 0.05. **p < 0.01. n.s., not significant (unpaired two-tailed Student's t-test).


C2C12 cells were transfected with 50 nM of indicated siRNAs in growth medium . After additional 24 - h incubation in growth medium , cells were transferred to differentiation medium . The levels of Myoparr and myogenin expression were quantified by qRT - PCR 24 h after differentiation induction . n = 3 , mean ± SD . * p < 0 . 05 . n . s . , not significant . Statistical analyses were performed using unpaired two - tailed Student ' s t - test . In cases of unequal variances , unpaired two - tailed Welch ' s t - test was used .

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C2C12 cells were transfected with 50 nM of indicated siRNAs in growth medium. After additional 24-h incubation in growth medium, cells were transferred to differentiation medium. The levels of Myoparr and myogenin expression were quantified by qRT-PCR 24 h after differentiation induction. n = 3, mean ± SD. *p < 0.05. n.s., not significant. Statistical analyses were performed using unpaired two-tailed Student's t-test. In cases of unequal variances, unpaired two-tailed Welch's t-test was used.


Western blot showing decreased expression of myogenin in differentiating C2C12 cells 48 h after Myoparr KD . Tubulin expression served as an internal control .

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Western blot showing decreased expression of myogenin in differentiating C2C12 cells 48 h after Myoparr KD. Tubulin expression served as an internal control.


The expression levels of myogenin and Myoparr evaluated by qRT - PCR in myogenin - depleted C2C12 cells . n = 3 , mean ± SD . * * p < 0 . 01 . * * * p < 0 . 001 ( unpaired two - tailed Student ' s t - test ) .

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The expression levels of myogenin and Myoparr evaluated by qRT-PCR in myogenin-depleted C2C12 cells. n = 3, mean ± SD. **p < 0.01. ***p < 0.001 (unpaired two-tailed Student's t-test).


ChIP - qPCR detection of Pol II occupancy and histone modification status at the myogenin locus in Myoparr - depleted differentiating C2C12 cells . The data were normalized to input values . n = 3 , mean ± SD . * p < 0 . 05 . * * p < 0 . 01 ( unpaired two - tailed Student ' s t - test or unpaired two - tailed Welch ' s t - test ) .

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ChIP-qPCR detection of Pol II occupancy and histone modification status at the myogenin locus in Myoparr-depleted differentiating C2C12 cells. The data were normalized to input values. n = 3, mean ± SD. *p < 0.05. **p < 0.01 (unpaired two-tailed Student's t-test or unpaired two-tailed Welch's t-test).


Retrieval rate of Myoparr using Myoparr - ChIRP probes from C2C12 cells quantified by qPCR is shown as percent of input values . GAPDH was amplified as a negative control . n = 3 , mean ± SD . ND , not detected .

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Retrieval rate of Myoparr using Myoparr-ChIRP probes from C2C12 cells quantified by qPCR is shown as percent of input values. GAPDH was amplified as a negative control. n = 3, mean ± SD. ND, not detected.


ChIRP - qPCR detection of the interaction between endogenous Myoparr and the myogenin promoter . The myogenin 3 ´ UTR and GAPDH promoter were amplified as negative controls . The data were normalized to input values . n = 3 , mean ± SD . * p < 0 . 05 . * * p < 0 . 01 ( unpaired two - tailed Welch ' s t - test ) . ND , not detected .

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ChIRP-qPCR detection of the interaction between endogenous Myoparr and the myogenin promoter. The myogenin 3´UTR and GAPDH promoter were amplified as negative controls. The data were normalized to input values. n = 3, mean ± SD. *p < 0.05. ** p < 0.01 (unpaired two-tailed Welch's t-test). ND, not detected.


Immunocytochemistry for the detection of myogenin at 24 h ( G ) or MHC at 72 h ( H ) in Myoparr - depleted C2C12 cells after differentiation induction . Nuclei were counterstained with DAPI . Bar , 100 μm . The percentage of myogenin - positive cells or fusion index is shown as percent of the control . n = 3 , mean ± SD . * * p < 0 . 01 . * * * p < 0 . 001 ( unpaired two - tailed Student ' s t - test or unpaired two - tailed Welch ' s t - test ) .

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Immunocytochemistry for the detection of myogenin at 24 h (G) or MHC at 72 h (H) in Myoparr-depleted C2C12 cells after differentiation induction. Nuclei were counterstained with DAPI. Bar, 100 μm. The percentage of myogenin-positive cells or fusion index is shown as percent of the control. n = 3, mean ± SD. **p < 0.01. ***p < 0.001 (unpaired two-tailed Student's t-test or unpaired two-tailed Welch's t-test).


Genes significantly differentially expressed in both Myoparr - and myogenin - depleted cells show correlated expression ( R = 0 . 63 , log 2 ratio scale ) .

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Genes significantly differentially expressed in both Myoparr- and myogenin-depleted cells show correlated expression (R = 0.63, log 2 ratio scale).


Following immunoprecipitation , the interaction between Myoparr and endogenous Ddx17 was confirmed by immunoblotting using a Ddx17 - specific antibody . Two different Ddx17 isoforms ( p72 and p82 ) were observed .

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Following immunoprecipitation, the interaction between Myoparr and endogenous Ddx17 was confirmed by immunoblotting using a Ddx17-specific antibody. Two different Ddx17 isoforms (p72 and p82) were observed.


Determination of the Ddx17 - binding region of Myoparr by RNA pull - down analyses . Schematic diagram of full - length or truncated Myoparr used for RNA pull - down ( top ) . In vitro transcribed / translated Ddx17 protein was pulled down by indicated Myoparr and then detected by western blot using a Ddx17 antibody ( bottom ) .

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Determination of the Ddx17-binding region of Myoparr by RNA pull-down analyses. Schematic diagram of full-length or truncated Myoparr used for RNA pull-down (top). In vitro transcribed/translated Ddx17 protein was pulled down by indicated Myoparr and then detected by western blot using a Ddx17 antibody (bottom).


Relative luciferase activities of indicated constructs in differentiating C2C12 cells .

Relative luciferase activities of indicated constructs in differentiating C2C12 cells.


Relative luciferase activities of indicated constructs in differentiating C2C12 cells transfected with control or Myoparr shRNA ( Myoparr KD ) .

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Relative luciferase activities of indicated constructs in differentiating C2C12 cells transfected with control or Myoparr shRNA (Myoparr KD).


Significantly decreased MHC expression in Ddx17 - depleted C2C12 cells is shown by immunocytochemistry . Nuclei were counterstained with DAPI . Bar , 100 μm . Fusion index is shown as percent of the control . n = 3 , mean ± SD . * * p < 0 . 01 ( unpaired two - tailed Student ' s t - test ) . Immunocytochemistry for myogenin in Ddx17 - depleted C2C12 cells . Bar , 100 μm . Myogenin - positive cells are shown as percent of the control . n = 4 , mean ± SD . * * p < 0 . 01 ( unpaired two - tailed Student ' s t - test ) .

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Significantly decreased MHC expression in Ddx17-depleted C2C12 cells is shown by immunocytochemistry. Nuclei were counterstained with DAPI. Bar, 100 μm. Fusion index is shown as percent of the control. n = 3, mean ± SD. **p < 0.01 (unpaired two-tailed Student's t-test). Immunocytochemistry for myogenin in Ddx17-depleted C2C12 cells. Bar, 100 μm. Myogenin-positive cells are shown as percent of the control. n = 4, mean ± SD. **p < 0.01 (unpaired two-tailed Student's t-test).


Relative luciferase activities of the - 1650 - Luc by the combination of MyoD and Ddx17 or Ddx17 mutant ( K142R ) . Bars indicate the average of two independent experiments , and open circles represent the values of each experiment .

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Relative luciferase activities of the -1650-Luc by the combination of MyoD and Ddx17 or Ddx17 mutant (K142R). Bars indicate the average of two independent experiments, and open circles represent the values of each experiment.


Ddx17 or Ddx17 mutant ( K142R ) was pulled down by full - length Myoparr and then detected by western blot .

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Ddx17 or Ddx17 mutant (K142R) was pulled down by full-length Myoparr and then detected by western blot.


Reduced interaction between endogenous Ddx17 and PCAF in Myoparr - depleted C2C12 cells . After Myoparr KD , the cell lysates were subjected to immunoprecipitation ( IP ) with a Ddx17 - specific antibody 36 h after differentiation induction ( right panel ) . Each lysate was loaded as an input ( left panel ) . Relative quantification of ( E ) is shown as IP / input ratio . n = 3 , mean ± SD . * p < 0 . 05 . n . s . , not significant ( unpaired two - tailed Student ' s t - test ) .

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Reduced interaction between endogenous Ddx17 and PCAF in Myoparr-depleted C2C12 cells. After Myoparr KD, the cell lysates were subjected to immunoprecipitation (IP) with a Ddx17-specific antibody 36 h after differentiation induction (right panel). Each lysate was loaded as an input (left panel). Relative quantification of (E) is shown as IP/input ratio. n = 3, mean ± SD. *p < 0.05. n.s., not significant (unpaired two-tailed Student's t-test).


Increased interaction between Ddx17 and PCAF by Myoparr . PCAF protein was pulled down by 3 × Flag - Ddx17 in the presence or absence of Myoparr using a Flag antibody and then detected by western blot .

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Increased interaction between Ddx17 and PCAF by Myoparr. PCAF protein was pulled down by 3×Flag-Ddx17 in the presence or absence of Myoparr using a Flag antibody and then detected by western blot.


ChIP - qPCR detection of Ddx17 ( H ) and PCAF ( I ) occupancies at the myogenin locus in Myoparr - depleted differentiating C2C12 cells . The data were normalized to input values . n = 3 , mean ± SD . * p < 0 . 05 ( unpaired two - tailed Student ' s t - test ) .

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ChIP-qPCR detection of Ddx17 (H) and PCAF (I) occupancies at the myogenin locus in Myoparr-depleted differentiating C2C12 cells. The data were normalized to input values. n = 3, mean ± SD. *p < 0.05 (unpaired two-tailed Student's t-test).


Heatmap displaying expression changes of 754 genes significantly altered either in Myoparr - or Ddx17 - depleted cells ( log 2 ratio scale ) .

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Heatmap displaying expression changes of 754 genes significantly altered either in Myoparr- or Ddx17-depleted cells (log 2 ratio scale).


Genes significantly differentially expressed in both Myoparr - and Ddx17 - depleted cells show correlated expression ( R = 0 . 94 , log 2 ratio scale ) .

Genes significantly differentially expressed in both Myoparr- and Ddx17-depleted cells show correlated expression (R = 0.94, log 2 ratio scale).


Myoparr and Ddx17 are required for C2C12 cell cycle withdrawal . C2C12 cells transfected with each siRNA were cultured in growth medium for 24 h . After differentiation induction , cells were maintained in differentiation medium for 40 h and then treated with EdU for 6 h . EdU - positive cells are shown as percent of the control . Nuclei were counterstained with Hoechst 33342 . n = 3 , mean ± SD . * * p < 0 . 01 ( unpaired two - tailed Student ' s t - test ) . Bar , 100 μm .

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Myoparr and Ddx17 are required for C2C12 cell cycle withdrawal. C2C12 cells transfected with each siRNA were cultured in growth medium for 24 h. After differentiation induction, cells were maintained in differentiation medium for 40 h and then treated with EdU for 6 h. EdU-positive cells are shown as percent of the control. Nuclei were counterstained with Hoechst 33342. n = 3, mean ± SD. **p < 0.01 (unpaired two-tailed Student's t-test). Bar, 100 μm.


qRT - PCR for pri - miR - 133b , pri - miR - 206 , and H19 expression in differentiating C2C12 cells transfected with indicated siRNAs . n = 3 , mean ± SD . * p < 0 . 05 . * * p < 0 . 01 .

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qRT-PCR for pri-miR-133b, pri-miR-206, and H19 expression in differentiating C2C12 cells transfected with indicated siRNAs. n = 3, mean ± SD. *p < 0.05. **p < 0.01.


ChIP - qPCR detection of Pol II occupancy at the indicated promoters in Myoparr - depleted C2C12 cells . The data were normalized to input values . n = 3 , mean ± SD . * p < 0 . 05 . Myoparr KD decreased Pol II occupancy at the miR - 133b promoter with a marginal trend toward significance ( p = 0 . 052 ) .

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ChIP-qPCR detection of Pol II occupancy at the indicated promoters in Myoparr-depleted C2C12 cells. The data were normalized to input values. n = 3, mean ± SD. *p < 0.05. Myoparr KD decreased Pol II occupancy at the miR-133b promoter with a marginal trend toward significance (p = 0.052).


ChIP - qPCR detection of Ddx17 ( C ) occupancies at the indicated promoters in Myoparr - depleted C2C12 cells . n = 3 , mean ± SD . * p < 0 . 05 . * * p < 0 . 01 .

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ChIP-qPCR detection of Ddx17 (C) occupancies at the indicated promoters in Myoparr-depleted C2C12 cells. n = 3, mean ± SD. *p < 0.05. **p < 0.01.


ChIP - qPCR detection of PCAF ( D ) occupancies at the indicated promoters in Myoparr - depleted C2C12 cells . n = 3 , mean ± SD . * p < 0 . 05 . * * p < 0 . 01 .

ChIP-qPCR detection of PCAF (D) occupancies at the indicated promoters in Myoparr-depleted C2C12 cells. n = 3, mean ± SD. *p < 0.05. **p < 0.01.


qRT - PCR showing decreased expression of miR - 133b and miR - 206 in both Myoparr - and Ddx17 - depleted C2C12 cells . n = 3 , mean ± SD . * p < 0 . 05 . * * p < 0 . 01 .

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qRT-PCR showing decreased expression of miR-133b and miR-206 in both Myoparr- and Ddx17-depleted C2C12 cells. n = 3, mean ± SD. *p < 0.05. **p < 0.01.


Western blots showing increased ERK1 / 2 activity ( pERK1 / 2 ) and Cdc6 expression in differentiating C2C12 cells 48 h after Myoparr and Ddx17 KD . Tubulin expression served as an internal control .

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Western blots showing increased ERK1/2 activity (pERK1/2) and Cdc6 expression in differentiating C2C12 cells 48 h after Myoparr and Ddx17 KD. Tubulin expression served as an internal control.


Increased Pola1 expression detected by qRT - PCR . n = 3 , mean ± SD . * * p < 0 . 01 .

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Increased Pola1 expression detected by qRT-PCR. n = 3, mean ± SD. **p < 0.01.


Seven days after denervation , weights of innervated ( - ) and denervated ( + ) tibialis anterior ( TA ) muscles were measured . n = 4 , mean ± SEM . * p < 0 . 05 ( unpaired two - tailed Student ' s t - test ) .

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Seven days after denervation, weights of innervated (-) and denervated (+) tibialis anterior (TA) muscles were measured. n = 4, mean ± SEM. *p < 0.05 (unpaired two-tailed Student's t-test).


Expression of myogenin in innervated and denervated TA muscles detected by qRT - PCR 7 d after denervation . n = 4 , mean ± SD . * * * p < 0 . 001 ( unpaired two - tailed Welch ' s t - test ) . qRT - PCR showing increased Myoparr expression in denervated TA muscles 7 d after denervation . n = 3 , mean ± SD . * * * p < 0 . 001 ( unpaired two - tailed Student ' s t - test ) .

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Expression of myogenin in innervated and denervated TA muscles detected by qRT-PCR 7 d after denervation. n = 4, mean ± SD. ***p < 0.001 (unpaired two-tailed Welch's t-test). qRT-PCR showing increased Myoparr expression in denervated TA muscles 7 d after denervation. n = 3, mean ± SD. ***p < 0.001 (unpaired two-tailed Student's t-test).


Immunoblot showing decreased expression of myogenin by Myoparr depletion in denervated TA muscle 7 d after denervation . Expression of tubulin served as an internal control .

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Immunoblot showing decreased expression of myogenin by Myoparr depletion in denervated TA muscle 7 d after denervation. Expression of tubulin served as an internal control.


Weights of innervated and denervated TA muscles electroporated either with control or Myoparr shRNA . Muscle weights were measured 7 d after denervation . n = 5 for each group , mean ± SEM . * * * p < 0 . 001 . n . s . , not significant ( unpaired two - tailed Student ' s t - test ) .

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Weights of innervated and denervated TA muscles electroporated either with control or Myoparr shRNA. Muscle weights were measured 7 d after denervation. n = 5 for each group, mean ± SEM. ***p < 0.001. n.s., not significant (unpaired two-tailed Student's t-test).


Representative immunostaining images for laminin ( red ) and EmGFP ( green ) of innervated or denervated TA muscles electroporated with control or Myoparr shRNA , both containing EmGFP . Bar , 100 μm .

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Representative immunostaining images for laminin (red) and EmGFP (green) of innervated or denervated TA muscles electroporated with control or Myoparr shRNA, both containing EmGFP. Bar, 100 μm.


Cross - sectional area ( CSA ) of electroporated EmGFP - positive myofibers ( 250 fibers per sample ) was analyzed 7 d after denervation . n = 5 for each group , mean ± SEM . * * p < 0 . 01 . n . s . , not significant ( unpaired two - tailed Student ' s t - test ) .

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Cross-sectional area (CSA) of electroporated EmGFP-positive myofibers (250 fibers per sample) was analyzed 7 d after denervation. n = 5 for each group, mean ± SEM. **p < 0.01. n.s., not significant (unpaired two-tailed Student's t-test).


A Immunofluorescence staining of endothelium ( CD31 , in red ) , pericytes ( NG2 , in green ) , smooth muscle cells ( α - SMA , in cyan ) , and nuclei ( DAPI , in blue ) on frozen sections of limbmuscles injected with myoblast clones expressing different VEGF levels ( V Low , V Med , and V High , respectively ) and treated with VEGF - Trap or saline after 10 and 17 days . Analysis was performed after 4 days of treatment ( 2 and 3 weeks , respectively ) . All normal vessels induced by low and medium VEGF doses displayed a similar coverage by normal pericytes , whereas aberrant structures induced by high VEGF were covered with α - SMA - positive smooth muscle cells . Asterisks indicate muscle fibers , which were mostly sectioned in longitudinal orientation , although some cross sections are visible depending on the orientation of muscle bundles . Scale bar = 25 μm . B , C Quantification of vessel length density ( VLD ) on the same samples showed that stabilization started already by 10 days for vessels induced by low VEGF , but not until 17 days for those induced by medium VEGF , despite similar pericyte coverage , while aberrant structures induced by high VEGF always regressed . Data represent the mean ± SEM of individual images ( n ) acquired from three muscles / group ; 2 weeks ( B ) : V Low saline , n = 9 ; V Med saline , n = 13 ; V High saline , n = 8 ; V Low TRAP , n = 28 ; V Med TRAP , n = 22 ; V High TRAP , n = 30 ; 3 weeks ( C ) : V Low saline , n = 9 ; V Med saline , n = 11 ; V High saline , n = 19 ; V Low TRAP , n = 43 ; V Med TRAP , n = 14 ; V High TRAP , n = 19 ; * P < 0 . 05 and * * * P < 0 . 001 ( all versus Ctrl ) by one - way ANOVA with Bonferroni multiple comparisons test ; 2 weeks ( B ) : V Low TRAP versus Ctrl P < 0 . 0001 ; 3 weeks ( C ) : V Low TRAP versus Ctrl P < 0 . 0001 ; V Med TRAP versus Ctrl P = 0 . 0158 .

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A Immunofluorescence staining of endothelium (CD31, in red), pericytes (NG2, in green), smooth muscle cells (α-SMA, in cyan), and nuclei (DAPI, in blue) on frozen sections of limbmuscles injected with myoblast clones expressing different VEGF levels (V Low, V Med, and V High, respectively) and treated with VEGF-Trap or saline after 10 and 17 days. Analysis was performed after 4 days of treatment (2 and 3 weeks, respectively). All normal vessels induced by low and medium VEGF doses displayed a similar coverage by normal pericytes, whereas aberrant structures induced by high VEGF were covered with α-SMA-positive smooth muscle cells. Asterisks indicate muscle fibers, which were mostly sectioned in longitudinal orientation, although some cross sections are visible depending on the orientation of muscle bundles. Scale bar = 25 μm.B, C Quantification of vessel length density (VLD) on the same samples showed that stabilization started already by 10 days for vessels induced by low VEGF, but not until 17 days for those induced by medium VEGF, despite similar pericyte coverage, while aberrant structures induced by high VEGF always regressed. Data represent the mean ± SEM of individual images (n) acquired from three muscles/group; 2 weeks (B): V Low saline, n = 9; V Med saline, n = 13; V High saline, n = 8; V Low TRAP,n = 28; V Med TRAP,n = 22; V High TRAP,n = 30; 3 weeks (C): V Low saline, n = 9; V Med saline, n = 11; V High saline, n = 19; V Low TRAP,n = 43; V Med TRAP,n = 14; V High TRAP,n = 19; *P < 0.05 and ***P < 0.001 (all versus Ctrl) by one-way ANOVA with Bonferroni multiple comparisons test; 2 weeks (B): V Low TRAP versus Ctrl P < 0.0001; 3 weeks (C): V Low TRAP versus Ctrl P < 0.0001; V Med TRAP versus Ctrl P = 0.0158.


Muscles were harvested 7 days after implantation of V Low , V Med , and V High clones or control cells ( Ctrl ) . Gene expression of Pdgfb , Ang1 , Ang2 , Tgfb1 , and Sema3a was quantified by qRT - PCR and expressed as fold - change versus control muscles . Data represent individual values , with mean ± SEM ( n = 3 or 4 , as indicated ) ; * P < 0 . 05 and * * P < 0 . 01 by one - way ANOVA with Bonferroni multiple comparisons test , after data normalization by logarithmic transformation . TGF - β1 : V Low versus V Med P = 0 . 0047 ; V Low versus V High P = 0 . 0379 ; Sema3A : V Low versus V High P = 0 . 0042 ; V Med versus V High P = 0 . 0179 .

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Muscles were harvested 7 days after implantation of V Low, V Med, and V High clones or control cells (Ctrl).Gene expression of Pdgfb,Ang1,Ang2,Tgfb1, and Sema3a was quantified by qRT-PCR and expressed as fold-change versus control muscles. Data represent individual values, with mean ± SEM (n = 3 or 4, as indicated); *P < 0.05 and **P < 0.01 by one-way ANOVA with Bonferroni multiple comparisons test, after data normalization by logarithmic transformation. TGF-β1: V Low versus V Med P = 0.0047; V Low versus V High P = 0.0379; Sema3A: V Low versus V High P = 0.0042; V Med versus V High P = 0.0179.


Muscles were harvested 7 days after implantation of V Low , V Med , and V High clones or control cells ( Ctrl ) . Immunohistochemistry for Sema3A on frozen muscle sections confirmed a decreasing protein expression in the areas implanted with myoblasts producing increasing VEGF doses ( n = 4 ) . The right panels represent higher - magnification views of the left panels , which encompass the majority of the implantation sites . Scale bar = 50 μm .

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Muscles were harvested 7 days after implantation of V Low, V Med, and V High clones or control cells (Ctrl). Immunohistochemistry for Sema3A on frozen muscle sections confirmed a decreasing protein expression in the areas implanted with myoblasts producing increasing VEGF doses (n = 4). The right panels represent higher-magnification views of the left panels, which encompass the majority of the implantation sites. Scale bar = 50 μm.