Representative false colour images of photosystem II ( PSII ) operating efficiency ( ΦPSII ) captured by chlorophyll fluorescence imaging from WT , stp1 - 1 , stp4 - 1 and stp1stp4 plants . ΦPSII was measured at a photosynthetically active radiation ( PAR ) of 440 µmol m - 2 s - 1 .

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Representative false colour images of photosystem II (PSII) operating efficiency (ΦPSII) captured by chlorophyll fluorescence imaging from WT, stp1-1, stp4-1 and stp1stp4 plants. ΦPSII was measured at a photosynthetically active radiation (PAR) of 440 µmol m-2 s-1.


ΦPSII quantified over the phenotyping period . Data shown are means ± SEM ; n = 10 per genotype and time point .

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ΦPSII quantified over the phenotyping period. Data shown are means ± SEM; n = 10 per genotype and time point.


Whole - plant recordings of changes in CO2 assimilation ( A ) from WT , stp1 - 1 , stp4 - 1 and stp1stp4 plants . Data shown are means ± SEM ; n ≥ 3 per genotype .

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Whole-plant recordings of changes in CO2 assimilation (A) from WT, stp1-1, stp4-1 and stp1stp4 plants. Data shown are means ± SEM; n ≥ 3 per genotype.


Whole - plant recordings of changes in CO2 assimilation ( A ) from self - grafted donor lines ( WT / WT , stp1stp4 / stp1stp4 ) and reciprocal grafting of shoot / root ( WT / stp1stp4 , stp1stp4 / WT ) plants . Data shown are means ± SEM ; n = 3 per genotype .

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Whole-plant recordings of changes in CO2 assimilation (A) from self-grafted donor lines (WT/WT, stp1stp4/stp1stp4) and reciprocal grafting of shoot/root (WT/stp1stp4, stp1stp4/WT) plants. Data shown are means ± SEM; n = 3 per genotype.


Quantification of leaf glucose ( Glc ) . Data shown are means ± SEM ; n = 8 per genotype and time point . Quantification of leaf fructose ( Fru ) . Data shown are means ± SEM ; n = 8 per genotype and time point . Quantification of leaf sucrose ( Suc ) . Data shown are means ± SEM ; n = 8 per genotype and time point .

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Quantification of leaf glucose (Glc). Data shown are means ± SEM; n = 8 per genotype and time point.   Quantification of leaf fructose (Fru). Data shown are means ± SEM; n = 8 per genotype and time point.   Quantification of leaf sucrose (Suc). Data shown are means ± SEM; n = 8 per genotype and time point.


Quantification of leaf starch . Data shown are means ± SEM ; n = 8 per genotype and time point .

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Quantification of leaf starch. Data shown are means ± SEM; n = 8 per genotype and time point.


Representative Red Green Blue ( RGB ) images of 3 - ( day 0 ) and 4 - week - old ( day 7 ) WT , stp1 - 1 , stp4 - 1 and stp1stp4 plants .

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Representative Red Green Blue (RGB) images of 3-(day 0) and 4-week-old (day 7) WT, stp1-1, stp4-1 and stp1stp4 plants.


Projected rosette area over the phenotyping period . Data shown are means ± SEM ; n = 10 per genotype and time point .

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Projected rosette area over the phenotyping period. Data shown are means ± SEM; n = 10 per genotype and time point.


Photosynthetic CO2 assimilation ( A ) in dark - adapted WT and stp1stp4 plants in response to a step increase in CO2 concentrations from 0 to 1000 ppm . Data shown are means ± SEM ; n = 3 per genotype .

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Photosynthetic CO2 assimilation (A) in dark-adapted WT and stp1stp4 plants in response to a step increase in CO2 concentrations from 0 to 1000 ppm. Data shown are means ± SEM; n=3 per genotype.


Quantification of leaf glucose ( Glc ) . Data for two independent experiments are shown ; means ± SEM ; n ≥ 17 per genotype and time point .

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Quantification of leaf glucose (Glc). Data for two independent experiments are shown; means ± SEM; n ≥ 17 per genotype and time point.


Quantification of leaf fructose ( Fru ) . Data for two independent experiments are shown ; means ± SEM ; n ≥ 17 per genotype and time point . Quantification of leaf sucrose ( Suc ) . Data for two independent experiments are shown ; means ± SEM ; n ≥ 17 per genotype and time point . Quantification of leaf starch . Data for two independent experiments are shown ; means ± SEM ; n ≥ 17 per genotype and time point .

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

Quantification of leaf fructose (Fru). Data for two independent experiments are shown; means ± SEM; n ≥ 17 per genotype and time point.   Quantification of leaf sucrose (Suc). Data for two independent experiments are shown; means ± SEM; n ≥ 17 per genotype and time point.   Quantification of leaf starch. Data for two independent experiments are shown; means ± SEM; n ≥ 17 per genotype and time point.


Representative Red Green Blue ( RGB ) images of 3 - ( day 0 ) and 4 - week - old ( day 6 ) WT and stp1stp4 plants grown under 600 ppm CO2 .

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Representative Red Green Blue (RGB) images of 3-(day 0) and 4-week-old (day 6) WT and stp1stp4 plants grown under 600 ppm CO2.


Projected rosette area over the phenotyping period . Data shown are from two independent experiments ; means ± SEM ; n ≥ 24 per genotype and time point .

Projected rosette area over the phenotyping period. Data shown are from two independent experiments; means ± SEM; n ≥ 24 per genotype and time point.


Representative confocal laser microscopy images of propidium iodide - stained guard cell starch granules of intact leaves of WT and stp1stp4 plants grown under 600 ppm CO2 . Scale bar , 10 µm .

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Representative confocal laser microscopy images of propidium iodide-stained guard cell starch granules of intact leaves of WT and stp1stp4 plants grown under 600 ppm CO2. Scale bar, 10 µm.


Starch dynamics in guard cells of intact leaves of WT and stp1stp4 plants grown under 600 ppm CO2 at the end of the night ( EoN ) and after one and three hours of illumination with 150 µmol m - 2 s - 1 of white light . Data for two independent experiments are shown ; means ± SEM ; n = 80 individual guard cells per genotype and time point .

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Starch dynamics in guard cells of intact leaves of WT and stp1stp4 plants grown under 600 ppm CO2 at the end of the night (EoN) and after one and three hours of illumination with 150 µmol m-2 s-1 of white light. Data for two independent experiments are shown; means ± SEM; n = 80 individual guard cells per genotype and time point.


A , Western blots in HCT116 cells treated for 24 hrs with increasing concentrations of MPA , or 5 nM Actinomycin D ( ActD ) . GAPDH served as a loading control .

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A, Western blots in HCT116 cells treated for 24 hrs with increasing concentrations of MPA, or 5 nM Actinomycin D (ActD). GAPDH served as a loading control.


B , HCT116 cells were treated for 24 hrs with vehicle alone ( - ) , 1 µM MPA , 10 µM MPA , or the combination of 10 µM MPA with 400 µM guanosine . Guanylate nucleotides levels were measured by LC - MS , and normalized to protein content . Mean ± SEM is representative of 3 independent experiments carried out in triplicate . Data information data are presented as relative to control . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 by two tail T - Student ' s test .

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B, HCT116 cells were treated for 24 hrs with vehicle alone (-), 1 µM MPA, 10 µM MPA, or the combination of 10 µM MPA with 400 µM guanosine. Guanylate nucleotides levels were measured by LC-MS, and normalized to protein content. Mean ± SEM is representative of 3 independent experiments carried out in triplicate. Data information data are presented as relative to control. *p<0.05, **p<0.01, ***p<0.001 by two tail T-Student's test.


Parental HCT116 cells expressing a stable tetracycline - inducible shRNA against IMPDH2 ( IM2iKD ) and either a stable inducible non - targeting ( NT ) shRNA ( IM2iKD - shNT ) or an shRNA targeting IMPDH1 ( IM2iKD - shIM1 ) , were grown for 7 days in the absence or presence of doxycycline ( 2 µg / mL ) ( - dox or + dox , respectively ) . mRNAs of IMPDH1 and IMPDH2 were quantified by real - time qPCR in 2 independent experiments carried out in triplicate and normalized to β - actin mRNA ( C ) . Data information : data are presented as relative to control . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 by two tail T - Student ' s test .

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Parental HCT116 cells expressing a stable tetracycline-inducible shRNA against IMPDH2 (IM2iKD) and either a stable inducible non-targeting (NT) shRNA (IM2iKD-shNT) or an shRNA targeting IMPDH1 (IM2iKD - shIM1), were grown for 7 days in the absence or presence of doxycycline (2 µg/mL) (- dox or + dox, respectively). mRNAs of IMPDH1 and IMPDH2 were quantified by real-time qPCR in 2 independent experiments carried out in triplicate and normalized to β-actin mRNA (C). Data information: data are presented as relative to control. *p<0.05, **p<0.01, ***p<0.001 by two tail T-Student's test.


Parental HCT116 cells expressing a stable tetracycline - inducible shRNA against IMPDH2 ( IM2iKD ) and either a stable inducible non - targeting ( NT ) shRNA ( IM2iKD - shNT ) or an shRNA targeting IMPDH1 ( IM2iKD - shIM1 ) , were grown for 7 days in the absence or presence of doxycycline ( 2 µg / mL ) ( - dox or + dox , respectively ) . Whole cell extracts were analyzed on Western blots with the indicated antibodies . GAPDH was used as a loading control . Quantification of band intensity of p53 and p21 is shown . Mean ± SD is representative of four independent experiments . ( right panel ) ( D ) . Data information : data are presented as relative to control . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 by two tail T - Student ' s test .

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Parental HCT116 cells expressing a stable tetracycline-inducible shRNA against IMPDH2 (IM2iKD) and either a stable inducible non-targeting (NT) shRNA (IM2iKD-shNT) or an shRNA targeting IMPDH1 (IM2iKD - shIM1), were grown for 7 days in the absence or presence of doxycycline (2 µg/mL) (- dox or + dox, respectively). Whole cell extracts were analyzed on Western blots with the indicated antibodies. GAPDH was used as a loading control. Quantification of band intensity of p53 and p21 is shown. Mean ± SD is representative of four independent experiments. (right panel) (D). Data information: data are presented as relative to control. *p<0.05, **p<0.01, ***p<0.001 by two tail T-Student's test.


E , Western blots in HCT116 cells treated for 24 hrs with the indicated concentration of MPA , in presence of dimethyl sulfoxide ( DMSO ) or 400 µM guanosine . GAPDH was used as a loading control .

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E, Western blots in HCT116 cells treated for 24 hrs with the indicated concentration of MPA, in presence of dimethyl sulfoxide (DMSO) or 400 µM guanosine. GAPDH was used as a loading control.


A , HCT116 cells were transfected with either a NT or a siRNA against RPL11 for 24 hrs and treated with the vehicle alone ( - ) or the indicated concentration of MPA for 24 hrs . The levels of p53 and p21 were analyzed on Western blots . GAPDH was used as a loading control . Quantification of band intensity of p53 and p21 of four independent experiments is shown ( right panel ) . Data information : All data are presented as Mean ± SD , relative to control . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , by two tail T - Student ' s test .

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A, HCT116 cells were transfected with either a NT or a siRNA against RPL11 for 24 hrs and treated with the vehicle alone (-) or the indicated concentration of MPA for 24 hrs. The levels of p53 and p21 were analyzed on Western blots. GAPDH was used as a loading control. Quantification of band intensity of p53 and p21 of four independent experiments is shown (right panel). Data information: All data are presented as Mean ± SD, relative to control. *p<0.05, **p<0.01, ***p<0.001, by two tail T-Student's test.


B , HCT116 cells were transfected with the indicated siRNA and 24 hrs later treated with the vehicle alone or 10 µM MPA for 6 hrs and the levels of p53 and p21 were analyzed on Western blots . GAPDH was used as a loading control . Quantification of band intensity of p53 of at least four independent experiments is shown ( right panel ) . Data information : All data are presented as Mean ± SD , relative to control . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , by two tail T - Student ' s test .

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B, HCT116 cells were transfected with the indicated siRNA and 24 hrs later treated with the vehicle alone or 10 µM MPA for 6 hrs and the levels of p53 and p21 were analyzed on Western blots. GAPDH was used as a loading control. Quantification of band intensity of p53 of at least four independent experiments is shown (right panel). Data information: All data are presented as Mean ± SD, relative to control. *p<0.05, **p<0.01, ***p<0.001, by two tail T-Student's test.


C , HCT116 cells were transfected with either a NT siRNA or a siRNA against RPL11 for 24 hrs and were pretreated for 30 mins in the absence ( - ) or the presence ( + ) of the combination of 10 µM of the ATR inhibitor VE - 821 ( ATRi ) and 10 µM the ATM inhibitor KU - 55933 ( ATMi ) , followed by addition of the vehicle alone ( - ) or MPA 10 µM for additional 24 hrs . The levels of p53 were analyzed on Western blots . GAPDH was used as a loading control . Quantification of band intensity of p53 of at least two independent experiments is shown ( right panel ) . Data information : All data are presented as Mean ± SD , relative to control . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , by two tail T - Student ' s test .

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C, HCT116 cells were transfected with either a NT siRNA or a siRNA against RPL11 for 24 hrs and were pretreated for 30 mins in the absence (-) or the presence (+) of the combination of 10 µM of the ATR inhibitor VE-821 (ATRi) and 10 µM the ATM inhibitor KU-55933 (ATMi), followed by addition of the vehicle alone (-) or MPA 10 µM for additional 24 hrs. The levels of p53 were analyzed on Western blots. GAPDH was used as a loading control. Quantification of band intensity of p53 of at least two independent experiments is shown (right panel). Data information: All data are presented as Mean ± SD, relative to control. *p<0.05, **p<0.01, ***p<0.001, by two tail T-Student's test.


A , HCT116 cells were treated with either vehicle alone ( - ) , 1 µM MPA , 10 µM MPA or 5 nM ActD for 24 hrs and mRNA levels of p21 were quantified by real - time qPCR in 2 independent experiments carried out in triplicate and normalized to 28S rRNA . Data information : , data are presented as mean ± SD , relative to control . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , by two tail T - Student ' s test .

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A, HCT116 cells were treated with either vehicle alone (-), 1 µM MPA, 10 µM MPA or 5 nM ActD for 24 hrs and mRNA levels of p21 were quantified by real-time qPCR in 2 independent experiments carried out in triplicate and normalized to 28S rRNA. Data information: , data are presented as mean ± SD, relative to control. *p<0.05, **p<0.01, ***p<0.001, by two tail T-Student's test.


B , Autoradiogram ( upper panel ) and Ethidium Bromide ( EtBr ) - stained agarose gel ( lower panel ) of 18S and 28S rRNA from HCT116 cells treated as indicated in the Methods section .

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B, Autoradiogram (upper panel) and Ethidium Bromide (EtBr)-stained agarose gel (lower panel) of 18S and 28S rRNA from HCT116 cells treated as indicated in the Methods section.


C , Autoradiogram ( upper panel ) and EtBr - stained TBE - urea polyacrylamide gel ( lower panel ) of 5S , 5 . 8S rRNA and tRNAs in

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C, Autoradiogram (upper panel) and EtBr-stained TBE-urea polyacrylamide gel (lower panel) of 5S, 5.8S rRNA and tRNAs in lysates


HCT116 cells were treated for 24 hrs . Cell lysates were collected , subjected to ultracentrifugation , spiked with firefly luciferase mRNA and immunoprecipitated with anti - HDM2 rabbit antibody ( IP HDM2 ) or the IgG control ( IgG ) . D , Levels of HDM2 , RPL5 , RPL11 , and the IP negative control mTOR in the whole cell extract ( WCE ) , the post - ribosomal lysates ( INPUT , left panel ) and in the HDM2 immunoprecipitated fraction ( right panel ) were analyzed on Western blots . The results are representative of two independent experiments . Quantification of band intensity of HDM2 , RPL5 and RPL11 of at least two independent experiments is shown ( lower right panel ) .

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HCT116 cells were treated for 24 hrs. Cell lysates were collected, subjected to ultracentrifugation, spiked with firefly luciferase mRNA and immunoprecipitated with anti-HDM2 rabbit antibody (IP HDM2) or the IgG control (IgG). D, Levels of HDM2, RPL5, RPL11, and the IP negative control mTOR in the whole cell extract (WCE), the post-ribosomal lysates (INPUT, left panel) and in the HDM2 immunoprecipitated fraction (right panel) were analyzed on Western blots. The results are representative of two independent experiments. Quantification of band intensity of HDM2, RPL5 and RPL11 of at least two independent experiments is shown (lower right panel).


HCT116 cells were treated for 24 hrs . Cell lysates were collected , subjected to ultracentrifugation , spiked with firefly luciferase mRNA and immunoprecipitated with anti - HDM2 rabbit antibody ( IP HDM2 ) or the IgG control ( IgG ) . E , the Levels of immunoprecipitated 5S rRNA were determined by real time qPCR and normalized to the firefly luciferase mRNA . Data are normalized and presented as relative to untreated cells immunoprecipitated with anti - HDM2 . The results are representative of three independent experiments carried out in triplicates . Data information : data are presented as mean ± SD , relative to control . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , by two tail T - Student ' s test .

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HCT116 cells were treated for 24 hrs. Cell lysates were collected, subjected to ultracentrifugation, spiked with firefly luciferase mRNA and immunoprecipitated with anti-HDM2 rabbit antibody (IP HDM2) or the IgG control (IgG). E, the Levels of immunoprecipitated 5S rRNA were determined by real time qPCR and normalized to the firefly luciferase mRNA. Data are normalized and presented as relative to untreated cells immunoprecipitated with anti-HDM2. The results are representative of three independent experiments carried out in triplicates. Data information: data are presented as mean ± SD, relative to control. *p<0.05, **p<0.01, ***p<0.001, by two tail T-Student's test.


F , HCT116 cells were pretreated with vehicle alone ( - ) , 1 µM , or 10 µM MPA for 24 hrs , and the levels of p53 and p21 were followed on Western blots in presence of 100 µg / mL cycloheximide ( CHX ) for the indicated time , in the continued presence of the treatment . GAPDH was used as a loading control . Quantification of band intensities is shown in the lower panels as relative to time 0 of CHX treatment .

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F, HCT116 cells were pretreated with vehicle alone (-), 1 µM, or 10 µM MPA for 24 hrs, and the levels of p53 and p21 were followed on Western blots in presence of 100 µg/mL cycloheximide (CHX) for the indicated time, in the continued presence of the treatment. GAPDH was used as a loading control. Quantification of band intensities is shown in the lower panels as relative to time 0 of CHX treatment.


G , HCT116 cells were pretreated as in ( F ) and 10 µM of the proteasome inhibitor MG132 was added for 4 hrs when indicated . The levels of p53 and p21 were analyzed on Western blots . GAPDH was used as a loading control . Quantification of p21 band intensity of at least four independent experiments is shown in the lower panel . Data information : In panels data are presented as mean ± SD , relative to control . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , by two tail T - Student ' s test .

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G, HCT116 cells were pretreated as in (F) and 10 µM of the proteasome inhibitor MG132 was added for 4 hrs when indicated. The levels of p53 and p21 were analyzed on Western blots. GAPDH was used as a loading control. Quantification of p21 band intensity of at least four independent experiments is shown in the lower panel. Data information: In panels data are presented as mean ± SD, relative to control. *p<0.05, **p<0.01, ***p<0.001, by two tail T-Student's test.


A , HCT116 were treated as indicated for 24 hrs , subjected to propidium iodide staining and analyzed by fluorescence - activated cell sorting ( FACS ) . Data are representative of at least three independent experiments .

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A, HCT116 were treated as indicated for 24 hrs, subjected to propidium iodide staining and analyzed by fluorescence-activated cell sorting (FACS). Data are representative of at least three independent experiments.


B , Colon adenocarcinoma cell lines HCT116 ( circles ) , LoVo ( squares ) , LS174 ( triangles ) , or RKO ( inversed triangles ) were treated with the vehicle alone ( - ) , 1 µM MPA or 10 µM MPA for 24 hrs and subjected to propidium iodide staining and FACS analysis . Data are shown as the fold induction of the cell number in G0 / G1 phases ( blue ) , S phase ( red ) , and G2 / M phases upon treatment with 1 µM MPA ( dark ) or 10 µM MPA ( clear ) over vehicle treated cells .

B, Colon adenocarcinoma cell lines HCT116 (circles), LoVo (squares), LS174 (triangles), or RKO (inversed triangles) were treated with the vehicle alone (-), 1 µM MPA or 10 µM MPA for 24 hrs and subjected to propidium iodide staining and FACS analysis. Data are shown as the fold induction of the cell number in G0/G1 phases (blue), S phase (red), and G2/M phases upon treatment with 1 µM MPA (dark) or 10 µM MPA (clear) over vehicle treated cells.


C , IdU track length distribution , and distribution of long fork / short fork IdU track length ratios ( insert ) . IdU track length of at least 300 fibers was measured for each condition . For fork symmetry analyses , IdU track length was measure in at least 38 bidirectional forks ( see methods ) . * * * p < 0 . 001 , * * * * p < 0 . 0001 by Kruskal - Wallis test of the means . D , Representative images from ( C ) .

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C, IdU track length distribution, and distribution of long fork / short fork IdU track length ratios (insert). IdU track length of at least 300 fibers was measured for each condition. For fork symmetry analyses, IdU track length was measure in at least 38 bidirectional forks (see methods). ***p<0.001, ****p<0.0001 by Kruskal-Wallis test of the means. D, Representative images from (C).


E , HCT116 cells , pre - incubated with 10 µM BrdU for 48 hrs were treated with Vehicle ( - ) , 10µM MPA for 6 or 24 hrs , or 20 µM Hydroxyurea for 4 hrs before cell permeabilization and fixation ( see Method ) . Cells were immunostained for native BrDU ( green ) , or RPA ( red ) . DNA was counterstained with DAPI ( blue ) . One representative z - confocal stack is shown per condition . Scale bars correspond to 5 μm .

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E, HCT116 cells, pre-incubated with 10 µM BrdU for 48 hrs were treated with Vehicle (-), 10µM MPA for 6 or 24 hrs, or 20 µM Hydroxyurea for 4 hrs before cell permeabilization and fixation (see Method). Cells were immunostained for native BrDU (green), or RPA (red). DNA was counterstained with DAPI (blue). One representative z-confocal stack is shown per condition. Scale bars correspond to 5 μm.


F , HCT116 cells treated with 10 µM MPA for increasing time were analyzed by Western blots for the indicated proteins . α - tubulin was used as a loading control .

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F, HCT116 cells treated with 10 µM MPA for increasing time were analyzed by Western blots for the indicated proteins. α-tubulin was used as a loading control.


A , HCT116 cells were treated with either the vehicle alone ( - ) , 1 µM MPA or 10 µM MPA for 24 hrs , before p21 immuno - labeling , propidium iodide staining and FACS analysis . Cell cycle profiles of the total cell population ( upper panel , left ) , the total intensity of p21 and propidium iodide were determined in 20 , 000 cells and plotted in a scatter diagram ( lower panel , left ) . The specific cell cycle profiles are shown of p21high ( upper panel , right ) and p21low ( lower panel , right ) cell populations . Mean ± SD of the percentage of cells in each cell cycle phase of at least two independent experiments is shown in the right panels .

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A, HCT116 cells were treated with either the vehicle alone (-), 1 µM MPA or 10 µM MPA for 24 hrs, before p21 immuno-labeling, propidium iodide staining and FACS analysis. Cell cycle profiles of the total cell population (upper panel, left), the total intensity of p21 and propidium iodide were determined in 20,000 cells and plotted in a scatter diagram (lower panel, left). The specific cell cycle profiles are shown of p21high (upper panel, right) and p21low (lower panel, right) cell populations. Mean ± SD of the percentage of cells in each cell cycle phase of at least two independent experiments is shown in the right panels.


B , HCT116 p21 - / - isogenic cell lines were transfected for 24 hrs with an empty plasmid ( EV ) or a plasmid encoding a p21wt cDNA ( p21 - OE ) , then cells were serum - deprived for 16 hrs before the re - addition of serum for additional 24 hrs in absence or presence of 10 µM MPA . Cells were subjected to p21 immunolabeling , propidium iodide staining and FACS analysis . The results are representative of two independent experiments .

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B, HCT116 p21-/- isogenic cell lines were transfected for 24 hrs with an empty plasmid (EV) or a plasmid encoding a p21wt cDNA (p21-OE), then cells were serum-deprived for 16 hrs before the re-addition of serum for additional 24 hrs in absence or presence of 10 µM MPA. Cells were subjected to p21 immunolabeling, propidium iodide staining and FACS analysis. The results are representative of two independent experiments.


A , HCT116 cells were transfected for 8 hrs with the indicated siRNA then treated and analyzed as in ( Fig 5B ) . Here are shown the cell cycle profiles after 16 hrs of serum deprivation ( top panels ) , or after an additional 24 hrs of serum re - addition in absence ( middle panels ) , or presence of 10 µM MPA ( bottom panels ) . The results are representative of at least three independent experiments .

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A, HCT116 cells were transfected for 8 hrs with the indicated siRNA then treated and analyzed as in (Fig 5B). Here are shown the cell cycle profiles after 16 hrs of serum deprivation (top panels), or after an additional 24 hrs of serum re-addition in absence (middle panels), or presence of 10 µM MPA (bottom panels). The results are representative of at least three independent experiments.


B , HCT116 cells transfected with a NT siRNA or a siRNA against RPL11 for 24 hrs were treated with 10 µM MPA for additional 72 hrs . The levels of γ - H2AX were analyzed on Western blots . GAPDH was used as a loading control .

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B, HCT116 cells transfected with a NT siRNA or a siRNA against RPL11 for 24 hrs were treated with 10 µM MPA for additional 72 hrs. The levels of γ-H2AX were analyzed on Western blots. GAPDH was used as a loading control.


Statistical analysis of the immunostaining of γ - H2AX ( C ) in HCT116 cells treated Total nuclear intensities were obtained on > 6000 ( γ - H2AX ) individual cells for each condition from four or two independent biological replicates , respectively . * * p < 0 . 01 , * * * * p < 0 . 0001 by Kruskal - Wallis test . A . U . Arbitrary Units .

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Statistical analysis of the immunostaining of γ-H2AX (C) in HCT116 cells treated Total nuclear intensities were obtained on >6000 (γ-H2AX) individual cells for each condition from four or two independent biological replicates, respectively. **p<0.01, ****p<0.0001 by Kruskal-Wallis test. A.U. Arbitrary Units.


Statistical analysis of the immunostaining of 53BP1 ( D ) in HCT116 cells treated Total nuclear intensities were obtained on > 1000 ( 53BP1 ) individual cells for each condition from four or two independent biological replicates , respectively . * * p < 0 . 01 , * * * * p < 0 . 0001 by Kruskal - Wallis test . A . U . Arbitrary Units .

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Statistical analysis of the immunostaining of 53BP1 (D) in HCT116 cells treated Total nuclear intensities were obtained on > 1000 (53BP1) individual cells for each condition from four or two independent biological replicates, respectively. **p<0.01, ****p<0.0001 by Kruskal-Wallis test. A.U. Arbitrary Units.


E , Representative images cells labeled with γ - H2AX ( green ) , 53BP1 ( red ) and DAPI ( blue ) . Scale bars correspond to 10

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E, Representative images cells labeled with γ-H2AX (green), 53BP1 (red) and DAPI (blue). Scale bars correspond to 10 μm


F , HCT116 cells treated were analyzed by the comet assay . The percentage of tail DNA / total DNA was analyzed in a total of 300 cells for each condition from three independent biological replicates . * * * * p < 0 . 0001 by Kruskal - Wallis

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F, HCT116 cells treated were analyzed by the comet assay. The percentage of tail DNA/total DNA was analyzed in a total of 300 cells for each condition from three independent biological replicates. ****p<0.0001 by Kruskal-Wallis test


( A - C ) Western blot analysis of THP - 1 cells infected with ( A ) HSV - 1 at MOI 5 for 3 h , 6 h , 12 h , 18 h , and 24 h ( B ) CHIKV at MOI 5 for 3 h , 6 h , 12 h , 18 h , and 24 h ( C ) JEV at MOI 5 for 3 h , 6 h , 12 h , and 24 h .

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(A-C) Western blot analysis of THP-1 cells infected with (A) HSV-1 at MOI 5 for 3 h, 6 h, 12 h, 18 h, and 24 h (B) CHIKV at MOI 5 for 3 h, 6 h, 12 h, 18 h, and 24 h (C) JEV at MOI 5 for 3 h, 6 h, 12 h, and 24 h.


( I - L ) Control and IRGM knockdown THP - 1 IFN reporter cells were treated with ( I ) Poly I : C ( 1 μg / ml ) for 8 h , 12 h and 24 h or ( J ) Poly dA : dT ( 1 μg / ml ) for 12 h and 24 h or ( K ) IFN - β ( 500 ng / ml ) for 8 h and 12 h or ( L ) cGAMP ( 1 μg / ml ) for 12 h and 24 h and the supernatant was subjected to luciferase reporter assay using QuantiLuc

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(I-L) Control and IRGM knockdown THP-1 IFN reporter cells were treated with (I) Poly I:C (1 μg/ml) for 8 h, 12 h and 24 h or (J) Poly dA:dT (1 μg/ml) for 12 h and 24 h or (K) IFN-β (500 ng/ml) for 8 h and 12 h or (L) cGAMP (1 μg/ml) for 12 h and 24 h and the supernatant was subjected to luciferase reporter assay using QuantiLuc reagent


( M - P ) Control and IRGM knockdown THP - 1 cells were untreated and treated with Poly I : C ( 1 μg / ml ) for 8 h and the total RNA was subjected to qRT PCR with primers of ( M ) MX2 ( N ) ISG15 ( O ) OAS1 and ( P ) IFN - β . ( Q - T ) Control and IRGM knockdown THP - 1 cells were untreated and treated with Poly dA : dT ( 1 μg / ml ) for 8 h and the total RNA was subjected to qRT PCR with primers of ( Q ) MX2 ( R ) ISG15 ( S ) OAS1 and ( T ) IFN - β .

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(M-P) Control and IRGM knockdown THP-1 cells were untreated and treated with Poly I:C (1 μg/ml) for 8 h and the total RNA was subjected to qRT PCR with primers of (M) MX2 (N) ISG15 (O) OAS1 and (P) IFN-β. (Q-T) Control and IRGM knockdown THP-1 cells were untreated and treated with Poly dA:dT (1 μg/ml) for 8 h and the total RNA was subjected to qRT PCR with primers of (Q) MX2 (R) ISG15 (S) OAS1 and (T) IFN-β.


( D ) RNA was isolated from irgm1 + / + and irgm1 - / - BMDMs and subjected to qRT - PCR with indicated viral restriction factor

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(D) RNA was isolated from irgm1+/+ and irgm1-/- BMDMs and subjected to qRT-PCR with indicated viral restriction factor genes


( H - J ) RNA isolated from control and IRGM knockdown HT - 29 cells and subjected to qRT - PCR with indicated genes of ( H ) Immunoproteasome complex ( I ) TAP complex ( J ) human leukocyte antigen ( HLA ) system .

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(H-J) RNA isolated from control and IRGM knockdown HT-29 cells and subjected to qRT-PCR with indicated genes of (H) Immunoproteasome complex (I) TAP complex (J) human leukocyte antigen (HLA) system.


( L , M ) Antigen processing assay shown by representative confocal images of control and IRGM siRNA transfected THP - 1 cells treated with DQ - OVA ( green ) ( 10 µg / ml , 30 min ) . Scale Bar , 10 µm . ( M ) Graph depicts percentage of control and IRGM knockdown THP - 1 cells with DQ - OVA puncta '

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(L, M) Antigen processing assay shown by representative confocal images of control and IRGM siRNA transfected THP-1 cells treated with DQ-OVA (green) (10 µg/ml, 30 min). Scale Bar, 10 µm. (M) Graph depicts percentage of control and IRGM knockdown THP-1 cells with DQ-OVA puncta's


( P ) Representative confocal images of H - 2Kb - SIINFEKL ( red ) on the surface of Irgm1 + / + and irgm1 - / - mouse BMDMs treated with OVA ( 2 mg / ml , 3 h ) , Scale bar 5 µm or 8 µm ( as indicated ) .

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(P) Representative confocal images of H-2Kb-SIINFEKL (red) on the surface of Irgm1+/+ and irgm1-/- mouse BMDMs treated with OVA (2 mg/ml, 3 h), Scale bar 5 µm or 8 µm (as indicated).


( C ) Representative images of the plaque assay in Vero cells performed from the culture supernatant of CHIKV ( MOI 1 , 24 h ) infected HT - 29 control and IRGM knockdown cells .

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(C) Representative images of the plaque assay in Vero cells performed from the culture supernatant of CHIKV (MOI 1, 24 h) infected HT-29 control and IRGM knockdown cells.


( F ) Representative images of plaque assay in Vero cells performed from the culture supernatant of HSV - 1 ( MOI 1 , 24 h ) infected HT - 29 control and IRGM knockdown cells .

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(F) Representative images of plaque assay in Vero cells performed from the culture supernatant of HSV-1 (MOI 1, 24 h) infected HT-29 control and IRGM knockdown cells.


( I ) Representative images of plaque assay in Vero cells performed from the culture supernatant of JEV ( MOI 1 , 24 h ) infected HT - 29 control and IRGM knockdown cells .

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(I) Representative images of plaque assay in Vero cells performed from the culture supernatant of JEV (MOI 1, 24 h) infected HT-29 control and IRGM knockdown cells.


( L ) Representative images of plaque assay in Vero cells were performed from the culture supernatant of WNV ( MOI 0 . 3 , 16 h ) infected HuH7 control and IRGM knockdown cells .

(L) Representative images of plaque assay in Vero cells were performed from the culture supernatant of WNV (MOI 0.3, 16 h) infected HuH7 control and IRGM knockdown cells.


( O ) Representative images of plaque assay in Vero cells were performed from the culture supernatant of ZIKV ( MOI 5 , 48 h ) infected Huh7 control and IRGM knockdown cells .

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(O) Representative images of plaque assay in Vero cells were performed from the culture supernatant of ZIKV (MOI 5, 48 h) infected Huh7 control and IRGM knockdown cells.


( S ) Representative images of plaque assay in Vero E6 cells were performed from the culture supernatant of SARS - CoV2 ( MOI 1 , 24 h ) infected THP - 1 control and IRGM knockdown cells .

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(S) Representative images of plaque assay in Vero E6 cells were performed from the culture supernatant of SARS-CoV2 (MOI 1, 24 h) infected THP-1 control and IRGM knockdown cells.


( D ) Kaplan - Meier survival graph depicts percentage survival of mock and CHIKV infected irgm1 + / + and irgm1 - / - neonates during the course of

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(D) Kaplan-Meier survival graph depicts percentage survival of mock and CHIKV infected irgm1+/+ and irgm1-/- neonates during the course of infection


( H , I ) Total RNA isolated from muscles and brains of mock and CHIKV infected ( MOI 1x107 PFU / mouse ) ( 6 dpi ) irgm1 + / + and irgm1 - / - mice and was subjected to qRT PCR with MX2 The total RNA used for qRT PCR with the brain is four times more than muscles .

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(H, I) Total RNA isolated from muscles and brains of mock and CHIKV infected (MOI 1x107 PFU/mouse) (6 dpi) irgm1+/+ and irgm1-/-mice and was subjected to qRT PCR with MX2 The total RNA used for qRT PCR with the brain is four times more than muscles.


( A - D ) Control and si - IRGM transfected THP - 1 IFN reporter cells were kept uninfected ( Mock ) or infected with ( A ) JEV ( MOI 5 ) or ( B ) CHIKV ( MOI 5 ) or ( C ) HSV - 1 ( MOI 2 . 5 ) or ( D ) VSV - eGFP ( MOI 1 ) and the supernatant collected 8 hpi were subjected to luciferase assay . The graphs depict fold change in interferon response .

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(A-D) Control and si-IRGM transfected THP-1 IFN reporter cells were kept uninfected (Mock) or infected with (A) JEV (MOI 5) or (B) CHIKV (MOI 5) or (C) HSV-1 (MOI 2.5) or (D) VSV-eGFP (MOI 1) and the supernatant collected 8 hpi were subjected to luciferase assay. The graphs depict fold change in interferon response.


( L ) Western blot analysis with cell lysates of mock and CHIKV ( MOI 5 , 24 h ) infected control and IRGM knockdown HT - 29 cells and probed with the indicated antibodies . ( M ) Western blot analysis with cell lysates of mock and JEV ( MOI 5 , 24 h ) infected control and si - IRGM transfected HT - 29 cells and probed with the indicated antibodies .

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(L) Western blot analysis with cell lysates of mock and CHIKV (MOI 5, 24 h) infected control and IRGM knockdown HT-29 cells and probed with the indicated antibodies. (M) Western blot analysis with cell lysates of mock and JEV (MOI 5, 24 h) infected control and si-IRGM transfected HT-29 cells and probed with the indicated antibodies.


( Q ) Western blot analysis with cell lysates of mock and CHIKV ( MOI 1 , 24 h ) infected control and IRGM knockdown HT - 29 cells transfected with indicated siRNA and probed with the indicated antibodies .

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(Q) Western blot analysis with cell lysates of mock and CHIKV (MOI 1, 24 h) infected control and IRGM knockdown HT-29 cells transfected with indicated siRNA and probed with the indicated antibodies.


A Representative immunofluorescence for PH3 ( green ) , combined with DAPI staining ( white ) , of E14 . 5 wildtype ( WT ) and 11B mouse dorsolateral neocortex rostrally . B Representative immunofluorescence for pVim ( yellow ) , combined with DAPI staining ( white ) , of E14 . 5 WT and 11B mouse dorsolateral neocortex rostrally . Boxes ( 50 µm x 65 µm ) in ( B ) indicate areas that are shown at higher magnification in ( B ' ) . Arrow , pVim + BP with basal process ( mitotic bRG ) ; notched arrowheads , basal process ; arrowhead , pVim + BP without basal process ( mitotic bIP ) . C - E Quantification of PH3 + APs ( C ) , PH3 + BPs ( D ) and pVim + bRG ( E left ) and bIPs ( E right ) in a 200 µm - wide field of E14 . 5 WT ( white ) and 11B ( black ) mouse dorsolateral neocortex rostrally . Note the difference in ordinate scales in ( E ) . F Data information : Images are single optical sections Scale bars , 50 µm 10 µm Data are the mean of 6 - 19 ( WT ) and 6 - 26 ( 11B ) littermate embryos , which were derived from 3 - 9 separate litters . Error bars indicate SD , two - tailed unpaired Student ' s t - test , * P < 0 . 05 ; * * P < 0 . 01 P = 0 . 0269 ( D ) ; P = 0 . 0302 ( E , left ) ; P = 0 . 0139 ( E , right )

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A Representative immunofluorescence for PH3 (green), combined with DAPI staining (white), of E14.5 wildtype (WT) and 11B mouse dorsolateral neocortex rostrally. B Representative immunofluorescence for pVim (yellow), combined with DAPI staining (white), of E14.5 WT and 11B mouse dorsolateral neocortex rostrally. Boxes (50 µm x 65 µm) in (B) indicate areas that are shown at higher magnification in (B'). Arrow, pVim+ BP with basal process (mitotic bRG); notched arrowheads, basal process; arrowhead, pVim+ BP without basal process (mitotic bIP). C-E Quantification of PH3+ APs (C), PH3+ BPs (D) and pVim+ bRG (E left) and bIPs (E right) in a 200 µm-wide field of E14.5 WT (white) and 11B (black) mouse dorsolateral neocortex rostrally. Note the difference in ordinate scales in (E). F Data information: Images are single optical sections Scale bars, 50 µm 10 µm Data are the mean of 6-19 (WT) and 6-26 (11B) littermate embryos, which were derived from 3-9 separate litters. Error bars indicate SD, two-tailed unpaired Student's t-test, * P < 0.05; ** P < 0.01 P = 0.0269 (D); P = 0.0302 (E, left); P = 0.0139 (E, right)


F Representative immunofluorescence for Pax6 ( cyan ) and Tbr2 ( magenta ) , combined with DAPI staining ( white ) , of E14 . 5 WT and 11B mouse dorsolateral neocortex rostrally . Boxes ( 25 µm x 25 µm ) in ( F ) indicate areas that are shown at higher magnification in ( F ' ) . Outlined by dashed white lines , Pax6 + & Tbr2 + BP ; outlined by solid white lines , Pax6 + & Tbr2 - BP . G - K Quantification of Pax6 + & Tbr2 - cells in VZ ( G ) , Pax6 + & Tbr2 + cells in VZ ( H ) , Pax6 + & Tbr2 - cells in SVZ and IZ ( I ) , Pax6 + & Tbr2 + cells in SVZ and IZ ( J ) and Pax6 - & Tbr2 + cells in SVZ and IZ ( K ) in a 200 µm - wide field of E14 . 5 WT ( white ) and 11B ( black ) mouse dorsolateral neocortex rostrally . L Data information : Images are Z projections of 4 optical sections Scale bars , 50 µm 10 µm Data are the mean of 6 - 19 ( WT ) and 6 - 26 ( 11B ) littermate embryos , which were derived from 3 - 9 separate litters . Error bars indicate SD , two - tailed unpaired Student ' s t - test , * P < 0 . 05 ; * * P < 0 . 01 P = 0 . 0281 ( I ) ; P = 0 . 0088 ( J ) ; P = 0 . 0014 ( K )

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

F Representative immunofluorescence for Pax6 (cyan) and Tbr2 (magenta), combined with DAPI staining (white), of E14.5 WT and 11B mouse dorsolateral neocortex rostrally. Boxes (25 µm x 25 µm) in (F) indicate areas that are shown at higher magnification in (F'). Outlined by dashed white lines, Pax6+ & Tbr2+ BP; outlined by solid white lines, Pax6+ & Tbr2- BP. G-K Quantification of Pax6+ & Tbr2- cells in VZ (G), Pax6+ & Tbr2+ cells in VZ (H), Pax6+ & Tbr2- cells in SVZ and IZ (I), Pax6+ & Tbr2+ cells in SVZ and IZ (J) and Pax6- & Tbr2+ cells in SVZ and IZ (K) in a 200 µm-wide field of E14.5 WT (white) and 11B (black) mouse dorsolateral neocortex rostrally. L Data information: Images are Z projections of 4 optical sections Scale bars, 50 µm 10 µm Data are the mean of 6-19 (WT) and 6-26 (11B) littermate embryos, which were derived from 3-9 separate litters. Error bars indicate SD, two-tailed unpaired Student's t-test, * P < 0.05; ** P < 0.01 P = 0.0281 (I); P = 0.0088 (J); P = 0.0014 (K)


L - N , Sequential BrdU - EdU labelling . BrdU was injected into pregnant mice at E13 . 5 , i . e . 24 hr before sacrifice at E14 . 5 , and EdU was injected into the same pregnant mice 0 . 5 hr before sacrifice at E14 . 5 . ( L ) Representative immunofluorescence for BrdU ( magenta ) , combined with EdU staining ( cyan ) and DAPI staining ( white ) , of E14 . 5 WT and 11B mouse dorsolateral neocortex rostrally . ( M , N ) Quantification of BrdU + & EdU + cells ( M ) and the percentage of BrdU + cells that are BrdU + & EdU + ( N ) in VZ and SVZ in a 200 µm - wide field of E14 . 5 WT ( white ) and 11B ( black ) mouse dorsolateral neocortex rostrally . Data information : Data are the mean of 6 - 19 ( WT ) and 6 - 26 ( 11B ) littermate embryos , which were derived from 3 - 9 separate litters . Error bars indicate SD , two - tailed unpaired Student ' s t - test , * P < 0 . 05 ; * * P < 0 . 01 P = 0 . 0049 ( M , SVZ ) ; P = 0 . 0323 ( N , SVZ ) .

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

L-N, Sequential BrdU-EdU labelling. BrdU was injected into pregnant mice at E13.5, i.e. 24 hr before sacrifice at E14.5, and EdU was injected into the same pregnant mice 0.5 hr before sacrifice at E14.5. (L) Representative immunofluorescence for BrdU (magenta), combined with EdU staining (cyan) and DAPI staining (white), of E14.5 WT and 11B mouse dorsolateral neocortex rostrally. (M, N) Quantification of BrdU+ & EdU+ cells (M) and the percentage of BrdU+ cells that are BrdU+ & EdU+ (N) in VZ and SVZ in a 200 µm-wide field of E14.5 WT (white) and 11B (black) mouse dorsolateral neocortex rostrally. Data information: Data are the mean of 6-19 (WT) and 6-26 (11B) littermate embryos, which were derived from 3-9 separate litters. Error bars indicate SD, two-tailed unpaired Student's t-test, * P < 0.05; ** P < 0.01 P = 0.0049 (M, SVZ); P = 0.0323 (N, SVZ).


H Representative immunofluorescence for Tbr1 ( cyan ) , Ctip2 ( magenta ) and Satb2 ( green ) , combined with DAPI staining ( white ) , of E18 . 5 WT and 11B mouse neocortex at the position where cortical thickness was measured ( A , red lines ) . I Quantification of zone thickness of E18 . 5 WT ( white ) and 11B ( black ) mouse neocortex at the position where cortical thickness was measured ( A , red lines ) . J Data information : Images are single optical sections Scale bar , 20 µm Data are the mean of 7 ( WT ) and 8 - 12 ( 11B ) littermate embryos , which were derived from 4 separate litters . Error bars indicate SD . Two - way ANOVA , followed by Bonferroni ' s multiple comparisons test ( B - D ) ; two - tailed unpaired Student ' s t - test P = 0 . 0287 ( I , IZ ) ; P = 0 .

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

H Representative immunofluorescence for Tbr1 (cyan), Ctip2 (magenta) and Satb2 (green), combined with DAPI staining (white), of E18.5 WT and 11B mouse neocortex at the position where cortical thickness was measured (A, red lines). I Quantification of zone thickness of E18.5 WT (white) and 11B (black) mouse neocortex at the position where cortical thickness was measured (A, red lines). J Data information: Images are single optical sections Scale bar, 20 µm Data are the mean of 7 (WT) and 8-12 (11B) littermate embryos, which were derived from 4 separate litters. Error bars indicate SD. Two-way ANOVA, followed by Bonferroni's multiple comparisons test (B-D); two-tailed unpaired Student's t-test P = 0.0287 (I, IZ); P = 0.0039


A Dorsal view of adult wildtype ( WT ) and 11B mouse brain at P56 . Orange lines illustrate the measurements of neocortex width and length ; red lines illustrate the measurements of neocortex area . B , C Quantification of neocortex width , length and area . D Data information : Scale bar , 1 mm Data are the mean of 8 ( WT ) and 8 ( 11B ) adult littermate mice . Error bars indicate SD , two - tailed unpaired Student ' s t - test , * P < 0 . 05 , * * P < 0 . 01 , * * * * P < 0 . 0001 . P < 0 . 0001 ( B , neocortex width ) ; P < 0 . 0001 ( C )

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

A Dorsal view of adult wildtype (WT) and 11B mouse brain at P56. Orange lines illustrate the measurements of neocortex width and length; red lines illustrate the measurements of neocortex area. B, C Quantification of neocortex width, length and area. D Data information: Scale bar, 1 mm Data are the mean of 8 (WT) and 8 (11B) adult littermate mice. Error bars indicate SD, two-tailed unpaired Student's t-test , * P < 0.05, ** P < 0.01, **** P < 0.0001. P < 0.0001 (B, neocortex width); P < 0.0001 (C)


G , H Representative immunofluorescence for Ctip2 ( magenta ) , Satb2 ( green ) and Brn2 ( yellow ) , combined with DAPI staining ( white ) , of P56 adult WT and 11B mouse neocortex at the position where cortical thickness was measured ( D , red lines ) . Data information : Images are single optical sections Scale bar , 20

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

G, H Representative immunofluorescence for Ctip2 (magenta), Satb2 (green) and Brn2 (yellow), combined with DAPI staining (white), of P56 adult WT and 11B mouse neocortex at the position where cortical thickness was measured (D, red lines). Data information: Images are single optical sections Scale bar, 20 µm


IntelliCage was used to evaluate the memory flexibility of adult 11B mice . Four separate sessions were carried out : ( i ) 5 WT males together with 5 11B males ; ( ii ) 5 other WT males together with 5 other 11B males ; ( iii ) 5 WT females together with 7 11B females ; ( iv ) 4 other WT females together with 8 other 11B females . Data are the mean of the total 10 males ( WT ) , 10 males ( 11B ) , 9 females ( WT ) and 15 females ( 11B ) adult littermate mice . Data points were obtained at consecutive days , error bars indicate SD . ( A , B ) Quantification of the discrimination error rate in the first 100 visits during the drinking phase of the acquisition stage of adult wildtype ( WT ) and 11B mice ( male , A ; female , B ) in the IntelliCage . Overall place learning performance of WT and 11B mice was analysed using two - way ANOVA followed by Fisher ' s LSD test ; all data points during the acquisition phase were included . For males ( A ) , WT vs . 11B , P > 0 . 05 ; data over time , * * * * P < 0 . 0001 ; interaction , P > 0 . 05 . For females ( B ) , WT vs . 11B , P > 0 . 05 ; data over time , * * * * P < 0 . 0001 ; interaction , P > 0 . 05 . Note that ( i ) the two male sessions and the two female sessions yielded essentially the same results , and ( ii ) not shown in panels the two separate male sessions yielded essentially the same results , and the two separate female sessions yielded essentially the same results .

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

IntelliCage was used to evaluate the memory flexibility of adult 11B mice. Four separate sessions were carried out: (i) 5 WT males together with 5 11B males; (ii) 5 other WT males together with 5 other 11B males; (iii) 5 WT females together with 7 11B females; (iv) 4 other WT females together with 8 other 11B females. Data are the mean of the total 10 males (WT), 10 males (11B), 9 females (WT) and 15 females (11B) adult littermate mice. Data points were obtained at consecutive days, error bars indicate SD. (A, B) Quantification of the discrimination error rate in the first 100 visits during the drinking phase of the acquisition stage of adult wildtype (WT) and 11B mice (male, A; female, B) in the IntelliCage. Overall place learning performance of WT and 11B mice was analysed using two-way ANOVA followed by Fisher's LSD test; all data points during the acquisition phase were included. For males (A), WT vs. 11B, P > 0.05; data over time, **** P < 0.0001; interaction, P > 0.05. For females (B), WT vs. 11B, P > 0.05; data over time, **** P < 0.0001; interaction, P > 0.05. Note that (i) the two male sessions and the two female sessions yielded essentially the same results, and (ii) not shown in panels the two separate male sessions yielded essentially the same results, and the two separate female sessions yielded essentially the same results.


Expression levels of the autophagosome marker LC3B - II were evaluated in wild - type ( WT ) and Ctns − / − fibroblasts by Western blot ( WB ) . Data are representative of five different experiments with similar results .

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

Expression levels of the autophagosome marker LC3B-II were evaluated in wild-type (WT) and Ctns−/− fibroblasts by Western blot (WB). Data are representative of five different experiments with similar results.


WT and Ctns − / − fibroblasts were stained using LC3B antibodies and analyzed by fluorescence microscopy . The number of LC3B - positive puncta was quantified by the ImagePro software and normalized per area arbitrary units . Results are mean ± SEM ( n = 27 WT and 24 Ctns − / − cells ) . * * * P < 0 . 001 ( unpaired t - test ) .

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

WT and Ctns−/− fibroblasts were stained using LC3B antibodies and analyzed by fluorescence microscopy. The number of LC3B-positive puncta was quantified by the ImagePro software and normalized per area arbitrary units. Results are mean ± SEM (n = 27 WT and 24 Ctns−/− cells). ***P < 0.001 (unpaired t-test).


Immunofluorescence analysis of liver ( C ) and kidney ( D ) tissues from WT and Ctns − / − GFP - LC3 transgenic mice using anti - GFP antibodies . Quantification of LC3 puncta was obtained by counting the number of GFP - positive structures relative to the number of nuclei in the same field , using the ImagePro software . GFP - LC3 puncta were counted by analyzing 6 to 10 fields per tissue section ( 200 - 400 nuclei per field ) , in a total of 3 WT and 3 Ctns − / − mice expressing GFP - LC3 . Results are mean ± SEM . In ( C ) , * * P = 0 . 0029 ; in ( D ) , * * P = 0 . 0025 ( unpaired t - test ) . Scale bar : 5 μm .

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

Immunofluorescence analysis of liver (C) and kidney (D) tissues from WT and Ctns−/−GFP-LC3 transgenic mice using anti-GFP antibodies. Quantification of LC3 puncta was obtained by counting the number of GFP-positive structures relative to the number of nuclei in the same field, using the ImagePro software. GFP-LC3 puncta were counted by analyzing 6 to 10 fields per tissue section (200-400 nuclei per field), in a total of 3 WT and 3 Ctns−/−mice expressing GFP-LC3. Results are mean ± SEM. In (C), **P = 0.0029; in (D), **P = 0.0025 (unpaired t-test). Scale bar: 5 μm.


Representative images of wild - type ( WT ) and Ctns − / − mouse fibroblasts transfected with the ptfLC3 vector under resting conditions ( fed ) , or after serum starvation ( Serum Starv . ) in the presence or absence of the alkalinizing drug chloroquine ( CQ ) . GFP and RFP staining was analyzed by confocal microscopy . Examples of red - only puncta ( mature autophagosomes ) are indicated with arrows . Scale bar : 5 μm . The percentage of mature autophagosomes ( red - only vesicles ) was calculated based on the ratio between the number of red - only puncta and the total number of autophagosomes ( number of green and red + red - only puncta ) . The graph is representative of three different experiments with similar results . Results are mean ± SEM ( n = 7 cells per condition ) . In addition to the experimental conditions shown in ( A ) , the Aa / serum starvation condition is included .

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

Representative images of wild-type (WT) and Ctns−/− mouse fibroblasts transfected with the ptfLC3 vector under resting conditions (fed), or after serum starvation (Serum Starv.) in the presence or absence of the alkalinizing drug chloroquine (CQ). GFP and RFP staining was analyzed by confocal microscopy. Examples of red-only puncta (mature autophagosomes) are indicated with arrows. Scale bar: 5 μm.The percentage of mature autophagosomes (red-only vesicles) was calculated based on the ratio between the number of red-only puncta and the total number of autophagosomes (number of green and red + red-only puncta). The graph is representative of three different experiments with similar results. Results are mean ± SEM (n = 7 cells per condition). In addition to the experimental conditions shown in (A), the Aa/serum starvation condition is included.


WT and Ctns − / − fibroblasts under resting conditions were treated with 100 nM bafilomycin A ( BafA ) for 2 h and LC3B - II levels were analyzed by Western blot ( WB ) .

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 O O O O O

WT and Ctns−/− fibroblasts under resting conditions were treated with 100 nM bafilomycin A (BafA) for 2 h and LC3B-II levels were analyzed by Western blot (WB).


Phosphorylation levels of the mTOR complex kinase 1 substrate S6K and LC3B - II levels in WT and Ctns − / − fibroblasts were measured by WB under resting conditions ( − ) , withdrawal of both amino acids and serum ( Aa / Ser . Starv . ) and subsequent recovery by replacement of starvation medium with normal cell growth medium ( Rec ) , in the presence or absence of 100 nM BafA for the indicated time .

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

Phosphorylation levels of the mTOR complex kinase 1 substrate S6K and LC3B-II levels in WT and Ctns−/− fibroblasts were measured by WB under resting conditions (−), withdrawal of both amino acids and serum (Aa/Ser. Starv.) and subsequent recovery by replacement of starvation medium with normal cell growth medium (Rec), in the presence or absence of 100 nM BafA for the indicated time.


WB analysis showing phosphorylation levels of the mTOR substrate S6K and expression of LC3B - II in resting or serum - starved WT , Ctns − / − and Ctns − / − fibroblasts expressing GFP - CTNS .

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WB analysis showing phosphorylation levels of the mTOR substrate S6K and expression of LC3B-II in resting or serum-starved WT,Ctns−/− and Ctns−/− fibroblasts expressing GFP-CTNS.


Analysis of the expression of endogenous SQSTM1 in wild - type or Ctns − / − mouse fibroblasts under fed ( serum ) or starved ( 15 h serum starvation ) conditions . In these assays , 20 μg of total protein lysates were resolved by SDS - PAGE and analyzed by Western blot ( WB ; A ) . Equal loading was established by silver staining ( B ) . Quantification ( mean ± SEM ) of five different areas of each silver - stained lane are shown in ( C ) .

O O O O O O B-MEASURED_VAR 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

Analysis of the expression of endogenous SQSTM1 in wild-type or Ctns−/− mouse fibroblasts under fed (serum) or starved (15 h serum starvation) conditions. In these assays, 20 μg of total protein lysates were resolved by SDS-PAGE and analyzed by Western blot (WB; A). Equal loading was established by silver staining (B). Quantification (mean ± SEM) of five different areas of each silver-stained lane are shown in (C).


Degradation of long - lived proteins using metabolic labeling was performed as described in Supplementary Materials and Methods . The data represent the mean ± SEM of nine independent samples analyzed in three independent experiments . * P = 0 . 009 for WT and * P = 0 . 0013 for Ctns − / − , respectively . NS , not significant ( unpaired t - test ) .

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

Degradation of long-lived proteins using metabolic labeling was performed as described in Supplementary Materials and Methods. The data represent the mean ± SEM of nine independent samples analyzed in three independent experiments. *P = 0.009 for WT and *P = 0.0013 for Ctns−/−, respectively. NS, not significant (unpaired t-test).


The expression levels of the endogenous LAMP2A and LAMP1 proteins were analyzed in wild - type ( WT ) and Ctns − / − mousefibroblasts ( A ) , kidney ( B ) and liverlysosomes ( C ) by Western blot . Quantitative densitometry analysis of immunoblots obtained from different experiments was performed by calculating the ratio between LAMP2A and actin signals in each lane . Results are mean ± SEM of four independent experiments with mousefibroblasts , 5 WT and 6 Ctns − / − mousekidneys , 3 WT and 3 Ctns − / − mouselivers ( lysosomal extracts ) .

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The expression levels of the endogenous LAMP2A and LAMP1 proteins were analyzed in wild-type (WT) and Ctns−/−mousefibroblasts (A), kidney (B) and liverlysosomes (C) by Western blot. Quantitative densitometry analysis of immunoblots obtained from different experiments was performed by calculating the ratio between LAMP2A and actin signals in each lane. Results are mean ± SEM of four independent experiments with mousefibroblasts, 5 WT and 6 Ctns−/−mousekidneys, 3 WT and 3 Ctns−/−mouselivers (lysosomal extracts).


Comparative analysis of the amount of LAMP2A in wild - type or Ctns − / − liverlysosomes was performed using two different anti - LAMP2A antibodies . 15 μg of lysosomal proteins were loaded in each lane . Ab1 , anti - LAMP2A antibody raised against the twelve amino acids of the cytosolic region of ratLAMP2A largely validated to recognize mouseLAMP2A but not other LAMP2 isoforms ( Cuervo Dice , 1996 ) . Ab2 , anti - LAMP2A Abcam Ab18528 . Decreased lysosomalLAMP2A was evident using either antibody . LAMP1 was used as a loading control . Representative of two independent experiments with similar results .

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Comparative analysis of the amount of LAMP2A in wild-type or Ctns−/−liverlysosomes was performed using two different anti-LAMP2A antibodies. 15 μg of lysosomal proteins were loaded in each lane. Ab1, anti-LAMP2A antibody raised against the twelve amino acids of the cytosolic region of ratLAMP2A largely validated to recognize mouseLAMP2A but not other LAMP2 isoforms (Cuervo Dice, 1996). Ab2, anti-LAMP2A Abcam Ab18528. Decreased lysosomalLAMP2A was evident using either antibody. LAMP1 was used as a loading control. Representative of two independent experiments with similar results.


Immunofluorescence analyses of endogenous LAMP1 , LAMP2A ( A , B ) , LAMP2B ( C ) and total LAMP2 ( D ) localization in wild - type ( WT ) and Ctns − / − mouse fibroblasts . ( A ) Confocal microscopy analysis . White arrows represent LAMP1 - positive circular structures ( lysosomes ) which were visible in both WT and Ctns − / − cells . Colocalization of LAMP1 and LAMP2A was observed in WT fibroblasts ( white arrows , merged panel ) but rarely in Ctns − / − cells ( red arrow ) . Most LAMP2A showed a punctate pattern and lack of colocalization with LAMP1 in Ctns − / − cells ( arrowheads , lower panels ) . Scale bars : 5 μm . Inset scale bars : 2 μm .

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Immunofluorescence analyses of endogenous LAMP1, LAMP2A (A, B), LAMP2B (C) and total LAMP2 (D) localization in wild-type (WT) and Ctns−/− mouse fibroblasts. (A) Confocal microscopy analysis. White arrows represent LAMP1-positive circular structures (lysosomes) which were visible in both WT and Ctns−/− cells. Colocalization of LAMP1 and LAMP2A was observed in WT fibroblasts (white arrows, merged panel) but rarely in Ctns−/− cells (red arrow). Most LAMP2A showed a punctate pattern and lack of colocalization with LAMP1 in Ctns−/− cells (arrowheads, lower panels). Scale bars: 5 μm. Inset scale bars: 2 μm.


Immunofluorescence analyses of endogenous LAMP1 , LAMP2A ( A , B ) , LAMP2B ( C ) and total LAMP2 ( D ) localization in wild - type ( WT ) and Ctns − / − mouse fibroblasts . ( B ) High - resolution stochastic optical reconstruction microscopy ( STORM ) analysis of the localization of LAMP1 and LAMP2A in wild - type and Ctns − / − cells . In Ctns − / − cells , LAMP2A was detected near ( arrowheads , estimated distance > 50 nm ) but not always adjacent ( 10 - 50 nm ) to LAMP1 or at LAMP1 - negative structures ( arrows ) . Scale bar : 0 . 5 μm .

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Immunofluorescence analyses of endogenous LAMP1, LAMP2A (A, B), LAMP2B (C) and total LAMP2 (D) localization in wild-type (WT) and Ctns−/− mouse fibroblasts.(B) High-resolution stochastic optical reconstruction microscopy (STORM) analysis of the localization of LAMP1 and LAMP2A in wild-type and Ctns−/− cells. In Ctns−/− cells, LAMP2A was detected near (arrowheads, estimated distance > 50 nm) but not always adjacent (10-50 nm) to LAMP1 or at LAMP1-negative structures (arrows). Scale bar: 0.5 μm.


Immunofluorescence analyses of endogenous LAMP1 , LAMP2A ( A , B ) , LAMP2B ( C ) and total LAMP2 ( D ) localization in wild - type ( WT ) and Ctns − / − mouse fibroblasts . ( C ) Immunofluorescence showing colocalization of endogenous LAMP1 and LAMP2B in both wild - type and CTNS - deficient fibroblasts ( arrows ) . Scale bar : 20 μm .

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Immunofluorescence analyses of endogenous LAMP1, LAMP2A (A, B), LAMP2B (C) and total LAMP2 (D) localization in wild-type (WT) and Ctns−/− mouse fibroblasts. (C) Immunofluorescence showing colocalization of endogenous LAMP1 and LAMP2B in both wild-type and CTNS-deficient fibroblasts (arrows). Scale bar: 20 μm.


Immunofluorescence analyses of endogenous LAMP1 , LAMP2A ( A , B ) , LAMP2B ( C ) and total LAMP2 ( D ) localization in wild - type ( WT ) and Ctns − / − mouse fibroblasts . ( D ) Immunofluorescence analysis of LAMP1 and total LAMP2 ( antibody ABL93 DSHB ) . Arrows show colocalization . Arrowheads show LAMP2 - positive LAMP1 - negative vesicles .

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Immunofluorescence analyses of endogenous LAMP1, LAMP2A (A, B), LAMP2B (C) and total LAMP2 (D) localization in wild-type (WT) and Ctns−/− mouse fibroblasts. (D) Immunofluorescence analysis of LAMP1 and total LAMP2 (antibody ABL93 DSHB). Arrows show colocalization. Arrowheads show LAMP2-positive LAMP1-negative vesicles.


Immunofluorescence analyses of endogenous LAMP2A , ER ( Grp78 / 94 ) ( A ) , which is expanded in cystinotic cells ( white arrow ) , VAMP7 - positive vesicles ( B ) and Rab11a - positive structures ( C ) was performed using wild - type or Ctns − / − fibroblasts as described in Materials and Methods . At least 2 independent experiments were performed for each vesicular trafficking or ER marker . ( B , C ) Results are expressed as the percentage of LAMP2A that colocalizes with VAMP7 or Rab11 . For Rab11 / LAMP2A colocalization , 29 wild - type and 15 Ctns − / − cells were quantified . For VAMP7 / LAMP2A colocalization ( white arrows ) , 49 WT and 57 Ctns − / − cells were analyzed . Data are expressed as mean ± SEM . * P < 0 . 001 ( unpaired t - test ) . Scale bars : 20 μm .

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

Immunofluorescence analyses of endogenous LAMP2A, ER (Grp78/94) (A), which is expanded in cystinotic cells (white arrow), VAMP7-positive vesicles (B) and Rab11a-positive structures (C) was performed using wild-type or Ctns−/− fibroblasts as described in Materials and Methods. At least 2 independent experiments were performed for each vesicular trafficking or ER marker. (B, C) Results are expressed as the percentage of LAMP2A that colocalizes with VAMP7 or Rab11. For Rab11/LAMP2A colocalization, 29 wild-type and 15 Ctns−/− cells were quantified. For VAMP7/LAMP2A colocalization (white arrows), 49 WT and 57 Ctns−/− cells were analyzed. Data are expressed as mean ± SEM. *P < 0.001 (unpaired t-test). Scale bars: 20 μm.


WT , Ctns − / − or Ctns − / − mouse fibroblasts treated for 20 h with either a combination of both leupeptin and chloroquine ( CQ / Leu ) or bafilomycin A ( BafA ) alone were fixed and immunostained with antibodies recognizing endogenous LAMP1 and LAMP2A proteins and samples were analyzed by confocal microscopy as described in Material and Methods . Lysosomal colocalization of LAMP1 and LAMP2A was evident in treated ( middle lower and bottom panels ) but not in untreated ( middle upper panels ) Ctns − / − cells . Scale bars : 5 μm . Inset scale bars : 2 μm .

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 B-MEASURED_VAR 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 B-MEASURED_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

WT,Ctns−/− or Ctns−/− mouse fibroblasts treated for 20 h with either a combination of both leupeptin and chloroquine (CQ/Leu) or bafilomycin A (BafA) alone were fixed and immunostained with antibodies recognizing endogenous LAMP1 and LAMP2A proteins and samples were analyzed by confocal microscopy as described in Material and Methods. Lysosomal colocalization of LAMP1 and LAMP2A was evident in treated (middle lower and bottom panels) but not in untreated (middle upper panels) Ctns−/− cells. Scale bars: 5 μm. Inset scale bars: 2 μm.


Lysosomes were isolated from livers of starved wild - type ( WT ) and Ctns − / − mice as described in Supplementary Materials and Methods and incubated at 37 ° C for 30 min with the CMA substrate GAPDH , in the presence or absence of ATP ( necessary for CMA ) and protease inhibitors ( Prot . Inhib . ) . A fraction of the CMA reactions was then mixed with sample buffer and boiled at 95 ° C for 5 min , followed by SDS - PAGE and GAPDH and LAMP1 immunoblotting . Quantitative densitometry analysis of CMA activity performed in independent experiments using lysosomes isolated from a total of 11 WT and 10 Ctns − / − mice . Results are mean ± SEM . * * * P = 0 . 0005 ; * * P = 0 . 0041 ; N . S . , not significant ( unpaired t - test ) .

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Lysosomes were isolated from livers of starved wild-type (WT) and Ctns−/− mice as described in Supplementary Materials and Methods and incubated at 37°C for 30 min with the CMA substrate GAPDH, in the presence or absence of ATP (necessary for CMA) and protease inhibitors (Prot. Inhib.). A fraction of the CMA reactions was then mixed with sample buffer and boiled at 95°C for 5 min, followed by SDS-PAGE and GAPDH and LAMP1 immunoblotting.Quantitative densitometry analysis of CMA activity performed in independent experiments using lysosomes isolated from a total of 11 WT and 10 Ctns−/− mice. Results are mean ± SEM. ***P = 0.0005; **P = 0.0041; N.S., not significant (unpaired t-test).


The ability of lysosomal proteases to mediate GAPDH degradation was assessed by incubating Triton X - 100 - treated WT and Ctns − / − lysosomes with GAPDH for 30 min in acidic reaction buffer and compared with input ( reaction with no lysosomes ) . Quantitative densitometry analysis of independent reactions performed with lysosomes from a total of 9 WT and 9 Ctns − / − mice shows no significant difference in protease activity . Results are mean ± SEM .

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The ability of lysosomal proteases to mediate GAPDH degradation was assessed by incubating Triton X-100-treated WT and Ctns−/− lysosomes with GAPDH for 30 min in acidic reaction buffer and compared with input (reaction with no lysosomes). Quantitative densitometry analysis of independent reactions performed with lysosomes from a total of 9 WT and 9 Ctns−/− mice shows no significant difference in protease activity. Results are mean ± SEM.


WT and Ctns − / − mouse fibroblasts were treated with either 50 μM H2O2 or 1 mM paraquat ( PQ ) for 4 h and stained with ( A ) FITC - annexin V or ( B ) propidium iodide ( PI ) and analyzed by FACS . Results from 3 different experiments are shown as mean ± SEM . In ( A ) , * P = 0 . 05 ; * * * P = 0 . 0002 . In ( B ) , * P = 0 . 046 ; * * P = 0 . 0065 . Unpaired t - test .

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WT and Ctns−/− mouse fibroblasts were treated with either 50 μM H2O2 or 1 mM paraquat (PQ) for 4 h and stained with (A) FITC-annexin V or (B) propidium iodide (PI) and analyzed by FACS. Results from 3 different experiments are shown as mean ± SEM. In (A), *P = 0.05; ***P = 0.0002. In (B), *P = 0.046; **P = 0.0065. Unpaired t-test.


WT and Ctns − / − cells were treated as described above and samples analyzed by Western blot using antibodies recognizing cleaved ( active ) caspase 3 . Data are representative of 2 independent experiments with similar results .

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WT and Ctns−/− cells were treated as described above and samples analyzed by Western blot using antibodies recognizing cleaved (active) caspase 3. Data are representative of 2 independent experiments with similar results.


Cystine content in WT , Ctns − / − , cysteamine - treated Ctns − / − fibroblasts or Ctns − / − fibroblasts expressing GFP - CTNS was assessed by mass spectrometry .

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Cystine content in WT,Ctns−/−, cysteamine-treated Ctns−/− fibroblasts or Ctns−/− fibroblasts expressing GFP-CTNS was assessed by mass spectrometry.


WT , Ctns − / − , Ctns − / − fibroblasts treated with 1 mM cysteamine ( 48 h ) , or Ctns − / − fibroblasts expressing GFP - CTNS were fixed and stained with anti - LAMP1 and LAMP2A antibodies as indicated . For better visualization of LAMP1 / LAMP2A colocalization , GFP - CTNS was pseudocolored as magenta and LAMP1 was pseudocolored as green ( lower panels ) . Some lysosomes are indicated with arrows . Arrowheads indicate LAMP2A distribution to structures different from lysosomes ( only observed in Ctns − / − cells and in cysteamine - treated Ctns − / − cells ) . Rescue of the LAMP2A localization to lysosomes was observed in Ctns − / − cells expressing GFP - CTNS . Scale bar : 2 μm . Quantification of the colocalization analysis described in ( B ) . Calculation of the Pearson ' s colocalization coefficient was done by analyzing 113 WT , 251 Ctns − / − , 88 cysteamine - treated and 164 GFP - CTNS - expressing Ctns − / − cells , by using the ZEN 2010 software . Results are mean ± SEM . * * * P < 0 . 001 ; NS , not significant ( one - way ANOVA , Bonferroni ' s multiple comparisons test ) .

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WT, Ctns−/−, Ctns−/− fibroblasts treated with 1 mM cysteamine (48 h), or Ctns−/− fibroblasts expressing GFP-CTNS were fixed and stained with anti-LAMP1 and LAMP2A antibodies as indicated. For better visualization of LAMP1/LAMP2A colocalization, GFP-CTNS was pseudocolored as magenta and LAMP1 was pseudocolored as green (lower panels). Some lysosomes are indicated with arrows. Arrowheads indicate LAMP2A distribution to structures different from lysosomes (only observed in Ctns−/− cells and in cysteamine-treated Ctns−/− cells). Rescue of the LAMP2A localization to lysosomes was observed in Ctns−/− cells expressing GFP-CTNS. Scale bar: 2 μm.Quantification of the colocalization analysis described in (B). Calculation of the Pearson's colocalization coefficient was done by analyzing 113 WT, 251 Ctns−/−, 88 cysteamine-treated and 164 GFP-CTNS-expressing Ctns−/− cells, by using the ZEN 2010 software. Results are mean ± SEM. ***P < 0.001; NS, not significant (one-way ANOVA, Bonferroni's multiple comparisons test).


Cystine content in WT , Ctns − / − and cysteamine - treated Ctns − / − mouse livers was assessed by mass spectrometry . Results are mean ± SEM ( n = 4 ) . * * * P < 0 . 001 ; * * P = 0 . 0026 ( one - way ANOVA , Bonferroni ' s multiple comparisons test ) .

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Cystine content in WT,Ctns−/− and cysteamine-treated Ctns−/− mouse livers was assessed by mass spectrometry. Results are mean ± SEM (n = 4). ***P < 0.001; **P = 0.0026 (one-way ANOVA, Bonferroni's multiple comparisons test).


Western blot analysis of LAMP2A expression in lysosomes isolated from WT , Ctns − / − and cysteamine - treated Ctns − / − mouse livers .

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Western blot analysis of LAMP2A expression in lysosomes isolated from WT,Ctns−/− and cysteamine-treated Ctns−/− mouse livers.


Lysosomes , isolated as in ( B ) , were incubated with the CMA substrate GAPDH , and CMA activity was evaluated as in Fig8 and as described in Materials and Methods . Quantitative analysis of substrate degradation is expressed as percent of the residual amount of GAPDH in the full reaction relative to its amount in the presence of protease inhibitors . Results are mean ± SEM ( n = 4 ) . * P = 0 . 0126 for WT versus Ctns − / − and * P = 0 . 0436 for WT versus Ctns − / − + cysteamine ; NS , not significant ; unpaired t - test .

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Lysosomes, isolated as in (B), were incubated with the CMA substrate GAPDH, and CMA activity was evaluated as in Fig8 and as described in Materials and Methods.Quantitative analysis of substrate degradation is expressed as percent of the residual amount of GAPDH in the full reaction relative to its amount in the presence of protease inhibitors. Results are mean ± SEM (n = 4). *P = 0.0126 for WT versus Ctns−/− and *P = 0.0436 for WT versus Ctns−/− + cysteamine; NS, not significant; unpaired t-test.


A The intrinsic fluorescence of NAD ( P ) H co - localizes extensively with mitochondria ( marked by Mitotracker CMXRos )

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A The intrinsic fluorescence of NAD(P)H co-localizes extensively with mitochondria (marked by Mitotracker CMXRos)


B 2P - FLIM detects the fraction of NAD ( P ) H bound to enzyme partners ( a2 % )

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B 2P-FLIM detects the fraction of NAD(P)H bound to enzyme partners (a2%)


a2 % values ( starved , and after insulin or R + L addition ) were recorded pixel by pixel for mouse cortical ( C neurons . Each colored line in the center panel graphs refers to a single field of view containing 2 - 15 cells , and each pair of solid and dotted lines ( same color ) refers to the same field of view before and after stimulation , respectively . Using the same raw data , the relative contributions of NADH and NADPH to the total a2 % values were calculated ( Blacker et al , 2014 ) and are shown in the right panel bar graphs . NADH and NAD ( P ) H contributed an average of 63 % and 37 % , respectively , of the total a2 % values . All statistical analyses were performed using Student ' s two - tailed unpaired t -

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a2% values (starved, and after insulin or R+L addition) were recorded pixel by pixel for mouse cortical (C neurons. Each colored line in the center panel graphs refers to a single field of view containing 2-15 cells, and each pair of solid and dotted lines (same color) refers to the same field of view before and after stimulation, respectively. Using the same raw data, the relative contributions of NADH and NADPH to the total a2% values were calculated (Blacker et al, 2014) and are shown in the right panel bar graphs. NADH and NAD(P)H contributed an average of 63% and 37%, respectively, of the total a2% values. All statistical analyses were performed using Student's two-tailed unpaired t-tests


D a2 % values ( starved , and after insulin or R + L addition ) were recorded pixel by pixel fo human ( D ) neurons . Each colored line in the center panel graphs refers to a single field of view containing 2 - 15 cells , and each pair of solid and dotted lines ( same color ) refers to the same field of view before and after stimulation , respectively . Using the same raw data , the relative contributions of NADH and NADPH to the total a2 % values were calculated ( Blacker et al , 2014 ) and are shown in the right panel bar graphs . NADH and NAD ( P ) H contributed an average of 63 % and 37 % , respectively , of the total a2 % values . All statistical analyses were performed using Student ' s two - tailed unpaired t -

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

D a2% values (starved, and after insulin or R+L addition) were recorded pixel by pixel fo human (D) neurons. Each colored line in the center panel graphs refers to a single field of view containing 2-15 cells, and each pair of solid and dotted lines (same color) refers to the same field of view before and after stimulation, respectively. Using the same raw data, the relative contributions of NADH and NADPH to the total a2% values were calculated (Blacker et al, 2014) and are shown in the right panel bar graphs. NADH and NAD(P)H contributed an average of 63% and 37%, respectively, of the total a2% values. All statistical analyses were performed using Student's two-tailed unpaired t-tests


Wild type ( WT ) mouse cortical neurons cultured for 10 days in vitro ( A were serum - starved in Hank ' s balanced salt solution for 2 hours . Next , the cells were imaged immediately ( starved ) , after which either amino acids ( R + L ) or insulin were added . Thirty minutes later the cells were imaged again . Finally , the mTOR inhibitor , Torin1 was added , and after an additional 30 minutes , the cells were imaged for the last time . Histograms represent changes in the fraction of enzyme - bound NAD ( P ) H ( a2 % ) for each condition . Note that insulin and amino acids significantly increased the a2 % values , which was reversed by Torin1 - mediated inhibition of mTORC1 . Average data from 5 fields of view per condition are shown . Statistical analyses were performed using Student ' s two - tailed unpaired t -

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

Wild type (WT) mouse cortical neurons cultured for 10 days in vitro (A were serum-starved in Hank's balanced salt solution for 2 hours. Next, the cells were imaged immediately (starved), after which either amino acids (R+L) or insulin were added. Thirty minutes later the cells were imaged again. Finally, the mTOR inhibitor, Torin1 was added, and after an additional 30 minutes, the cells were imaged for the last time. Histograms represent changes in the fraction of enzyme-bound NAD(P)H (a2%) for each condition. Note that insulin and amino acids significantly increased the a2% values, which was reversed by Torin1-mediated inhibition of mTORC1. Average data from 5 fields of view per condition are shown. Statistical analyses were performed using Student's two-tailed unpaired t-test