qRT - PCR analysis of the three parasite lines at day 7 and 12 after infection . Error bars correspond to the standard deviation from two biological replicates .

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qRT-PCR analysis of the three parasite lines at day 7 and 12 after infection. Error bars correspond to the standard deviation from two biological replicates.


TEM reveals α1cm & ∆ introns - expressing sporozoites with smaller ( A and B ) and larger ( C ) diameters than wild type sporozoites . See also Fig S7 . Scale bar : 0 . 2 µm .

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TEM reveals α1cm&∆introns-expressing sporozoites with smaller (A and B) and larger (C) diameters than wild type sporozoites. See also Fig S7. Scale bar: 0.2 µm.


α1cm & ∆ introns - expressing sporozoites show a reduced number of subpellicular microtubules ( green arrowheads ) by TEM .

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α1cm&∆introns-expressing sporozoites show a reduced number of subpellicular microtubules (green arrowheads) by TEM.


Expression of different α1 - α2 chimeric α - tubulins instead of α1 - tubulin leads to sporozoite shape changes as revealed by SEM . See also Fig EV5 . Scale bar : 1 µm . α1 ∆ c - term lacks the last 3 amino acids of α1 - tubulin . α2 + parasites contain an α1 - tubulin with all amino acids that are different between the two α - tubulins changed into those of α2 - tubulin ; see also Fig S4 . α2 + + parasites contain α2 - tubulin ( exons , introns , 3 ' UTR ) and α2 + + + parasites express the exons of α2 - tubulin and introns of α1 - tubulin from the α1 - tubulin promoter .

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Expression of different α1-α2 chimeric α-tubulins instead of α1-tubulin leads to sporozoite shape changes as revealed by SEM. See also Fig EV5. Scale bar: 1 µm. α1∆c-term lacks the last 3 amino acids of α1-tubulin. α2+ parasites contain an α1-tubulin with all amino acids that are different between the two α-tubulins changed into those of α2-tubulin; see also Fig S4. α2++ parasites contain α2-tubulin (exons, introns, 3'UTR) and α2+++ parasites express the exons of α2-tubulin and introns of α1-tubulin from the α1-tubulin promoter.


SiR - tubulin staining reveals differences in microtubule lengths and intensity . Between 69 and 156 sporozoites were analysed per line . Box plots represent 50 % ( boxes ) and 95 % ( bars ) of data with horizontal bar showing the median . * * * , and * * * * indicate p < 0 . 001 and p < 0 . 0001 , respectively ; ns : not significant ; Kruskal - Wallis - test .

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SiR-tubulin staining reveals differences in microtubule lengths and intensity. Between 69 and 156 sporozoites were analysed per line. Box plots represent 50% (boxes) and 95% (bars) of data with horizontal bar showing the median. ***, and **** indicate p<0.001 and p<0.0001, respectively; ns: not significant; Kruskal-Wallis-test.


qRT - PCR analysis of the parasite lines at day 12 after infection shows different expression level . Note the subtly higher expression of α2 + + compared to α2 + + + . Error bars show standard deviation from the mean of two biological replicates .

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qRT-PCR analysis of the parasite lines at day 12 after infection shows different expression level. Note the subtly higher expression of α2++ compared to α2+++. Error bars show standard deviation from the mean of two biological replicates.


TEM along sporozoites shows a decrease of microtubule numbers towards the nucleus in parasites expressing α - tubulin chimeras . Green arrowheads point to microtubules . See also Fig S8 and S9 . Note that the WT control panel is shown already in Figure 4E to indicate that here we perform an extended analysis to the dataset generated earlier . Scale bars : 0 . 2 µm .

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TEM along sporozoites shows a decrease of microtubule numbers towards the nucleus in parasites expressing α-tubulin chimeras. Green arrowheads point to microtubules. See also Fig S8 and S9. Note that the WT control panel is shown already in Figure 4E to indicate that here we perform an extended analysis to the dataset generated earlier. Scale bars: 0.2 µm.


TEM reveals different numbers of microtubules at the apical end of sporozoites for the different lines . Each dot represents the numbers of microtubules counted per section . Red lines indicate median from 23 to 40 analysed sections per line .

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TEM reveals different numbers of microtubules at the apical end of sporozoites for the different lines. Each dot represents the numbers of microtubules counted per section. Red lines indicate median from 23 to 40 analysed sections per line.


Microtubule quantification along the sporozoite reveals the presence of fewer microtubules in central and nuclear sections of α2 + and α1 ∆ c - term parasites compared to wild type sporozoites .

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Microtubule quantification along the sporozoite reveals the presence of fewer microtubules in central and nuclear sections of α2+ and α1∆c-term parasites compared to wild type sporozoites.


Semilog correlation of gene expression as determined by qRT - PCR with microtubule numbers as determine by TEM . Bars show range of values in x and standard deviation in y around the mean ( dots ) of two biological replicates ( x ) and all determined values ( y ) .

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Semilog correlation of gene expression as determined by qRT-PCR with microtubule numbers as determine by TEM. Bars show range of values in x and standard deviation in y around the mean (dots) of two biological replicates (x) and all determined values (y).


The microtubule number increases with the increase in the concentration of tubulin dimers . Microtubule number scales with tubulin concentration at low expression . At high expression , microtubule number is limited by the number of nucleation sites . Shown are mean + / - standard deviation from all determined values .

The microtubule number increases with the increase in the concentration of tubulin dimers. Microtubule number scales with tubulin concentration at low expression. At high expression, microtubule number is limited by the number of nucleation sites. Shown are mean +/- standard deviation from all determined values.


The average microtubule length remains close to the maximum length 6 μm . The circles are obtained by averaging results from 200 stochastic simulations of the model equations and stars are the experimental data ( blue stars are for ( α2 + , α2 + + , α2 + + + ) strains ) . The error bar shows the standard deviation in the data and simulation results .

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The average microtubule length remains close to the maximum length 6 μm. The circles are obtained by averaging results from 200 stochastic simulations of the model equations and stars are the experimental data (blue stars are for (α2+, α2++, α2+++) strains). The error bar shows the standard deviation in the data and simulation results.


For the WT expression level ( c0 = 36 . 6 ( m0 + p + n ) ) , the rate of nucleation is faster than the microtubule growth . Then the microtubule number is determined by the number of nucleation sites . The average microtubule length is around 1 μm ( shown by dashed arrow ) when the last nucleus forms . At intermediate tubulin numbers ( c0 = 16 ( m0 + p + n ) ) , corresponding to the α1c - term mutant , the microtubule formation rate is comparable to nucleation rate . The average microtubule length is around 4 μm ( shown by dashed arrow ) when the last nucleus forms .

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For the WT expression level (c0 = 36.6 (m0+p+n)), the rate of nucleation is faster than the microtubule growth. Then the microtubule number is determined by the number of nucleation sites. The average microtubule length is around 1 μm (shown by dashed arrow) when the last nucleus forms. At intermediate tubulin numbers (c0 = 16 (m0+p+n)), corresponding to the α1c-term mutant, the microtubule formation rate is comparable to nucleation rate. The average microtubule length is around 4 μm (shown by dashed arrow) when the last nucleus forms.


Quantitative analysis of salivary gland sporozoite lengths from the different lines . n indicates numbers of investigated sporozoites . Box plots represent 50 % ( boxes ) and 95 % ( bars ) of data with horizontal bar showing the median . * * * and * * * * indicate p < 0 . 001 , and p < 0 . 0001 , respectively ; ns : not significant ; Kruskal - Wallis - test ; scale bar : 1 µm .

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Quantitative analysis of salivary gland sporozoite lengths from the different lines. n indicates numbers of investigated sporozoites. Box plots represent 50% (boxes) and 95% (bars) of data with horizontal bar showing the median. *** and **** indicate p<0.001, and p<0.0001, respectively; ns: not significant; Kruskal-Wallis-test; scale bar: 1 µm.


Quantification of SiR - tubulin fluorescence from 69 - 127 salivary gland derived sporozoites . Note the decrease in the fluorescence intensity of sporozoites expressing α1 - tubulin without introns as well as the decreased intensity and length of the chimeras . Linear correlation of sporozoite length and microtubule length reveals a R2 = 0 . 94 . * , * * , and * * * * indicate p < 0 . 05 , p < 0 . 01 , and p < 0 . 0001 , respectively ; ns : not significant ; Kruskal - Wallis - test .

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Quantification of SiR-tubulin fluorescence from 69-127 salivary gland derived sporozoites. Note the decrease in the fluorescence intensity of sporozoites expressing α1-tubulin without introns as well as the decreased intensity and length of the chimeras. Linear correlation of sporozoite length and microtubule length reveals a R2=0.94. *, **, and **** indicate p<0.05, p<0.01, and p<0.0001, respectively; ns: not significant; Kruskal-Wallis-test.


Shorter sporozoites exhibit stronger curvatures and hence tighter circular motility on glass . Scale bar : 20 µm . Box plots represent 50 % ( boxes ) and 95 % ( bars ) of data with horizontal bar showing the median . * and * * * * indicate p < 0 . 05 , and p < 0 . 0001 , respectively ; ns : not significant ; Kruskal - Wallis - test .

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Shorter sporozoites exhibit stronger curvatures and hence tighter circular motility on glass. Scale bar: 20 µm. Box plots represent 50% (boxes) and 95% (bars) of data with horizontal bar showing the median. * and **** indicate p<0.05, and p<0.0001, respectively; ns: not significant; Kruskal-Wallis-test.


Sporozoites with fewest microtubules ( α2 + + + ) show a motility defect . n indicates numbers of investigated sporozoites ; Numbers on grey bars indicate percentage of partially and persistently moving sporozoites .

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Sporozoites with fewest microtubules (α2+++) show a motility defect. n indicates numbers of investigated sporozoites; Numbers on grey bars indicate percentage of partially and persistently moving sporozoites.


Sporozoites from lines with fewer microtubules move more frequently in apparent clockwise ( CW ) direction . Numbers on black bars indicate percentage of CW moving sporozoites ; numbers in movie stills indicate time in seconds . CW moving sporozoites move at lower speed than CCW moving ones .

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Sporozoites from lines with fewer microtubules move more frequently in apparent clockwise (CW) direction. Numbers on black bars indicate percentage of CW moving sporozoites; numbers in movie stills indicate time in seconds. CW moving sporozoites move at lower speed than CCW moving ones.


Salivary gland sporozoites contain at least 10 microtubules as revealed by TEM of α2 + + infected salivary glands . Green arrowheads point to microtubules . Scale bar : 0 . 2 µm . Error bars show the full range and boxes 50 % of values . Red crosses indicate the mean , red lines the median . Fisher ' s exact test ( two - tailed ) .

Salivary gland sporozoites contain at least 10 microtubules as revealed by TEM of α2++ infected salivary glands. Green arrowheads point to microtubules. Scale bar: 0.2 µm. Error bars show the full range and boxes 50% of values. Red crosses indicate the mean, red lines the median. Fisher's exact test (two-tailed).


( C ) Representative FACS plot showing loss of hCD2 marker expression of Ch14 ICL MEFs after treatment with 4 - hydroxy tamoxifen ( 4OHT ) for 14 days to induce Cre , compared to Ethanol treated control cells ( See also Appendix figure S1A ) .

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(C) Representative FACS plot showing loss of hCD2 marker expression of Ch14 ICL MEFs after treatment with 4-hydroxy tamoxifen (4OHT) for 14 days to induce Cre, compared to Ethanol treated control cells (See also Appendix figure S1A).


( D ) Shallow whole genome sequencing ( copy number profiles ) of large T antigen immortalized , MEFs after exposure to Cre recombinase and sorted for control ( hCD2 plus ) and ICL ( hCD2 Minus ) cells for chromosomes 12 , 14 and 9 and 10 , without any in vitro culturing , post FACS sort .

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(D) Shallow whole genome sequencing (copy number profiles) of large T antigen immortalized, MEFs after exposure to Cre recombinase and sorted for control (hCD2 plus) and ICL (hCD2 Minus) cells for chromosomes 12, 14 and 9 and 10, without any in vitro culturing, post FACS sort.


( A ) Shallow whole genome sequencing ( copy number profiles ) of large T antigen immortalized , early passage MEFs after exposure to Cre recombinase and sorted for control ( hCD2 plus ) and ICL ( hCD2 Minus ) cells for chromosomes 12 , 14 and 9 and 10 .

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(A) Shallow whole genome sequencing (copy number profiles) of large T antigen immortalized, early passage MEFs after exposure to Cre recombinase and sorted for control (hCD2 plus) and ICL (hCD2 Minus) cells for chromosomes 12, 14 and 9 and 10.


( B ) Tumor growth curve after early passage ICL cells were injected into flanks of athymic nude mice ( n = 5 per group and error bars denote SEM , p < 0 . 05 for Ch9 and Ch14 ) .

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(B) Tumor growth curve after early passage ICL cells were injected into flanks of athymic nude mice (n = 5 per group and error bars denote SEM, p<0.05 for Ch9 and Ch14).


Representative karyotypes of late passage , large T antigen immortalized MEFs after exposure to Cre recombinase and serially sorted for control ( hCD2 plus ) and ICL ( hCD2 Minus ) cells for chromosome lines ( A ) Ch12 Boxed panels denote targeted chromosome loss ; arrows and asterisks denote shared and unshared , non - targeted chromosomal copy number variations respectively .

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Representative karyotypes of late passage, large T antigen immortalized MEFs after exposure to Cre recombinase and serially sorted for control (hCD2 plus) and ICL (hCD2 Minus) cells for chromosome lines (A) Ch12 Boxed panels denote targeted chromosome loss; arrows and asterisks denote shared and unshared, non-targeted chromosomal copy number variations respectively.


Representative karyotypes of late passage , large T antigen immortalized MEFs after exposure to Cre recombinase and serially sorted for control ( hCD2 plus ) and ICL ( hCD2 Minus ) cells for chromosome lines ( B ) Ch14 . Boxed panels denote targeted chromosome loss ; arrows and asterisks denote shared and unshared , non - targeted chromosomal copy number variations respectively .

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Representative karyotypes of late passage, large T antigen immortalized MEFs after exposure to Cre recombinase and serially sorted for control (hCD2 plus) and ICL (hCD2 Minus) cells for chromosome lines (B) Ch14. Boxed panels denote targeted chromosome loss; arrows and asterisks denote shared and unshared, non-targeted chromosomal copy number variations respectively.


Representative karyotypes of late passage , large T antigen immortalized MEFs after exposure to Cre recombinase and serially sorted for control ( hCD2 plus ) and ICL ( hCD2 Minus ) cells for chromosome lines ( C ) Ch9 Boxed panels denote targeted chromosome loss ; arrows and asterisks denote shared and unshared , non - targeted chromosomal copy number variations respectively .

Representative karyotypes of late passage, large T antigen immortalized MEFs after exposure to Cre recombinase and serially sorted for control (hCD2 plus) and ICL (hCD2 Minus) cells for chromosome lines (C) Ch9 Boxed panels denote targeted chromosome loss; arrows and asterisks denote shared and unshared, non-targeted chromosomal copy number variations respectively.


Representative karyotypes of late passage , large T antigen immortalized MEFs after exposure to Cre recombinase and serially sorted for control ( hCD2 plus ) and ICL ( hCD2 Minus ) cells for chromosome lines ( D ) Ch10 . Boxed panels denote targeted chromosome loss ; arrows and asterisks denote shared and unshared , non - targeted chromosomal copy number variations respectively .

Representative karyotypes of late passage, large T antigen immortalized MEFs after exposure to Cre recombinase and serially sorted for control (hCD2 plus) and ICL (hCD2 Minus) cells for chromosome lines (D) Ch10. Boxed panels denote targeted chromosome loss; arrows and asterisks denote shared and unshared, non-targeted chromosomal copy number variations respectively.


Karyotypic analysis of later passage , control and ICL of a replicate ( # 1 ) of Ch12 ( A ) . All chromosomes are shown for 20 metaphases . Target chromosomes are boxed in red .

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Karyotypic analysis of later passage, control and ICL of a replicate (#1) of Ch12 (A). All chromosomes are shown for 20 metaphases. Target chromosomes are boxed in red.


Karyotypic analysis of later passage , control and ICL of a replicate ( # 1 ) of Ch10 ( B ) All chromosomes are shown for 20 metaphases . Target chromosomes are boxed in red .

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Karyotypic analysis of later passage, control and ICL of a replicate (#1) of Ch10 (B) All chromosomes are shown for 20 metaphases. Target chromosomes are boxed in red.


Karyotypic analysis of later passage , control and ICL of a replicate ( # 1 ) of Ch14 ( C ) All chromosomes are shown for 20 metaphases . Target chromosomes are boxed in red .

Karyotypic analysis of later passage, control and ICL of a replicate (#1) of Ch14 (C) All chromosomes are shown for 20 metaphases. Target chromosomes are boxed in red.


Karyotypic analysis of later passage , control and ICL of a replicate ( # 1 ) of Ch9 ( D ) lines . All chromosomes are shown for 20 metaphases . Target chromosomes are boxed in red .

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Karyotypic analysis of later passage, control and ICL of a replicate (#1) of Ch9 (D) lines. All chromosomes are shown for 20 metaphases. Target chromosomes are boxed in red.


( A ) Tumor growth curve after later passage ICL cells were injected into flanks of athymic nude mice ( n = 5 per group and error bars denote SD , p < 0 . 0005 for Ch9 , p < 0 . 05 for Ch10 and p < 0 . 005 for Ch14 ) .

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(A) Tumor growth curve after later passage ICL cells were injected into flanks of athymic nude mice (n = 5 per group and error bars denote SD, p<0.0005 for Ch9, p<0.05 for Ch10 and p<0.005 for Ch14).


( B ) Tumors sections were stained with H & E to assess tumor histology ( scale bars denote 5mm for the inset and 200μm for the zoomed image ) Arrows in the ICL tumors sections indicate regions where tumors have invaded into the adjacent tissue .

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(B) Tumors sections were stained with H&E to assess tumor histology (scale bars denote 5mm for the inset and 200μm for the zoomed image) Arrows in the ICL tumors sections indicate regions where tumors have invaded into the adjacent tissue.


( C ) Tumor growth curve after later passage ICL cells were injected into flanks of NOD / SCID mice ( n = 5 per group and error bars denote SD p < 0 . 0005 for Ch10 and p < 0 . 005 for Ch14 ) .

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(C) Tumor growth curve after later passage ICL cells were injected into flanks of NOD/SCID mice (n = 5 per group and error bars denote SD p<0.0005 for Ch10 and p<0.005 for Ch14).


( A ) Growth curve of the later passage ICL lines Ch10 and Ch14 under adherent in vitro culture conditions ( n = 3 for each data set and error bars denote SD , p < 0 . 005 for Ch10 and p < 0 . 0001 for Ch14 ) . ( See also Appendix Figure S3A )

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(A) Growth curve of the later passage ICL lines Ch10 and Ch14 under adherent in vitro culture conditions (n=3 for each data set and error bars denote SD, p<0.005 for Ch10 and p<0.0001 for Ch14). (See also Appendix Figure S3A)


( B ) Colony formation in ICL and control MEFs . 5000 cells were seeded in 10 cm plates and stained with Methylene blue after three days . Representative colony formation images in Ch10 and Ch14 are shown ( left ) and quantification of colonies formed ( right ) in the 4 ICL lines ( n = 3 for each data set and error bars denote SD , p < 0 . 005 for Ch10 , Ch12 and Ch14 and ns for Ch9 ) .

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(B) Colony formation in ICL and control MEFs. 5000 cells were seeded in 10 cm plates and stained with Methylene blue after three days. Representative colony formation images in Ch10 and Ch14 are shown (left) and quantification of colonies formed (right) in the 4 ICL lines (n=3 for each data set and error bars denote SD, p<0.005 for Ch10, Ch12 and Ch14 and ns for Ch9).


( C ) Quantification of growth in ultra - low adherent , sphere forming conditions for the 4 ICL lines ( n = 3 for Ch10 and Ch14 , n = 6 for Ch9 and Ch12 and error bars denote SD , p < 0 . 0001 for Ch9 , p < 0 . 001 for Ch10 and p < 0 . 05 for Ch12 and Ch14 ) .

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(C) Quantification of growth in ultra-low adherent, sphere forming conditions for the 4 ICL lines (n=3 for Ch10 and Ch14, n=6 for Ch9 and Ch12 and error bars denote SD, p<0.0001 for Ch9, p<0.001 for Ch10 and p<0.05 for Ch12 and Ch14).


( D ) Analysis of metabolites ( glucose consumed , lactate produced and glutamine consumed ) for the ICL lines . Spent media was analyzed after each ICL & control line was grown in culture for three days . Media in identical culture conditions , but with no plated cells , was used as a baseline for all samples .

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(D) Analysis of metabolites (glucose consumed, lactate produced and glutamine consumed) for the ICL lines. Spent media was analyzed after each ICL & control line was grown in culture for three days. Media in identical culture conditions, but with no plated cells, was used as a baseline for all samples.


( A ) Flow cytometry analysis of tail blood from Ch10 / + ( control ) at 30 days post tamoxifen administration , Ch10 / + ; Id1 Cre / + at 30 days post tamoxifen administration , and Ch10 / + ; Id1Cre / + at 80 days post tamoxifen administration .

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(A) Flow cytometry analysis of tail blood from Ch10/+ (control) at 30 days post tamoxifen administration, Ch10/+; Id1 Cre/+ at 30 days post tamoxifen administration, and Ch10/+; Id1Cre/+ at 80 days post tamoxifen administration.


( B ) Analysis from the Mitelman database for the fraction of human solid tumors that have lost ( top ) or gained ( bottom ) individual chromosomes , that have modal chromosome number > 2N or ~ 2N ( top , p < 0 . 0001 for chromosome 1 - 22 , X and p < 0 . 05 for chromosome Y ; bottom , p < 0 . 0001 for all chromosomes ) .

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(B) Analysis from the Mitelman database for the fraction of human solid tumors that have lost (top) or gained (bottom) individual chromosomes, that have modal chromosome number >2N or ~2N (top, p<0.0001 for chromosome 1-22, X and p<0.05 for chromosome Y; bottom, p<0.0001 for all chromosomes).


( C ) Analysis from the Mitelman database for the fraction of human solid tumors that have lost or gained any chromosome , that have modal chromosome number > 2N or ~ 2N ( p < 0 . 0001 for both modal number > 2N and ~ 2N ) .

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(C) Analysis from the Mitelman database for the fraction of human solid tumors that have lost or gained any chromosome, that have modal chromosome number >2N or ~2N (p<0.0001 for both modal number >2N and ~2N).


( A ) Chromosomal instability readouts , of later passage lines , assessed by average chromosomes loss per metaphase , average number of structural rearrangements per metaphase and percentage of metaphases with marker chromosomes ( n = 20 for each dataset and error bars denote SD ) .

(A) Chromosomal instability readouts, of later passage lines, assessed by average chromosomes loss per metaphase, average number of structural rearrangements per metaphase and percentage of metaphases with marker chromosomes (n=20 for each dataset and error bars denote SD).


( B ) Determination of number of γH2AX foci in ICL and control nuclei and quantification ( scale bars denote 10μm , p < 0 . 0001 for Ch9 , Ch10 and Ch12 , p < 0 . 05 for Ch14 ; n = 25 for each dataset and error bars denote SD ) . ( See also Figures S5A and S5B )

(B) Determination of number of γH2AX foci in ICL and control nuclei and quantification (scale bars denote 10μm, p<0.0001 for Ch9, Ch10 and Ch12, p<0.05 for Ch14; n=25 for each dataset and error bars denote SD). (See also Figures S5A and S5B)


( C ) Number of γH2AX positive cells in formalin fixed ICL and control tumor sections by immunofluorescence staining and quantification ( Scale bars denote 20μm , p < 0 . 0001 for Ch10 and p < 0 . 05 for Ch14 ) .

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(C) Number of γH2AX positive cells in formalin fixed ICL and control tumor sections by immunofluorescence staining and quantification (Scale bars denote 20μm, p<0.0001 for Ch10 and p<0.05 for Ch14).


( A , B ) Airyscan confocal analysis of PC1 ( 568 , red ) co - localization with GFP - LC3 ( green ) in ( A ) MEF ( B ) Saos2 . Scale bars = 10 µm . ( C ) Airyscan confocal analysis of PC2 ( 647 , red ) co - localization with LC3 ( 488 , green ) in RCS cells . Scale bars = 10 µm . ( A - C ) The insets show higher magnification ( A = x4 . 68 ; B = x6 . 76 ; C = x7 . 33 ) and single colour channels of the boxed area . ( D ) Quantification of GFP ( A , B ) or LC3 ( C ) vesicles positive for PC1 or PC2 , expressed as % of total LC3 ( mean + / - SEM ) , n = 18 cells ( MEFs and Saos2 ) ; n = 12 ( RCS ) from 3 independent experiments .

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(A, B) Airyscan confocal analysis of PC1 (568, red) co-localization with GFP-LC3 (green) in (A) MEF (B) Saos2. Scale bars = 10 µm. (C) Airyscan confocal analysis of PC2 (647, red) co-localization with LC3 (488, green) in RCS cells. Scale bars = 10 µm. (A-C) The insets show higher magnification (A = x4.68; B = x6.76; C = x7.33) and single colour channels of the boxed area. (D) Quantification of GFP (A,B) or LC3 (C) vesicles positive for PC1 or PC2, expressed as % of total LC3 (mean +/- SEM), n = 18 cells (MEFs and Saos2); n=12 (RCS) from 3 independent experiments.


( E - G ) Scanning confocal microscopy analysis of MEFs , Saos2 and RCS cells treated with BafA1 , immunolabeled for PC1 or PC2 and LAMP1 . Nuclei were stained with Hoechst . ( E , F ) Scale bars = 10 µm , ( G ) Scale bars = 5 µm . The insets show higher magnification ( E = x4 . 99 ; F = x6 . 49 ; G = x2 . 01 ) and single colour channels of the boxed area .

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(E-G) Scanning confocal microscopy analysis of MEFs, Saos2 and RCS cells treated with BafA1, immunolabeled for PC1 or PC2 and LAMP1. Nuclei were stained with Hoechst. (E, F) Scale bars = 10 µm, (G) Scale bars = 5 µm. The insets show higher magnification (E = x4.99; F = x6.49; G = x2.01) and single colour channels of the boxed area.


( H ) Transmission EM analysis in Saos2 cells , treated with BafA1 , showing in detail a lysosome which contains immunolabeled PC1 ( with nanogold particles ) , as indicated by arrows .

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(H) Transmission EM analysis in Saos2 cells, treated with BafA1, showing in detail a lysosome which contains immunolabeled PC1 (with nanogold particles), as indicated by arrows.


( I ) Scanning confocal microscopy analysis of Saos2 WT and CRISPR - Cas9 IDUA Saos2 at steady state , immunolabeled for PC1 and LAMP1 . Nuclei were stained with Hoechst . Scale bar = 10 µm . The insets show higher magnification ( left = x3 . 09 ; right = x3 . 12 ) and single colour channels of the boxed area . Bar graph shows quantification of lysosomes containing PC1 expressed as % of total LAMP1 per cell ( mean + / - SEM ) . n = 31 WT cells , n = 33 CRISPR cells counted ; 3 independent experiments . Student ' s unpaired , two - tailed T - Test * * * P < 0 . 0001 .

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(I) Scanning confocal microscopy analysis of Saos2 WT and CRISPR-Cas9 IDUA Saos2 at steady state, immunolabeled for PC1 and LAMP1. Nuclei were stained with Hoechst. Scale bar = 10 µm. The insets show higher magnification (left = x3.09; right = x3.12) and single colour channels of the boxed area. Bar graph shows quantification of lysosomes containing PC1 expressed as % of total LAMP1 per cell (mean +/- SEM). n= 31 WT cells, n=33 CRISPR cells counted; 3 independent experiments. Student's unpaired, two-tailed T-Test *** P<0.0001.


( J ) WT and CRISPR IDUA Saos2 lysed and analysed by western blot . Data are representative of 3 independent experiments .

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(J) WT and CRISPR IDUA Saos2 lysed and analysed by western blot. Data are representative of 3 independent experiments.


U2OS expressing ( A ) GFP - LC3 mCherry - PC2 ( red ) and RDEL - HALO ( blue ) were imaged live by spinning disc microscopy . Single and merge channels time - lapse stills at higher magnification ( A = x3 . 93 from the boxed region are shown on the right . Scale bar = 10 µm .

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

U2OS expressing (A) GFP-LC3 mCherry-PC2 (red) and RDEL-HALO (blue) were imaged live by spinning disc microscopy. Single and merge channels time-lapse stills at higher magnification (A = x3.93 from the boxed region are shown on the right. Scale bar = 10 µm.


U2OS expressing ( B ) GFP - 2 - FYVE ( green ) , mCherry - PC2 ( red ) and RDEL - HALO ( blue ) were imaged live by spinning disc microscopy . Single and merge channels time - lapse stills at higher magnification B = x3 . 42 from the boxed region are shown on the right . Scale bar = 10 µm .

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U2OS expressing (B) GFP-2-FYVE (green), mCherry-PC2 (red) and RDEL-HALO (blue) were imaged live by spinning disc microscopy. Single and merge channels time-lapse stills at higher magnification B = x3.42 from the boxed region are shown on the right. Scale bar = 10 µm.


( C ) Airyscan analysis of Saos2 cells expressing GFP - LC3 ( green ) and immunolabeled for PC1 ( 405 , blue ) and CANX ( 647 , red ) . The insets show higher magnification ( x5 . 26 ) and single colour channels of the boxed area . Scale bar = 10 µm .

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

(C) Airyscan analysis of Saos2 cells expressing GFP-LC3 (green) and immunolabeled for PC1 (405, blue) and CANX (647, red). The insets show higher magnification (x5.26) and single colour channels of the boxed area. Scale bar = 10 µm.


( D ) Correlative Light Electron Microscopy ( CLEM ) and Electron Tomography of Saos2 cells transfected with GFP - LC3 ( green ) and labelled for PC1 ( 568 , red and nanogold particles ) and CANX ( 647 , blue ) . Cells were first imaged by confocal microscopy ( top left panel ) and then the same region was retraced in EM ( upper middle panel ) and overlay is shown ( upper right panel ) . Arrow indicates a LC3 positive vesicle containing CANX and PC1 molecules .

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(D) Correlative Light Electron Microscopy (CLEM) and Electron Tomography of Saos2 cells transfected with GFP-LC3 (green) and labelled for PC1 (568, red and nanogold particles) and CANX (647, blue). Cells were first imaged by confocal microscopy (top left panel) and then the same region was retraced in EM (upper middle panel) and overlay is shown (upper right panel). Arrow indicates a LC3 positive vesicle containing CANX and PC1 molecules.


( E ) Single tomography slice ( left panel , taken from boxed are in D at a magnification of x2 . 84 ) , overlay with immunofluorescence ( IF ) ( central panel ) and IF 3D rendering of AV ( green ) and the CANX positive vesicle containing gold particles of labelled collagen ( blue and white respectively ) inside an AV ( right panel ) .

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(E) Single tomography slice (left panel, taken from boxed are in D at a magnification of x2.84), overlay with immunofluorescence (IF) (central panel) and IF 3D rendering of AV (green) and the CANX positive vesicle containing gold particles of labelled collagen (blue and white respectively) inside an AV (right panel).


( A ) . Bar graph shows quantification of lysosomes ( LAMP1 + ) containing PC1 expressed as % of total number of lysosomes ( mean + / - SEM ) in Saos2 cells mock transfected or transfected with SiRNA against the indicated genes and treated with 100 nM BafA1 for 9h . n = 20 cells per condition ; three independent experiments . One - way ANOVA ( P < 0 . 0001 ) with Dunnett ' s multiple comparisons test performed , * * * P < 0 . 0001 .

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(A). Bar graph shows quantification of lysosomes (LAMP1+) containing PC1 expressed as % of total number of lysosomes (mean +/- SEM) in Saos2 cells mock transfected or transfected with SiRNA against the indicated genes and treated with 100 nM BafA1 for 9h. n=20 cells per condition; three independent experiments. One-way ANOVA (P<0.0001) with Dunnett's multiple comparisons test performed, ***P<0.0001.


( B ) MEF cell lines lacking the expression of indicated genes were treated for 12h with 50 nM BafA1 , fixed and immunolabeled for PC1 ( 568 , red ) and LAMP1 ( 488 , green ) . Scale bar = 10 µm . Insets show magnification of the boxed area . Bar graph ( on the left ) shows quantification of LAMP1 vesicles positive for PC1 , expressed as % of total lysosomes ( mean + / - SEM ) , n = 12 , 10 , 12 , 10 , 7 , 10 cells per genotype respectively ; 3 independent experiments . One - way ANOVA with Dunnett ' s multiple comparisons test performed and P value adjusted for multiple comparisons . ns ≥ 0 . 05 , * * * P < 0 . 0001 .

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(B) MEF cell lines lacking the expression of indicated genes were treated for 12h with 50 nM BafA1, fixed and immunolabeled for PC1 (568, red) and LAMP1 (488, green). Scale bar = 10 µm. Insets show magnification of the boxed area. Bar graph (on the left) shows quantification of LAMP1 vesicles positive for PC1, expressed as % of total lysosomes (mean +/- SEM), n=12, 10, 12, 10, 7, 10 cells per genotype respectively; 3 independent experiments. One-way ANOVA with Dunnett's multiple comparisons test performed and P value adjusted for multiple comparisons. ns ≥0.05, *** P<0.0001.


( A ) WT and CRISPR - Cas9 Fam134b knockout MEFs were treated as indicated , lysed and analysed by western blot with the indicated antibodies . Western blots are representative of 4 independent experiments .

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(A) WT and CRISPR-Cas9 Fam134b knockout MEFs were treated as indicated, lysed and analysed by western blot with the indicated antibodies. Western blots are representative of 4 independent experiments.


( B ) WT and CRISPR - Cas9 Fam134b MEFs were immunolabeled for PC1 ( 568 , red ) , nuclei stained with Hoechst ( blue ) and analysed by scanning confocal microscopy . Scale bar = 10 µm .

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(B) WT and CRISPR-Cas9 Fam134b MEFs were immunolabeled for PC1 (568, red), nuclei stained with Hoechst (blue) and analysed by scanning confocal microscopy. Scale bar = 10 µm.


( C ) CRISPR FAM134B MEF mock , wild type FAM134B - HA or FAM134Blir - HA transfected were immunolabeled for PC1 ( 568 , red ) , LAMP1 ( 488 , green ) and HA ( 647 , violet ) and analysed by scanning confocal microscopy . Scale bar = 10 µm . Inset panels show magnification of the boxed area . Bar graph shows quantification of LAMP1 vesicles positive for PC1 , expressed as % of total lysosomes ( mean + / - SEM ) , quantification of n = 10 cells per treatment ; 3 independent experiments . One - way ANOVA with Dunnett ' s multiple comparisons test was performed . ns ≥ 0 . 05 , * * * P < 0 . 0001 .

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(C) CRISPR FAM134B MEF mock, wild type FAM134B-HA or FAM134Blir-HA transfected were immunolabeled for PC1 (568, red), LAMP1 (488, green) and HA (647, violet) and analysed by scanning confocal microscopy. Scale bar = 10 µm. Inset panels show magnification of the boxed area. Bar graph shows quantification of LAMP1 vesicles positive for PC1, expressed as % of total lysosomes (mean +/- SEM), quantification of n=10 cells per treatment; 3 independent experiments. One-way ANOVA with Dunnett's multiple comparisons test was performed. ns ≥0.05, *** P<0.0001.


( A ) Bar graph shows quantification of lysosomes ( LAMP1 + ) containing PC1 expressed as % of lysosomes ( mean + / - SEM ) in Saos2 cells mock transfected or transfected with SiRNA against the indicated genes , treated with 100 nM BafA1 for 9h . n = 18 cells / treatment ; 3 independent experiments . One - way ANOVA ( P < 0 . 0001 ) with Dunnett ' s multiple comparisons test performed , * * P < 0 . 005 , * * * P < 0 . 0001 .

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(A) Bar graph shows quantification of lysosomes (LAMP1+) containing PC1 expressed as % of lysosomes (mean +/- SEM) in Saos2 cells mock transfected or transfected with SiRNA against the indicated genes, treated with 100 nM BafA1 for 9h. n=18 cells/treatment; 3 independent experiments. One-way ANOVA (P<0.0001) with Dunnett's multiple comparisons test performed, ** P<0.005, *** P<0.0001.


( B ) WT and Canx - / - MEFs were untreated or treated with BafA1 ( 10 μM ) for 6 h , lysed and analysed by western blot with indicated antibodies . Filamin and β - actin were used as loading control . Dashed line indicates that unnecessary lanes were removed . Western blot is representative of 3 independent experiments .

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

(B) WT and Canx-/- MEFs were untreated or treated with BafA1 (10 μM) for 6 h, lysed and analysed by western blot with indicated antibodies. Filamin and β-actin were used as loading control. Dashed line indicates that unnecessary lanes were removed. Western blot is representative of 3 independent experiments.


( C ) MEF cell lines lacking the indicated genes were treated for 12h with 50 nM BafA1 fixed and immunolabeled for PC1 ( 568 , red ) and LAMP1 ( 488 , green ) . CST was added where indicated . Scale bar = 10 µm . Inset panels show magnification of the boxed area . Bar graph on the right shows quantification of LAMP1 vesicles positive for PC1 , expressed as % of total lysosomes ( mean + / - SEM ) , n = 8 , 8 , 12 , 8 , 8 , 8 cells respectively ; 3 independent experiments . One - way ANOVA with Dunnett ' s multiple comparisons test performed and P value adjusted for multiple comparisons . * * * P < 0 . 0001 .

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

(C) MEF cell lines lacking the indicated genes were treated for 12h with 50 nM BafA1 fixed and immunolabeled for PC1 (568, red) and LAMP1 (488, green). CST was added where indicated. Scale bar = 10 µm. Inset panels show magnification of the boxed area. Bar graph on the right shows quantification of LAMP1 vesicles positive for PC1, expressed as % of total lysosomes (mean +/- SEM), n=8, 8, 12, 8, 8, 8 cells respectively; 3 independent experiments. One-way ANOVA with Dunnett's multiple comparisons test performed and P value adjusted for multiple comparisons. *** P<0.0001.


( A , B ) Volcano plot comparing protein fold changes between WT versus Fam134b - / - ( A ) and WT versus Canx - / - MEFs ( B ) . Significantly regulated proteins are labelled in red ( log2 fold change > 1 , - log10 p > 1 . 3 ) . Red dots with blue ring indicate collagen 1 peptides . Graphs represent statistics from three separate experiments for each genotype .

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(A, B) Volcano plot comparing protein fold changes between WT versus Fam134b-/- (A) and WT versus Canx-/- MEFs (B). Significantly regulated proteins are labelled in red (log2 fold change > 1, -log10 p>1.3). Red dots with blue ring indicate collagen 1 peptides. Graphs represent statistics from three separate experiments for each genotype.


( B ) U2OS cells were transfected with empty vector control ( pcDNA3 ) , FAM134B - HA WT or mutant constructs as indicated . Complexes were immune - isolated with HA - magnetic beads , separated by western blot and visualised with antibodies against CANX , FAM134B 5 % of the input is shown . Western blots are representative of 3 independent experiments . Dashed line indicates that unnecessary lanes were removed .

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(B) U2OS cells were transfected with empty vector control (pcDNA3), FAM134B-HA WT or mutant constructs as indicated. Complexes were immune-isolated with HA-magnetic beads, separated by western blot and visualised with antibodies against CANX, FAM134B 5% of the input is shown. Western blots are representative of 3 independent experiments. Dashed line indicates that unnecessary lanes were removed.


U2OS cells were transfected with HALO - PC2 , FAM134B - HA or FAM134Blir - HA constructs , treated with 100 nM BafA1 for 6h and with CST where indicated . Complexes were immune - isolated with HA - magnetic beads , separated by western blot and visualised with antibodies against HALO , CANX , FAM134B , LC3 5 % of the input is shown . Western blots are representative of 3 independent experiments . Dashed line indicates that unnecessary lanes were removed .

O O O 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

U2OS cells were transfected with HALO-PC2, FAM134B-HA or FAM134Blir-HA constructs, treated with 100 nM BafA1 for 6h and with CST where indicated. Complexes were immune-isolated with HA-magnetic beads, separated by western blot and visualised with antibodies against HALO, CANX, FAM134B, LC3 5% of the input is shown. Western blots are representative of 3 independent experiments. Dashed line indicates that unnecessary lanes were removed.


B Immunoblot showing CDK5RAP2 levels in Cdk5rap2 wild - type ( WT ) , heterozygous ( HET ) and null erythroid progenitors ( EPs ) isolated from fetal livers . Actin was used as loading control . * * indicates nonspecific band . C Quantification of mean protein levels from ( B ) . Numbers in brackets correspond to number of embryos analyzed . Data information : Bar graphs display mean ± s . d . Statistical analysis was based on the number of embryos ( C , Statistical significances were determined by One - way ANOVA test with Tukey ' s ( C , test * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 .

B Immunoblot showing CDK5RAP2 levels in Cdk5rap2 wild-type (WT), heterozygous (HET) and null erythroid progenitors (EPs) isolated from fetal livers. Actin was used as loading control. ** indicates nonspecific band. C Quantification of mean protein levels from (B). Numbers in brackets correspond to number of embryos analyzed. Data information: Bar graphs display mean ± s.d. Statistical analysis was based on the number of embryos (C, Statistical significances were determined by One-way ANOVA test with Tukey's (C, test *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001.


D Immunofluorescence images of Cdk5rap2 WT , HET and null erythroid progenitors isolated from fetal livers . Progenitors were stained for CDK5RAP2 ( grey ) , γ - tubulin ( magenta ) and DNA ( Hoechst , blue ) . Images are maximum intensity projections of deconvolved z - stacks . Scale bar , 3 μm . Insets show higher magnification of centrosomes . Scale bar , 1 μm . E Quantification of mean centrosomal signal intensities of CDK5RAP2 and γ - tubulin from ( D ) . Numbers in brackets correspond to number of embryos analyzed with a total number of 470 ( WT ) , 406 ( HET ) and 379 ( null ) progenitors . Data information : Bar graphs display mean ± s . d . Statistical analysis was based on the number of embryos E , Statistical significances were determined by One - way ANOVA test with Tukey ' s E , test * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 .

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D Immunofluorescence images of Cdk5rap2 WT, HET and null erythroid progenitors isolated from fetal livers. Progenitors were stained for CDK5RAP2 (grey), γ-tubulin (magenta) and DNA (Hoechst, blue). Images are maximum intensity projections of deconvolved z-stacks. Scale bar, 3 μm. Insets show higher magnification of centrosomes. Scale bar, 1 μm. E Quantification of mean centrosomal signal intensities of CDK5RAP2 and γ-tubulin from (D). Numbers in brackets correspond to number of embryos analyzed with a total number of 470 (WT), 406 (HET) and 379 (null) progenitors. Data information: Bar graphs display mean ± s.d. Statistical analysis was based on the number of embryos E, Statistical significances were determined by One-way ANOVA test with Tukey's E, test *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001.


I ImageStream images of ex vivo cultured enucleating EBs and reticulocytes . Cells were stained for TER119 ( erythroid marker , green ) and DNA ( Hoechst , blue ) . BF : brightfield . Scale bar , 5 μm . J Quantification of enucleating EBs and reticulocytes after 48 hours ( T48 ) in ex vivo culture . Genotypes are as indicated . The numbers in brackets correspond to the number of embryos analyzed . K Quantification of enucleating EB size from ( I ) . The numbers in brackets refer to the number of embryos analyzed . Data information : Box plots show 5th , 95th ( whiskers ) and 25th , 50th and 75th percentile ( boxes ) . Statistical analysis was based on the number of embryos I and J ) Statistical significances were determined by One - way ANOVA test with Tukey ' s J ) or Kruskal Wallis test with Dunn ' s ( K ) multiple comparisons . * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 .

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I ImageStream images of ex vivo cultured enucleating EBs and reticulocytes. Cells were stained for TER119 (erythroid marker, green) and DNA (Hoechst, blue). BF: brightfield. Scale bar, 5 μm. J Quantification of enucleating EBs and reticulocytes after 48 hours (T48) in ex vivo culture. Genotypes are as indicated. The numbers in brackets correspond to the number of embryos analyzed. K Quantification of enucleating EB size from (I). The numbers in brackets refer to the number of embryos analyzed. Data information: Box plots show 5th,95th (whiskers) and 25th, 50th and 75th percentile (boxes). Statistical analysis was based on the number of embryos I and J) Statistical significances were determined by One-way ANOVA test with Tukey's J) or Kruskal Wallis test with Dunn's (K) multiple comparisons.*P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001.


A Deconvolved STED images of centrosomes in ex vivo cultured wild - type non - enucleating EBs ( non - enucl . ) or enucleating EBs / reticulocytes ( enucl . / RetiC ) . Cells were stained for γ - tubulin ( magenta ) and centriolar ( CP110 , CEP135 ) or PCM ( CDK5RAP2 , PCNT ) proteins ( cyan ) . Scale bar , 400 nm .

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

A Deconvolved STED images of centrosomes in ex vivo cultured wild-type non-enucleating EBs (non-enucl.) or enucleating EBs/reticulocytes (enucl./RetiC). Cells were stained for γ-tubulin (magenta) and centriolar (CP110, CEP135) or PCM (CDK5RAP2, PCNT) proteins (cyan). Scale bar, 400 nm.


E ImageStream images showing EBs enucleating with 2N or 4N DNA content at different stages from ( B ) . EBs were stained for DNA ( Hoechst , blue ) and TER119 ( erythroid marker , green ) . Scale bar , 5 μm . BF : brightfield . F Quantification of enucleation stages according to DNA content from ( B ) . 4 ( WT ) , 14 ( HET ) and 8 ( null ) embryos were analyzed . Data information : Bar graphs display mean ± s . d . All statistical analysis were based on the number of embryos . Statistical significance was determined by Two - way ANOVA with Tukey ' s ( F ) multiple comparisons . * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 .

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

E ImageStream images showing EBs enucleating with 2N or 4N DNA content at different stages from (B). EBs were stained for DNA (Hoechst, blue) and TER119 (erythroid marker, green). Scale bar, 5 μm. BF: brightfield. F Quantification of enucleation stages according to DNA content from (B). 4 (WT), 14 (HET) and 8 (null) embryos were analyzed. Data information: Bar graphs display mean ± s.d. All statistical analysis were based on the number of embryos. Statistical significance was determined by Two-way ANOVA with Tukey's (F) multiple comparisons. **P≤0.01, ***P≤0.001, ****P≤0.0001.


B Immunofluorescence images of untreated ( untx ) or CB - treated erythroid progenitors at stated differentiation stages after 24 ( T24 ) and 48 ( T48 ) hours in ex vivo culture . Cells were stained for γ - tubulin ( magenta ) , TER119 ( erythroid marker , green ) and DNA ( Hoechst , blue ) . Scale bar , 3 μm .

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-MEASURED_VAR O O O O O O O O O O O O

B Immunofluorescence images of untreated (untx) or CB-treated erythroid progenitors at stated differentiation stages after 24 (T24) and 48 (T48) hours in ex vivo culture. Cells were stained for γ-tubulin (magenta), TER119 (erythroid marker, green) and DNA (Hoechst, blue). Scale bar, 3 μm.


C Quantification of centrosome numbers ( marked by γ - tubulin ) upon CB treatment from ( B ) . For the left panel , in total 632 ( untx , T24 ) , 637 ( CB , T24 ) , 544 ( untx , T48 ) and 467 ( CB , T48 ) cells from two litters were analyzed . T24 = 24 hours . T48 = 48 hours . For the right panel , in total 799 ( untx , non - enucl ) , 142 ( untx , enucl ) , 235 ( untx , RetiC ) , 839 ( CB , non - enucl ) , 127 ( CB , enucl ) and 131 ( CB , RetiC ) cells from two litters were analysed . Data information : Bar graphs display mean ± s . d . Statistical analysis was based on the number of litters ( C * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 .

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

C Quantification of centrosome numbers (marked by γ-tubulin) upon CB treatment from (B). For the left panel, in total 632 (untx, T24), 637 (CB, T24), 544 (untx, T48) and 467 (CB, T48) cells from two litters were analyzed. T24 = 24 hours. T48 = 48 hours. For the right panel, in total 799 (untx, non-enucl), 142 (untx, enucl), 235 (untx, RetiC), 839 (CB, non-enucl), 127 (CB, enucl) and 131 (CB, RetiC) cells from two litters were analysed. Data information: Bar graphs display mean ± s.d. Statistical analysis was based on the number of litters (C *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001.


A Immunofluorescence images of untreated ( untx ) or centrinone - B ( CB ) - treated cells at 36 hours ( T36 ) of ex vivo culture . Representative examples for different mitotic spindle morphologies are shown . Cells were stained for α - tubulin ( magenta ) , γ - tubulin ( grey ) , phospho - Histone H3 ( pHH3 , green ) and DNA ( Hoechst , blue ) . Images are maximum intensity projections of deconvolved z - stacks . Scale bar , 2 μm . B Quantification of mitotic spindle morphologies in untreated ( untx ) or centrinone - B ( CB ) - treated cells at T24 or T36 of ex vivo culture . Graph depicts percentage of spindle phenotypes . In total 315 ( untx , T24 ) , 367 ( CB , T24 ) , 295 ( untx , T36 ) , 282 ( CB , T36 ) cells were analyzed from 3 litters . Data information : Bar graphs display mean ± s . d . Statistical analysis was based on the number of litters ( B ) Statistical significance was determined by One - way ANOVA with Tukey ' s multiple comparisons test ( B , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 .

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

A Immunofluorescence images of untreated (untx) or centrinone-B (CB)-treated cells at 36 hours (T36) of ex vivo culture. Representative examples for different mitotic spindle morphologies are shown. Cells were stained for α-tubulin (magenta), γ-tubulin (grey), phospho-Histone H3 (pHH3, green) and DNA (Hoechst, blue). Images are maximum intensity projections of deconvolved z-stacks. Scale bar, 2 μm. B Quantification of mitotic spindle morphologies in untreated (untx) or centrinone-B (CB)-treated cells at T24 or T36 of ex vivo culture. Graph depicts percentage of spindle phenotypes. In total 315 (untx, T24), 367 (CB, T24), 295 (untx, T36), 282 (CB, T36) cells were analyzed from 3 litters. Data information: Bar graphs display mean ± s.d. Statistical analysis was based on the number of litters (B) Statistical significance was determined by One-way ANOVA with Tukey's multiple comparisons test (B, *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001.


C Immunofluorescence images of Cdk5rap2HET and Cdk5rap2null cells at 36 hours ( T36 ) of ex vivo culture . Representative examples for different mitotic spindle morphologies are shown . Cells were stained for α - tubulin ( magenta ) , γ - tubulin ( grey ) , pHH3 ( green ) and DNA ( Hoechst , blue ) . Images are maximum intensity projections of deconvolved z - stacks . Scale bar , 2 μm . D Quantification of mitotic spindle morphology at T24 or T36 of ex vivo culture . Graph depicts percentage of spindle phenotypes . At T24 , 3 Cdk5rap2WT ( 323 cells ) , 3 Cdk5rap2HET ( 296 cells ) and 2 Cdk5rap2null ( 197 cells ) embryos were analyzed . At T36 , 5 Cdk5rap2WT ( 387 cells ) , 6 Cdk5rap2HET ( 384 cells ) and 3 Cdk5rap2null ( 202 cells ) embryos were analyzed . Data information : Bar graphs display mean ± s . d . Statistical analysis was based on the number of embryos ( D , Statistical significance was determined by One - way ANOVA with Tukey ' s multiple comparisons test D , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 .

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

C Immunofluorescence images of Cdk5rap2HET and Cdk5rap2null cells at 36 hours (T36) of ex vivo culture. Representative examples for different mitotic spindle morphologies are shown. Cells were stained for α-tubulin (magenta), γ-tubulin (grey), pHH3 (green) and DNA (Hoechst, blue). Images are maximum intensity projections of deconvolved z-stacks. Scale bar, 2 μm. D Quantification of mitotic spindle morphology at T24 or T36 of ex vivo culture. Graph depicts percentage of spindle phenotypes. At T24, 3 Cdk5rap2WT (323 cells), 3 Cdk5rap2HET (296 cells) and 2 Cdk5rap2null (197 cells) embryos were analyzed. At T36, 5 Cdk5rap2WT (387 cells), 6 Cdk5rap2HET (384 cells) and 3 Cdk5rap2null (202 cells) embryos were analyzed. Data information: Bar graphs display mean ± s.d. Statistical analysis was based on the number of embryos (D, Statistical significance was determined by One-way ANOVA with Tukey's multiple comparisons test D, *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001.


E Schematic ( left ) shows experimental outline for nocodazole treatment of ex vivo cultured erythroid progenitors at indicated time points . Representative ImageStream images of ex vivo cultured cells at different mitotic stages ( right ) . Cells were stained for phospho - Histone H3 ( pHH3 , green ) , TER119 ( erythroid marker , magenta ) and DNA ( Hoechst , blue ) . BF : brightfield . Scale bar , 10 μm . F and G Quantification of pHH3pos , TER119pos cells and mitotic stages ( see text for details ) after 36 hours ( T36 ) upon DMSO or nocodazole treatment according to ( E ) using ImageStream . Number of embryos analyzed is shown in brackets . Data information : Box plots show 5th , 95th ( whiskers ) and 25th , 50th and 75th percentile ( boxes ) . Statistical analysis was based on the number of embryos F and G ) . Statistical significance was determined by One - way ANOVA with Tukey ' s multiple comparisons test F and G ) . * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 .

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

E Schematic (left) shows experimental outline for nocodazole treatment of ex vivo cultured erythroid progenitors at indicated time points. Representative ImageStream images of ex vivo cultured cells at different mitotic stages (right). Cells were stained for phospho-Histone H3 (pHH3, green), TER119 (erythroid marker, magenta) and DNA (Hoechst, blue). BF: brightfield. Scale bar, 10 μm. F and G Quantification of pHH3pos, TER119pos cells and mitotic stages (see text for details) after 36 hours (T36) upon DMSO or nocodazole treatment according to (E) using ImageStream. Number of embryos analyzed is shown in brackets. Data information: Box plots show 5th,95th (whiskers) and 25th, 50th and 75th percentile (boxes). Statistical analysis was based on the number of embryos F and G). Statistical significance was determined by One-way ANOVA with Tukey's multiple comparisons test F and G).*P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001.


B Immunofluorescence images of cells in interphase ( I ) and mitosis ( M ) at 24 ( T24 ) and 36 ( T36 ) hours of ex vivo culture . Cells were stained for γ - tubulin ( magenta ) , protein of interest ( POI , CDK5RAP2 , PCNT or CEP192 in grey ) and DNA ( Hoechst , blue ) . Images are maximum intensity projections of deconvolved z - stacks . Scale bar , 2 μm . C Quantification of mean centrosomal signal intensities of PCM proteins from ( B ) . Three litters were analyzed with the following number of cells for I , T24 ; M , T24 ; I , T36 and M , T36 : 290 , 157 , 259 and 174 ( CDK5RAP2 ) ; 291 , 190 , 203 and 181 ( PCNT ) ; 314 , 122 , 283 and 152 ( CEP192 ) ; 895 , 465 , 720 and 493 ( γ - tubulin ) . Data information : Bar graphs display mean ± s . d . Statistical analysis was based on the number of litters ( C Statistical significance was determined by One - way ANOVA with Tukey ' s multiple comparisons test * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Immunofluorescence images of cells in interphase (I) and mitosis (M) at 24 (T24) and 36 (T36) hours of ex vivo culture. Cells were stained for γ-tubulin (magenta), protein of interest (POI, CDK5RAP2, PCNT or CEP192 in grey) and DNA (Hoechst, blue). Images are maximum intensity projections of deconvolved z-stacks. Scale bar, 2 μm. C Quantification of mean centrosomal signal intensities of PCM proteins from (B). Three litters were analyzed with the following number of cells for I, T24; M, T24; I, T36 and M, T36: 290, 157, 259 and 174 (CDK5RAP2); 291, 190, 203 and 181 (PCNT); 314, 122, 283 and 152 (CEP192); 895, 465, 720 and 493 (γ-tubulin). Data information: Bar graphs display mean ± s.d. Statistical analysis was based on the number of litters (C Statistical significance was determined by One-way ANOVA with Tukey's multiple comparisons test *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001.


A Immunoblot analysis of TP53 , P27 and P21 levels in ex vivo cultured Cdk5rap2 wild - type ( WT ) , heterozygous ( HET ) and null EBs after 24 ( T24 ) and 48 ( T48 ) hours . GAPDH was used as loading control . * * indicates unspecific band . B Quantification of mean protein levels from ( A ) . Numbers in brackets represent number of embryos analyzed . Data information : Box plots show 5th , 95th ( whiskers ) and 25th , 50th and 75th percentile ( boxes ) . All statistical analysis were based on number of embryos . All statistical significances were determined by One - way ANOVA with Tukey ' s multiple comparisons test . * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 .

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

A Immunoblot analysis of TP53, P27 and P21 levels in ex vivo cultured Cdk5rap2 wild-type (WT), heterozygous (HET) and null EBs after 24 (T24) and 48 (T48) hours. GAPDH was used as loading control. ** indicates unspecific band. B Quantification of mean protein levels from (A). Numbers in brackets represent number of embryos analyzed. Data information: Box plots show 5th,95th (whiskers) and 25th, 50th and 75th percentile (boxes). All statistical analysis were based on number of embryos. All statistical significances were determined by One-way ANOVA with Tukey's multiple comparisons test.*P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001.


A . Expression levels of various GPI - APs in different clones of PIGG - KO HEK293 cells . Fluorescence intensities of NT5E in PIGG - KO clones # 4 , # 10 , # 17 , # 19 and wild type cells are 575 , 910 , 696 , 530 and 1548 while those of EphrinA5 in clones # 4 , # 17 , # 19 and wild type cells are 1431 , 2080 , 1868 and 3336 , respectively . Data information : Each FACS analysis was performed at least three times .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Expression levels of various GPI-APs in different clones of PIGG-KO HEK293 cells. Fluorescence intensities of NT5E in PIGG-KO clones #4, #10, #17, #19 and wild type cells are 575, 910, 696, 530 and 1548 while those of EphrinA5 in clones #4, #17, #19 and wild type cells are 1431, 2080, 1868 and 3336, respectively. Data information: Each FACS analysis was performed at least three times.


B . Expression levels of GPI - APs in PIGG - KO clone # 19 cells stably transfected with PIGG cDNA compared to levels in empty vector transfected cells . Fluorescence intensities of NT5E in PIGG cDNA and in vector transfected cells are 1332 and 886 , respectively . Data information : Each FACS analysis was performed at least three times .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Expression levels of GPI-APs in PIGG-KO clone #19 cells stably transfected with PIGG cDNA compared to levels in empty vector transfected cells. Fluorescence intensities of NT5E in PIGG cDNA and in vector transfected cells are 1332 and 886, respectively. Data information: Each FACS analysis was performed at least three times.


A . Expression levels of different GPI - APs in PIGG - KO HEK293 cells transiently co - transfected with PIGG cDNA or empty vector . Vertical bars represent % of levels of GPI - APs expression in non - rescued PIGG - KO cells relative to those in PIGG cDNA rescued PIGG - KO cells . Analysis was repeated 3 to 6 times and SD bars were shown .

A. Expression levels of different GPI-APs in PIGG-KO HEK293 cells transiently co-transfected with PIGG cDNA or empty vector. Vertical bars represent % of levels of GPI-APs expression in non-rescued PIGG-KO cells relative to those in PIGG cDNA rescued PIGG-KO cells. Analysis was repeated 3 to 6 times and SD bars were shown.


B . HA tagged NTNG2 and FLAG tagged DAF were transiently co - transfected together with PIGG cDNA or empty vector to PIGG - KO cells and were analyzed by FACS . Fluorescence intensity levels of NTNG2 in PIGG - KO cells and in PIGG rescued cells are 639 and 3426 , while those of DAF in PIGG - KO cells and in PIGG rescued cells are 562 and 599 , respectively . Each FACS analysis was performed at least three times .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. HA tagged NTNG2 and FLAG tagged DAF were transiently co-transfected together with PIGG cDNA or empty vector to PIGG-KO cells and were analyzed by FACS. Fluorescence intensity levels of NTNG2 in PIGG-KO cells and in PIGG rescued cells are 639 and 3426, while those of DAF in PIGG-KO cells and in PIGG rescued cells are 562 and 599, respectively. Each FACS analysis was performed at least three times.


( A ) IP of GFP - Atg8 ( α ‐ GFP ) , Atg30 - Flag ( α ‐ Flag ) and Pex3 ( α ‐ Pex3 ) under pexophagy conditions . The abundant peroxisome matrix protein , AOX , was used as a negative control . Input : total lysate ; ϕ : IP without antibody .

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

(A) IP of GFP-Atg8 (α‐GFP), Atg30-Flag (α‐Flag) and Pex3 (α‐Pex3) under pexophagy conditions. The abundant peroxisome matrix protein, AOX, was used as a negative control. Input: total lysate; ϕ: IP without antibody.


( B ) Two multiple sequence alignments , including 11 Atg30 homologues and 11 Atg32 homologues ( identical residues are indicated with black boxes , and similar residues with grey boxes ) , and a sequence logo of the combined multiple sequence alignments from Atg30 and Atg32 homologues listed currently in GenBank .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Two multiple sequence alignments, including 11 Atg30 homologues and 11 Atg32 homologues (identical residues are indicated with black boxes, and similar residues with grey boxes), and a sequence logo of the combined multiple sequence alignments from Atg30 and Atg32 homologues listed currently in GenBank.


( C , D ) Pexophagy experiments of Δatg30 ( ϕ ) , wild ‐ type ( Atg30 ) and Atg30 AIM mutant ( Atg30W73A F76A ) cells were done by fluorescence microscopy , following the degradation of peroxisomes labelled with BFP fused at its C ‐ terminus to the Ser - Lys - Leu peroxisomal targeting signal 1 ( BFP - SKL ) and biochemically by monitoring peroxisomal thiolase degradation . Vacuoles were labelled with FM4 ‐ 64 . Scale bar , 5 μm .

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

(C,D) Pexophagy experiments of Δatg30 (ϕ), wild‐type (Atg30) and Atg30 AIM mutant (Atg30W73A F76A) cells were done by fluorescence microscopy, following the degradation of peroxisomes labelled with BFP fused at its C‐terminus to the Ser-Lys-Leu peroxisomal targeting signal 1 (BFP-SKL) and biochemically by monitoring peroxisomal thiolase degradation. Vacuoles were labelled with FM4‐64. Scale bar, 5 μm.


( E ) AH109 cells were transformed with two yeast two ‐ hybrid assay plasmids , AD and BD , which encode the indicated domains fused with Atg30 , Atg32 and Atg8 or an empty vector , as negative controls and grown on + His and − His + 40 mM 3 ‐ AT plates .

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

(E) AH109 cells were transformed with two yeast two‐hybrid assay plasmids, AD and BD, which encode the indicated domains fused with Atg30, Atg32 and Atg8 or an empty vector, as negative controls and grown on +His and −His+40 mM 3‐AT plates.


( F ) . Δatg30 ( ϕ ) and Δatg30 cells complemented with Atg30 - HA ( Atg30 ) and several Atg30 - HA mutants were immunoprecipitated ( α ‐ HA IP ) under pexophagy conditions . In addition , α ‐ HA IP of Δatg30 cells ( ϕ ) and Δatg30 cells complemented with Atg30 - HA ( Atg30 ) were incubated with ( + ) and without ( − ) λPP . Input : total lysate .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) . Δatg30 (ϕ) and Δatg30 cells complemented with Atg30-HA (Atg30) and several Atg30-HA mutants were immunoprecipitated (α‐HA IP) under pexophagy conditions. In addition, α‐HA IP of Δatg30 cells (ϕ) and Δatg30 cells complemented with Atg30-HA (Atg30) were incubated with (+) and without (−) λPP. Input: total lysate.


( G ) Pexophagy in atg30 mutants was monitored by following thiolase levels of oleate ‐ induced peroxisomes after shifting cells to SD ‐ N . aa , amino acid ; AD , activation domain ; AIM , Atg8 ‐ family ‐ interacting motif ; AOX , alcohol oxidase ; BD , binding domain ; GFP , green fluorescent protein ; HA , haemagglutinin ; IP , immunoprecipitation ; λPP , λ protein phosphatase .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Pexophagy in atg30 mutants was monitored by following thiolase levels of oleate‐induced peroxisomes after shifting cells to SD‐N. aa, amino acid; AD, activation domain; AIM, Atg8‐family‐interacting motif; AOX, alcohol oxidase; BD, binding domain; GFP, green fluorescent protein; HA, haemagglutinin; IP, immunoprecipitation; λPP, λ protein phosphatase.


( A1 ) Atg30 sequences from aa 68 - 122 of wild ‐ type ( Atg30 ) , the S71A mutant ( Atg30S71A ) and two different deletions of the sequence between the AIM and phosphosite required for Atg11 binding in Atg30 ( Atg30WDILSSS and Atg30WSILSSS ) .

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

(A1) Atg30 sequences from aa 68-122 of wild‐type (Atg30), the S71A mutant (Atg30S71A) and two different deletions of the sequence between the AIM and phosphosite required for Atg11 binding in Atg30 (Atg30WDILSSS and Atg30WSILSSS).


( A2 ) Pexophagy experiments of Δatg30 cells complemented with appropriate wild ‐ type or mutant Atg30 proteins described in A1 .

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

(A2) Pexophagy experiments of Δatg30 cells complemented with appropriate wild‐type or mutant Atg30 proteins described in A1.


( A3 ) Two ‐ hybrid assays between Atg30 wild ‐ type or mutants ( described in A1 ) and Atg8 . Phosphomimic S71E was included to detect the interaction with Atg8 .

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(A3) Two‐hybrid assays between Atg30 wild‐type or mutants (described in A1) and Atg8. Phosphomimic S71E was included to detect the interaction with Atg8.


( B2 ) Pexophagy experiments of Δatg30 cells complemented with the two Atg30 molecules described in B1 .

(B2) Pexophagy experiments of Δatg30 cells complemented with the two Atg30 molecules described in B1.


( C2 ) Pexophagy assays of Δatg30 cells complemented with Atg30 wild ‐ type and mutants . aa , amino acids ; AD , activation domain ; AIM , Atg8 ‐ family ‐ interacting motif ; BD , binding domain ; HA , haemagglutinin .

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(C2) Pexophagy assays of Δatg30 cells complemented with Atg30 wild‐type and mutants. aa, amino acids; AD, activation domain; AIM, Atg8‐family‐interacting motif; BD, binding domain; HA, haemagglutinin.


( A ) Large phagophore membrane formation in WT and the Atg30 mutant cells monitored by GFP - Atg8 during pexophagy conditions .

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(A) Large phagophore membrane formation in WT and the Atg30 mutant cells monitored by GFP-Atg8 during pexophagy conditions.


( B ) Localization of GFP - Atg11 during pexophagy of methanol ‐ induced peroxisomes in cells expressing WT or mutant Atg30 proteins . White arrows indicate correct localization and yellow arrows indicate mislocalization , or in case of Atg8 localization , indicate absence of phagophore membrane elongation . Peroxisomes were labelled with BFP - SKL and vacuoles with FM4 ‐ 64 . Scale bar , 5 μm . GFP , green fluorescent protein ; WT , wild type .

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(B) Localization of GFP-Atg11 during pexophagy of methanol‐induced peroxisomes in cells expressing WT or mutant Atg30 proteins. White arrows indicate correct localization and yellow arrows indicate mislocalization, or in case of Atg8 localization, indicate absence of phagophore membrane elongation. Peroxisomes were labelled with BFP-SKL and vacuoles with FM4‐64. Scale bar, 5 μm. GFP, green fluorescent protein; WT, wild type.


( A ) Mitophagy experiments of WT ( PPY12 ) , Δatg5 , Δatg32 ( ϕ ) and Δatg32 complemented with WT Atg32 ( Atg32 ) , Atg32 with a deletion of the sequence between the AIM and phosphosite required for Atg11 binding ( Atg32WQVLSSS ) , Atg32 AIM mutant ( Atg32W121A V124A ) , mutants of the Thr upstream of the Atg32 AIM ( Atg32T119A ) and mutants altered in the Ser required for Atg11 binding ( Atg32S159A ) . The cells were grown in YPL medium and shifted to SD ‐ N . Mitophagy was followed by the transport of Tom20 - mCherry to the vacuole by fluorescence microscopy . Mitophagy was classified as − no mitophagy , + few cells show mitophagy , + + + most cells show mitophagy and + + + + almost all the cells show mitophagy ( the intensity and numbers of cells containing Tom20 - mCherry in the vacuole was considered ) . Scale bar , 5 μm .

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(A) Mitophagy experiments of WT (PPY12), Δatg5, Δatg32 (ϕ) and Δatg32 complemented with WT Atg32 (Atg32), Atg32 with a deletion of the sequence between the AIM and phosphosite required for Atg11 binding (Atg32WQVLSSS), Atg32 AIM mutant (Atg32W121A V124A), mutants of the Thr upstream of the Atg32 AIM (Atg32T119A) and mutants altered in the Ser required for Atg11 binding (Atg32S159A). The cells were grown in YPL medium and shifted to SD‐N. Mitophagy was followed by the transport of Tom20-mCherry to the vacuole by fluorescence microscopy. Mitophagy was classified as −no mitophagy,+few cells show mitophagy, +++ most cells show mitophagy and ++++ almost all the cells show mitophagy (the intensity and numbers of cells containing Tom20-mCherry in the vacuole was considered). Scale bar, 5 μm.


( B ) P . pastoris Δatg32 cells ( ΔPpatg32 ) expressing Tom20 - GFP and expressing the indicated Atg32 mutants were cultured in YPL medium for 12 , 18 and 36 h . Mitophagy was monitored by GFP appearance by immunoblotting with α ‐ GFP antibodies .

(B) P. pastoris Δatg32 cells (ΔPpatg32) expressing Tom20-GFP and expressing the indicated Atg32 mutants were cultured in YPL medium for 12, 18 and 36 h. Mitophagy was monitored by GFP appearance by immunoblotting with α‐GFP antibodies.


( C ) N ‐ terminal sequence of Atg36 manually aligned against the several sequence alignments of Atg30 , Atg32 and ScAtg32 . Identical residues are indicated with black boxes , and similar residues with grey boxes .

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(C) N‐terminal sequence of Atg36 manually aligned against the several sequence alignments of Atg30, Atg32 and ScAtg32. Identical residues are indicated with black boxes, and similar residues with grey boxes.


( D ) Two ‐ hybrid protein - protein interaction analysis of ScAtg36 , ScAtg8 and ScAtg11 . The receptors were mutated at the AIM ( Atg36F33A L36A ) , at serine ( s ) upstream of the AIM ( Atg36S31A ) and at the Atg11 ‐ binding site ( Atg36S97A ) .

(D) Two‐hybrid protein-protein interaction analysis of ScAtg36, ScAtg8 and ScAtg11. The receptors were mutated at the AIM (Atg36F33A L36A), at serine(s) upstream of the AIM (Atg36S31A) and at the Atg11‐binding site (Atg36S97A).


( E ) S . cerevisiae Δatg36 cells ( ΔScatg36 ) expressing thiolase - GFP and expressing the indicated Atg36 mutants were cultured in oleate medium until mid ‐ log growth and then shifted to SD ‐ N . Pexophagy was monitored by GFP appearance by immunoblotting with α ‐ GFP antibodies .

(E) S. cerevisiae Δatg36 cells (ΔScatg36) expressing thiolase-GFP and expressing the indicated Atg36 mutants were cultured in oleate medium until mid‐log growth and then shifted to SD‐N. Pexophagy was monitored by GFP appearance by immunoblotting with α‐GFP antibodies.


( F ) Y3H analysis of ScAtg36 with ScAtg8 and ScAtg11 . The Y3H technology is on the basis of the yeast two ‐ hybrid system but with the co ‐ expression of third protein as a competitor and indicated in the figure ( NLS - ScAtg11 or NLS - ScAtg8 ) . The positive control was the ScAtg36 mutant affected in Atg11 binding ( ScAtg36S97A ) or in Atg8 binding ( ScAtg36S31A ) , which should be unaffected by the competition of NLS - ScAtg11 or NLS - ScAtg8 , respectively . Appropriate auto ‐ activation and interaction controls were also included . AD , activation domain ; AIM , Atg8 ‐ family ‐ interacting motif ; BD , binding domain ; GFP , green fluorescent protein ; WT , wild type ; Y3H , yeast three ‐ hybrid .

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(F) Y3H analysis of ScAtg36 with ScAtg8 and ScAtg11. The Y3H technology is on the basis of the yeast two‐hybrid system but with the co‐expression of third protein as a competitor and indicated in the figure (NLS-ScAtg11 or NLS-ScAtg8). The positive control was the ScAtg36 mutant affected in Atg11 binding (ScAtg36S97A) or in Atg8 binding (ScAtg36S31A), which should be unaffected by the competition of NLS-ScAtg11 or NLS-ScAtg8, respectively. Appropriate auto‐activation and interaction controls were also included. AD, activation domain; AIM, Atg8‐family‐interacting motif; BD, binding domain; GFP, green fluorescent protein; WT, wild type; Y3H, yeast three‐hybrid.


( A ) 786 - 0 or 769 - P cells stably expressing shNT or shGDH1 were treated with or without aa starvation for the indicated times , and then cell viability was assessed . ( B ) 786 - 0 or 769 - P cells stably expressing shNT or shGDH1 were reconstituted with or without various amounts of rGDH1 WT ( 3 * WT indicates 3 - fold expression WT ) and then treated with aa starvation for the indicated time . Cell viability was subsequently assessed . ( Data information : The data are represented as means ± SEM of three independent experiments and all statistical analyses were conducted using unpaired t - test . ( * * p < 0 . 01 ; * * * p < 0 . 001 . )

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(A) 786-0 or 769-P cells stably expressing shNT or shGDH1 were treated with or without aa starvation for the indicated times, and then cell viability was assessed. (B) 786-0 or 769-P cells stably expressing shNT or shGDH1 were reconstituted with or without various amounts of rGDH1 WT (3*WT indicates 3-fold expression WT) and then treated with aa starvation for the indicated time. Cell viability was subsequently assessed. ( Data information: The data are represented as means ± SEM of three independent experiments and all statistical analyses were conducted using unpaired t-test. (**p < 0.01; ***p < 0.001.)


( I ) IHC staining of GDH1 in primary KIRC tumors and adjacent normal tissues collected by us was performed ( left ) . Quantitative analysis of the GDH1 protein levels was performed using unpaired t - test ( right ) . * * * p < 0 . 001 . Scale bars : 50 μm .

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(I) IHC staining of GDH1 in primary KIRC tumors and adjacent normal tissues collected by us was performed (left). Quantitative analysis of the GDH1 protein levels was performed using unpaired t-test (right). ***p < 0.001. Scale bars: 50 μm.


( K ) Overall survival of patients in different tumor stages in the TCGA - KIRC cohort . The R package " survminer " - based maximally selected log - rank statistic was used to determine the best cutoff . The log - rank test was used to determine the p value .

(K) Overall survival of patients in different tumor stages in the TCGA-KIRC cohort. The R package "survminer"-based maximally selected log-rank statistic was used to determine the best cutoff. The log-rank test was used to determine the p value.


( D ) Immunofluorescence ( IF ) assay was performed to determine the subcellular location of GDH1 with or without aa starvation . GDH1 localization was indicated by GDH1 antibody and MitoTracker ( left ) . Signal intensities and distances were quantified ( right ) . Scale bars : 20 μm .

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(D) Immunofluorescence (IF) assay was performed to determine the subcellular location of GDH1 with or without aa starvation. GDH1 localization was indicated by GDH1 antibody and MitoTracker (left). Signal intensities and distances were quantified (right). Scale bars: 20 μm.


( E ) Cytoplasm - mitochondrial fractionation assay was performed to determine the subcellular location of GDH1 with or without aa starvation . The fractionation efficiency and GDH1 location were determined by WB using antibodies against VDAC1 ( mitochondrial membrane marker ) and GDH1 . Tubulin was used as loading control .

(E) Cytoplasm-mitochondrial fractionation assay was performed to determine the subcellular location of GDH1 with or without aa starvation. The fractionation efficiency and GDH1 location were determined by WB using antibodies against VDAC1 (mitochondrial membrane marker) and GDH1. Tubulin was used as loading control.


( G ) Immunofluorescence ( IF ) assay was performed to determine the subcellular location of WT or mitochondrial signal peptide deleted ( dMSP ) GDH1 . GDH1 localization was indicated by GDH1 antibody and MitoTracker ( left ) . Signal intensities and distances were quantified ( right ) . Scale bars : 20 μm .

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(G) Immunofluorescence (IF) assay was performed to determine the subcellular location of WT or mitochondrial signal peptide deleted (dMSP) GDH1. GDH1 localization was indicated by GDH1 antibody and MitoTracker (left). Signal intensities and distances were quantified (right). Scale bars: 20 μm.


( H ) Cytoplasm - mitochondrial fractionation assay was performed to determine the subcellular location of WT or mitochondrial signal peptide deleted ( dMSP ) GDH1 . The fractionation efficiency and GDH1 location were determined by WB using antibodies against VDAC1 ( mitochondrial membrane marker ) and GDH1 . Tubulin was used as loading control .

(H) Cytoplasm-mitochondrial fractionation assay was performed to determine the subcellular location of WT or mitochondrial signal peptide deleted (dMSP) GDH1. The fractionation efficiency and GDH1 location were determined by WB using antibodies against VDAC1 (mitochondrial membrane marker) and GDH1. Tubulin was used as loading control.


( D ) FLAG - tagged GDH1 ( including WT and mutants with different lysine mutations to arginine ) was co - transfected with HA - tagged ubiquitin for 48 hours , and then cells were subjected to aa starvation for 12 hours before collecting cells . WB and qPCR separately tested the levels of GDH1 protein and mRNA , respectively . Data information : The data are represented as the mean ± SEM of three independent experiments . The one - way ANOVA test ( N . S . : not significant ; * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ) .

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(D) FLAG-tagged GDH1 (including WT and mutants with different lysine mutations to arginine) was co-transfected with HA-tagged ubiquitin for 48 hours, and then cells were subjected to aa starvation for 12 hours before collecting cells. WB and qPCR separately tested the levels of GDH1 protein and mRNA, respectively. Data information: The data are represented as the mean ± SEM of three independent experiments. The one-way ANOVA test (N.S.: not significant; *p < 0.05; **p < 0.01; ***p < 0.001).


( H ) 786 - 0 or 769 - P cells stably expressing shGDH1 were reconstituted with rGDH1 WT or 3KR mutant and then treated with or without aa starvation for the indicated time . Cell viability was determined . Unpaired two - tailed t - test was used . Data information : The data are represented as the mean ± SEM of three independent experiments . The unpaired t - test were used ( N . S . : not significant ; * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ) .

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(H) 786-0 or 769-P cells stably expressing shGDH1 were reconstituted with rGDH1 WT or 3KR mutant and then treated with or without aa starvation for the indicated time. Cell viability was determined. Unpaired two-tailed t-test was used. Data information: The data are represented as the mean ± SEM of three independent experiments. The unpaired t-test were used (N.S.: not significant; *p < 0.05; **p < 0.01; ***p < 0.001).


786 - 0 cells were treated with MG132 ( 10 μM ) under aa starvation . Antibodies against GDH1 ( C ) were used to enrich protein complexes in cells with or without GDH1 . IgG was used as a control . WB was performed using the indicated antibodies .

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786-0 cells were treated with MG132 (10 μM) under aa starvation. Antibodies against GDH1 (C) were used to enrich protein complexes in cells with or without GDH1. IgG was used as a control. WB was performed using the indicated antibodies.


( H ) 786 - 0 or 769 - P cells ( WT or stably expressing shGDH1 ) were transfected with siNC or siRNF213 for 48 hours and then were treated with or without aa starvation for the indicated times . Cell viability was determined . Unpaired t - test was used with * * * p < 0 . 001 .

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(H) 786-0 or 769-P cells (WT or stably expressing shGDH1) were transfected with siNC or siRNF213 for 48 hours and then were treated with or without aa starvation for the indicated times. Cell viability was determined. Unpaired t-test was used with ***p < 0.001.


( I ) IHCs of KIRC clinical samples were performed using antibodies against GDH1 or RNF213 ( upper panel ) . The correlated IHC signal between GDH1 and RNF213 in KIRC samples was calculated ( lower panel ) . The cor . test was used to determine the p - value . Scale bars : 50 μm .

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(I) IHCs of KIRC clinical samples were performed using antibodies against GDH1 or RNF213 (upper panel). The correlated IHC signal between GDH1 and RNF213 in KIRC samples was calculated (lower panel). The cor.test was used to determine the p-value. Scale bars: 50 μm.


786 - 0 cells expressing WT or enzymatically dead ( ED ) GDH1 were transfected with siNC or siRNF213 for 48 hours and then were treated with or without aa starvation for 12 hours . In the above cells in ( A ) , qPCR was used to measure the mRNA of the indicated RP genes ( C ) .

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786-0 cells expressing WT or enzymatically dead (ED) GDH1 were transfected with siNC or siRNF213 for 48 hours and then were treated with or without aa starvation for 12 hours. In the above cells in (A), qPCR was used to measure the mRNA of the indicated RP genes (C).


( J ) KIRC clinical samples were collected for αKG measurement , and we assessed overall survival ( left panel ) with log - rank test according to the dividing groups of high and low intracellular levels of αKG with the best separation ( right panel ) . Unpaired t - test was used in the right panel with * * * p < 0 . 001 .

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(J) KIRC clinical samples were collected for αKG measurement, and we assessed overall survival (left panel) with log-rank test according to the dividing groups of high and low intracellular levels of αKG with the best separation (right panel). Unpaired t-test was used in the right panel with ***p < 0.001.


( G ) An in vitro demethylation assay was performed with recombinant KDM4A ( 0 . 05 mM ) or KDM6A ( 0 . 05 mM ) , and the indicated histone H3 lysine methylation peptides under 50 - μM αKG conditions ( no αKG was used as a blank control ) . Various concentrations of 2HG from 0 to 200 μM was used to compete with αKG in the reaction system .

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(G) An in vitro demethylation assay was performed with recombinant KDM4A (0.05 mM) or KDM6A (0.05 mM), and the indicated histone H3 lysine methylation peptides under 50-μM αKG conditions (no αKG was used as a blank control). Various concentrations of 2HG from 0 to 200 μM was used to compete with αKG in the reaction system.