( Left ) The 25 - kDa neddylated species induced by oxidative stress increases with time in WT U2OS cells . U2OS WT or NEDP1 KO cells were treated with 600 μM H2O2 for the indicated time and then lysed in LDS sample loading buffer and processed for Western blot analysis . ( Right ) The same 25 kDa NEDD8 species , which is strongly induced in WT cells , is present in untreated NEDP1 KO cells and does not further increase after oxidative stress induced by H2O2 ( as indicated by the arrow ) .

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 B-MEASURED_VAR 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

(Left) The 25-kDa neddylated species induced by oxidative stress increases with time in WT U2OS cells. U2OS WT or NEDP1 KO cells were treated with 600 μM H2O2 for the indicated time and then lysed in LDS sample loading buffer and processed for Western blot analysis. (Right) The same 25 kDa NEDD8 species, which is strongly induced in WT cells, is present in untreated NEDP1 KO cells and does not further increase after oxidative stress induced by H2O2 (as indicated by the arrow).


U2OS NEDP1 KO cells are resistant to the PARP - 1 inducer , H2O2 . WT and NEDP1 U2OS cells were plated in 96 - well plates and after 24 hours were treated with the indicated concentration of H2O2 . They were assessed for viability 24 hours later by the CellTiter - Glo assay . Graphs represent the mean ± S . E . M . of the percent survival compared to untreated cells .

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

U2OS NEDP1 KO cells are resistant to the PARP-1 inducer, H2O2. WT and NEDP1 U2OS cells were plated in 96-well plates and after 24 hours were treated with the indicated concentration of H2O2. They were assessed for viability 24 hours later by the CellTiter-Glo assay. Graphs represent the mean ± S.E.M. of the percent survival compared to untreated cells.


NEDP1 KO cells are re - sensitized to H2O2 , following re - expression of NEDP1 by transient transfection . U2OS cells were plated in 96 well plates and reversed transfected with NEDP1 or an empty vector . 48 hours after plating , cells were challenged with the indicated amount of H2O2 and cell viability was assessed 24 hours later by the CellTiter - Glo assay . Graphs represent the mean ± S . E . M . of the percent survival compared to untreated cells .

B-CONTROLLED_VAR 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 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

NEDP1 KO cells are re-sensitized to H2O2, following re-expression of NEDP1 by transient transfection. U2OS cells were plated in 96 well plates and reversed transfected with NEDP1 or an empty vector. 48 hours after plating, cells were challenged with the indicated amount of H2O2 and cell viability was assessed 24 hours later by the CellTiter-Glo assay. Graphs represent the mean ± S.E.M. of the percent survival compared to untreated cells.


Neddylation of cullins is decreased in NEDP1 KO cells and is rescued by re - expression of NEDP1 by transient transfection . Western blot analysis from HEK 293 WT , NEDP1 KO cells and NEDP1 KO cells rescued with transient transfection of NEDP1 reveals that upon NEDP1 re - expression , the intensity of the NEDD8 reactive bands is reduced and the levels of free NEDD8 are increased . NEDP1 re - expression also increased the neddylation of each Cullin . The neddylated band of each Cullin is denoted with a star . Quantification and graph of the mean ± S . E . M . of the percentage neddylation of each Cullin in ( E ) .

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

Neddylation of cullins is decreased in NEDP1 KO cells and is rescued by re-expression of NEDP1 by transient transfection. Western blot analysis from HEK 293 WT, NEDP1 KO cells and NEDP1 KO cells rescued with transient transfection of NEDP1 reveals that upon NEDP1 re-expression, the intensity of the NEDD8 reactive bands is reduced and the levels of free NEDD8 are increased. NEDP1 re-expression also increased the neddylation of each Cullin. The neddylated band of each Cullin is denoted with a star. Quantification and graph of the mean ± S.E.M. of the percentage neddylation of each Cullin in (E).


Deletion of Nedp1 does not lead to induction Nrf2 response genes . RNA was harvested from WT and NEDP1 KO U2OS cells , reversed transcribed to cDNA , and analysed by qPCR for Nrf2 and NQO1 expression .

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

Deletion of Nedp1 does not lead to induction Nrf2 response genes. RNA was harvested from WT and NEDP1 KO U2OS cells, reversed transcribed to cDNA, and analysed by qPCR for Nrf2 and NQO1 expression.


Deletion of Nedp1 but not of Cand1 in HEK 293 cells results in resistance to cell death from H2O2 . WT , NEDP1 KO , and CAND1 KO cells were plated in 96 wells plates and treated with the indicated amount of H2O2 . 24 hours after treatment , cell viability was measured using the CellTiter - Glo assay . Graphs represent the mean ± S . E . M . of the percent survival compared to untreated cells .

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

Deletion of Nedp1 but not of Cand1 in HEK 293 cells results in resistance to cell death from H2O2. WT, NEDP1 KO, and CAND1 KO cells were plated in 96 wells plates and treated with the indicated amount of H2O2. 24 hours after treatment, cell viability was measured using the CellTiter-Glo assay. Graphs represent the mean ± S.E.M. of the percent survival compared to untreated cells.


PARP - 1 activity is reduced in NEDP1 KO cells . Western blot analysis of whole cell lysates from WT and NEDP1 KO U2OS cells after treatment with 600 μM H2O2 for the indicated amount of time . PAR polymer generation is induced in WT cells after H2O2 treatment but induction is reduced in NEDP1 KO cells .

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

PARP-1 activity is reduced in NEDP1 KO cells. Western blot analysis of whole cell lysates from WT and NEDP1 KO U2OS cells after treatment with 600 μM H2O2 for the indicated amount of time. PAR polymer generation is induced in WT cells after H2O2 treatment but induction is reduced in NEDP1 KO cells.


PARP - 1 activity is rescued by re - expression of NEDP1 in NEDP1 KO cells . Western blot analysis of whole cell lysates from WT and NEDP1 KO U2OS cells transfected with the indicated constructs . 72 hours after transfection , cells were treated with the indicated amount of H2O2 and processed for Western blot analysis . PAR polymer generation is reduced in NEDP1 KO cells but can be rescued by the transient re - expression of NEDP1 .

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

PARP-1 activity is rescued by re-expression of NEDP1 in NEDP1 KO cells. Western blot analysis of whole cell lysates from WT and NEDP1 KO U2OS cells transfected with the indicated constructs. 72 hours after transfection, cells were treated with the indicated amount of H2O2 and processed for Western blot analysis. PAR polymer generation is reduced in NEDP1 KO cells but can be rescued by the transient re-expression of NEDP1.


PARP - 1 inhibitor Olaparib protects WT cells from H2O2 treatment to the same extent as NEDP1 KO . U2OS cells were plated in 96 well plates and 24 hours later pre - treated with Olaparib ( 10 μM ) for 1 hour before treatment with the indicated concentration of H2O2 . 24 hours later , cell viability was measured using the CellTiterGlo assay . NEDP1 deletion protects cells from H2O2 treatment to the same extent as PARP - 1 inhibition in WT cells . Olaparib does not provide additional protection to NEDP1 cells from H2O2 treatment .

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

PARP-1 inhibitor Olaparib protects WT cells from H2O2 treatment to the same extent as NEDP1 KO. U2OS cells were plated in 96 well plates and 24 hours later pre-treated with Olaparib (10 μM) for 1 hour before treatment with the indicated concentration of H2O2. 24 hours later, cell viability was measured using the CellTiterGlo assay. NEDP1 deletion protects cells from H2O2 treatment to the same extent as PARP-1 inhibition in WT cells. Olaparib does not provide additional protection to NEDP1 cells from H2O2 treatment.


AIF translocation to the nucleus is impaired in NEDP1 KO cells . WT and NEDP1 KO cells were plated on coverslips and 24 hours later were treated with H2O2 ( 600 μM ) for the indicated amount of time and then processed for immunofluorescence analysis using α - AIF antibodies and DAPI staining . PARP - 1 dependent cell death is induced in WT U2OS cells , as indicated by translocation of AIF from the mitochondria to the nucleus ( Scale bar 10 μm ) .

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

AIF translocation to the nucleus is impaired in NEDP1 KO cells. WT and NEDP1 KO cells were plated on coverslips and 24 hours later were treated with H2O2 (600 μM) for the indicated amount of time and then processed for immunofluorescence analysis using α-AIF antibodies and DAPI staining. PARP-1 dependent cell death is induced in WT U2OS cells, as indicated by translocation of AIF from the mitochondria to the nucleus (Scale bar 10 μm).


Total nuclear AIF intensity from ( D ) was measured with ImageJ and plotted as a vertical scatter plot with the group mean intensity indicated with a black bar . AIF has translocated to the nucleus after WT cells were exposed to H2O2 but not in NEDP1 KO cells .

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

Total nuclear AIF intensity from (D) was measured with ImageJ and plotted as a vertical scatter plot with the group mean intensity indicated with a black bar. AIF has translocated to the nucleus after WT cells were exposed to H2O2 but not in NEDP1 KO cells.


NEDP1 KO cells express normal levels of PARP - 1 and AIF . Western blot analysis of whole cell lysates from WT and NEDP1 KO U2OS cells shows that the proteins necessary for the induction of PARP - 1 dependent cell death are present at equal levels in both cell lines .

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

NEDP1 KO cells express normal levels of PARP-1 and AIF. Western blot analysis of whole cell lysates from WT and NEDP1 KO U2OS cells shows that the proteins necessary for the induction of PARP-1 dependent cell death are present at equal levels in both cell lines.


NEDP1 KO U2OS cells are not resistant to induction of apoptosis from combined treatment with TNF - α and cycloheximide . U2OS cells were plated in 96 well plates and incubated with cycloheximide ( 10 μg / mL ) before addition of the indicated amount of TNF - α . Cell survival was determined 24 hours later using the CellTiter - Glo assay . Graphs represent the mean ± S . E . M . of the percent survival compared to untreated cells .

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

NEDP1 KO U2OS cells are not resistant to induction of apoptosis from combined treatment with TNF-α and cycloheximide. U2OS cells were plated in 96 well plates and incubated with cycloheximide (10 μg/mL) before addition of the indicated amount of TNF-α. Cell survival was determined 24 hours later using the CellTiter-Glo assay. Graphs represent the mean ± S.E.M. of the percent survival compared to untreated cells.


NEDP1 KO U2OS cells are not resistant to treatment with camptothecin ( CPT ) . WT U2OS and NEDP1 KO cells were plated in 96 - well plates and treated with the indicated amount of CPT . 48 hours after exposure cell survival was measured using the CellTiterGlo assay . Graphs represent the mean ± S . E . M . of the percent survival compared to untreated cells .

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

NEDP1 KO U2OS cells are not resistant to treatment with camptothecin (CPT). WT U2OS and NEDP1 KO cells were plated in 96-well plates and treated with the indicated amount of CPT. 48 hours after exposure cell survival was measured using the CellTiterGlo assay. Graphs represent the mean ± S.E.M. of the percent survival compared to untreated cells.


NEDD8 trimers co - immunoprecipitate with endogenous PARP - 1 in NEDP1 KO cells . Lysates were prepared from U2OS NEDP1 KO cells and immunoprecipitation was performed with PARP - 1 - Trap or GFP - Trap as a negative control . Bound proteins were resolved by SDS - PAGE and processed for Western blot analysis with the indicated antibodies .

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

NEDD8 trimers co-immunoprecipitate with endogenous PARP-1 in NEDP1 KO cells. Lysates were prepared from U2OS NEDP1 KO cells and immunoprecipitation was performed with PARP-1-Trap or GFP-Trap as a negative control. Bound proteins were resolved by SDS-PAGE and processed for Western blot analysis with the indicated antibodies.


NEDD8 trimers bind to GFP - PARP - 1 . NEDP1 U2OS KO cells were transfected with GFP or with GFP - PARP - 1 and 24 hours later cell lysates were collected . Immunoprecipitation was then performed with GFP - Trap , and bound proteins were resolved by SDS - PAGE and processed for Western blot analysis with the indicated antibodies . GFP - PARP - 1 but not GFP alone can co - immunoprecipitate NEDD8 trimers from NEDP1 KO cells .

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

NEDD8 trimers bind to GFP-PARP-1. NEDP1 U2OS KO cells were transfected with GFP or with GFP-PARP-1 and 24 hours later cell lysates were collected. Immunoprecipitation was then performed with GFP-Trap, and bound proteins were resolved by SDS-PAGE and processed for Western blot analysis with the indicated antibodies. GFP-PARP-1 but not GFP alone can co-immunoprecipitate NEDD8 trimers from NEDP1 KO cells.


NEDD8 trimers form in WT U2OS cells after treatment with H2O2 and co - immunoprecipitate with endogenous PARP - 1 but not with immunoglobulin control ( IGG ) . WT U2OS cells were left untreated of treated with H2O2 ( 600 μM ) for 30 min and were harvested for immunoprecipitation and Western blot

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

NEDD8 trimers form in WT U2OS cells after treatment with H2O2 and co-immunoprecipitate with endogenous PARP-1 but not with immunoglobulin control (IGG). WT U2OS cells were left untreated of treated with H2O2 (600 μM) for 30 min and were harvested for immunoprecipitation and Western blot analysis


NEDD8 trimers bind to GFP - PARP - 1 . WT or NEDP1 U2OS KO cells were transfected with GFP or with full - length GFP - PARP - 1 and 24 hours later treated as indicated with H2O2 ( 600 μM ) for 30 min before cell lysates were collected . Immunoprecipitation was then performed with GFP - Trap , and bound proteins were resolved by SDS - PAGE and processed for Western blot analysis with the indicated antibodies . GFP - PARP - 1 but not GFP alone can co - immunoprecipitate NEDD8 trimers from WT cells only after H2O2 treatment or from both treated or untreated NEDP1 KO cells .

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

NEDD8 trimers bind to GFP-PARP-1. WT or NEDP1 U2OS KO cells were transfected with GFP or with full-length GFP-PARP-1 and 24 hours later treated as indicated with H2O2 (600 μM) for 30 min before cell lysates were collected. Immunoprecipitation was then performed with GFP-Trap, and bound proteins were resolved by SDS-PAGE and processed for Western blot analysis with the indicated antibodies. GFP-PARP-1 but not GFP alone can co-immunoprecipitate NEDD8 trimers from WT cells only after H2O2 treatment or from both treated or untreated NEDP1 KO cells.


NEDD8 trimers bind to the DNA - binding domain of PARP - 1 ( Zn1 - 3 ) but not to its automodification and catalytic domain ( CAT ) . NEDP1 KO U2OS cells were transfected with GFP , GFP - PARP - 1 , GFP fused to the DNA - binding domain of PARP - 1 ( AA 1 - 336 ) or with GFP fused to the PARP - 1 automodification and catalytic domain ( AA 336 - 1014 ) . 24 hours post transfection , cell lysates were collected and immunoprecipitation with GFP - Trap was performed . Bound proteins were eluted and resolved by SDS - PAGE analysis , followed by Western blot analysis with the indicated antibodies .

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

NEDD8 trimers bind to the DNA-binding domain of PARP-1 (Zn1-3) but not to its automodification and catalytic domain (CAT). NEDP1 KO U2OS cells were transfected with GFP, GFP-PARP-1, GFP fused to the DNA-binding domain of PARP-1 (AA 1-336) or with GFP fused to the PARP-1 automodification and catalytic domain (AA 336-1014). 24 hours post transfection, cell lysates were collected and immunoprecipitation with GFP-Trap was performed. Bound proteins were eluted and resolved by SDS-PAGE analysis, followed by Western blot analysis with the indicated antibodies.


NEDD8 trimers specifically bind to the second zinc finger of PARP - 1 . NEDP1 KO U2OS cells were transfected with GFP , GFP - PARP - 1 , or with GFP fused to the Zn1 domain ( AA 1 - 96 ) , the Zn2 domain ( AA 97 - 215 ) , the Zn3 domain ( AA 216 - 336 ) or the Zn1 + 2 domains ( AA 1 - 215 ) of PARP - 1 . 24 hours post transfection , cell lysates were collected and immunoprecipitation with GFP - Trap was performed . Bound proteins were eluted and resolved by SDS - PAGE analysis , followed by Western blot analysis with the indicated antibodies .

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

NEDD8 trimers specifically bind to the second zinc finger of PARP-1. NEDP1 KO U2OS cells were transfected with GFP, GFP-PARP-1, or with GFP fused to the Zn1 domain (AA 1-96), the Zn2 domain (AA 97-215), the Zn3 domain (AA 216-336) or the Zn1+2 domains (AA 1-215) of PARP-1. 24 hours post transfection, cell lysates were collected and immunoprecipitation with GFP-Trap was performed. Bound proteins were eluted and resolved by SDS-PAGE analysis, followed by Western blot analysis with the indicated antibodies.


2D gel electrophoresis of lysate from WT cells followed by Western blot analysis with α - NEDD8 antibody indicates free NEDD8 migrates near its predicted isoelectic point ( pI ) of 6 .

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

2D gel electrophoresis of lysate from WT cells followed by Western blot analysis with α-NEDD8 antibody indicates free NEDD8 migrates near its predicted isoelectic point (pI) of 6.59


2D Gel electrophoresis of pulldowns from NEDP1 KO lysate with GST - Zn1 + 2 followed by Western blot analysis with α - NEDD8 antibody . Some of the 25 - kDa NEDD8 band migrates to the same isoelectric point as unconjugated NEDD8 ( denoted by white triangle ) , indicating the species is most likely an unanchored NEDD8 trimer . However , the majority of NEDD8 trimer migrates to multiple spots of lower pI ( indicated in the brackets ) , which suggests that NEDD8 trimers undergo a post - translational modification that either adds a negative charge or blocks a positive charge on the NEDD8 trimer .

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

2D Gel electrophoresis of pulldowns from NEDP1 KO lysate with GST-Zn1+2 followed by Western blot analysis with α-NEDD8 antibody. Some of the 25-kDa NEDD8 band migrates to the same isoelectric point as unconjugated NEDD8 (denoted by white triangle), indicating the species is most likely an unanchored NEDD8 trimer. However, the majority of NEDD8 trimer migrates to multiple spots of lower pI (indicated in the brackets), which suggests that NEDD8 trimers undergo a post-translational modification that either adds a negative charge or blocks a positive charge on the NEDD8 trimer.


Overexpression of HDAC1 or HDAC2 decreases PARP - 1 - NEDD8 trimer binding . NEDP1 KO U2OS cells were transfected with GFP or GFP fused to the first two zinc finger domains of PARP - 1 ( GFP - Zn1 + 2 ) . Cells were also co - transfected , as indicated , with empty FLAG vector , HDAC1 - FLAG , or HDAC2 - FLAG . 24 hours post transfection , cells were harvested and immunoprecipitation was performed on the lysates with GFP - Trap . Immunoprecipitated proteins were resolved by SDS - PAGE followed by Western blot analysis with the indicated antibodies .

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

Overexpression of HDAC1 or HDAC2 decreases PARP-1-NEDD8 trimer binding. NEDP1 KO U2OS cells were transfected with GFP or GFP fused to the first two zinc finger domains of PARP-1 (GFP-Zn1+2). Cells were also co-transfected, as indicated, with empty FLAG vector, HDAC1-FLAG, or HDAC2-FLAG. 24 hours post transfection, cells were harvested and immunoprecipitation was performed on the lysates with GFP-Trap. Immunoprecipitated proteins were resolved by SDS-PAGE followed by Western blot analysis with the indicated antibodies.


HDAC inhibition increases PARP - 1 - NEDD8 trimer binding . NEDP1 KO U2OS cells were transfected with GFP or GFP fused to the first two zinc finger domains of PARP - 1 ( GFP - Zn1 + 2 ) . 24 hours later , cells were left untreated or treated with the HDAC inhibitor sodium butyrate ( NaB ) ( 10 mM ) for 4 hours . Cells were harvested and immunoprecipitation was performed on the lysates with GFP - Trap . Immunoprecipitated proteins were resolved by SDS - PAGE followed by Western blot analysis with the indicated antibodies .

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

HDAC inhibition increases PARP-1-NEDD8 trimer binding. NEDP1 KO U2OS cells were transfected with GFP or GFP fused to the first two zinc finger domains of PARP-1 (GFP-Zn1+2). 24 hours later, cells were left untreated or treated with the HDAC inhibitor sodium butyrate (NaB) (10 mM) for 4 hours. Cells were harvested and immunoprecipitation was performed on the lysates with GFP-Trap. Immunoprecipitated proteins were resolved by SDS-PAGE followed by Western blot analysis with the indicated antibodies.


HDAC inhibition increases PARP - 1 - NEDD8 trimer binding . NEDP1 KO U2OS cells were left untreated or treated with the HDAC inhibitor sodium butyrate ( NaB ) ( 10 mM ) for 4 hours . Cells were harvested and lysates were incubated with recombinant Zn1 - GFP or Zn2 - GFP ( 25 nM ) for 1 hour followed by immunoprecipitation with GFP - Trap . Immunoprecipitated proteins were resolved by SDS - PAGE followed by Western blot analysis with the indicated antibodies .

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

HDAC inhibition increases PARP-1-NEDD8 trimer binding. NEDP1 KO U2OS cells were left untreated or treated with the HDAC inhibitor sodium butyrate (NaB) (10 mM) for 4 hours. Cells were harvested and lysates were incubated with recombinant Zn1-GFP or Zn2-GFP (25 nM) for 1 hour followed by immunoprecipitation with GFP-Trap. Immunoprecipitated proteins were resolved by SDS-PAGE followed by Western blot analysis with the indicated antibodies.


Overexpression of HDAC1 or HDAC2 in NEDP1 KO cells can rescue the induction of PAR polymer following H2O2 treatment . U2OS WT or NEDP1 KO cells were transfected with the indicated FLAG vectors . 48 post - transfection cells , were treated with H2O2 ( 600 μM ) for the indicated amount of time and harvested directly in sample loading buffer . Lysates were resolved by SDS - PAGE and processed for Western blot analysis with the indicated antibodies .

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

Overexpression of HDAC1 or HDAC2 in NEDP1 KO cells can rescue the induction of PAR polymer following H2O2 treatment. U2OS WT or NEDP1 KO cells were transfected with the indicated FLAG vectors. 48 post-transfection cells, were treated with H2O2 (600 μM) for the indicated amount of time and harvested directly in sample loading buffer. Lysates were resolved by SDS-PAGE and processed for Western blot analysis with the indicated antibodies.


In vitro de - neddylation by NEDP1 is sensitive to oxidation . Poly - NEDD8 reactions were prepared with NAE , UBE2M , and NEDD8 as in Figure 1E . NEDD8 reactions were stopped by the addition of NAE inhibitor MLN4924 ( 3 μM ) . NEDD8 reactions were incubated with GST - NEDP1 at 30 ° C for the indicated amount of time under reducing ( 50 μM DTT ) or oxidizing conditions ( 50 μM DTT + 150 μM H2O2 ) . Reactions were stopped by the addition of LDS sample loading buffer and resolved by SDS - PAGE before processing for Western blot analysis with the indicated antibodies .

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

In vitro de-neddylation by NEDP1 is sensitive to oxidation. Poly-NEDD8 reactions were prepared with NAE, UBE2M, and NEDD8 as in Figure 1E. NEDD8 reactions were stopped by the addition of NAE inhibitor MLN4924 (3 μM). NEDD8 reactions were incubated with GST-NEDP1 at 30°C for the indicated amount of time under reducing (50 μM DTT) or oxidizing conditions (50 μM DTT + 150 μM H2O2). Reactions were stopped by the addition of LDS sample loading buffer and resolved by SDS-PAGE before processing for Western blot analysis with the indicated antibodies.


In vitro neddylation reactions with UBE2M are insensitive to H2O2 . Poly - NEDD8 reactions were prepared with NAE , UBE2M , and NEDD8 as in Figure 1E . Before reactions were started , increasing concentrations of H2O2 ( as indicated ) were added to the NEDD8 reactions . Subsequently the reactions were incubated at 30 ° C for the indicated amount of time . Reactions were stopped by the addition of LDS sample loading buffer and resolved by SDS - PAGE before processing for Western blot analysis with the indicated antibodies .

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 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

In vitro neddylation reactions with UBE2M are insensitive to H2O2. Poly-NEDD8 reactions were prepared with NAE, UBE2M, and NEDD8 as in Figure 1E. Before reactions were started, increasing concentrations of H2O2 (as indicated) were added to the NEDD8 reactions. Subsequently the reactions were incubated at 30°C for the indicated amount of time. Reactions were stopped by the addition of LDS sample loading buffer and resolved by SDS-PAGE before processing for Western blot analysis with the indicated antibodies.


Representative BLI traces showing decreased binding of Nxph1 to the Nrxn1 LNS2SS2 - ( I401Q ) mutant compared to that of wild - type Nrxn1 LNS2SS2 - . Replicate numbers are shown in E .

O O O O O O O B-MEASURED_VAR 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

Representative BLI traces showing decreased binding of Nxph1 to the Nrxn1 LNS2SS2- (I401Q) mutant compared to that of wild-type Nrxn1 LNS2SS2-. Replicate numbers are shown in E.


Comparison of binding affinities for Nxph1 binding to mouse Nrxn1 - 3 LNS2SS2 - and LNS2SS2 - ( I401Q ) , plotted on a logarithmic scale . Error bars represent SEM and replicate numbers are indicated in bars . Significance values were calculated using Welch ' s t - test , * P < 0 . 05 , * * P < 0 . 01 .

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

Comparison of binding affinities for Nxph1 binding to mouse Nrxn1-3 LNS2SS2- and LNS2SS2- (I401Q), plotted on a logarithmic scale. Error bars represent SEM and replicate numbers are indicated in bars. Significance values were calculated using Welch's t-test, *P < 0.05, **P < 0.01.


Representative BLI traces showing increased binding of Nxph1 to Nrxn1 LNS2SS2A + and Nrxn1 LNS2SS2AB + compared to that of Nrxn1 LNS2SS2 - . Replicate numbers are shown in E .

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

Representative BLI traces showing increased binding of Nxph1 to Nrxn1 LNS2SS2A+ and Nrxn1 LNS2SS2AB+ compared to that of Nrxn1 LNS2SS2-. Replicate numbers are shown in E.


Comparison of binding affinities for Nxph1 binding to mouse Nrxn1 LNS2 splice variants and splice - insert containing Nrxn1 LNS2 with the I401Q mutation ; error bars represent SEM and replicate numbers are indicated in or above bars . Significance values were calculated using Welch ' s t - test , * P < 0 . 05 , * * P < 0 . 01 , * * * * P < 0 . 0001 .

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

Comparison of binding affinities for Nxph1 binding to mouse Nrxn1 LNS2 splice variants and splice-insert containing Nrxn1 LNS2 with the I401Q mutation; error bars represent SEM and replicate numbers are indicated in or above bars. Significance values were calculated using Welch's t-test, *P < 0.05, **P < 0.01, ****P < 0.0001.


Representative fluorescence polarization binding curves are shown for LNS2SS2 - and LNS2SS2A + interacting with Nxph1 with or without Ca2 + added .

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

Representative fluorescence polarization binding curves are shown for LNS2SS2- and LNS2SS2A+ interacting with Nxph1 with or without Ca2+ added.


Comparison of binding affinities of Nxph1 for Nrxn1 LNS2SS2 - and Nrxn1 LNS2SS2A + with or without Ca2 + added ; replicate numbers are indicated in bars . Error bars represent SEM , and significance values were calculated using Welch ' s t - test . ns , not significant .

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

Comparison of binding affinities of Nxph1 for Nrxn1 LNS2SS2- and Nrxn1 LNS2SS2A+ with or without Ca2+ added; replicate numbers are indicated in bars. Error bars represent SEM, and significance values were calculated using Welch's t-test. ns, not significant.


Comparison of binding affinities of Nxph1 binding to different Nrxn1 - 3 LNS2 isoforms .

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

Comparison of binding affinities of Nxph1 binding to different Nrxn1-3 LNS2 isoforms.


Comparison of binding affinities for Nxph3 binding to Nrxn1 - 3 LNS2SS2 - .

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

Comparison of binding affinities for Nxph3 binding to Nrxn1-3 LNS2SS2-.


Representative current traces from oocytes expressing wild - type ( WT ) and double cysteine mutant NMDARs before and after DTE treatment . For each mutant , traces are normalized ( in height , not in current amplitude ) to the maximal response obtained after DTE treatment on wild - type ( WT )

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

Representative current traces from oocytes expressing wild-type (WT) and double cysteine mutant NMDARs before and after DTE treatment. For each mutant, traces are normalized (in height, not in current amplitude) to the maximal response obtained after DTE treatment on wild-type (WT) receptors


Summary of the DTE - induced potentiation of WT and double cysteine

Summary of the DTE-induced potentiation of WT and double cysteine mutants


Immunoblots from Xenopus oocytes expressing either wt or mutant subunits . M1 indicates the GluN1 monomer ( ~ 110 kDa ) ; M2 the GluN2B monomer ( ~ 180 kDa ) ; D1 / 1 the GluN1 homodimer ( ~ 220 kDa ) ; and D1 / 2 the GluN1 / GluN2B heterodimer ( ~ 290 kDa ) . Lower panels : immunoblots in reducing conditions ( + β - mercaptoethanol ) . N . I . , non - injected

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

Immunoblots from Xenopus oocytes expressing either wt or mutant subunits. M1 indicates the GluN1 monomer (~110 kDa); M2 the GluN2B monomer (~180 kDa); D1/1 the GluN1 homodimer (~220 kDa); and D1/2 the GluN1/GluN2B heterodimer (~290 kDa). Lower panels: immunoblots in reducing conditions (+ β-mercaptoethanol). N.I., non-injected oocytes


Assessment of receptor channel activity using MK - 801 inhibition kinetics . Representative current traces from oocytes expressing either wild - type ( WT ) or GluN1 - E698C / GluN2B - L795C mutant receptors in response to 10 nM MK - 801 during agonist application . Responses were scaled to the current amplitude obtained before MK - 801 application . Inset , mono - exponential fits of MK - 801 wash - in and wash - out . Note the strikingly faster kinetics in mutant receptors , both at the onset and offset of MK -

O O 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 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

Assessment of receptor channel activity using MK-801 inhibition kinetics. Representative current traces from oocytes expressing either wild-type (WT) or GluN1-E698C/GluN2B-L795C mutant receptors in response to 10 nM MK-801 during agonist application. Responses were scaled to the current amplitude obtained before MK-801 application. Inset, mono-exponential fits of MK-801 wash-in and wash-out. Note the strikingly faster kinetics in mutant receptors, both at the onset and offset of MK-801


Relative MK - 801 inhibition on - and off - rate constants ( kon and koff ) . All values were normalized to the value obtained for WT GluN1 / GluN2B

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

Relative MK-801 inhibition on- and off-rate constants (kon and koff). All values were normalized to the value obtained for WT GluN1/GluN2B receptors


Zinc , ifenprodil and pH dose - response curves of wild - type ( WT ) GluN1 / GluN2B receptors and mutant GluN1 - E698C / GluN2B - L795C receptors . For comparison , zinc and ifenprodil dose - response curves of GluN1 / GluN2B receptors lacking the whole GluN2B NTD ( GluN1 WT / GluN2B - delNTD ) are also shown ( dashed lines ; data from Rachline et al , 2005 )

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

Zinc, ifenprodil and pH dose-response curves of wild-type (WT) GluN1/GluN2B receptors and mutant GluN1-E698C/GluN2B-L795C receptors. For comparison, zinc and ifenprodil dose-response curves of GluN1/GluN2B receptors lacking the whole GluN2B NTD (GluN1 WT/GluN2B-delNTD) are also shown (dashed lines; data from Rachline et al, 2005)


Spermine ( 200 µM , pH 6 . 3 ) potentiation of WT GluN1 / GluN2B and mutant GluN1 - E698C / GluN - L795C receptors . Spermine ( 200 µM , pH 6 . 5 ) sensitivity is also shown for WT GluN1 / GluN2B receptors and receptors lacking either the GluN1 ( GluN1 - delNTD / GluN2B WT ) or GluN2B ( GluN1 WT / GluN2B - delNTD ) NTD ( data from Mony et al , 2011 )

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

Spermine (200 µM, pH 6.3) potentiation of WT GluN1/GluN2B and mutant GluN1-E698C/GluN-L795C receptors. Spermine (200 µM, pH 6.5) sensitivity is also shown for WT GluN1/GluN2B receptors and receptors lacking either the GluN1 (GluN1-delNTD/GluN2B WT) or GluN2B (GluN1 WT/GluN2B-delNTD) NTD (data from Mony et al, 2011)


Glutamate and glycine dose - response curves of WT GluN1 / GluN2B receptors and mutant GluN1 - E698C / GluN2B - L795C

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

Glutamate and glycine dose-response curves of WT GluN1/GluN2B receptors and mutant GluN1-E698C/GluN2B-L795C receptors


MK - 801 inhibition kinetics . On - rate constants ( kon ) of inhibition by 10 nM MK - 801 on wild - type ( WT ) and mutant receptors . All values are normalized to that obtained for WT GluN1 / GluN2B

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

MK-801 inhibition kinetics. On-rate constants (kon) of inhibition by 10 nM MK-801 on wild-type (WT) and mutant receptors. All values are normalized to that obtained for WT GluN1/GluN2B receptors


Representative recordings of patches containing one single wild - type ( WT ) or GluN1 - E698C / GluN2B - L795C receptor . The bottom trace is an expanded view . C , closed channel ; O , open

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

Representative recordings of patches containing one single wild-type (WT) or GluN1-E698C/GluN2B-L795C receptor. The bottom trace is an expanded view. C, closed channel; O, open channel


Single channel properties ( equilibrium channel open probability , mean open time , mean closed time ) of the WT and super - active GluN1 - E698C / GluN2B - L795C

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 B-CONTROLLED_VAR O O

Single channel properties (equilibrium channel open probability, mean open time, mean closed time) of the WT and super-active GluN1-E698C/GluN2B-L795C receptors


Closed ( left ) and open ( right ) time histograms for WT ( upper panels ) or super - active GluN1 - E698C / GluN2B - L795C ( lower panels ) receptors . Closed time histograms were best fit with 5 exponentials , whereas open time histograms were best fit with 2 exponentials ( see Online Methods and Appendix Fig S8 ) . Smooth lines are associated exponential

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 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

Closed (left) and open (right) time histograms for WT (upper panels) or super-active GluN1-E698C/GluN2B-L795C (lower panels) receptors. Closed time histograms were best fit with 5 exponentials, whereas open time histograms were best fit with 2 exponentials (see Online Methods and Appendix Fig S8). Smooth lines are associated exponential fits


( A ) H1 - Hela cells were mock infected or infected with poliovirus ( MOI = 50 pfu / cell ) and cells were lysed at the indicated hours post - infection ( h . p . i . ) . Three independent experiments were measured for quantification of p62 levels , and the blot shown is representative .

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

(A) H1-Hela cells were mock infected or infected with poliovirus (MOI = 50 pfu/cell) and cells were lysed at the indicated hours post-infection (h.p.i.). Three independent experiments were measured for quantification of p62 levels, and the blot shown is representative.


( B ) H1 - Hela cells were mock infected or infected with poliovirus ( MOI = 50 pfu / cell ) and cells were lysed at 6 h . p . i . Cycloheximide ( Cy . ) was added to mock infected cells at a final concentration of 50 µg / mL , for 4 hours . Cells were then pre - treated with 0 . 1 µM bafilomycin A1 ( Baf . A1 ) for 14 hours prior to infection and kept under treatment throughout infection . MG132 was used at a final concentration of 20 µM and added to the media at the time of infection . The blot shown is representative of three independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) H1-Hela cells were mock infected or infected with poliovirus (MOI = 50 pfu/cell) and cells were lysed at 6 h.p.i. Cycloheximide (Cy.) was added to mock infected cells at a final concentration of 50 µg/mL, for 4 hours. Cells were then pre-treated with 0.1 µM bafilomycin A1 (Baf.A1) for 14 hours prior to infection and kept under treatment throughout infection. MG132 was used at a final concentration of 20 µM and added to the media at the time of infection. The blot shown is representative of three independent experiments.


( A ) Cells were transfected with scrambled ( SCR ) or anti - LC3 ( LC3 ) siRNAs , and 48 h later infected with PV at an MOI of 50 pfu / cell , or mock infected . Cells were collected at 6 h . p . i . , and immunoblots were performed on lysates for p62 , GAPDH , and LC3 .

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

(A) Cells were transfected with scrambled (SCR) or anti-LC3 (LC3) siRNAs, and 48 h later infected with PV at an MOI of 50 pfu/cell, or mock infected. Cells were collected at 6 h.p.i., and immunoblots were performed on lysates for p62, GAPDH, and LC3.


( B ) Cells were treated with 10 mM 3 - MA for 12 h , then infected with PV at an MOI of 50 pfu / cell , or mock infected . Cells were collected at 6 h . p . i . and lysates were used for immunoblots .

O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Cells were treated with 10 mM 3-MA for 12 h, then infected with PV at an MOI of 50 pfu/cell, or mock infected. Cells were collected at 6 h.p.i. and lysates were used for immunoblots.


Triplicate samples of H1 - Hela cells were infected with PV at an MOI of 0 . 1 pfu / cell ( A ) or MOI of 50 pfu / cell ( B ) . Cells were pre - treated with 20 µM leupeptin for 14 hours prior to infection and kept under treatment throughout infection . Cell - associated virus was collected at the indicated times post - infection , and virus titers were determined by plaque assay . Parallel infections were collected for Western blots of LC3 ( A ) or p62 ( B ) In ( B ) cell - associated virus was collected at 6 h . p . i . for plaque assay .

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

Triplicate samples of H1-Hela cells were infected with PV at an MOI of 0.1 pfu/cell (A) or MOI of 50 pfu/cell (B). Cells were pre-treated with 20 µM leupeptin for 14 hours prior to infection and kept under treatment throughout infection. Cell-associated virus was collected at the indicated times post-infection, and virus titers were determined by plaque assay. Parallel infections were collected for Western blots of LC3 (A) or p62 (B) In (B) cell-associated virus was collected at 6 h.p.i. for plaque assay.


( C ) Infections were performed as in ( A ) , then 10 µg / mL each of E64d and Pepstatin A ( Pep . A ) were added to the media at the time of infection . Inhibition of lysosomal degradation was confirmed by immunoblot for LC3 and GAPDH . ( D ) Infection at an MOI of 50 pfu / cell , with E64 / Pep . A as in ( C ) .

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

(C) Infections were performed as in (A), then 10 µg/mL each of E64d and Pepstatin A (Pep.A) were added to the media at the time of infection. Inhibition of lysosomal degradation was confirmed by immunoblot for LC3 and GAPDH. (D) Infection at an MOI of 50 pfu/cell, with E64/Pep.A as in (C).


( A ) Triplicate samples of H1 - Hela cells were infected with PV at an MOI of 0 . 1 pfu / cell , and cell - associated virus was collected at the indicated times post - infection . NH4Cl ( 20 mM ) was added to the media at the time of infection ( solid line ) . Virus titers were then determined by plaque assay . ( B ) Infection as in ( A ) , carried out to 16 h to represent a multiple - cycle infection .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Triplicate samples of H1-Hela cells were infected with PV at an MOI of 0.1 pfu/cell, and cell-associated virus was collected at the indicated times post-infection. NH4Cl (20 mM) was added to the media at the time of infection (solid line). Virus titers were then determined by plaque assay. (B) Infection as in (A), carried out to 16 h to represent a multiple-cycle infection.


( C ) Triplicate H1 - Hela infections with PV at an MOI of 50 pfu / cell , with cell - associated virus collected at 6 h . p . i . ( D ) Cells were infected as in ( A ) , then NH4Cl was added to half of the samples at 3 . 5 hours post - infection , and cell - associated virus was collected immediately or at 7 h . p . i .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Triplicate H1-Hela infections with PV at an MOI of 50 pfu/cell, with cell-associated virus collected at 6 h.p.i. (D) Cells were infected as in (A), then NH4Cl was added to half of the samples at 3.5 hours post-infection, and cell-associated virus was collected immediately or at 7 h.p.i.


( E ) Cells were pre - treated for 14 hours with 0 . 1 µM bafilomycin A1 ( Baf . A1 ) and kept under treatment throughout infection ( MOI = 0 . 1 pfu / cell ) . Cell - associated virus was collected at 6 or 12 h . p . i . and virus titers were determined by plaque assay . * p < 0 . 05 , * * p < 0 . 01 , * * * 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

(E) Cells were pre-treated for 14 hours with 0.1 µM bafilomycin A1 (Baf.A1) and kept under treatment throughout infection (MOI = 0.1 pfu/cell). Cell-associated virus was collected at 6 or 12 h.p.i. and virus titers were determined by plaque assay. * p<0.05, ** p<0.01, *** p<0.0001.


( A ) H1 - HeLa cells were infected with PV at an MOI of 50 pfu / cell . Cells were pulsed at the indicated h . p . i with 35S - labeled methionine for 1 h then lysed . Lysates were run on SDS - PAGE . Expected viral proteins are labeled according to recognized banding patterns .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) H1-HeLa cells were infected with PV at an MOI of 50 pfu/cell. Cells were pulsed at the indicated h.p.i with 35S-labeled methionine for 1 h then lysed. Lysates were run on SDS-PAGE. Expected viral proteins are labeled according to recognized banding patterns.


( B ) Triplicate plates of H1 - Hela cells were infected with PV at an MOI of 0 . 1 pfu / cell , virus RNA and host GAPDH RNA were measured by qRT - PCR . Virus RNA levels were normalized to GAPDH levels using the delta - Ct method . NH4Cl treatment was as described in Figure 4 , and Guanidine HCl ( 2 mM ) was added to the media at the time of infection . The data shown are pooled from three replicate experiments , and the titer of cell - associated virus collected at 6h . p . i . from each replicate was determined by plaque assay .

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

(B) Triplicate plates of H1-Hela cells were infected with PV at an MOI of 0.1 pfu/cell, virus RNA and host GAPDH RNA were measured by qRT-PCR. Virus RNA levels were normalized to GAPDH levels using the delta-Ct method. NH4Cl treatment was as described in Figure 4, and Guanidine HCl (2 mM) was added to the media at the time of infection. The data shown are pooled from three replicate experiments, and the titer of cell-associated virus collected at 6h.p.i. from each replicate was determined by plaque assay.


( C ) Triplicate plates of 293T cells were treated with 20 mM 3 - MA for 2 hours prior to infection , and kept under treatment throughout infection . PV infections were done at an MOI of 0 . 1 pfu / cell . RNA levels and virus titers were analyzed as in ( B ) . * * p < 0 . 01 , * * * 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

(C) Triplicate plates of 293T cells were treated with 20 mM 3-MA for 2 hours prior to infection, and kept under treatment throughout infection. PV infections were done at an MOI of 0.1 pfu/cell. RNA levels and virus titers were analyzed as in (B). ** p<0.01, *** p<0.0001.


( A ) H1 - Hela cells were infected at an MOI of 50 pfu / cell , and half of the samples were treated with NH4Cl . Cells were labeled with 35S - Methionine from 3 h . p . i . until collection at 5 or 6 h . p . i . , and lysates were then separated on a 15 - 30 % sucrose gradients . Fractions were then collected and the counts per minute ( CPM ) were analyzed for each fraction . Representative gradients from three independent experiments are shown .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) H1-Hela cells were infected at an MOI of 50 pfu/cell, and half of the samples were treated with NH4Cl. Cells were labeled with 35S-Methionine from 3 h.p.i. until collection at 5 or 6 h.p.i., and lysates were then separated on a 15-30% sucrose gradients. Fractions were then collected and the counts per minute (CPM) were analyzed for each fraction. Representative gradients from three independent experiments are shown.


. ( B ) The three fractions representing the 150S peak in each experiment were pooled for plaque assay analysis . Data shown are the averages from three independent experiments . (

O O O O O O O O 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) The three fractions representing the 150S peak in each experiment were pooled for plaque assay analysis. Data shown are the averages from three independent experiments. (


( C ) Three fractions representing the 150S and 75S peaks were pooled and run on SDS - PAGE , and the 35S - Methionine labeled bands were visualized . The bands are labeled according to expected relative migration pattern , and VP2 is identified by its absence in the 75S peak . The ratio of VP0 to VP3 bands was analyzed from four independent experiments and plotted in arbitrary units . * p < 0 . 05 , * * p < 0 . 01 , * * * 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 B-MEASURED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Three fractions representing the 150S and 75S peaks were pooled and run on SDS-PAGE, and the 35S-Methionine labeled bands were visualized. The bands are labeled according to expected relative migration pattern, and VP2 is identified by its absence in the 75S peak. The ratio of VP0 to VP3 bands was analyzed from four independent experiments and plotted in arbitrary units. * p<0.05, ** p<0.01, *** p<0.0001.


( A ) H1 - Hela cells were infected at an MOI of 50 pfu / cell , and half of the samples were treated with bafilomycin A1 as described in Figure 4 . Cells were labeled with 35S - Methionine from 3 h . p . i . until collection at 5 h . p . i . , and lysates were then separated on a 15 - 30 % sucrose gradients . Fractions were then collected and the counts per minute ( CPM ) were analyzed for each fraction .

(A) H1-Hela cells were infected at an MOI of 50 pfu/cell, and half of the samples were treated with bafilomycin A1 as described in Figure 4. Cells were labeled with 35S-Methionine from 3 h.p.i. until collection at 5 h.p.i., and lysates were then separated on a 15-30% sucrose gradients. Fractions were then collected and the counts per minute (CPM) were analyzed for each fraction.


( B ) Three fractions representing the 150S and 75S peaks were pooled and run on SDS - PAGE as in Figure 6 . The ratio of VP0 to VP3 bands was analyzed and plotted in arbitrary units . The titer of the infectious virus in the pooled 150S fractions was determined by plaque assay . * p < 0 . 05 .

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

(B) Three fractions representing the 150S and 75S peaks were pooled and run on SDS-PAGE as in Figure 6. The ratio of VP0 to VP3 bands was analyzed and plotted in arbitrary units. The titer of the infectious virus in the pooled 150S fractions was determined by plaque assay. * p<0.05.


C ) Immunofluorescence image of dCas9 - EGFP or CENP ‑ T ∆ C ‑ dCas9 ‑ EGFP targeted cells stained with antibodies against CENP - T . Images in C maximum intensity projections taken across the depth of the EGFP foci .

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

C) Immunofluorescence image of dCas9-EGFP or CENP‑T∆C‑dCas9‑EGFP targeted cells stained with antibodies against CENP-T. Images in C maximum intensity projections taken across the depth of the EGFP foci.


E ) Quantification of CENP - T signal intensity at MUC4 ectopic foci vs . endogenous centromeres , normalised to CENP - T signal intensity at endogenous centromeres ( = 1 , red line ) . LinesLines = Mean ± SD , 3 experiments , each with ≥ 10 metaphases analysed per condition . Each point = 1 focus or centromere . ns = p > 0 . 05 , * * * = p < 0 . 01 , * * * * = p < 0 . 001 ( Kruskal Wallis test with Dunn ' s multiple comparison correction ) .

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

E) Quantification of CENP-T signal intensity at MUC4 ectopic foci vs. endogenous centromeres, normalised to CENP-T signal intensity at endogenous centromeres (=1, red line). LinesLines = Mean ± SD, 3 experiments, each with ≥ 10 metaphases analysed per condition. Each point = 1 focus or centromere. ns = p>0.05, *** = p<0.01, **** = p<0.001 (Kruskal Wallis test with Dunn's multiple comparison correction).


H ) Quantification of KNL ‑ 1 signal intensity at MUC4 foci , normalised to KNL ‑ 1 signal intensity at centromeres ( = 1 , red line ) . LinesLines = Mean ± SD , 3 experiments , each with ≥ 10 metaphases analysed per condition . Each point = 1 focus or centromere . ns = p > 0 . 05 , * * * = p < 0 . 01 , * * * * = p < 0 . 001 ( Kruskal Wallis test with Dunn ' s multiple comparison correction ) .

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

H) Quantification of KNL‑1 signal intensity at MUC4 foci, normalised to KNL‑1 signal intensity at centromeres (=1, red line). LinesLines = Mean ± SD, 3 experiments, each with ≥ 10 metaphases analysed per condition. Each point = 1 focus or centromere. ns = p>0.05, *** = p<0.01, **** = p<0.001 (Kruskal Wallis test with Dunn's multiple comparison correction).


Targeting of dCas9 - EGFP or CENP ‑ T ∆ C ‑ dCas9 ‑ EGFP to Chr9 - CEN and Chr1 ‑ TELO in HEK293T cells . C ) Immunofluorescence images of dCas9 - EGFP or CENP ‑ T ∆ C ‑ dCas9 ‑ EGFP targeted cells stained with antibodies against CENP - T . Images in are maximum intensity projections taken across the depth of the EGFP foci .

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

Targeting of dCas9-EGFP or CENP‑T∆C‑dCas9‑EGFP to Chr9-CEN and Chr1‑TELO in HEK293T cells. C) Immunofluorescence images of dCas9-EGFP or CENP‑T∆C‑dCas9‑EGFP targeted cells stained with antibodies against CENP-T. Images in are maximum intensity projections taken across the depth of the EGFP foci.


Targeting of dCas9 - EGFP or CENP ‑ T ∆ C ‑ dCas9 ‑ EGFP to Chr9 - CEN and Chr1 ‑ TELO in HEK293T cells . F ) Immunofluorescence images of CENP ‑ T ∆ C ‑ dCas9 ‑ EGFP targeted cells stained with antibodies against KNL - 1 . Images in , F are maximum intensity projections taken across the depth of the EGFP foci .

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

Targeting of dCas9-EGFP or CENP‑T∆C‑dCas9‑EGFP to Chr9-CEN and Chr1‑TELO in HEK293T cells. F) Immunofluorescence images of CENP‑T∆C‑dCas9‑EGFP targeted cells stained with antibodies against KNL-1. Images in ,F are maximum intensity projections taken across the depth of the EGFP foci.


Targeting of dCas9 - EGFP or CENP ‑ T ∆ C ‑ dCas9 ‑ EGFP to Chr9 - CEN and Chr1 ‑ TELO in HEK293T cells . I ) Immunofluorescence images of dCas9 - EGFP or CENP ‑ T ∆ C ‑ dCas9 ‑ EGFP targeted cells stained with antibodies against Ndc80 . Images in I ) are maximum intensity projections taken across the depth of the EGFP foci .

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

Targeting of dCas9-EGFP or CENP‑T∆C‑dCas9‑EGFP to Chr9-CEN and Chr1‑TELO in HEK293T cells. I) Immunofluorescence images of dCas9-EGFP or CENP‑T∆C‑dCas9‑EGFP targeted cells stained with antibodies against Ndc80. Images in I) are maximum intensity projections taken across the depth of the EGFP foci.


Targeting of dCas9 - EGFP or CENP ‑ T ∆ C ‑ dCas9 ‑ EGFP to Chr9 - CEN and Chr1 ‑ TELO in HEK293T cells . A ) Immunofluorescence images of dCas9 - EGFP or CENP ‑ T ∆ C ‑ dCas9 ‑ EGFP targeted metaphase cells stained with antibodies against alpha - tubulin . B ) Immunofluorescence images of mitotic cells with CENP ‑ T ∆ C ‑ dCas9 ‑ EGFP targeting after nocodazole treatment . C ) Length measurements of individual EGFP foci in µm . Example measurements are indicated on zooms in A - B ) in orange . Data is ≥ 20 cells per condition , from 1 experiment . Lines = Mean ± SEM . * = p < 0 . 05 , * * = p < 0 . 01 , * * * * = p < 0 . 001 ( Kruskal - Wallis with multiple comparison correction ) . Images in A , B are maximum intensity projections taken across the depth of the EGFP foci . In A , B ) Orange bars = measured lengths in C .

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

Targeting of dCas9-EGFP or CENP‑T∆C‑dCas9‑EGFP to Chr9-CEN and Chr1‑TELO in HEK293T cells. A) Immunofluorescence images of dCas9-EGFP or CENP‑T∆C‑dCas9‑EGFP targeted metaphase cells stained with antibodies against alpha-tubulin. B) Immunofluorescence images of mitotic cells with CENP‑T∆C‑dCas9‑EGFP targeting after nocodazole treatment. C) Length measurements of individual EGFP foci in µm. Example measurements are indicated on zooms in A-B) in orange. Data is ≥20 cells per condition, from 1 experiment. Lines = Mean ± SEM. * = p<0.05, ** = p<0.01,**** = p<0.001 (Kruskal-Wallis with multiple comparison correction). Images in A,B are maximum intensity projections taken across the depth of the EGFP foci. In A,B) Orange bars = measured lengths in C.


Targeting of dCas9 - EGFP or CENP ‑ T ∆ C ‑ dCas9 ‑ EGFP to Chr9 - CEN and Chr1 ‑ TELO in HEK293T cells . D ) Immunofluorescence images of metaphase cells with CENP - T ∆ C - dCas9 ‑ EGFP targeting after cold treatment , stained with antibodies against alpha - tubulin . E ) Percentage of metaphase cells showing microtubule ( MT ) recruitment . ≥ 50 metaphases per condition , from 1 experiment . . are maximum intensity projections taken across the depth of the EGFP foci .

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

Targeting of dCas9-EGFP or CENP‑T∆C‑dCas9‑EGFP to Chr9-CEN and Chr1‑TELO in HEK293T cells. D) Immunofluorescence images of metaphase cells with CENP-T∆C-dCas9‑EGFP targeting after cold treatment, stained with antibodies against alpha-tubulin. E) Percentage of metaphase cells showing microtubule (MT) recruitment. ≥50 metaphases per condition, from 1 experiment.. are maximum intensity projections taken across the depth of the EGFP foci.


Targeting of dCas9 - EGFP or CENP ‑ T ∆ C ‑ dCas9 ‑ EGFP to Chr9 - CEN in HEK293T cells . F ) Frames from live cell imaging of cells with dCas9 targeted to Chr9 - CEN , or an EGFP negative cell . are maximum intensity projections taken across the depth of the EGFP foci .

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

Targeting of dCas9-EGFP or CENP‑T∆C‑dCas9‑EGFP to Chr9-CEN in HEK293T cells. F) Frames from live cell imaging of cells with dCas9 targeted to Chr9-CEN, or an EGFP negative cell. are maximum intensity projections taken across the depth of the EGFP foci.


Targeting of dCas9 - EGFP or CENP ‑ T ∆ C ‑ dCas9 ‑ EGFP to Chr9 - CEN and Chr1 ‑ TELO in HEK293T cells . A ) Immunofluorescence images of paired EGFP foci for each attachment status category . Images were chosen where both the EGFP foci and any of their attachments lay in the same Z - plane for clarity of presentation . B ) Percentage of paired EGFP foci showing each attachment status . are from ≥ 75 EGFP foci pairs from n ≥ 15 metaphases per condition 1 experiment

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

Targeting of dCas9-EGFP or CENP‑T∆C‑dCas9‑EGFP to Chr9-CEN and Chr1‑TELO in HEK293T cells. A) Immunofluorescence images of paired EGFP foci for each attachment status category. Images were chosen where both the EGFP foci and any of their attachments lay in the same Z-plane for clarity of presentation. B) Percentage of paired EGFP foci showing each attachment status. are from ≥75 EGFP foci pairs from n≥15 metaphases per condition 1 experiment for


Targeting of dCas9 - EGFP or CENP ‑ T ∆ C ‑ dCas9 ‑ EGFP to Chr9 - CEN and Chr1 ‑ TELO in HEK293T cells . C ) Immunofluorescence images of CENP ‑ T ∆ C ‑ dCas9 ‑ EGFP targeted metaphase cells stained with antibodies against Mad2 . D ) Percentage of individual EGFP foci showing EGFP and Mad2 signal co - localisation , in metaphase cells with CENP ‑ T ∆ C - dCas9 - targeting . 2 experiments for ( D ) . Image in C ) is a maximum intensity projection taken across the depth of the EGFP signal . Scale bars = 1µm on small images and zooms , 5µm on larger images in C ) .

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

Targeting of dCas9-EGFP or CENP‑T∆C‑dCas9‑EGFP to Chr9-CEN and Chr1‑TELO in HEK293T cells. C) Immunofluorescence images of CENP‑T∆C‑dCas9‑EGFP targeted metaphase cells stained with antibodies against Mad2. D) Percentage of individual EGFP foci showing EGFP and Mad2 signal co-localisation, in metaphase cells with CENP‑T∆C-dCas9-targeting. 2 experiments for (D). Image in C) is a maximum intensity projection taken across the depth of the EGFP signal. Scale bars = 1µm on small images and zooms, 5µm on larger images in C).


Immunofluorescence images from CENP ‑ T ∆ C ‑ dCas9 ‑ EGFP targeted metaphase cells stained with antibodies against Mad2 and alpha - Tubulin , showing examples of microtubule attached ( G ) EGFP foci . H ) Percentage of individual EGFP foci with EGFP and Mad2 signal co ‑ localisation attached ( H ) EGFP foci . are from ≥ 75 EGFP foci pairs from n ≥ 15 metaphases per condition 1 experiment for H ) are single Z - slices .

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

Immunofluorescence images from CENP‑T∆C‑dCas9‑EGFP targeted metaphase cells stained with antibodies against Mad2 and alpha-Tubulin, showing examples of microtubule attached (G) EGFP foci. H) Percentage of individual EGFP foci with EGFP and Mad2 signal co‑localisation attached (H) EGFP foci. are from ≥75 EGFP foci pairs from n≥15 metaphases per condition 1 experiment for H) are single Z-slices.


C , D ) Example images of chromosome mis - segregation events involving the target locus ( EGFP signal ) in anaphase HEK293T ( C ) or HCT116 ( D ) cells with CENP - T ∆ C - dCas9 ‑ EGFP targeting after Mps1i pulse . E , F ) Mis ‑ segregation rate in fixed dCas9 - targeted HEK293T ( E ) or HCT116 ( F ) cells after Mps1i pulse , involving only EGFP + chromosomes , only EGFP - chromosomes or both . Bars = mean ± SD , 3 experiments for each cell line , each with ≥ 25 anaphase cells per condition . * = p < 0 . 05 , * * = p < 0 . 01 , * * * = p < 0 . 001 , * * * * = p < 0 . 0001 ( Oneway ANOVA with Šidák ' s multiple comparison correction , in E / F ) comparing the mis - segregation rate for any events involved EGFP ) .

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

C,D) Example images of chromosome mis-segregation events involving the target locus (EGFP signal) in anaphase HEK293T (C) or HCT116 (D) cells with CENP-T∆C-dCas9‑EGFP targeting after Mps1i pulse. E,F) Mis‑segregation rate in fixed dCas9-targeted HEK293T (E) or HCT116 (F) cells after Mps1i pulse, involving only EGFP+ chromosomes, only EGFP- chromosomes or both. Bars = mean ± SD, 3 experiments for each cell line, each with ≥25 anaphase cells per condition. * = p<0.05, **= p<0.01, *** = p<0.001, **** = p<0.0001 (Oneway ANOVA with Šidák's multiple comparison correction, in E/F) comparing the mis-segregation rate for any events involved EGFP).


G - H ) Frames from live cell imaging of HEK293T H2B - RFP cells after treatment with Mps1i ( t = 0 ) , with CENP ‑ T ∆ C ‑ dCas9 ‑ EGFP targeted to Chr9 - CEN or Chr1 - TELO . Frames are either across the duration of the 4 h movie ( G ) or show examples of the different categories of fate ( H ) . I ) Percentage of CENP - T ∆ C - dCas9 ‑ EGFP targeted cells that made an EGFP + error in anaphase , categorised by subsequent fate of EGFP signal in daughter cells 3 h after Mps1i addition . Assessed by live cell imaging . ≥ 22 cells with error per condition , combining data from ≥ 3 independent experiments .

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

G-H) Frames from live cell imaging of HEK293T H2B-RFP cells after treatment with Mps1i (t=0), with CENP‑T∆C‑dCas9‑EGFP targeted to Chr9-CEN or Chr1-TELO. Frames are either across the duration of the 4 h movie (G) or show examples of the different categories of fate (H). I) Percentage of CENP-T∆C-dCas9‑EGFP targeted cells that made an EGFP+ error in anaphase, categorised by subsequent fate of EGFP signal in daughter cells 3 h after Mps1i addition. Assessed by live cell imaging. ≥22 cells with error per condition, combining data from ≥3 independent experiments.


( B ) Slice through a tomogram showing EBOV virions inside a late endosomal compartment .

(B) Slice through a tomogram showing EBOV virions inside a late endosomal compartment.


( I - J ) Magnified areas highlighted in ( F ) and ( H ) , respectively , showing the viral membrane and VP40 densities at the luminal side . For comparison , line profiles at 3 nm distance from the inner membrane monolayer , visualized by dotted profiles ( magenta and blue , respectively ) , were determined . ( K ) Line profiles adjacent to the inner viral membrane leaflet of a virion inside an endosome and a purified virion before infection .

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

(I-J) Magnified areas highlighted in (F) and (H), respectively, showing the viral membrane and VP40 densities at the luminal side. For comparison, line profiles at 3 nm distance from the inner membrane monolayer, visualized by dotted profiles (magenta and blue, respectively), were determined. (K) Line profiles adjacent to the inner viral membrane leaflet of a virion inside an endosome and a purified virion before infection.


( G - J ) Slices of a tomogram showing a filamentous EBOV VLP composed of VP40 and GP after incubation at low pH ( n = 18 ) . White arrows in ( H ) and ( J ) highlight areas adjacent to the VLP membrane devoid of protein densities in contrast to corresponding slices of VLPs at neutral pH .

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

(G-J) Slices of a tomogram showing a filamentous EBOV VLP composed of VP40 and GP after incubation at low pH (n=18). White arrows in (H) and (J) highlight areas adjacent to the VLP membrane devoid of protein densities in contrast to corresponding slices of VLPs at neutral pH.


( K ) Slices of tomograms showing filamentous VLPs composed of VP40 after incubation at neutral ( n = 22 ) and low pH ( n = 8 ) , respectively . ( L ) Line density profiles determined adjacent to the inner membrane monolayer of VLPs incubated at neutral ( blue ) and low pH ( magenta ) . At neutral pH , the VP40 matrix detectable as regular densities in ( D ) have a 5 - 6 nm pitch .

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

(K) Slices of tomograms showing filamentous VLPs composed of VP40 after incubation at neutral (n=22) and low pH (n=8), respectively. (L) Line density profiles determined adjacent to the inner membrane monolayer of VLPs incubated at neutral (blue) and low pH (magenta). At neutral pH, the VP40 matrix detectable as regular densities in (D) have a 5-6 nm pitch.


( E ) Free energy profiles of VP40 - lipid interactions at pH 7 . 4 and pH 4 . 5 determined from MD simulations . The plot shows free energy ( in kBT ) at increasing membrane - VP40 distances ( nm ) with indicated three states shown in ( A ) .

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

(E) Free energy profiles of VP40-lipid interactions at pH 7.4 and pH 4.5 determined from MD simulations. The plot shows free energy (in kBT) at increasing membrane-VP40 distances (nm) with indicated three states shown in (A).


( F ) EBOV VLP lipid composition showing highly abundant lipids determined by mass spectrometry in mol % . Lipid abbreviations : phosphatidylcholine ( PC ) , phosphatidylserine ( PS ) , phosphatidylethanolamine ( PE ) , phosphatidylinositol ( PI ) , lyso - phosphatidylcholine ( LPC ) , sphingomyelin ( SM ) . Prefix " a " indicates acyl - linked glycerophospholipids , prefix " e " indicates ether - linked ( plasmanyl ) or the presence of one odd and one even chain fatty acyl . Bars represent mean and error bars represent standard error of the mean ( red ) ; n = 3 biological replicates .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) EBOV VLP lipid composition showing highly abundant lipids determined by mass spectrometry in mol%. Lipid abbreviations: phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidylinositol (PI), lyso-phosphatidylcholine (LPC), sphingomyelin (SM). Prefix "a" indicates acyl-linked glycerophospholipids, prefix "e" indicates ether-linked (plasmanyl) or the presence of one odd and one even chain fatty acyl. Bars represent mean and error bars represent standard error of the mean (red); n=3 biological replicates.


( B ) Overview confocal fluorescence microscopy image showing pleomorphic pHluorin - labelled VLPs composed of VP40 , pHluorin - VP40 ( ratio 10 : 1 ) and GP . ( C ) Magnified images of representative VLPs acquired during time - lapse microscopy at neutral pH and after acidification to approximately pH 5 . Frames are exemplarily shown at 0 , 1 , 3 , 5 and 10 min after lowering the external pH .

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

(B) Overview confocal fluorescence microscopy image showing pleomorphic pHluorin-labelled VLPs composed of VP40, pHluorin-VP40 (ratio 10:1) and GP. (C) Magnified images of representative VLPs acquired during time-lapse microscopy at neutral pH and after acidification to approximately pH 5. Frames are exemplarily shown at 0, 1, 3, 5 and 10 min after lowering the external pH.


( D ) Plot showing the mean relative fluorescence intensities and standard deviation of VLPs imaged at neutral pH , low pH and in the presence of T - X100 at low pH over time . Data was obtained from 3 independent VLP preparations . Number of technical replicates : n = 42 ( pH 7 . 4 ) ; n = 19 ( pH 4 . 5 and pH 5 + T - X100 ) .

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

(D) Plot showing the mean relative fluorescence intensities and standard deviation of VLPs imaged at neutral pH, low pH and in the presence of T-X100 at low pH over time. Data was obtained from 3 independent VLP preparations. Number of technical replicates: n=42 (pH 7.4); n=19 (pH 4.5 and pH 5 + T-X100).


( E ) Plot showing the drop of pH inside VLPs over time after lowering the pH of the surrounding buffer to 5 . The dots represent the mean values , and the dashed lines are the theoretical fit to Eq . 3 .

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

(E) Plot showing the drop of pH inside VLPs over time after lowering the pH of the surrounding buffer to 5. The dots represent the mean values, and the dashed lines are the theoretical fit to Eq. 3.


( B ) Stalk formation energy as a function of VP40 - membrane interaction energy . The stalk energy for non - interacting VP40 matrix ( u0 = 0 ) is 65 kBT . VP40 matrix layer Young ' s modulus legend - red 5 . 6 MPa , blue 11 . 2 MPa , black 16 . 8 MPa , and orange is infinitely rigid . The bending rigidity ratio between the VP40 matrix layer and lipid monolayer are 1 , 2 , 3 , and infinity , respectively . The line represents an infinitely rigid layer .

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

(B) Stalk formation energy as a function of VP40-membrane interaction energy. The stalk energy for non-interacting VP40 matrix (u0 = 0) is 65 kBT. VP40 matrix layer Young's modulus legend - red 5.6 MPa, blue 11.2 MPa, black 16.8 MPa, and orange is infinitely rigid. The bending rigidity ratio between the VP40 matrix layer and lipid monolayer are 1, 2, 3, and infinity, respectively. The line represents an infinitely rigid layer.


( D ) Fusion pore formation energy as a function of VP40 - membrane interaction energy . The discontinuity in the energy is located at the phase line between configurations ( see C ) . The change in pore formation energy , ΔEpore is defined as the difference between fusion - pore formation energy at interaction energy density 0 . 2 kBT / nm2 ( the value found using MD simulations ) to the matrix - free case ( no interaction energy ) . A - D ) Dotted lines serve as a guide to the eye .

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 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

(D) Fusion pore formation energy as a function of VP40-membrane interaction energy. The discontinuity in the energy is located at the phase line between configurations (see C). The change in pore formation energy, ΔEpore is defined as the difference between fusion-pore formation energy at interaction energy density 0.2 kBT/nm2 (the value found using MD simulations) to the matrix-free case (no interaction energy). A-D) Dotted lines serve as a guide to the eye.


( F ) Quantification of FACS data showing EBOV VLP entry as measured by a fluorescence shift of infected cells from emission at 510 nm ( no entry ) to 450 nm ( entry ) . VLPs were treated prior to infection as indicated on the x - axis , with control : uninfected control cells , - : no treatment , T : thermolysin - treatment at neutral pH , Lp : low pH treatment . Target cells were treated with media or ammonium chloride ( NH4Cl ) , n = 3 with 10 ' 000 cells measured per sample .

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

(F) Quantification of FACS data showing EBOV VLP entry as measured by a fluorescence shift of infected cells from emission at 510 nm (no entry) to 450 nm (entry). VLPs were treated prior to infection as indicated on the x-axis, with control: uninfected control cells, - : no treatment, T: thermolysin-treatment at neutral pH, Lp: low pH treatment. Target cells were treated with media or ammonium chloride (NH4Cl), n= 3 with 10'000 cells measured per sample.


Immunofluorescence of early pluripotent marker Sall4 in DMSO - and R406 - treated cells on d12 . n = 5 . Scale bar , 100 μm .

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

Immunofluorescence of early pluripotent marker Sall4 in DMSO- and R406- treated cells on d12. n = 5. Scale bar, 100 μm.


RT - qPCR analysis of Sall4 , Cdh1 , Epcam and Esrrb gene expression in MEFs , intermediate cells on d12 treated with DMSO and R406 , and R1 ( mESCs ) . n = 3 .

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

RT-qPCR analysis of Sall4, Cdh1, Epcam and Esrrb gene expression in MEFs, intermediate cells on d12 treated with DMSO and R406, and R1 (mESCs). n = 3.


Immunofluorescence of Sall4 in reprogramming intermediates infected with shNC and shSyk . n = 3 . Scale bar , 100 μm .

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

Immunofluorescence of Sall4 in reprogramming intermediates infected with shNC and shSyk. n = 3. Scale bar, 100 μm.


ciPSCs express key pluripotent markers Oct4 , Sox2 and Nanog . Scale bar , 100 μm .

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

ciPSCs express key pluripotent markers Oct4, Sox2 and Nanog. Scale bar, 100 μm.


Immunofluorescence of Sall4 in WT cells and Ppp3ca knockdown cells on d12 . n = 6 . Scale bar , 100 μm . RT - qPCR analysis of pluripotent genes expression in cells treated with shNC and shPpp3ca . n = 3 .

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

Immunofluorescence of Sall4 in WT cells and Ppp3ca knockdown cells on d12. n = 6. Scale bar, 100 μm. RT-qPCR analysis of pluripotent genes expression in cells treated with shNC and shPpp3ca. n = 3.


Immunofluorescence of Sall4 in WT cells and Nfatc1 knockdown cells on d12 . n = 3 . Scale bar , 100 μm . RT - qPCR analysis of pluripotent genes expression in cells treated with shNC and shNfatc1 . n = 3 .

Immunofluorescence of Sall4 in WT cells and Nfatc1 knockdown cells on d12. n = 3. Scale bar, 100 μm. RT-qPCR analysis of pluripotent genes expression in cells treated with shNC and shNfatc1. n = 3.


RT - qPCR analysis of Gldc , Cbs and Tdh expression in R406 - and shSyk - treated cells ( E ) and FK506 - , shPpp3ca - and shNfatc1 - treated cells ( F ) on d8 . n = 3 .

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

RT-qPCR analysis of Gldc, Cbs and Tdh expression in R406- and shSyk-treated cells (E) and FK506-, shPpp3ca- and shNfatc1-treated cells (F) on d8. n = 3.


Metabolite abundance of SAM , SAH in samples treated with DMSO and R406 . n = 6 .

Metabolite abundance of SAM, SAH in samples treated with DMSO and R406. n = 6.


Immunofluorescence of Sall4 in reprogramming intermediates treated by R406 + HA and R406 + DPBS on d12 . n = 3 . Scale bar , 100 μm . Immunofluorescence of Sall4 in NAC - and DPBS - treated cells on d12 . n = 5 . Scale bar , 100 μm .

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

Immunofluorescence of Sall4 in reprogramming intermediates treated by R406 + HA and R406 + DPBS on d12. n = 3. Scale bar, 100 μm. Immunofluorescence of Sall4 in NAC- and DPBS-treated cells on d12. n = 5. Scale bar, 100 μm.


RT - qPCR analysis of Sall4 , Cdh1 , Epcam and Esrrb gene expression in DPBS - and HA - treated cells on d12 . n = 3 . RT - qPCR analysis of Sall4 , Cdh1 , Epcam and Esrrb gene expression in DPBS - and NAC - treated cells on d12 . n = 3 .

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

RT-qPCR analysis of Sall4, Cdh1, Epcam and Esrrb gene expression in DPBS- and HA-treated cells on d12. n = 3. RT-qPCR analysis of Sall4, Cdh1, Epcam and Esrrb gene expression in DPBS- and NAC-treated cells on d12. n = 3.


Immunofluorescence of Sall4 in WT cells and Cbs overexpressing cells on d12 . n = 4 . Scale bar , 100 μm .

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

Immunofluorescence of Sall4 in WT cells and Cbs overexpressing cells on d12. n = 4. Scale bar, 100 μm.


Quantification of oxygen consumption rate ( OCR ) of reprogramming intermediates treated with small molecules ( A , C ) and the indicated OCR parameters ( B , D ) . n = 9 .

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

Quantification of oxygen consumption rate (OCR) of reprogramming intermediates treated with small molecules (A, C) and the indicated OCR parameters (B, D). n = 9.


Fluorescence images of DCFH - DA staining for ROS levels in cells treated with DMSO , HA , NAC , and R406 on d12 ( E ) and the quantification of the ROS levels ( F ) . n = 4 . Scale bar , 100 μm . MDA quantification in cells treated with DMSO , HA , NAC , and R406 on d12 . n = 9 .

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

Fluorescence images of DCFH-DA staining for ROS levels in cells treated with DMSO, HA, NAC, and R406 on d12 (E) and the quantification of the ROS levels (F). n = 4. Scale bar, 100 μm. MDA quantification in cells treated with DMSO, HA, NAC, and R406 on d12. n = 9.


A qRT - PCR analysis of LINC00941 repression during keratinocyte differentiation in primary keratinocyte cultures from three different donors compared to undifferentiated keratinocytes ( D0 ) ( n = 6 biological replicates ) . Data is presented as mean ± SD .

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

A qRT-PCR analysis of LINC00941 repression during keratinocyte differentiation in primary keratinocyte cultures from three different donors compared to undifferentiated keratinocytes (D0) (n = 6 biological replicates). Data is presented as mean ± SD.


siPool mediated LINC00941 knockdown ( B ) in calcium - induced keratinocyte differentiation ( n = 3 biological replicates / knockdown group ) . Data is presented as mean ± SD . Statistical significance was tested by an unpaired t - test and corrected for multiple testing after Bonferroni ( * = adj . p - value < 0 . 05 , * * = adj . p - value < 0 . 01 , * * * = adj . p ‑ value < 0 . 001 , and n . s . = not significant ) .

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

siPool mediated LINC00941 knockdown (B) in calcium-induced keratinocyte differentiation (n = 3 biological replicates/knockdown group). Data is presented as mean ± SD. Statistical significance was tested by an unpaired t-test and corrected for multiple testing after Bonferroni (* = adj. p-value < 0.05, ** = adj. p-value < 0.01, *** = adj. p‑value < 0.001, and n.s. = not significant).


siPool mediated LINC00941 knockdown in calcium - induced keratinocyte differentiation , results in increased abundance of early and late differentiation marker transcripts on day 3 of differentiation ( C ) ( n = 3 biological replicates / knockdown group ) . Data is presented as mean ± SD . Statistical significance was tested by an unpaired t - test and corrected for multiple testing after Bonferroni ( * = adj . p - value < 0 . 05 , * * = adj . p - value < 0 . 01 , * * * = adj . p ‑ value < 0 . 001 , and n . s . = not significant ) .

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

siPool mediated LINC00941 knockdown in calcium-induced keratinocyte differentiation, results in increased abundance of early and late differentiation marker transcripts on day 3 of differentiation (C) (n = 3 biological replicates/knockdown group). Data is presented as mean ± SD. Statistical significance was tested by an unpaired t-test and corrected for multiple testing after Bonferroni (* = adj. p-value < 0.05, ** = adj. p-value < 0.01, *** = adj. p‑value < 0.001, and n.s. = not significant).


LINC00941 knockdown in day 3 ( D3 ) organotypic epidermal tissue cultures ( D ) ( n = 3 - 4 tissue cultures / knockdown group ) . Data is presented as mean ± SD . Statistical significance was tested by an unpaired t - test and corrected for multiple testing after Bonferroni ( * = adj . p - value < 0 . 05 , * * = adj . p - value < 0 . 01 , * * * = adj . p ‑ value < 0 . 001 , and n . s . = not significant ) .

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

LINC00941 knockdown in day 3 (D3) organotypic epidermal tissue cultures (D) (n = 3-4 tissue cultures/knockdown group). Data is presented as mean ± SD. Statistical significance was tested by an unpaired t-test and corrected for multiple testing after Bonferroni (* = adj. p-value < 0.05, ** = adj. p-value < 0.01, *** = adj. p‑value < 0.001, and n.s. = not significant).


LINC00941 knockdown in day 3 ( D3 ) organotypic epidermal tissue cultures results in increased expression of key differentiation genes ( E ) ( n = 3 - 4 tissue cultures / knockdown group ) . Data is presented as mean ± SD . Statistical significance was tested by an unpaired t - test and corrected for multiple testing after Bonferroni ( * = adj . p - value < 0 . 05 , * * = adj . p - value < 0 . 01 , * * * = adj . p ‑ value < 0 . 001 , and n . s . = not significant ) .

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

LINC00941 knockdown in day 3 (D3) organotypic epidermal tissue cultures results in increased expression of key differentiation genes (E) (n = 3-4 tissue cultures/knockdown group). Data is presented as mean ± SD. Statistical significance was tested by an unpaired t-test and corrected for multiple testing after Bonferroni (* = adj. p-value < 0.05, ** = adj. p-value < 0.01, *** = adj. p‑value < 0.001, and n.s. = not significant).


F Immunofluorescence analysis shows increased levels of early and late differentiation proteins . Collagen VII ( col VII , green ) stain indicates the basement membrane , nuclei are shown in blue and the differentiation proteins Keratin 10 and Loricrin are shown in red ( n = 3 - 4 tissue cultures / knockdown group ; one exemplary picture for each group is depicted ) . Scale bar : 50 µm .

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

F Immunofluorescence analysis shows increased levels of early and late differentiation proteins. Collagen VII (col VII, green) stain indicates the basement membrane, nuclei are shown in blue and the differentiation proteins Keratin 10 and Loricrin are shown in red (n = 3-4 tissue cultures/knockdown group; one exemplary picture for each group is depicted). Scale bar: 50 µm.


B LINC00941 knockdown efficiency ( siLINC00941 ) in epidermal tissue on day 3 of differentiation as obtained by qRT - PCR measurements ( n = 4 - 5 epidermal tissue cultures / knockdown group ) . Data is presented as mean ± SD . Statistical significance was tested by an unpaired t - test and corrected for multiple testing after Bonferroni ( * = adj . p - value < 0 . 05 , * * = adj . p - value < 0 . 01 , * * * = adj . p ‑ value < 0 . 001 , and n . s . = not significant ) .

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

B LINC00941 knockdown efficiency (siLINC00941) in epidermal tissue on day 3 of differentiation as obtained by qRT-PCR measurements (n = 4-5 epidermal tissue cultures/knockdown group). Data is presented as mean ± SD. Statistical significance was tested by an unpaired t-test and corrected for multiple testing after Bonferroni (* = adj. p-value < 0.05, ** = adj. p-value < 0.01, *** = adj. p‑value < 0.001, and n.s. = not significant).


C Heatmap of differentially expressed genes ( padj < 0 . 05 and - 1 > log2FC > 1 ) upon LINC00941 depletion on day 3 in organotypic epidermal tissue with marked keratinocyte differentiation genes ( n = 4 - 5 epidermal tissue cultures / knockdown group ) .

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

C Heatmap of differentially expressed genes (padj < 0.05 and -1 > log2FC > 1) upon LINC00941 depletion on day 3 in organotypic epidermal tissue with marked keratinocyte differentiation genes (n = 4-5 epidermal tissue cultures/knockdown group).


A Differentially expressed genes in day 3 LINC00941 depleted epidermal tissue cluster within the epidermal differentiation complex ( EDC ) ( n = 4 - 5 biological replicates / knockdown group ) .

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

A Differentially expressed genes in day 3 LINC00941 depleted epidermal tissue cluster within the epidermal differentiation complex (EDC) (n = 4-5 biological replicates/knockdown group).