( E , left and middle ) Brightfield images of the indicated myotubes treated or not with 3 - MA at DM - 8 . ( right ) Immunostaining for MHC ( red ) at DM - 14 in the presence of 3 - MA . Arrowheads label 3 - MA - rescued Rb − / − myotubes at DM - 8 . 3 - MA - rescued , twitching Rb − / − myotubes are shown in Video 4 .

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 B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR 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

(E, left and middle) Brightfield images of the indicated myotubes treated or not with 3-MA at DM-8. (right) Immunostaining for MHC (red) at DM-14 in the presence of 3-MA. Arrowheads label 3-MA-rescued Rb−/− myotubes at DM-8. 3-MA-rescued, twitching Rb−/− myotubes are shown in Video 4.


( F ) Immunostaining for BrdU ( green ) and MHC ( red ) of 3 - MA - treated cultures at DM - 2 or DM - 10 . Rb − / − myotubes incorporated BrdU at DM - 2 ( arrowheads ) but not DM - 10 .

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(F) Immunostaining for BrdU (green) and MHC (red) of 3-MA-treated cultures at DM-2 or DM-10. Rb−/− myotubes incorporated BrdU at DM-2 (arrowheads) but not DM-10.


( G ) Brightfield images ( left and middle ) and MHC staining ( right ) of the indicated cultures treated or not with bezafibrate . Arrowheads point to bezafibrate - rescued Rb − / − myotubes .

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 B-CONTROLLED_VAR O O O O O O O O

(G) Brightfield images (left and middle) and MHC staining (right) of the indicated cultures treated or not with bezafibrate. Arrowheads point to bezafibrate-rescued Rb−/− myotubes.


( A ) Immunostaining for MHC ( green ) in the indicated myoblasts induced to differentiate under normoxia or hypoxia treated or not with lonidamine . Note the shortened myotubes in the presence of lonidamine .

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 B-CONTROLLED_VAR O

(A) Immunostaining for MHC (green) in the indicated myoblasts induced to differentiate under normoxia or hypoxia treated or not with lonidamine. Note the shortened myotubes in the presence of lonidamine.


( B ) Representative immunoblot ( left ) and mean expression of myogenin and MCK normalized for MHC expression ( right ; n = 3 ) .

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(B) Representative immunoblot (left) and mean expression of myogenin and MCK normalized for MHC expression (right; n = 3).


( C ) Rbf / f myoblasts transduced with Ad . GFP or Ad . Cre and immunoblotted for pRb . Tubulin was used as a loading control .

O O O O O 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) Rbf/f myoblasts transduced with Ad.GFP or Ad.Cre and immunoblotted for pRb. Tubulin was used as a loading control.


( D ) Rbf / f myoblasts transduced with Ad . GFP or Ad . Cre and immunostained for pRb ( red ) . ( insets ) DAPI staining for nuclei ( blue ) .

O O O O O O O O O O O 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) Rbf/f myoblasts transduced with Ad.GFP or Ad.Cre and immunostained for pRb (red). (insets) DAPI staining for nuclei (blue).


( E ) Immunostaining for MHC in Rbf / f myoblasts transduced with Ad . Cre and induced to differentiate in normoxia ( top and middle ) or hypoxia ( bottom ) .

O O O O O O O O O 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) Immunostaining for MHC in Rbf/f myoblasts transduced with Ad.Cre and induced to differentiate in normoxia (top and middle) or hypoxia (bottom).


( F , top ) Brightfield images of Rbf / f myoblasts transduced with Ad . EV or Ad . Cre and induced to differentiate under hypoxia . ( bottom ) Larger view of the boxed field . Arrowheads point to rescued Rb − / − myotubes . This field is shown in Video 6 .

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(F, top) Brightfield images of Rbf/f myoblasts transduced with Ad.EV or Ad.Cre and induced to differentiate under hypoxia. (bottom) Larger view of the boxed field. Arrowheads point to rescued Rb−/− myotubes. This field is shown in Video 6.


( G ) Quantification of myotube formation in Rbf / f myoblasts transduced with Ad . EV or Ad . Cre and induced to differentiate under hypoxia . Counts are mean of six representative fields ( n = 3 ) . * , P = 0 . 004 .

O O O O O O O O O O O O O O O O O O O O O O O O O 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) Quantification of myotube formation in Rbf/f myoblasts transduced with Ad.EV or Ad.Cre and induced to differentiate under hypoxia. Counts are mean of six representative fields (n = 3). *, P = 0.004.


( H ) Quantification of myotube formation in the indicated myoblasts induced to differentiate under the indicated conditions . Myotube counts are the mean of six fields ( n = 4 ) .

(H) Quantification of myotube formation in the indicated myoblasts induced to differentiate under the indicated conditions. Myotube counts are the mean of six fields (n = 4).


( I ) MHC staining ( red ) of the indicated myoblasts transduced with Ad . NF - κB / HIF - 1α and induced to differentiate in normoxia .

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(I) MHC staining (red) of the indicated myoblasts transduced with Ad.NF-κB/HIF-1α and induced to differentiate in normoxia.


( J ) Quantification of myotube formation in the indicated myoblasts transduced with Ad . EV or Ad . NF - κB / HIF - 1α and induced to differentiate in normoxia . Myotube counts are the mean of six fields ( n = 3 ) . Error bars represent SD .

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

(J) Quantification of myotube formation in the indicated myoblasts transduced with Ad.EV or Ad.NF-κB/HIF-1α and induced to differentiate in normoxia. Myotube counts are the mean of six fields (n = 3). Error bars represent SD.


( B , C ) Levels of anti - HEL IgG in serum as measured by ELISA at the indicated days post immunization with HEL - OVA / CFA following adoptive transfer of BHEL cells without CD4OT - II cells ( no T cell control ) or with control CD4OT - II cells or NEF - expressing CD4OT - II cells . ( B ) NEF WT of HIV - 1SF2 ( C ) patient - derived NEF variants . Shown are relative levels of HEL IgG production with the mean of IgG levels in mice receiving control cells between days 12 - 14 post immunization arbitrarily set to 1 . Each dot represents one animal . Bars represent mean values with SD from 2 - 10 mice analyzed from 3 ( B ) and 2 ( C ) independent experiments . Statistical significance was assessed by Mann Whitney U test . * * * P ≤ 0 . 001 , * P ≤ 0 .

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(B, C) Levels of anti-HEL IgG in serum as measured by ELISA at the indicated days post immunization with HEL-OVA/CFA following adoptive transfer of BHEL cells without CD4OT-II cells (no T cell control) or with control CD4OT-II cells or NEF-expressing CD4OT-II cells. (B) NEF WT of HIV-1SF2 (C) patient-derived NEF variants. Shown are relative levels of HEL IgG production with the mean of IgG levels in mice receiving control cells between days 12 - 14 post immunization arbitrarily set to 1. Each dot represents one animal. Bars represent mean values with SD from 2-10 mice analyzed from 3 (B) and 2 (C) independent experiments. Statistical significance was assessed by Mann Whitney U test. ***P ≤0.001, *P ≤0.05


( E ) Representative confocal micrographs of p - tyrosine ( p - Tyr ) polarization at B - T cell contacts after 1 - 2h of co - culture . Cell conjugates were stained with anti - pTyr antibody . Scale bar , 5µm . The top panels display only the p - Tyr signal in grey , the bottom panel depicts p - Tyr ( red ) , BHEL cells ( blue ) and CD4OT - II cells ( green ) . ( F ) Quantification of p - Tyr accumulation at the B - T interface as shown in E . Shown are mean values with SD from three independent experiments with at least 50 conjugates analyzed per experiment .

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 O O O O 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 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 O O 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) Representative confocal micrographs of p-tyrosine (p-Tyr) polarization at B-T cell contacts after 1-2h of co-culture. Cell conjugates were stained with anti-pTyr antibody. Scale bar, 5µm. The top panels display only the p-Tyr signal in grey, the bottom panel depicts p-Tyr (red), BHEL cells (blue) and CD4OT-II cells (green). (F) Quantification of p-Tyr accumulation at the B-T interface as shown in E. Shown are mean values with SD from three independent experiments with at least 50 conjugates analyzed per experiment.


( G ) NEF prevents induction of F - actin remodeling in BHEL during IS . BHEL cells ( blue ) and CD4OT - II cells ( green ) were placed on PLL - coated slides and subjected to live - cell time - lapse imaging . Shown are representative still images from the indicated time points post contact formation ( see Movies EV1 and EV2 ) . Red signals indicate actin polymerization specifically in BHEL cells . Scale bars , 5µm . Filled and empty arrowheads indicate examples of accumulation of polarized actin or p - Tyr signal at B - T interface during IS formation or lack thereof , respectively . Asterisks mark pairs of interacting T and B cell . ( H ) Quantification of B cell actin polymerization as shown in G with the frequency of actin polymerization in B cells co - cultured with control T cells arbitrarily set to 100 % . Shown are mean values with SD from three independent experiments with at least 50 conjugates counted per experiment .

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

(G) NEF prevents induction of F-actin remodeling in BHEL during IS. BHEL cells (blue) and CD4OT-II cells (green) were placed on PLL-coated slides and subjected to live-cell time-lapse imaging. Shown are representative still images from the indicated time points post contact formation (see Movies EV1 and EV2). Red signals indicate actin polymerization specifically in BHEL cells. Scale bars, 5µm. Filled and empty arrowheads indicate examples of accumulation of polarized actin or p-Tyr signal at B-T interface during IS formation or lack thereof, respectively. Asterisks mark pairs of interacting T and B cell. (H) Quantification of B cell actin polymerization as shown in G with the frequency of actin polymerization in B cells co-cultured with control T cells arbitrarily set to 100%. Shown are mean values with SD from three independent experiments with at least 50 conjugates counted per experiment.


( L ) Relative B cell proliferation ( quantified by gating on the last 3 - 4 generations ) is plotted as mean values with SD from four independent experiments . Control T cells + Ag were arbitrarily set to 1 .

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(L) Relative B cell proliferation (quantified by gating on the last 3-4 generations) is plotted as mean values with SD from four independent experiments. Control T cells +Ag were arbitrarily set to 1.


( B ) Production of anti - HEL IgG at the indicated day post immunization with HEL - OVA / CFA following adoptive transfer of BHEL and CD4OT - II cells expressing NEF or various NEF mutants . Shown are relative levels of HEL IgG production with the median of IgG levels in mice receiving control cells on day 14 post immunization arbitrarily set to 1 . Each dot represents one animal . Shown are mean values with SD from 5 - 12 mice from 4 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

(B) Production of anti-HEL IgG at the indicated day post immunization with HEL-OVA/CFA following adoptive transfer of BHEL and CD4OT-II cells expressing NEF or various NEF mutants. Shown are relative levels of HEL IgG production with the median of IgG levels in mice receiving control cells on day 14 post immunization arbitrarily set to 1. Each dot represents one animal. Shown are mean values with SD from 5-12 mice from 4 experiments.


( D ) Quantification of B cell actin polymerization as shown in C with frequency of actin polymerization in B cells co - cultured with control T cells arbitrarily set to 100 % . Shown are mean values with SD from three independent experiments with at least 30 conjugates evaluated per condition .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Quantification of B cell actin polymerization as shown in C with frequency of actin polymerization in B cells co-cultured with control T cells arbitrarily set to 100%. Shown are mean values with SD from three independent experiments with at least 30 conjugates evaluated per condition.


( H ) Relative surface expression of CD4 , CD28 , OX40 , ICOS , CD45 and CD54 on T cells expressing NEF WT or the indicated NEF mutants . Analysis of surface expression was performed using flow cytometry 3 - 7 d post transduction of T cells . Depicted are mean values with SD from 3 - 6 independent experiments , with control T cells arbitrarily set to 100 % as indicated by the dashed line .

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(H) Relative surface expression of CD4, CD28, OX40, ICOS, CD45 and CD54 on T cells expressing NEF WT or the indicated NEF mutants. Analysis of surface expression was performed using flow cytometry 3-7 d post transduction of T cells. Depicted are mean values with SD from 3-6 independent experiments, with control T cells arbitrarily set to 100% as indicated by the dashed line.


( I ) Representative confocal images of the subcellular distribution of IL - 4 in CD4OT - II cells expressing the indicated proteins engaged in an IS with BHEL cells . B cells and T cells were placed on PLL - coated cover glasses for 60 - 90min , fixed and stained for intracellular IL - 4 . Filled and empty arrowheads indicate presence or absence of IL - 4 accumulation at the IS , respectively . Scale bars , 5µm . ( J ) Quantification of IL - 4 accumulation at the IS as depicted in I . Shown are mean values with SD from 3 independent experiments relative to control cells set to 100 % .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative confocal images of the subcellular distribution of IL-4 in CD4OT-II cells expressing the indicated proteins engaged in an IS with BHEL cells. B cells and T cells were placed on PLL-coated cover glasses for 60-90min, fixed and stained for intracellular IL-4. Filled and empty arrowheads indicate presence or absence of IL-4 accumulation at the IS, respectively. Scale bars, 5µm. (J) Quantification of IL-4 accumulation at the IS as depicted in I. Shown are mean values with SD from 3 independent experiments relative to control cells set to 100%.


( A ) Representative cross - sections of draining LNs ( dLNs ) from mice 7d post immunization with HEL - OVA / CFA following adoptive transfer of BHEL and transduced CD4OT - II cells . dLN were cryosectioned and stained with fluorophore - conjugated antibodies against GL - 7 to reveal GCs ( green ) , CD21 / 35 to stain for follicular dendritic cells ( FDC , red ) and Hoechst to stain for cell nuclei ( blue ) . Scale bar , 250µm . ( B , C ) Quantification of the number of GCs relative to B cell follicles ( B ) and size of GC ( C ) on confocal micrographs as shown in A . Shown are mean values with SD from dLNs from three mice in two independent experiment . Each dot represents one LN ( 3 LNs / mouse ) in B and one GC 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative cross-sections of draining LNs (dLNs) from mice 7d post immunization with HEL-OVA/CFA following adoptive transfer of BHEL and transduced CD4OT-II cells. dLN were cryosectioned and stained with fluorophore-conjugated antibodies against GL-7 to reveal GCs (green), CD21/35 to stain for follicular dendritic cells (FDC, red) and Hoechst to stain for cell nuclei (blue). Scale bar, 250µm. (B, C) Quantification of the number of GCs relative to B cell follicles (B) and size of GC (C) on confocal micrographs as shown in A. Shown are mean values with SD from dLNs from three mice in two independent experiment. Each dot represents one LN (3 LNs/mouse) in B and one GC in C.


( A ) Still images of the Movies EV3 ( Control , upper panel ) and EV4 ( NEF , lower panel ) , obtained by intravital time lapse 2PM imaging of draining popliteal LNs 24hrs post immunization with HEL - OVA / CFA , following adoptive transfer of GFP BHEL cells ( green ) and mTomato CD4OT - II cells ( red ) expressing empty vector ( Control ; upper panel ) or NEF WT ( lower panel ) , respectively . High endothelial venules ( HEVs ) ( white ) were labeled by intravenous injection of Alexa660 - labeled Meca - 79 antibody and collagen ( blue ) is visualized by second harmonic generation ( SHG ) . Yellow and orange arrows indicate stable and transient ISs , respectively . Numbering of ISs corresponds to that in the respective Supplemental movies . Scale bar 20µm . ( B ) Quantification of the duration of interactions as indicated by the arrows in A . Touching T and B cells were manually quantification using virtual reality ( InViewR , Arivis ) . Each dot represents one T - B interaction pair . Red bar shows median . Data is derived from 5 to 6 movies , acquired from 4 mice in 2 independent experiments . Percentages on top indicate frequency of interactions that lasted 20min or longer .

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

(A) Still images of the Movies EV3 (Control, upper panel) and EV4 (NEF, lower panel), obtained by intravital time lapse 2PM imaging of draining popliteal LNs 24hrs post immunization with HEL-OVA/CFA, following adoptive transfer of GFP BHEL cells (green) and mTomato CD4OT-II cells (red) expressing empty vector (Control; upper panel) or NEF WT (lower panel), respectively. High endothelial venules (HEVs) (white) were labeled by intravenous injection of Alexa660-labeled Meca-79 antibody and collagen (blue) is visualized by second harmonic generation (SHG). Yellow and orange arrows indicate stable and transient ISs, respectively. Numbering of ISs corresponds to that in the respective Supplemental movies. Scale bar 20µm. (B) Quantification of the duration of interactions as indicated by the arrows in A. Touching T and B cells were manually quantification using virtual reality (InViewR, Arivis). Each dot represents one T-B interaction pair. Red bar shows median. Data is derived from 5 to 6 movies, acquired from 4 mice in 2 independent experiments. Percentages on top indicate frequency of interactions that lasted 20min or longer.


( C ) Still images of the Movies EV5 ( Control , upper panel ) and EV6 ( NEF , lower panel ) , obtained by intravital time lapse 2PM imaging of draining popliteal LNs 8d post immunization with HEL - OVA / CFA , following adoptive transfer of GFP BHEL cells ( green ) and mTomato CD4OT - II cells ( red ) expressing empty vector ( Control ; upper panel ) or NEF WT ( lower panel ) . Follicular dendritic cells ( FDCs ) ( white ) were labeled by subcutaneous injection of Alexa647 - labeled anti - CD21 / CD35 antibody and collagen ( blue ) is visualized by SHG . Yellow and orange arrows indicate stable and transient ISs , respectively . Numbering of ISs corresponds to that in the respective Supplemental movies . Scale bar 20µm . ( D ) Quantification of the duration of interactions as indicated by the arrows in C . Touching T and B cells were manually quantification using virtual reality ( InViewR , Arivis ) . Each dot represents one T - B interaction pair . Red bar shows median . Shown is one representative dataset out of two , derived from 5 to 8 movies , acquired from 4 mice . Percentages on top indicate frequency of interactions that lasted 20min or longer .

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

(C) Still images of the Movies EV5 (Control, upper panel) and EV6 (NEF, lower panel), obtained by intravital time lapse 2PM imaging of draining popliteal LNs 8d post immunization with HEL-OVA/CFA, following adoptive transfer of GFP BHEL cells (green) and mTomato CD4OT-II cells (red) expressing empty vector (Control; upper panel) or NEF WT (lower panel). Follicular dendritic cells (FDCs) (white) were labeled by subcutaneous injection of Alexa647-labeled anti-CD21/CD35 antibody and collagen (blue) is visualized by SHG. Yellow and orange arrows indicate stable and transient ISs, respectively. Numbering of ISs corresponds to that in the respective Supplemental movies. Scale bar 20µm. (D) Quantification of the duration of interactions as indicated by the arrows in C. Touching T and B cells were manually quantification using virtual reality (InViewR, Arivis). Each dot represents one T-B interaction pair. Red bar shows median. Shown is one representative dataset out of two, derived from 5 to 8 movies, acquired from 4 mice. Percentages on top indicate frequency of interactions that lasted 20min or longer.


( B ) Virus replication as assessed by quantification of RT activity in the cell culture supernatant . Shown are mean values with SD from 6 donors .

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(B) Virus replication as assessed by quantification of RT activity in the cell culture supernatant. Shown are mean values with SD from 6 donors.


( C ) Production of IgG antibodies against TT from infected blocks at indicated time point . Each dot represents one donor , bar indicates mean value . Tonsillar blocks were infected with indicated virus for 24h . After washing off the virus , blocks were stimulated with TT antigen for 24h . Samples were collected at different time intervals to estimate viral replication and antibody production .

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(C) Production of IgG antibodies against TT from infected blocks at indicated time point. Each dot represents one donor, bar indicates mean value. Tonsillar blocks were infected with indicated virus for 24h. After washing off the virus, blocks were stimulated with TT antigen for 24h. Samples were collected at different time intervals to estimate viral replication and antibody production.


Comparison of structural preservation in cryosections picked up with sucrose ( a ) and MU ( b ) . In isolated hepatocytes , autophagy was induced for 30 min in suspension buffer before fixation . ( a ) Cryosection picked up with 2 . 3 M sucrose and labeled for SOD . Note the presence of large , " empty " gaps between sequestered contents of the AV and the surrounding cytoplasm . ( b ) A similar section as a , but picked up with an MU mixture . The AV are no longer distorted and the overall membranous structures are sharply delineated . The SOD - labeling density of AVi is as that of the cytosol , whereas AVd show much more intense labeling . ( Inset ) An AVi / d that is distinct from AVi by its single limiting membrane . The highly pleiomorphic AVd are also bound by a distinctive single membrane , but in contrast to AVi / d , their contents are characterized by debris of digested material . Bars , 250 nm .

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Comparison of structural preservation in cryosections picked up with sucrose (a) and MU (b). In isolated hepatocytes, autophagy was induced for 30 min in suspension buffer before fixation. (a) Cryosection picked up with 2.3 M sucrose and labeled for SOD. Note the presence of large, "empty" gaps between sequestered contents of the AV and the surrounding cytoplasm. (b) A similar section as a, but picked up with an MU mixture. The AV are no longer distorted and the overall membranous structures are sharply delineated. The SOD-labeling density of AVi is as that of the cytosol, whereas AVd show much more intense labeling. (Inset) An AVi/d that is distinct from AVi by its single limiting membrane. The highly pleiomorphic AVd are also bound by a distinctive single membrane, but in contrast to AVi/d, their contents are characterized by debris of digested material. Bars, 250 nm.


Labeling patterns of cathepsin D and CAIII in MU picked - up cryosections . ( a ) Cells treated as in Fig . 1 b , but the section was immunolabeled for cathepsin D , which is not detectable in the AVi , moderately present in the AVi / d , and abundant in AVd . Cells in b and c had endocytosed 5 - nm gold tracer for 20 and 40 min , respectively , before fixation and CAIII labeling . ( b ) Complex fusion profile of a CAIII - positive AVi , an AVd which is without CAIII labeling , and a VE that contains an endocytosed gold probe ( arrow ) . Close to the AVd is another tracer - bearing VE . ( c ) Fusion profile of a tracer - bearing MVE and an AVi / d that exhibits a single limiting membrane and a CAIII - labeling concentration similar to that of the cytoplasm . Bars , 200 nm .

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Labeling patterns of cathepsin D and CAIII in MU picked-up cryosections. (a) Cells treated as in Fig. 1 b, but the section was immunolabeled for cathepsin D, which is not detectable in the AVi, moderately present in the AVi/d, and abundant in AVd. Cells in b and c had endocytosed 5-nm gold tracer for 20 and 40 min, respectively, before fixation and CAIII labeling. (b) Complex fusion profile of a CAIII-positive AVi, an AVd which is without CAIII labeling, and a VE that contains an endocytosed gold probe (arrow). Close to the AVd is another tracer-bearing VE. (c) Fusion profile of a tracer-bearing MVE and an AVi/d that exhibits a single limiting membrane and a CAIII-labeling concentration similar to that of the cytoplasm. Bars, 200 nm.


Appearance of tracer in AV . Cells were allowed to endocytose tracer particles continuously for the designated periods of time . At each time , random sections were searched for AV + , and AV + / 500 cell profiles were plotted . The number of cell profiles examined ( denominator ) and the respective number of AV + scored ( numerator ) are listed above the graph .

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Appearance of tracer in AV. Cells were allowed to endocytose tracer particles continuously for the designated periods of time. At each time, random sections were searched for AV+, and AV+/500 cell profiles were plotted. The number of cell profiles examined (denominator) and the respective number of AV+ scored (numerator) are listed above the graph.


Tracer distribution in AV + types . Cells were allowed to endocytose tracer particles continuously for the designated periods of time . At each time , random sections were searched for AV + . The results are categorized according to AV types and plotted for the relative distribution of tracers . The total tracer counts are given on top of each bar . The number in each block represents the actual number of AV profiles , from which tracer counts are scored .

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Tracer distribution in AV+ types. Cells were allowed to endocytose tracer particles continuously for the designated periods of time. At each time, random sections were searched for AV+. The results are categorized according to AV types and plotted for the relative distribution of tracers. The total tracer counts are given on top of each bar. The number in each block represents the actual number of AV profiles, from which tracer counts are scored.


Fusion profiles . Cells exposed to 5 - nm gold tracers for 20 ( a and c ) and 25 min ( b ) before fixation . Sections were picked up by MU and air - dried ( c ) or underwent immunolabeling with anti - SOD antibodies ( a and b ) . ( a ) Fusion between a small VE ( arrow ) and an AVi . A cluster of two gold particles ( arrowhead ) is also seen at another location . ( b ) Fusion between an electron lucent MVE ( arrow ) and an AVd . ( c ) Fusion between an MVE ( arrow ) and an AVd . Bars , 200 nm .

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Fusion profiles. Cells exposed to 5-nm gold tracers for 20 (a and c) and 25 min (b) before fixation. Sections were picked up by MU and air-dried (c) or underwent immunolabeling with anti-SOD antibodies (a and b). (a) Fusion between a small VE (arrow) and an AVi. A cluster of two gold particles (arrowhead) is also seen at another location. (b) Fusion between an electron lucent MVE (arrow) and an AVd. (c) Fusion between an MVE (arrow) and an AVd. Bars, 200 nm.


Endosome types involved in FP + . The FP + were classified according to MVE and VE involved . The actual numbers of FP + analyzed are given above the bars .

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Endosome types involved in FP+. The FP+ were classified according to MVE and VE involved. The actual numbers of FP+ analyzed are given above the bars.


ASGP - R labeling in FP + and endosomes . Cells exposed for 40 min to 5 - nm gold tracer . Section immunolabeled for ASGP - R with 10 - nm gold . ( a ) Low ASGP - R labeling in tracer - carrying endocytic vesicles ( arrowheads ) , adjacent to ( top ) or fusing with ( bottom ) an AVi / d . ( b ) ASGP - R labeling at the plasma membrane and in endosomal elements near the cell surface ( ex , extra cellular space ) . Vesicles of the MVE ( asterisk ) and VE ( arrowhead ) type , containing endocytosed gold , are present in the same area . ( c ) As b . A VE - like vesicle with endocytosed gold and low ASGP - R labeling ( arrowhead ) is shown connected to an ASGP - R rich element ( arrow ) , possibly a recycling endosome . Bars , 200 nm .

ASGP-R labeling in FP+ and endosomes. Cells exposed for 40 min to 5-nm gold tracer. Section immunolabeled for ASGP-R with 10-nm gold. (a) Low ASGP-R labeling in tracer-carrying endocytic vesicles (arrowheads), adjacent to (top) or fusing with (bottom) an AVi/d. (b) ASGP-R labeling at the plasma membrane and in endosomal elements near the cell surface (ex, extra cellular space). Vesicles of the MVE (asterisk) and VE (arrowhead) type, containing endocytosed gold, are present in the same area. (c) As b. A VE-like vesicle with endocytosed gold and low ASGP-R labeling (arrowhead) is shown connected to an ASGP-R rich element (arrow), possibly a recycling endosome. Bars, 200 nm.


AV types involved in FP + . The same FP + data as used in Fig . 6 were classified according to the AV types .

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AV types involved in FP+. The same FP+ data as used in Fig. 6 were classified according to the AV types.


( b ) PC12 cells transfected with either 60Q or 60Q - HSC70bm . The presence of two HSC70bm motifs in 60Q - HSC70bm reduced the number of inclusions as compared to 60Q ( scale bar , 20 μm ) . ( c ) Quantification of the PC12 cells with inclusions 24 h after transfection with either 60Q or 60Q - HSC70bm . 10 mM 3 - methyladenine ( 3 - MA ) , 10 μM pepstatin A and 10 μM leupeptin were added to inhibit macroautophagy , the proteases , cathepsins D and E , and cathepsin A , respectively . Bars in c represent relative mean values ± s . e . m . from four independent experiments , with levels of aggregation observed for 60Q normalized to a value of 1 . Quantifications were performed by ArrayScan .

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(b) PC12 cells transfected with either 60Q or 60Q-HSC70bm. The presence of two HSC70bm motifs in 60Q-HSC70bm reduced the number of inclusions as compared to 60Q (scale bar, 20 μm). (c) Quantification of the PC12 cells with inclusions 24 h after transfection with either 60Q or 60Q-HSC70bm. 10 mM 3-methyladenine (3-MA), 10 μM pepstatin A and 10 μM leupeptin were added to inhibit macroautophagy, the proteases, cathepsins D and E, and cathepsin A, respectively. Bars in c represent relative mean values ± s.e.m. from four independent experiments, with levels of aggregation observed for 60Q normalized to a value of 1. Quantifications were performed by ArrayScan.


( b ) Confocal images of the 150Q Neuro2a cells transfected with R , RH , RS , RHS , RQ and RHQ . Molecules containing QBP1 co - localized with the tNHTT - 150Q - EGFP inclusions ( bar , 5 μm ) . Cells with inclusions were chosen to illustrate the co - localization of QBP1 - containing molecules with tNHTT - 150Q - EGFP .

(b) Confocal images of the 150Q Neuro2a cells transfected with R, RH, RS, RHS, RQ and RHQ. Molecules containing QBP1 co-localized with the tNHTT-150Q-EGFP inclusions (bar, 5 μm). Cells with inclusions were chosen to illustrate the co-localization of QBP1-containing molecules with tNHTT-150Q-EGFP.


( c ) The expression of RQ and RHQ in 150Q Neuro2a cells decreased the accumulation of insoluble tNHTT - 150Q - EGFP on the gel top . Note the shift of tNHTT - 150Q - EGFP from an insoluble to a soluble form when RQ was co - expressed . The levels of tNHTT - 16Q - EGFP were not affected . Full - length western blots are presented in Supplementary Figure 13 .

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(c) The expression of RQ and RHQ in 150Q Neuro2a cells decreased the accumulation of insoluble tNHTT-150Q-EGFP on the gel top. Note the shift of tNHTT-150Q-EGFP from an insoluble to a soluble form when RQ was co-expressed. The levels of tNHTT-16Q-EGFP were not affected. Full-length western blots are presented in Supplementary Figure 13.


( d ) RHQ reduced the levels of aggregated and soluble forms of tNHTT - 150Q - EGFP by 81 . 8 % and 33 . 6 % , respectively . WB , western blot . ( e ) Quantification of tNHTT - 16Q - EGFP protein levels .

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(d) RHQ reduced the levels of aggregated and soluble forms of tNHTT-150Q-EGFP by 81.8% and 33.6%, respectively. WB, western blot. (e) Quantification of tNHTT-16Q-EGFP protein levels.


( f ) ArrayScan analysis revealed 28 % and 56 % reductions in inclusion formation in 150Q Neuro2a cells by RQ and RHQ , respectively , after 24 h of differentiation and induction . ( g ) In 150Q - nls Neuro2a cells , expression of RQ and RHQ reduced the number of tNHTT - 150Q - nls - EGFP inclusion by 23 % ( RQ ) and 53 % ( RHQ ) after 48 h of differentiation and induction compared to RS and RHS , respectively . Bars in d and e represent mean values ± s . e . m . from four independent experiments . Quantifications were performed by ArrayScan .

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(f) ArrayScan analysis revealed 28% and 56% reductions in inclusion formation in 150Q Neuro2a cells by RQ and RHQ, respectively, after 24 h of differentiation and induction. (g) In 150Q-nls Neuro2a cells, expression of RQ and RHQ reduced the number of tNHTT-150Q-nls-EGFP inclusion by 23% (RQ) and 53% (RHQ) after 48 h of differentiation and induction compared to RS and RHS, respectively. Bars in d and e represent mean values ± s.e.m. from four independent experiments. Quantifications were performed by ArrayScan.


( a ) Western blot analysis of the chase experiment in 60Q Neuro2a cells . Full - length western blots are presented in Supplementary Figure 13 . ( b ) RHQ enhanced the tNHTT - 60Q - EGFP degradation during the chase period . 5 μM MG132 , 10 mM 3 - methyladenine and 15 mM ammonium chloride ( NH4Cl ) were used to inhibit proteasome , macroautophagy and lysosomal degradation , respectively . NH4Cl had no effect on proteasome activity and did not affect cell viability ( Supplementary Fig . 4a , b ) . Bars in b represent relative mean values ± s . e . m . from three independent experiments . Protein levels at time 0 represent the control condition ( arbitrarily set as 1 ) .

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(a) Western blot analysis of the chase experiment in 60Q Neuro2a cells. Full-length western blots are presented in Supplementary Figure 13. (b) RHQ enhanced the tNHTT-60Q-EGFP degradation during the chase period. 5 μM MG132, 10 mM 3-methyladenine and 15 mM ammonium chloride (NH4Cl) were used to inhibit proteasome, macroautophagy and lysosomal degradation, respectively. NH4Cl had no effect on proteasome activity and did not affect cell viability (Supplementary Fig. 4a,b). Bars in b represent relative mean values ± s.e.m. from three independent experiments. Protein levels at time 0 represent the control condition (arbitrarily set as 1).


( a , b ) Constructs identical to those described in Figure 2a , but lacking mRFP ( representations of S , HS , Q , HQ are shown in Supplementary Fig . 3a ) were co - transfected with tNHTT - 16Q - EGFP ( a ) and tNHTT - 150Q - EGFP ( b ) constructs into Neuro2a cells ( bar , 4 μm ) .

(a,b) Constructs identical to those described in Figure 2a, but lacking mRFP (representations of S, HS, Q, HQ are shown in Supplementary Fig. 3a) were co-transfected with tNHTT-16Q-EGFP (a) and tNHTT-150Q-EGFP (b) constructs into Neuro2a cells (bar, 4 μm).


( c ) Dot blot analysis of the lumenal fraction of purified lysosomes from Neuro2a cells co - transfected with tNHTT - 60Q - EGFP and R , RS , RHS , RQ or RHQ . PNS , post - nuclear supernatant .

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(c) Dot blot analysis of the lumenal fraction of purified lysosomes from Neuro2a cells co-transfected with tNHTT-60Q-EGFP and R, RS, RHS, RQ or RHQ. PNS, post-nuclear supernatant.


( d ) Quantification of HTT translocation into lysosomes . Levels of HTT detected by EM48 ( anti - huntingtin antibody ) were normalized to levels of β - tubulin ( in PNS ) and levels of cathepsin D ( in lysosomes ) .

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(d) Quantification of HTT translocation into lysosomes. Levels of HTT detected by EM48 (anti-huntingtin antibody) were normalized to levels of β-tubulin (in PNS) and levels of cathepsin D (in lysosomes).


( e ) Compound images generated by ArrayScan illustrating inclusion formation in 150Q Neuro2a cells transfected with R , RS , RHS , RQ and RHQ and shRNA specific for HSC70 and / or Lamp2a . ( blue , Hoechst 33258 ; green , tNHTT - 150Q - EGFP ; red , tested molecule ) .

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(e) Compound images generated by ArrayScan illustrating inclusion formation in 150Q Neuro2a cells transfected with R, RS, RHS, RQ and RHQ and shRNA specific for HSC70 and/or Lamp2a. (blue, Hoechst 33258; green, tNHTT-150Q-EGFP; red, tested molecule).


( f ) RNAi of HSC70 , Lamp2a or both alleviated the inhibitory effect of RHQ on the formation of inclusions containing tNHTT - 150Q - EGFP inclusions .

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(f) RNAi of HSC70, Lamp2a or both alleviated the inhibitory effect of RHQ on the formation of inclusions containing tNHTT-150Q-EGFP inclusions.


( g ) Western blot analysis of soluble polyQ protein in 150Q Neuro2a cells transfected with tested constructs and shRNA for HSC70 and / or Lamp2a . Full - length western blots are presented in Supplementary Figure 13 .

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(g) Western blot analysis of soluble polyQ protein in 150Q Neuro2a cells transfected with tested constructs and shRNA for HSC70 and/or Lamp2a. Full-length western blots are presented in Supplementary Figure 13.


( h ) Silencing of HSC70 , Lamp2a or both alleviated the inhibitory effect of RHQ on levels of soluble tNHTT - 150Q - EGFP . Bars in d and f represent mean values ± s . e . m . from three independent experiments . Bars in h represent relative mean values ± s . e . m . from three independent experiments . For h , values for the R construct were arbitrarily set to 1 .

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(h) Silencing of HSC70, Lamp2a or both alleviated the inhibitory effect of RHQ on levels of soluble tNHTT-150Q-EGFP. Bars in d and f represent mean values ± s.e.m. from three independent experiments. Bars in h represent relative mean values ± s.e.m. from three independent experiments. For h, values for the R construct were arbitrarily set to 1.


( a ) Filter trap assay analysis of HTT - polyQaggregation in nine R6 / 2 mouse brains injected with three different combinations of rAAV . ( b ) Quantification of the SDS - insoluble material detected by filter trap assay ( * , P = 0 . 037 ; * * , P = 0 . 0083 ; * * * , P = 0 . 0015 ) .

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(a) Filter trap assay analysis of HTT-polyQaggregation in nine R6/2 mouse brains injected with three different combinations of rAAV. (b) Quantification of the SDS-insoluble material detected by filter trap assay (*, P = 0.037; **, P = 0.0083; ***, P = 0.0015).


( c ) Western blot analysis of HTT aggregation ( gel top , EM48 antibody ) , soluble HTT ( 1C2 antibody ) , and higher molecular polyQ complexes by agarose gel electrophoresis for resolving aggregates ( AGERA ) . Full - length western blots are presented in Supplementary Figure 13 . ( d ) Quantification of the gel top - detected aggregation ( * , P = 0 . 093 ; * * , P = 0 . 0027 ; * * * , P = 0 . 0045 ) . ( e ) Quantification of soluble HTT - polyQ ( * , P = 0 . 034 ; * * , P = 0 . 00003 ; * * * , P = 0 . 0001 ) .

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(c) Western blot analysis of HTT aggregation (gel top, EM48 antibody), soluble HTT (1C2 antibody), and higher molecular polyQ complexes by agarose gel electrophoresis for resolving aggregates (AGERA). Full-length western blots are presented in Supplementary Figure 13. (d) Quantification of the gel top-detected aggregation (*, P = 0.093; **, P = 0.0027; ***, P = 0.0045). (e) Quantification of soluble HTT-polyQ (*, P = 0.034; **, P = 0.00003; ***, P = 0.0001).


( f ) Brain sections stained with anti - RFP , EM48 and anti - ubiquitin antibodies ( bar , 40 μm ) . ( g ) Numbers of ubiquitin - positive inclusions in the transduced areas ( * , P = 0 . 0026 ; * * , P = 5 . 535 × 10 − 8 ; * * * , P = 2 . 009 × 10 − 8 ) . Bars in b , d , e and g represent the relative mean values ± s . e . m . from three brains injected with different rAAV in each striatum . R in R / Q and R / HQ , and Q in Q / HQ brains represent the control value of 1 .

(f) Brain sections stained with anti-RFP, EM48 and anti-ubiquitin antibodies (bar, 40 μm). (g) Numbers of ubiquitin-positive inclusions in the transduced areas (*, P = 0.0026; **, P = 5.535 × 10−8; ***, P = 2.009 × 10−8). Bars in b, d, e and g represent the relative mean values ± s.e.m. from three brains injected with different rAAV in each striatum. R in R/Q and R/HQ, and Q in Q/HQ brains represent the control value of 1.


( a ) Relative body weight changes in mice after bilateral injection with rAAV - R , rAAV - Q and rAAV - HQ at 6 - 14 weeks of age ( n = 11 for all three groups until 12 weeks ; at 14 weeks , n = 7 for R / R , n = 10 for Q / Q , n = 11 for HQ / HQ ) ( * , P = 0 . 0401 ; * * , P = 0 . 0081 ; * * * , P = 0 . 0182 ; * * * * , P = 0 . 0225 ) .

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(a) Relative body weight changes in mice after bilateral injection with rAAV-R, rAAV-Q and rAAV-HQ at 6-14 weeks of age (n = 11 for all three groups until 12 weeks; at 14 weeks, n = 7 for R/R, n = 10 for Q/Q, n = 11 for HQ/HQ) (*, P = 0.0401; **, P = 0.0081; ***, P = 0.0182; ****, P = 0.0225).


( b ) Clasping scores . n = 11 for all three groups ( scale : 0 , no clasping ; 1 , clasping of the forelimbs only ; 2 , clasping of both fore and hind limbs once or twice ; 3 , clasping of both fore and hind limbs more than 3 times or more than 5 s ) .

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(b) Clasping scores. n = 11 for all three groups (scale: 0, no clasping; 1, clasping of the forelimbs only; 2, clasping of both fore and hind limbs once or twice; 3, clasping of both fore and hind limbs more than 3 times or more than 5 s).


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(c) Rotarod performance; n = 11 for all three groups (*, P = 0.0055; **, P = 0.002; ***, P = 0.0455; ****, P = 0.0242).


( d ) Survival curves for mice after bilateral injection with rAAV - R , rAAV - Q , and rAAV - HQ ; n = 11 for all groups . Median survival ( days ) : R / R - 108 ; Q / Q - 123 ; HQ / HQ - 140 . Mean survival ( days ) : R / R - 105 . 5 ± 3 . 9 ; Q / Q - 122 . 3 ± 4 . 8 ; HQ / HQ - 136 . 2 ± 4 . 4 . Log - rank test , P 0 . 0001 ; Wilcoxon test , P 0 . 0001 .

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(d) Survival curves for mice after bilateral injection with rAAV-R, rAAV-Q, and rAAV-HQ; n = 11 for all groups. Median survival (days): R/R-108; Q/Q-123; HQ/HQ-140. Mean survival (days): R/R-105.5 ± 3.9; Q/Q-122.3 ± 4.8; HQ/HQ-136.2 ± 4.4.Log-rank test, P 0.0001; Wilcoxon test, P 0.0001.


F Minimal inhibitory concentrations of DHA treated wild - type , heterozygous ( YME1 / yme1 ) and homozygous ( yme1 / yme1 ) mutant strains . Plates were imaged after 72h .

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F Minimal inhibitory concentrations of DHA treated wild-type, heterozygous (YME1/yme1) and homozygous (yme1/yme1) mutant strains. Plates were imaged after 72h.


D Competitive growth assay of DHA resistant single cell clones . Wild - type ( mCherry + _ Cre _ Puro ) and knockout ( GFP + _ Puro ) sister clones were derived from mutant ( retrovirus - intron RE , darker shading , Tol2 transposon - intron T2 , lighter shading ) resistant colonies , treated with DHA , analyzed with flow cytometry and Sanger sequenced for insertion site mapping .

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D Competitive growth assay of DHA resistant single cell clones. Wild-type (mCherry+_Cre_Puro) and knockout (GFP+_Puro) sister clones were derived from mutant (retrovirus - intron RE, darker shading, Tol2 transposon - intron T2, lighter shading) resistant colonies, treated with DHA, analyzed with flow cytometry and Sanger sequenced for insertion site mapping.


A , B Cell survival of dihydroartemisinin treated ESCs in combination with ( A ) the Ppox inhibitor acifluorfen or upon ( B ) 5 - ALA administration . Alamar Blue staining was used to assess viability after 72h of treatment .

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A, B Cell survival of dihydroartemisinin treated ESCs in combination with (A) the Ppox inhibitor acifluorfen or upon (B) 5-ALA administration. Alamar Blue staining was used to assess viability after 72h of treatment.


C , D Cell survival of DHA treated primary human glioblastoma cells from cancer patients and in combination with ( C ) the Ppox inhibitor acifluorfen ( BTL90 cells ) or upon ( D ) 5 - ALA administration ( VBT12 cells ) . Viability was assessed using CellTiter - Glo after 72h . Experiments were performed in triplicate .

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C, D Cell survival of DHA treated primary human glioblastoma cells from cancer patients and in combination with (C) the Ppox inhibitor acifluorfen (BTL90 cells) or upon (D) 5-ALA administration (VBT12 cells). Viability was assessed using CellTiter-Glo after 72h. Experiments were performed in triplicate.


E , F ( E ) ROS levels ( DHE staining , PE 582 / 15 nm - MFI ) and ( F ) cell survival of DHA ( 0 . 5μM ) , 5 - ALA ( 0 . 25mM ) or Ppox inhibitor ( 10μM ) treated human neuroblastoma cells ( SHSY5Y ) . Fluorescence of DHE ( ROS levels ) and cell numbers ( survival ) were assessed by flow cytometry and automated cell counting . The experiments were performed two times , in triplicate each .

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E, F (E) ROS levels (DHE staining, PE 582/15 nm - MFI) and (F) cell survival of DHA (0.5μM), 5-ALA (0.25mM) or Ppox inhibitor (10μM) treated human neuroblastoma cells (SHSY5Y). Fluorescence of DHE (ROS levels) and cell numbers (survival) were assessed by flow cytometry and automated cell counting. The experiments were performed two times, in triplicate each.


C , D ( C ) Fluorescence and ( D ) brightfield images of DMSO ( control ) , 5 - ALA ( 0 . 0625mM ) , DHA ( 1μM ) , or 5 - ALA and DHA ( 0 . 0625mM and 1μM ) treated tumor organoids ( CNS - PNET - like , c - MYC overexpression ) . Organoids were imaged on day 1 , day 5 and day 7 . One experiment is shown , representative of four independent experiments .

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C, D (C) Fluorescence and (D) brightfield images of DMSO (control), 5-ALA (0.0625mM), DHA (1μM), or 5-ALA and DHA (0.0625mM and 1μM) treated tumor organoids (CNS-PNET-like, c-MYC overexpression). Organoids were imaged on day 1, day 5 and day 7. One experiment is shown, representative of four independent experiments.


G ROS / DHE staining and flow cytometry analysis of dissociated tumor organoids ( PE , 582 / 15 nm ) . Mean fluorescence intensities ( MFI ) of DHE stained GFP + tumor cells and GFP - control cells are indicated . n = 2 , values are mean ± SD .

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G ROS/ DHE staining and flow cytometry analysis of dissociated tumor organoids (PE, 582/15 nm). Mean fluorescence intensities (MFI) of DHE stained GFP+ tumor cells and GFP- control cells are indicated. n=2, values are mean ± SD.


H Quantification of γH2AX + cells in cerebral tumor organoids . 6 cryo - sections of 3 organoids each were stained for γH2AX , scanned and 25 regions of interest ( ROI 2 . 500μm2 ) were analyzed per condition . Raw data were normalized to organoid area ( per ROI ) and are shown as box plots ( 25th - 75th percentiles , median ) .

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H Quantification of γH2AX+ cells in cerebral tumor organoids. 6 cryo-sections of 3 organoids each were stained for γH2AX, scanned and 25 regions of interest (ROI 2.500μm2) were analyzed per condition. Raw data were normalized to organoid area (per ROI) and are shown as box plots (25th-75th percentiles, median).


A Fluorescence ( left panel ) and brightfield ( right panel ) images of 5 - ALA and DHA treated human cerebral tumor organoids ( glioblastoma - like ) . Organoids were monitored on day 1 , day 5 , day 8 and day 11 . One experiment is shown , representative of two independent experiments . Scale bar 500μm . B , C Image analysis and quantification of GFP - positive tumor areas of the treated organoids . Four organoids per group were analyzed on day ( B ) d5 and ( C ) d11 , normalized to day 1 . Data is shown as box plots ( 25th - 75th percentiles , median ) n = 4 .

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A Fluorescence (left panel) and brightfield (right panel) images of 5-ALA and DHA treated human cerebral tumor organoids (glioblastoma-like). Organoids were monitored on day 1, day 5, day 8 and day 11. One experiment is shown, representative of two independent experiments. Scale bar 500μm. B, C Image analysis and quantification of GFP-positive tumor areas of the treated organoids. Four organoids per group were analyzed on day (B) d5 and (C) d11, normalized to day 1. Data is shown as box plots (25th-75th percentiles, median) n=4. D


A Representative brightfield images of 5 - ALA and DHA treated patient - derived glioblastoma spheroids ( VBT92 ) . Images were taken on day 3 of culture left untreated or exposed to the indicated treatments . Scale bar 500μm .

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A Representative brightfield images of 5-ALA and DHA treated patient-derived glioblastoma spheroids (VBT92). Images were taken on day 3 of culture left untreated or exposed to the indicated treatments. Scale bar 500μm.


B Re - plating of spheroids after 3 days of 5 - ALA and DHA treatment ( duplicates ) . Representative brightfield images are shown for glioblastoma VBT92 .

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B Re-plating of spheroids after 3 days of 5-ALA and DHA treatment (duplicates). Representative brightfield images are shown for glioblastoma VBT92.


C Quantification of the re - adhesion capacity of re - plated VBT92 spheroids as assessed by crystal violet staining and absorbance measurement . Data is shown as mean ± SD . Experiments were performed in quadruplicates .

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C Quantification of the re-adhesion capacity of re-plated VBT92 spheroids as assessed by crystal violet staining and absorbance measurement. Data is shown as mean ± SD. Experiments were performed in quadruplicates.


D Lipid peroxidation in VBT92 cells treated with 5 - ALA and DHA . Lipid peroxidation was measured using BODIPY ™ 581 / 591 C11 staining and green ( from red ) fluorescence shift was determined using flow cytometry . Experiments were performed in triplicate ; values are shown as mean ± SD .

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D Lipid peroxidation in VBT92 cells treated with 5-ALA and DHA. Lipid peroxidation was measured using BODIPY™ 581/591 C11 staining and green (from red) fluorescence shift was determined using flow cytometry. Experiments were performed in triplicate; values are shown as mean ± SD.


B In vivo efficacy comparison of solvents ( control ) , 5 - ALA , artesunate ( ARS ) and combined treatment in an orthotopic glioblastoma model . 11 - 14 days after stereotactic intracerebral injection of 2 x 105 VBT529 cells , mice received the following treatments : Control ( solvents ) , 5 - ALA ( 80 mg / kg ) , ARS ( 40 mg / kg ) , or 5 - ALA plus ARS 5 times per week via intraperitoneal injection ( n ≥ 5 ) .

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B In vivo efficacy comparison of solvents (control), 5-ALA, artesunate (ARS) and combined treatment in an orthotopic glioblastoma model. 11-14 days after stereotactic intracerebral injection of 2 x 105 VBT529 cells, mice received the following treatments: Control (solvents), 5-ALA (80 mg/kg), ARS (40 mg/kg), or 5-ALA plus ARS 5 times per week via intraperitoneal injection (n ≥ 5).


C , D Quantification and representative images of the VBT529 flank tumor luminescence signal . 6 - 7 days after injection of 1 . 5 x 105 VBT529 cells into the flank , mice received the following treatments : Control ( solvents ) or 5 - ALA plus ARS 4 - 5 times per week for 5 weeks via intraperitoneal injection ( n = 6 ) . After 4 days of treatment , concentrations of 5 - ALA ( 100 mg / kg ) and ARS ( 50 mg / kg ) were increased to 120 mg / kg and 60 mg / kg , respectively . The 20 % dosage increase of both drugs was used as the body - weight , behaviour and overall health of the mice were not affected by both , the lower as well as higher dosages . E , F Quantification and representative images of VBT529 brain tumor luminescence signals . 6 - 7 days after stereotactic intracerebral injection of 1 . 5 x 105 VBT529 cells , mice received the treatments as described in ( C and D ) ( n = 6 ) .

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C, D Quantification and representative images of the VBT529 flank tumor luminescence signal. 6-7 days after injection of 1.5 x 105 VBT529 cells into the flank, mice received the following treatments: Control (solvents) or 5-ALA plus ARS 4-5 times per week for 5 weeks via intraperitoneal injection (n = 6). After 4 days of treatment, concentrations of 5-ALA (100 mg/kg) and ARS (50 mg/kg) were increased to 120 mg/kg and 60 mg/kg, respectively. The 20% dosage increase of both drugs was used as the body-weight, behaviour and overall health of the mice were not affected by both, the lower as well as higher dosages. E, F Quantification and representative images of VBT529 brain tumor luminescence signals. 6-7 days after stereotactic intracerebral injection of 1.5 x 105 VBT529 cells, mice received the treatments as described in (C and D) (n = 6).


G Kaplan - Meier survival curves of mice injected with VBT529 glioblastoma cells into the flank and brain ( same mice as in C - F ; n = 6 ) . Mice were treated with solvents ( control ) or 5 - ALA plus ARS ( same mice as in C - F ) .

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G Kaplan-Meier survival curves of mice injected with VBT529 glioblastoma cells into the flank and brain (same mice as in C-F; n = 6). Mice were treated with solvents (control) or 5-ALA plus ARS (same mice as in C-F).


A : Immunoblot analysis of wild type ( WT ) , FAM83D - / - knockout ( KO ) and FAM83DGFP / GFP knockin ( KI ) U2OS cell lines . Data information : All blots are representative of at least 3 independent experiments .

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A: Immunoblot analysis of wild type (WT), FAM83D-/- knockout (KO) and FAM83DGFP/GFP knockin (KI) U2OS cell lines. Data information: All blots are representative of at least 3 independent experiments.


D : AS or nocodazole - synchronised M KI cells were lysed and subjected to GFP TRAP immunoprecipitations ( IP ) . Extracts ( input ) and IP samples were analysed by immunoblotting ( IB ) with the indicated antibodies . Data information : All blots are representative of at least 3 independent experiments .

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D: AS or nocodazole-synchronised M KI cells were lysed and subjected to GFP TRAP immunoprecipitations (IP). Extracts (input) and IP samples were analysed by immunoblotting (IB) with the indicated antibodies. Data information: All blots are representative of at least 3 independent experiments.


E : KI cells synchronised in mitosis with either nocodazole ( M Noc . ) or STLC ( M STLC ) were collected by shake - off , and drug - treated cells that remained adherent after shake - off ( AS Noc . ; AS STLC ) were lysed and subjected to GFP TRAP IP . AS cells and free - GFP - expressing 2G - PAI1 U2OS cells were included as controls . Input and IP samples were analysed by IB with the indicated antibodies . Data information : All blots are representative of at least 3 independent experiments .

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E: KI cells synchronised in mitosis with either nocodazole (M Noc.) or STLC (M STLC) were collected by shake-off, and drug-treated cells that remained adherent after shake-off (AS Noc.; AS STLC) were lysed and subjected to GFP TRAP IP. AS cells and free-GFP-expressing 2G-PAI1 U2OS cells were included as controls. Input and IP samples were analysed by IB with the indicated antibodies. Data information: All blots are representative of at least 3 independent experiments.


F : Propidium iodide staining analyses revealing cell cycle distribution profiles for the

F: Propidium iodide staining analyses revealing cell cycle distribution profiles for the samples


G , H : AS or nocodazole - synchronised ( M ) WT U2OS cells were subjected to IP with IgG and either anti - FAM83D - coupled sepharose beads ( G ) , or anti - CK1α - coupled sepharose beads ( H ) . Input and IP samples were analysed by IB with the indicated antibodies . Data information : All blots are representative of at least 3 independent experiments .

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G, H: AS or nocodazole-synchronised (M) WT U2OS cells were subjected to IP with IgG and either anti-FAM83D-coupled sepharose beads (G), or anti-CK1α-coupled sepharose beads (H). Input and IP samples were analysed by IB with the indicated antibodies. Data information: All blots are representative of at least 3 independent experiments.


I : KI cells were synchronised in G2 with RO - 3306 , or arrested in mitosis ( M ) using STLC . STLC - treated shake - off cells were washed and re - plated , and cells lysed at the indicated time points after STLC wash - out . Cell lysates were subjected to GFP TRAP IP and input and IP extracts analysed by IB with the indicated antibodies . Data information : All blots are representative of at least 3 independent experiments .

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I: KI cells were synchronised in G2 with RO-3306, or arrested in mitosis (M) using STLC. STLC-treated shake-off cells were washed and re-plated, and cells lysed at the indicated time points after STLC wash-out. Cell lysates were subjected to GFP TRAP IP and input and IP extracts analysed by IB with the indicated antibodies. Data information: All blots are representative of at least 3 independent experiments.


J : Propidium iodide staining analyses revealing cell cycle distribution profiles for the

J: Propidium iodide staining analyses revealing cell cycle distribution profiles for the samples


K : AS or STLC - synchronised ( M ) HeLa , A549 and HaCaT cells were subjected to IP with either IgG - or anti - FAM83D - coupled sepharose beads . Input and IP samples were analysed by IB with the indicated antibodies . Data information : All blots are representative of at least 3 independent experiments .

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K: AS or STLC-synchronised (M) HeLa, A549 and HaCaT cells were subjected to IP with either IgG- or anti-FAM83D-coupled sepharose beads. Input and IP samples were analysed by IB with the indicated antibodies. Data information: All blots are representative of at least 3 independent experiments.


A : STLC - synchronised mitotic ( M ) wild - type ( WT ) , FAM83D - / - knockout ( KO ) and FAM83DGFP / GFP knockin ( KI ) U2OS cells were subjected to anti - CK1α immunofluorescence and GFP fluorescence microscopy . DNA is stained with DAPI . Scale bars , 20 µm .

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A: STLC-synchronised mitotic (M) wild-type (WT), FAM83D-/- knockout (KO) and FAM83DGFP/GFP knockin (KI) U2OS cells were subjected to anti-CK1α immunofluorescence and GFP fluorescence microscopy. DNA is stained with DAPI. Scale bars, 20 µm.


B : STLC - synchronised mitotic ( M ) FAM83D - / - knockout ( KO ) , FAM83DGFP / GFP knockin ( KI ) and FAM83DGFP / GFP ( F283A ) knockin ( FA ) U2OS cells were subjected to anti - CK1α immunofluorescence and GFP fluorescence microscopy . DNA is stained with DAPI . Scale bars , 20

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B: STLC-synchronised mitotic (M) FAM83D-/- knockout (KO), FAM83DGFP/GFP knockin (KI) and FAM83DGFP/GFP(F283A) knockin (FA) U2OS cells were subjected to anti-CK1α immunofluorescence and GFP fluorescence microscopy. DNA is stained with DAPI. Scale bars, 20 µ


C : Quantification of CK1α spindle localisation for the cells Cell images denote the measured regions used to calculate the ratios on the box plot . Box plot whiskers denote the minimum and maximum measured values . The middle line represents the median , and the box ranges depict the 25th / 75th percentiles . * * * , P < 0 . 0001 ; ANOVA . Analysis was performed on the indicated number of cells , n = 2 .

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C: Quantification of CK1α spindle localisation for the cells Cell images denote the measured regions used to calculate the ratios on the box plot. Box plot whiskers denote the minimum and maximum measured values. The middle line represents the median, and the box ranges depict the 25th/75th percentiles. ***, P<0.0001; ANOVA. Analysis was performed on the indicated number of cells, n=2.


D : The cell lines were STLC - synchronised and mitotic cells ( M ) isolated by shake off . Asynchronous ( AS ) cells were included as a control . Cells were lysed and subjected to GFP TRAP immunoprecipitation ( IP ) and subsequent immunoblotting ( IB ) with the indicated antibodies . Data information : All blots are representative of at least 3 independent experiments .

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D: The cell lines were STLC-synchronised and mitotic cells (M) isolated by shake off. Asynchronous (AS) cells were included as a control. Cells were lysed and subjected to GFP TRAP immunoprecipitation (IP) and subsequent immunoblotting (IB) with the indicated antibodies. Data information: All blots are representative of at least 3 independent experiments.


F : KI cells were infected with retroviruses encoding either VHL , aGFP . 16 , or VHL - aGFP . 16 . Uninfected cells were used as a control . Cells were lysed and subjected to IB with the indicated antibodies . Data information : All blots are representative of at least 3 independent experiments .

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F: KI cells were infected with retroviruses encoding either VHL, aGFP.16, or VHL-aGFP.16. Uninfected cells were used as a control. Cells were lysed and subjected to IB with the indicated antibodies. Data information: All blots are representative of at least 3 independent experiments.


G : The cell lines were subjected to anti - CK1α immunofluorescence and GFP fluorescence microscopy . DNA is stained with DAPI . Scale bars , 20 µm .

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G: The cell lines were subjected to anti-CK1α immunofluorescence and GFP fluorescence microscopy. DNA is stained with DAPI. Scale bars, 20 µm.


B : FAM83D - / - ( KO ) , wild - type ( WT ) , and two independent clones from a CRISPR / Cas9 - mediated knockin rescue of FAM83D cDNA into FAM83D - / - cells ( c . 6 and c . 11 ) , were synchronized in mitosis ( M ) with STLC . Asynchronous ( AS ) cells were included as a control . Cells were lysed and subjected to immunoblotting ( IB ) with the indicated antibodies . Data information : All blots are representative of at least 3 independent experiments .

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B: FAM83D-/- (KO), wild-type (WT), and two independent clones from a CRISPR/Cas9-mediated knockin rescue of FAM83D cDNA into FAM83D-/- cells (c. 6 and c. 11), were synchronized in mitosis (M) with STLC. Asynchronous (AS) cells were included as a control. Cells were lysed and subjected to immunoblotting (IB) with the indicated antibodies. Data information: All blots are representative of at least 3 independent experiments.


The cell lines were STLC - synchronized in mitosis , fixed , and stained with antibodies recognizing FAM83D ( C ) Representative images of mitotic cells are included . Scale bars , 20 µm .

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The cell lines were STLC-synchronized in mitosis, fixed, and stained with antibodies recognizing FAM83D (C) Representative images of mitotic cells are included. Scale bars, 20 µm.


The cell lines were STLC - synchronized in mitosis , fixed , and stained with antibodies recognizing CK1 ⍺ ( D ) . Representative images of mitotic cells are included . Scale bars , 20 µm .

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The cell lines were STLC-synchronized in mitosis, fixed, and stained with antibodies recognizing CK1⍺ (D). Representative images of mitotic cells are included. Scale bars, 20 µm.


E : Quantification of CK1 ⍺ spindle localization Box plot whiskers denote the minimum and maximum measured values . The middle line represents the median , and the box ranges depict the 25th / 75th percentiles . * * * , P < 0 . 0001 ; ANOVA . Analysis was performed on the indicated number of cells , n = 1 .

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E: Quantification of CK1⍺ spindle localization Box plot whiskers denote the minimum and maximum measured values. The middle line represents the median, and the box ranges depict the 25th/75th percentiles. ***, P<0.0001; ANOVA. Analysis was performed on the indicated number of cells, n=1.


F : The cell lines were STLC - synchronized in mitosis ( M ) or left AS , lysed and subjected to immunoprecipitation ( IP ) with anti - FAM83D - coupled sepharose beads , before IB with the indicated antibodies . Data information : All blots are representative of at least 3 independent experiments .

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F: The cell lines were STLC-synchronized in mitosis (M) or left AS, lysed and subjected to immunoprecipitation (IP) with anti-FAM83D-coupled sepharose beads, before IB with the indicated antibodies. Data information: All blots are representative of at least 3 independent experiments.


G : Asynchronous ( AS ) FAM83DGFP / GFP / mCherry / mCherryCSNK1A1 knockin U2OS cells were fixed and imaged . Representative images from the indicated cell cycle stages are included . Scale bars , 10 µm .

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G: Asynchronous (AS) FAM83DGFP/GFP/ mCherry/mCherryCSNK1A1 knockin U2OS cells were fixed and imaged. Representative images from the indicated cell cycle stages are included. Scale bars, 10 µm.


A : Nocodazole - synchronised mitotic FAM83DGFP / GFP knockin U2OS cells were lysed and subjected to GFP TRAP immunoprecipitation ( IP ) , followed by incubation ± λ - phosphatase . Asynchronous ( AS ) cells were used as a control . Whole cell extracts ( input ) and IP samples were subjected to SDS - PAGE and subsequent immunoblotting ( IB ) with the indicated antibodies . Data information : All blots are representative of at least 3 independent experiments .

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A: Nocodazole-synchronised mitotic FAM83DGFP/GFP knockin U2OS cells were lysed and subjected to GFP TRAP immunoprecipitation (IP), followed by incubation ± λ-phosphatase. Asynchronous (AS) cells were used as a control. Whole cell extracts (input) and IP samples were subjected to SDS-PAGE and subsequent immunoblotting (IB) with the indicated antibodies. Data information: All blots are representative of at least 3 independent experiments.


STLC - synchronised mitotic wild - type U2OS cells were lysed at the indicated time points following STLC washout and release into medium ( B ) AS cells were used as a control . Lysed extracts were subjected to IB with the indicated antibodies . Data information : All blots are representative of at least 3 independent experiments .

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STLC-synchronised mitotic wild-type U2OS cells were lysed at the indicated time points following STLC washout and release into medium (B) AS cells were used as a control. Lysed extracts were subjected to IB with the indicated antibodies. Data information: All blots are representative of at least 3 independent experiments.


STLC - synchronised mitotic wild - type U2OS cells were lysed at the indicated time points following STLC washout and release into medium ± MG132 ( C ) AS cells were used as a control . Lysed extracts were subjected to IB with the indicated antibodies . Data information : All blots are representative of at least 3 independent experiments .

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STLC-synchronised mitotic wild-type U2OS cells were lysed at the indicated time points following STLC washout and release into medium ± MG132 (C) AS cells were used as a control. Lysed extracts were subjected to IB with the indicated antibodies. Data information: All blots are representative of at least 3 independent experiments.


STLC - synchronised mitotic wild - type U2OS cells were lysed at the indicated time points following STLC washout and release into medium ± ProTAME ( D ) . AS cells were used as a control . Lysed extracts were subjected to IB with the indicated antibodies . Data information : All blots are representative of at least 3 independent experiments .

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 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

STLC-synchronised mitotic wild-type U2OS cells were lysed at the indicated time points following STLC washout and release into medium ± ProTAME (D). AS cells were used as a control. Lysed extracts were subjected to IB with the indicated antibodies. Data information: All blots are representative of at least 3 independent experiments.


E : Wild - type U2OS cells were either left asynchronous ( AS ) , or arrested in mitosis with STLC and collected by shake - off ( M ) . AS and M cells were incubated in media containing combinations of RO - 3306 and MG132 as indicated , prior to lysis . MG132 was applied for 1 . 5 h , whereas RO - 3306 was applied for the last 1 h of incubation prior to lysis . Samples were lysed and extracts were subjected to SDS - PAGE , before IB with the indicated antibodies . Data information : All blots are representative of at least 3 independent experiments .

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E: Wild-type U2OS cells were either left asynchronous (AS), or arrested in mitosis with STLC and collected by shake-off (M). AS and M cells were incubated in media containing combinations of RO-3306 and MG132 as indicated, prior to lysis. MG132 was applied for 1.5 h, whereas RO-3306 was applied for the last 1 h of incubation prior to lysis. Samples were lysed and extracts were subjected to SDS-PAGE, before IB with the indicated antibodies. Data information: All blots are representative of at least 3 independent experiments.


F : STLC - synchronised mitotic wild - type U2OS cells were subjected to qRT - PCR analysis using primers for FAM83D , HMMR , CSNK1A1 , and CCNB1 . AS cells were used as a control . Error bars , SEM ; * , P < 0 . 01 ; Student ' s T - test . n =

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F: STLC-synchronised mitotic wild-type U2OS cells were subjected to qRT-PCR analysis using primers for FAM83D, HMMR, CSNK1A1, and CCNB1. AS cells were used as a control. Error bars, SEM; *, P<0.01; Student's T-test. n=3


H : STLC - synchronised mitotic wild - type ( WT ) and HMMR knockout ( KO ) mouse embryonic fibroblasts ( MEFs ) were lysed and subjected to IB with the indicated antibodies . AS cells were included as a control . Data information : All blots are representative of at least 3 independent experiments .

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H: STLC-synchronised mitotic wild-type (WT) and HMMR knockout (KO) mouse embryonic fibroblasts (MEFs) were lysed and subjected to IB with the indicated antibodies. AS cells were included as a control. Data information: All blots are representative of at least 3 independent experiments.


The cells were STLC - synchronised in mitosis and subjected to immunofluorescence microscopy with an anti - HMMR antibody ( I ) DNA is stained with DAPI . Scale bars , 20 µm .

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The cells were STLC-synchronised in mitosis and subjected to immunofluorescence microscopy with an anti-HMMR antibody (I) DNA is stained with DAPI. Scale bars, 20 µm.


The cells were STLC - synchronised in mitosis and subjected to immunofluorescence microscopy with an anti - CK1α antibody ( J ) . DNA is stained with DAPI . Scale bars , 20 µm .

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The cells were STLC-synchronised in mitosis and subjected to immunofluorescence microscopy with an anti-CK1α antibody (J). DNA is stained with DAPI. Scale bars, 20 µm.


B : STLC - synchronised mitotic FAM83DGFP / GFP knockin ( KI ) , FAM83DGFP / GFP ( F283A ) ( FA ) , and FAM83DGFP / GFP ( F283A ) stably expressing aGFP . 16 - CK1α ( FA + aGFP . 16 - CK1α ) U2OS cells were subjected to GFP TRAP immunoprecipitation ( IP ) , followed by immunoblotting ( IB ) with the indicated antibodies . Asynchronous ( AS ) cells were used as controls . Data information : All blots are representative of at least 3 independent experiments .

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B: STLC-synchronised mitotic FAM83DGFP/GFP knockin (KI), FAM83DGFP/GFP (F283A) (FA), and FAM83DGFP/GFP(F283A) stably expressing aGFP.16-CK1α (FA + aGFP.16-CK1α) U2OS cells were subjected to GFP TRAP immunoprecipitation (IP), followed by immunoblotting (IB) with the indicated antibodies. Asynchronous (AS) cells were used as controls. Data information: All blots are representative of at least 3 independent experiments.


C : FA cells were infected with retroviruses encoding wild - type aGFP . 16 - CK1α ( WT ) , or aGFP . 16 - CK1α with one of two distinct CK1α kinase - inactive mutants ( D136N or K46D ) . Uninfected cells ( - ) were included as a control . Cells were lysed , subjected to anti - GFP IP and IB with the indicated antibodies . Data information : All blots are representative of at least 3 independent experiments .

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C: FA cells were infected with retroviruses encoding wild-type aGFP.16-CK1α (WT), or aGFP.16-CK1α with one of two distinct CK1α kinase-inactive mutants (D136N or K46D). Uninfected cells (-) were included as a control. Cells were lysed, subjected to anti-GFP IP and IB with the indicated antibodies. Data information: All blots are representative of at least 3 independent experiments.


D : Immunofluorescence analysis for the cells described in ( B ) following synchronisation with STLC . Cells were stained using anti - CK1α antibody , and DNA is stained with DAPI . Scale bars , 20 µm .

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D: Immunofluorescence analysis for the cells described in (B) following synchronisation with STLC. Cells were stained using anti-CK1α antibody, and DNA is stained with DAPI. Scale bars, 20 µm.


F : FAM83D - / - knockout ( KO ) , KI , and FA cells were synchronised in mitosis ( M ) using STLC . Lysed extracts were subjected to anti - GFP IPs , followed by ATP [ γ - 32P ] kinase assays , using an optimised CK1 substrate peptide ( CK1tide ) . AS cells were used as controls . Error bars , SEM ; P < 0 . 01 ; ANOVA . n = 3 . Input and IP samples were analysed by IB with the indicated antibodies . Data information : All blots are representative of at least 3 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 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

F: FAM83D-/- knockout (KO), KI, and FA cells were synchronised in mitosis (M) using STLC. Lysed extracts were subjected to anti-GFP IPs, followed by ATP [γ-32P] kinase assays, using an optimised CK1 substrate peptide (CK1tide). AS cells were used as controls. Error bars, SEM; P<0.01; ANOVA. n=3. Input and IP samples were analysed by IB with the indicated antibodies. Data information: All blots are representative of at least 3 independent experiments.


A . Representative images initiating at metaphase for mitotic U2OS cell lines stained with Hoechst , taken every 5 min as they progress through division . Mitotic stage was determined by chromosome condensation and is indicated by the coloured boxes . Scale bar , 20 μm .

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A. Representative images initiating at metaphase for mitotic U2OS cell lines stained with Hoechst, taken every 5 min as they progress through division. Mitotic stage was determined by chromosome condensation and is indicated by the coloured boxes. Scale bar, 20 μm.


B . Graphical representation for the kinetics of transition from metaphase alignment ( yellow ) to anaphase ( green ) , and cytokinesis ( blue ) . 100 mitotic cells per genotype are plotted ; n = 2 .

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B. Graphical representation for the kinetics of transition from metaphase alignment (yellow) to anaphase (green), and cytokinesis (blue). 100 mitotic cells per genotype are plotted; n=2.


C . Length of time needed to transition from metaphase to anaphase . Mean of 100 mitotic cells per genotype are plotted ; n = 2 . Error bars , SEM . * * * , P < 0 . 0001 ; ANOVA .

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C. Length of time needed to transition from metaphase to anaphase. Mean of 100 mitotic cells per genotype are plotted; n=2. Error bars, SEM. ***, P< 0.0001; ANOVA.


D . Representative bright field images for mitotic U2OS cells indicating the long axis during interphase ( white line ) preceding mitosis , and the division axis determined at anaphase ( yellow line ) . Scale bar , 20 μm .

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D. Representative bright field images for mitotic U2OS cells indicating the long axis during interphase (white line) preceding mitosis, and the division axis determined at anaphase (yellow line). Scale bar, 20 μm.


E . Percentage of oriented divisions for U2OS cells grown in subconfluent cultures . An oriented division axis was defined as being less than 30 ° removed from the long axis of the interphase cell . Mean of 50 cells per genotype are plotted , n = 2 . Error bars , SEM . * * * , P < 0 . 0001 ; ANOVA .

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E. Percentage of oriented divisions for U2OS cells grown in subconfluent cultures. An oriented division axis was defined as being less than 30° removed from the long axis of the interphase cell. Mean of 50 cells per genotype are plotted, n=2. Error bars, SEM. ***, P< 0.0001; ANOVA.


F . Representative images of mitotic U2OS cells stained with Hoechst and grown on L - shaped micropatterns previously coated with fibronectin . The position of metaphase chromosomes is indicated ( yellow line ) as is the presence of cortical blebbing ( red arrows ) . Scale bar , 20 μm .

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F. Representative images of mitotic U2OS cells stained with Hoechst and grown on L-shaped micropatterns previously coated with fibronectin. The position of metaphase chromosomes is indicated (yellow line) as is the presence of cortical blebbing (red arrows). Scale bar, 20 μm.


G . Circular graphs , superimposed on L - shaped micropattern , show the distribution of cell division angles measured at anaphase . Angles for 100 U2OS cells are plotted per genotype , n = 2 . Metaphase position is indicated ( yellow line ) and the percentages of division angles ± 15 ° from the expected axis ( red line ) are indicated .

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G. Circular graphs, superimposed on L-shaped micropattern, show the distribution of cell division angles measured at anaphase. Angles for 100 U2OS cells are plotted per genotype, n=2. Metaphase position is indicated (yellow line) and the percentages of division angles ± 15° from the expected axis (red line) are indicated.


H . Percentage of metaphase U2OS cells that align chromosomes outside of the expected axis ( angles ± 15 ° ) . Mean of 100 cells per genotype are plotted , n = 2 . Error bars , SEM . * * * , P < 0 . 0001 ; ANOVA .

H. Percentage of metaphase U2OS cells that align chromosomes outside of the expected axis (angles ± 15°). Mean of 100 cells per genotype are plotted, n=2. Error bars, SEM. ***, P< 0.0001; ANOVA.


I . Representative images of RFP - actin localization in mitotic U2OS cells grown on fibronectin - coated , L - shaped micropatterns , which is superimposed . Arrowheads indicate polarized cortical actin . Heatmap shows the intensity of RFP - actin localization ( Image J z - projection standard deviation ) as the cell progresses from prophase to metaphase ( 12 - 24 min ) . Scale bar , 20 μm .

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I. Representative images of RFP-actin localization in mitotic U2OS cells grown on fibronectin-coated, L-shaped micropatterns, which is superimposed. Arrowheads indicate polarized cortical actin. Heatmap shows the intensity of RFP-actin localization (Image J z-projection standard deviation) as the cell progresses from prophase to metaphase (12- 24 min). Scale bar, 20 μm.


J . Heatmap additive intensities of RFP - actin localization in two representative mitotic U2OS cells for each genotype grown on fibronectin - coated , L - shaped micropatterns . Arrowheads indicate polarized cortical actin .

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J. Heatmap additive intensities of RFP-actin localization in two representative mitotic U2OS cells for each genotype grown on fibronectin-coated, L-shaped micropatterns. Arrowheads indicate polarized cortical actin.


A ) ALS patients from the UPenn Biobank neuroimaging cohort with higher wPRS exhibited greater reduction of cortical thickness in the orbital prefrontal cortex , anterior cingulate cortex , premotor cortex , lateral temporal cortex , and hippocampus . The heatmap indicates the associated T - statistic for each voxel , with light blue representing the highest value .

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A) ALS patients from the UPenn Biobank neuroimaging cohort with higher wPRS exhibited greater reduction of cortical thickness in the orbital prefrontal cortex, anterior cingulate cortex, premotor cortex, lateral temporal cortex, and hippocampus. The heatmap indicates the associated T-statistic for each voxel, with light blue representing the highest value.


( C ) Change in the expression levels of 36 genes defining the host - targeted anti - viral signature . log2 fold change at 24 hours post SARS - CoV - 2 infection from A549 cells are shown .

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(C) Change in the expression levels of 36 genes defining the host-targeted anti-viral signature. log2 fold change at 24 hours post SARS-CoV-2 infection from A549 cells are shown.


( D ) Change in the expression levels of 17 genes defining anti - cytokine signature ; log2 fold change at 24 hours post high SARS - CoV - 2 infection from A549 - ACE2 cells are shown .

(D) Change in the expression levels of 17 genes defining anti-cytokine signature; log2 fold change at 24 hours post high SARS-CoV-2 infection from A549-ACE2 cells are shown.


( A ) Representative fluorescence images of Mock , SARS - CoV - 2 infected ( Ctrl ) , and Salmeterol - treated wells analyzed with the Multiwavelength Cell Scoring application in MetaXpress . Grayscales of the images were adjusted to enable direct comparison of the relative levels of fluorescence among the treatments : Segmentation images show how cells were segmented and identified as spike positive . Purple , nuclei ; cyan , spike . Scale bar , 100 μm .

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(A) Representative fluorescence images of Mock, SARS-CoV-2 infected (Ctrl), and Salmeterol-treated wells analyzed with the Multiwavelength Cell Scoring application in MetaXpress. Grayscales of the images were adjusted to enable direct comparison of the relative levels of fluorescence among the treatments: Segmentation images show how cells were segmented and identified as spike positive. Purple, nuclei; cyan, spike. Scale bar, 100 μm.