Genes associated with indicated GO terms plotted by fold change in expression in Mta123 ∆ ES cells ( x - axis ) or Mbd3 ∆ ES cells ( y - axis ) . Genes are coloured if they are differentially expressed ( log2 fold - change > 1 and adjusted p - value < 0 . 05 ) in either comparison as indicated . The dotted lines show the fold - change cut - off of 2 . GO - terms were identified using David v . 6 . 8 using a Benjamini score with a cutoff of 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Genes associated with indicated GO terms plotted by fold change in expression in Mta123∆ ES cells (x-axis) or Mbd3∆ ES cells (y-axis). Genes are coloured if they are differentially expressed (log2 fold-change > 1 and adjusted p-value < 0.05) in either comparison as indicated. The dotted lines show the fold-change cut-off of 2. GO-terms were identified using David v.6.8 using a Benjamini score with a cutoff of 0.05.


Genes associated with core NuRD peaks , with an MTA - only peak , with a Chd4 peak and an MTA - peak but not an Mbd3 peak ( Chd4 + Mta - Mbd3 genes ) , or with an MTA - peak but not a Chd4 peak ( MTA - only genes ) were divided into quartiles based upon expression levels in wild type cells . Change in gene expression is plotted on the x - axis and calculated significance of the expression change on the y - axis . Those genes significantly misexpressed ( | log2FC | > 1 ; p ≤ 0 . 05 ) are indicated in red , genes showing no significant change are plotted in black . The percentage of genes within each quartile which is significantly misexpressed is indicated at the top of each plot .

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

Genes associated with core NuRD peaks, with an MTA-only peak, with a Chd4 peak and an MTA-peak but not an Mbd3 peak (Chd4+Mta-Mbd3 genes), or with an MTA-peak but not a Chd4 peak (MTA-only genes) were divided into quartiles based upon expression levels in wild type cells. Change in gene expression is plotted on the x-axis and calculated significance of the expression change on the y-axis. Those genes significantly misexpressed (|log2FC|>1; p≤0.05) are indicated in red, genes showing no significant change are plotted in black. The percentage of genes within each quartile which is significantly misexpressed is indicated at the top of each plot.


Phase contrast pictures of ES cells of indicated genotypes after 5 days in neuroectoderm differentiation conditions ( N2B27 ) . White arrowheads in the Mbd3 ∆ panel indicate pockets of ES - like cells . Scale bars represent 100 µm .

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

Phase contrast pictures of ES cells of indicated genotypes after 5 days in neuroectoderm differentiation conditions (N2B27). White arrowheads in the Mbd3∆ panel indicate pockets of ES-like cells. Scale bars represent 100 µm.


Expression of indicated genes in the neuroectoderm differentiation time course was measured by RT - qPCR in Control ( green ) Mta123 ∆ ( magenta ) or Mbd3 ∆ ( blue ) ES cells . N ≥ 3 biological replicates , error bars indicate SEM .

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

Expression of indicated genes in the neuroectoderm differentiation time course was measured by RT-qPCR in Control (green) Mta123∆ (magenta) or Mbd3∆ (blue) ES cells. N ≥ 3 biological replicates, error bars indicate SEM.


Phase contrast images of embryoid body outgrowths of indicated genotypes after 12 days in differentiation conditions ( N2B27 + 10 % serum ) . Scale bars represent 100µm .

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

Phase contrast images of embryoid body outgrowths of indicated genotypes after 12 days in differentiation conditions (N2B27 + 10% serum). Scale bars represent 100µm.


Expression of indicated genes in embryoid body cultures over time was measured by RT -

Expression of indicated genes in embryoid body cultures over time was measured by RT-qPCR


Comparison of expression data for wild type , Mbd3 ∆ or Mta123 ∆ ES cells in self - renewing ( 2i ) conditions or after 48 hours in differentiation conditions ( 48h N2B27 ) with mouse embryonic single cell RNA - seq data Larger shading encloses biological replicates , smaller circles represent individual cells .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 expression data for wild type, Mbd3∆ or Mta123∆ ES cells in self-renewing (2i) conditions or after 48 hours in differentiation conditions (48h N2B27) with mouse embryonic single cell RNA-seq data Larger shading encloses biological replicates, smaller circles represent individual cells.


Composite images of representative chimaeric embryos made with control ( Mta1Flox / ∆ Mta2 + / + Mta3Flox / Flox ) or Mta123 ∆ ES cells . ES - derived cells express the Kusabira Orange fluorescent marker . Sox2 indicates epiblast cells and Cdx2 is expressed in trophectoderm cells . Arrows indicate examples of K - Orange expressing cells in the mutant embryos . Scale bars = 20µm .

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

Composite images of representative chimaeric embryos made with control (Mta1Flox/∆Mta2+/+Mta3Flox/Flox) or Mta123∆ ES cells. ES-derived cells express the Kusabira Orange fluorescent marker. Sox2 indicates epiblast cells and Cdx2 is expressed in trophectoderm cells. Arrows indicate examples of K-Orange expressing cells in the mutant embryos. Scale bars = 20µm.


( Top ) Number of K - Orange expressing cells observed in chimaeric embryos obtained using control ES cells , Mta123 ∆ ES cells , or Mta123 ∆ ES cells in which Mta2 was reintroduced on a constitutively expressed transgene ( Mta123 ∆ + Mta2TG ) . P - values calculated using a two - tailed t - test . ( Middle ) Mean number of K - Orange cells per embryo separated by Sox2 expression . P - values calculated using a two - tailed t - test . ( Bottom ) Number of K - Orange and Caspase - 3 positive cells per embryo . P - values calculated using a one - tailed t - test : * P < 0 . 05 , * * * * P < 0 . 0001 , " ns " = not significant .

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

(Top) Number of K-Orange expressing cells observed in chimaeric embryos obtained using control ES cells, Mta123∆ ES cells, or Mta123∆ ES cells in which Mta2 was reintroduced on a constitutively expressed transgene (Mta123∆+Mta2TG). P-values calculated using a two-tailed t-test. (Middle) Mean number of K-Orange cells per embryo separated by Sox2 expression. P-values calculated using a two-tailed t-test. (Bottom) Number of K-Orange and Caspase-3 positive cells per embryo. P-values calculated using a one-tailed t-test: *P < 0.05, ****P < 0.0001, "ns" = not significant.


FP titrations show binding of WT α - actinin - 1 _ EF34 to IQ peptides WT ( black ) and K1647E ( red ) , of α - actinin - 1 _ EF34 mutant E847K / E851K to IQ peptides WT ( green ) and K1647E ( purple ) , of full - length α - actinin - 1 to IQ WT ( o ) , and lack of IQ binding to α - actinin - 1 _ CH1 - CH2 ( + ) and α - actinin - 1 _ EF12 ( x ; see Table 2 for binding parameters and standard errors ) . FP titrations showing binding of wild type α - actinin - 1 _ EF34 to IQ peptides WT ( black ) and I1654A ( purple ) and of ACTN1 _ EF34 mutant F833 to IQ WT ( red ; see Table 2 for binding parameters and standard errors ) .

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

FP titrations show binding of WT α-actinin-1_EF34 to IQ peptides WT (black) and K1647E (red), of α-actinin-1_EF34 mutant E847K/E851K to IQ peptides WT (green) and K1647E (purple), of full-length α-actinin-1 to IQ WT (o), and lack of IQ binding to α-actinin-1_CH1-CH2 (+) and α-actinin-1_EF12 (x; see Table 2 for binding parameters and standard errors). FP titrations showing binding of wild type α-actinin-1_EF34 to IQ peptides WT (black) and I1654A (purple) and of ACTN1_EF34 mutant F833 to IQ WT (red; see Table 2 for binding parameters and standard errors).


FP titrations showing binding of apoCaM to IQ peptides WT ( black ) and K1662E ( red ) and of apoCaM mutant E88K to IQ WT ( green ) and IQ peptide K1662E ( purple , " charge inversion " ; see Table 4 for binding parameters and standard errors ) . FP titrations showing binding of apo - CaM to IQ peptides WT ( black ) , I1654A ( red ) , and F1658A ( green ) and of apoCaM mutant F90A to IQ WT ( blue ; see Table 4 for binding parameters and standard errors ) .

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

FP titrations showing binding of apoCaM to IQ peptides WT (black) and K1662E (red) and of apoCaM mutant E88K to IQ WT (green) and IQ peptide K1662E (purple, "charge inversion"; see Table 4 for binding parameters and standard errors). FP titrations showing binding of apo-CaM to IQ peptides WT (black), I1654A (red), and F1658A (green) and of apoCaM mutant F90A to IQ WT (blue; see Table 4 for binding parameters and standard errors).


HEK293 cells were transfected with α11 . 2 , α2δ - 1 , and β2A and cultured for 22 - 24h before cell attached patch recording in 110 mM Ba2 + . Single CaV1 . 2 channel recordings of CaV1 . 2 WT and K1647A . Holding potential ( HP ) was - 80 mV and test potential ( TP ) 0 mV . Shown are 10 consecutive sweeps from representative experiments ( see Fig . EV 4 for more sweeps ) . Dot plots and means + SEM for availability ( i . e . , likelihood that a sweep had at least one event ; c ) , NPo ( d ) , Po ( e ) , unitary current amplitude i ( e ) , and the mean of the current of a single channel at any point in time calculated from the ensemble averages of each experiment ( g ) ( * * p < 0 . 01 , * * * p < 0 . 001 compared to WT ; one - way ANOVA with Bonferroni post - hoc test ( c ) or Welch ANOVA with Tamhane T2 test ( d - g ) ; n = 7 to 35 see Table 5 for more details ) .

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

HEK293 cells were transfected with α11.2, α2δ-1, and β2A and cultured for 22-24h before cell attached patch recording in 110 mM Ba2+. Single CaV1.2 channel recordings of CaV1.2 WT and K1647A. Holding potential (HP) was -80 mV and test potential (TP) 0 mV. Shown are 10 consecutive sweeps from representative experiments (see Fig. EV 4 for more sweeps). Dot plots and means + SEM for availability (i.e., likelihood that a sweep had at least one event; c), NPo (d), Po (e), unitary current amplitude i (e), and the mean of the current of a single channel at any point in time calculated from the ensemble averages of each experiment (g) (**p<0.01, ***p<0.001 compared to WT; one-way ANOVA with Bonferroni post-hoc test (c) or Welch ANOVA with Tamhane T2 test (d-g); n = 7 to 35 see Table 5 for more details).


HEK293 cells were transfected with α11 . 2 , α2δ - 1 , and β2A plus , if indicated , α - actinin - 1 before cell attached patch recording . Representative single CaV1 . 2 channel recordings of WT CaV1 . 2 alone or with WT or E847K / E851K mutant α - actinin - 1 . Mean assemble averages for all experiments for each combination . Dot plots and means + SEM for availability ( c ) , NPo ( d ) , Po ( e ) , unitary current amplitude i ( f ) , and the mean of the current of a single channel calculated from the ensemble averages of each experiment ( g ) ( * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 001 ; unpaired t - test ; n = 13 to 35 ; see Table 6 for more details ) .

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

HEK293 cells were transfected with α11.2, α2δ-1, and β2A plus, if indicated, α-actinin-1 before cell attached patch recording. Representative single CaV1.2 channel recordings of WT CaV1.2 alone or with WT or E847K/E851K mutant α-actinin-1. Mean assemble averages for all experiments for each combination. Dot plots and means + SEM for availability (c), NPo (d), Po (e), unitary current amplitude i (f), and the mean of the current of a single channel calculated from the ensemble averages of each experiment (g) (*p<0.05, **p<0.01, ****p<0.001; unpaired t-test; n = 13 to 35; see Table 6 for more details).


HEK293 cells were transfected with α11 . 2 with the K1647E mutation , α2δ - 1 , and β2A plus α - actinin - 1 WT or E847K / E851K before cell attached patch recording . Representative single CaV1 . 2 channel recordings of CaV1 . 2 K1647E with WT or E847K / E851K α - actinin - 1 . Mean assemble averages for all experiments for both combinations . Dot plots and means + SEM for availability ( c ) , NPo ( d ) , Po ( e ) , unitary current amplitude i ( f ) , and mean of the current of a single channel calculated from the ensemble averages of each experiment ( g ) ( * * p < 0 . 01 ; unpaired t - test ; n = 16 to 30 ; see Suppl . Fig . 8 and Table 6 for more details ) .

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

HEK293 cells were transfected with α11.2 with the K1647E mutation, α2δ-1, and β2A plus α-actinin-1 WT or E847K/E851K before cell attached patch recording. Representative single CaV1.2 channel recordings of CaV1.2 K1647E with WT or E847K/E851K α-actinin-1. Mean assemble averages for all experiments for both combinations. Dot plots and means + SEM for availability (c), NPo (d), Po (e), unitary current amplitude i (f), and mean of the current of a single channel calculated from the ensemble averages of each experiment (g) (**p<0.01; unpaired t-test; n = 16 to 30; see Suppl. Fig. 8 and Table 6 for more details).


HEK293 cells were transfected with WT α11 . 2 , α2δ - 1 , and β2A + WT α - actinin - 1 and cultured for 22 - 24h before surface biotinylation . Shown are representative immunoblots of NeutrAvidin pull - down samples ( from lysate containing 600 μg protein ) and total lysate samples containing 20 μg protein . α11 . 2 was detected with an antibody against its HA tag ( top ) , which is present in all constructs used throughout this work . Pull - down and lysate samples are from the same blot but different exposures because signals from lysate samples were much stronger than from pull - down samples . Immunoblotting for vinculin ( middle ) and tubulin ( bottom ) indicated that comparable amounts of these intracellular control proteins were present in lysate samples . Their absence in pull - down samples as seen on the same blots showed that these prominent intracellular proteins did not undergo biotinylation as control for membrane integrity during surface biotinylation . Bar graph shows means + SEM of the pull - down immunosignals in mutants relative to surface labelling of control α11 . 2 samples lacking α - actinin - 1 co - expression ( mean set to 100 % ; see Methods ; * * p < 0 . 01 two - tailed t - test ; n = 7 ) .

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

HEK293 cells were transfected with WT α11.2, α2δ-1, and β2A + WT α-actinin-1 and cultured for 22-24h before surface biotinylation. Shown are representative immunoblots of NeutrAvidin pull-down samples (from lysate containing 600 μg protein) and total lysate samples containing 20 μg protein. α11.2 was detected with an antibody against its HA tag (top), which is present in all constructs used throughout this work. Pull-down and lysate samples are from the same blot but different exposures because signals from lysate samples were much stronger than from pull-down samples. Immunoblotting for vinculin (middle) and tubulin (bottom) indicated that comparable amounts of these intracellular control proteins were present in lysate samples. Their absence in pull-down samples as seen on the same blots showed that these prominent intracellular proteins did not undergo biotinylation as control for membrane integrity during surface biotinylation. Bar graph shows means + SEM of the pull-down immunosignals in mutants relative to surface labelling of control α11.2 samples lacking α-actinin-1 co-expression (mean set to 100%; see Methods; **p<0.01 two-tailed t-test; n = 7).


HEK293 cells were transfected with WT or K1647E mutant α11 . 2 , α2δ - 1 , and β2A , and WT or E847K / E851K mutant α - actinin - 1 , or CFP alone as negative control and cultured for 22 - 24h before surface biotinylation . Shown are representative immunoblots of pull - down and lysate samples as in ( a ) . Bar graph shows means + SEM of the pull - down immunosignals in mutants relative to surface labelling in the WT / WT control ( mean set to 100 % ; see Methods ) . * * p < 0 . 01 , * * * p < 0 . 001 ; one - way ANOVA with Tukey post hoc test ; n = 4 - 5 ) .

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

HEK293 cells were transfected with WT or K1647E mutant α11.2, α2δ-1, and β2A, and WT or E847K/E851K mutant α-actinin-1, or CFP alone as negative control and cultured for 22-24h before surface biotinylation. Shown are representative immunoblots of pull-down and lysate samples as in (a). Bar graph shows means + SEM of the pull-down immunosignals in mutants relative to surface labelling in the WT/WT control (mean set to 100%; see Methods). **p<0.01, ***p < 0.001; one-way ANOVA with Tukey post hoc test; n = 4-5).


HEK293 cells were transfected with α11 . 2 , α2δ - 1 , and β2A before whole cell patch recording in 20 mM Ba2 + . Representative current traces of the first 2 ms obtained from recordings upon depolarizations from a holding potential of - 80 mV to the indicated potentials ( the voltage protocol is schematized in the upper left corner ) . Representative current traces upon step depolarizations to the reversal potential ( Erev ) for 20 ms to obtain movement of the ON - gating charges ( Qon ) , and subsequent to - 50 mV for 10 ms to obtain tail currents ( Itail ) . Insets : magnifications of exemplary Qon for CaV1 . 2 WT , K1647A , Y1649A , I1654A , F1658A and K1662E . Plots of Itail ( in this panel corrected for variations in cell capacitance ) versus total detectable charge transfer for Qon . Slopes of regression curves are strongly reduced for CaV1 . 2 K1647A , Y1649A and I1654A versus WT ( see Table 7 for more details ) . Dot plots and means + SEM of Qon ( in this panel corrected for variations in cell capacitance ; * p < 0 . 05 ; * * p < 0 . 01 ; one - way ANOVA with Bonferroni post - hoc test ; n = 13 - 18 ; see Table 7 for more details ) . The reduction in slope of the regression curve of combined population data for K1647A , Y1649A and I1654A versus WT indicates reduced coupling of Itail with Qon when α - actinin binding to the IQ motif is diminished .

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

HEK293 cells were transfected with α11.2, α2δ-1, and β2A before whole cell patch recording in 20 mM Ba2+. Representative current traces of the first 2 ms obtained from recordings upon depolarizations from a holding potential of -80 mV to the indicated potentials (the voltage protocol is schematized in the upper left corner). Representative current traces upon step depolarizations to the reversal potential (Erev) for 20 ms to obtain movement of the ON-gating charges (Qon), and subsequent to -50 mV for 10 ms to obtain tail currents (Itail). Insets: magnifications of exemplary Qon for CaV1.2 WT, K1647A, Y1649A, I1654A, F1658A and K1662E. Plots of Itail (in this panel corrected for variations in cell capacitance) versus total detectable charge transfer for Qon. Slopes of regression curves are strongly reduced for CaV1.2 K1647A, Y1649A and I1654A versus WT (see Table 7 for more details). Dot plots and means + SEM of Qon (in this panel corrected for variations in cell capacitance; *p<0.05; **p<0.01; one-way ANOVA with Bonferroni post-hoc test; n = 13-18; see Table 7 for more details). The reduction in slope of the regression curve of combined population data for K1647A, Y1649A and I1654A versus WT indicates reduced coupling of Itail with Qon when α-actinin binding to the IQ motif is diminished.


( C ) Fluorescence polarization assays demonstrating that a peptide containing the BH3 ‐ like domain of Beclin ‐ 1 interacts with Bcl ‐ XL . Recombinant Bcl ‐ XL ΔTM was incubated with carboxyfluorescein ‐ labeled Bak BH3 peptide , in the absence or presence of wild ‐ type ( WT ) Beclin ‐ 1 BH3 or either of the two mutant peptides described in B .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Fluorescence polarization assays demonstrating that a peptide containing the BH3‐like domain of Beclin‐1 interacts with Bcl‐XL. Recombinant Bcl‐XL ΔTM was incubated with carboxyfluorescein‐labeled Bak BH3 peptide, in the absence or presence of wild‐type (WT) Beclin‐1 BH3 or either of the two mutant peptides described in B.


( D ) Affinity determination of the interaction between recombinant Bcl ‐ XLΔTM and fluorescent Beclin ‐ 1 BH3 , by fluorescence polarization . A comparison between WT Bcl ‐ XL ( squares ) and G138A Bcl ‐ XL ( triangles ) is shown ( means ± s . d . , n = 3 separate 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

(D) Affinity determination of the interaction between recombinant Bcl‐XLΔTM and fluorescent Beclin‐1 BH3, by fluorescence polarization. A comparison between WT Bcl‐XL(squares) and G138A Bcl‐XL (triangles) is shown (means±s.d., n=3 separate experiments).


( E ) Co ‐ immunoprecipitation of Bcl ‐ XL and WT or mutant Beclin ‐ 1 . HeLa cells were transfected with the indicated constructs , followed by immunoprecipitation of Flag ‐ tagged Bcl ‐ XL and immunochemical detection of Beclin ‐ 1 .

O O O O O O O O O O O O O O O O O O O O O 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) Co‐immunoprecipitation of Bcl‐XL and WT or mutant Beclin‐1. HeLa cells were transfected with the indicated constructs, followed by immunoprecipitation of Flag‐tagged Bcl‐XL and immunochemical detection of Beclin‐1.


( F ) Co ‐ immunoprecipitation of Beclin ‐ 1 with ‐ WT and mutant ( G138A ) Bcl ‐ XL . Results are representative of at least three experiments yielding similar results .

O O O O O O O O O O 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) Co‐immunoprecipitation of Beclin‐1 with‐WT and mutant (G138A) Bcl‐XL. Results are representative of at least three experiments yielding similar results.


( A ) Competition between Beclin ‐ 1 BH3 and ABT737 for Bcl ‐ XL binding . A fluorescent 25 ‐ mer peptide containing the BH3 ‐ like domain of Beclin ‐ 1 ( Figure 1D ) was docked to recombinant Bcl ‐ XL ΔTM protein in the absence or presence of ABT737 . The IC50 of ABT737 , as measured in the presence of 15 nM of peptide and 100 nM of Bcl ‐ XL ΔTM , was 1 . 7 μM .

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 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

(A) Competition between Beclin‐1 BH3 and ABT737 for Bcl‐XL binding. A fluorescent 25‐mer peptide containing the BH3‐like domain of Beclin‐1 ( Figure 1D) was docked to recombinant Bcl‐XL ΔTM protein in the absence or presence of ABT737. The IC50 of ABT737, as measured in the presence of 15 nM of peptide and 100 nM of Bcl‐XL ΔTM, was 1.7 μM.


( B , C ) Abolition of the interaction between Bcl ‐ XL and Beclin ‐ 1 by ABT737 in intact cells . Co ‐ immunoprecipitation assays were performed on HeLa cells transfected 48 h earlier with the indicated constructs as in Figure 1E . Sixteen hours before the immunoprecipitation , cells were exposed to ABT737 . Similar results were obtained for co ‐ transfected Bcl ‐ XL and Beclin ‐ 1 ( B ) and for endogenous Beclin ‐ 1 interacting with Flag ‐ tagged Bcl ‐ XL ( C ) .

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(B, C) Abolition of the interaction between Bcl‐XL and Beclin‐1 by ABT737 in intact cells. Co‐immunoprecipitation assays were performed on HeLa cells transfected 48 h earlier with the indicated constructs as in Figure 1E. Sixteen hours before the immunoprecipitation, cells were exposed to ABT737. Similar results were obtained for co‐transfected Bcl‐XL and Beclin‐1 (B) and for endogenous Beclin‐1 interacting with Flag‐tagged Bcl‐XL (C).


( D , E ) Differential effect of ABT737 on the interaction between Bcl ‐ 2 ( D ) or Mcl ‐ 1 ( E ) and Beclin ‐ 1 . This experiment was designed as ( B ) . All experiments have been performed at least three times , with similar results .

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(D, E) Differential effect of ABT737 on the interaction between Bcl‐2 (D) or Mcl‐1 (E) and Beclin‐1. This experiment was designed as (B). All experiments have been performed at least three times, with similar results.


( A , B ) Detection of autophagic vacuoles by LC3 ‐ GFP and their modulation by ABT737 and by Beclin ‐ 1 ‐ specific siRNAs . HeLa cells were transfected with control or Beclin ‐ 1 ‐ specific siRNAs , 24 h later re ‐ transfected with LC3 ‐ GFP , cultured in complete medium ( CM ) for 24 h , and finally kept 12 h either in CM or in nutrient ‐ free ( NF ) conditions , in the presence or absence of 1 μM ABT737 . Representative microphotographs of cells cultured in NF medium are shown in ( A ) and the percentage ( means ± s . d . , n = 3 separate experiments ) of LC3 ‐ GFP ‐ transfected cells bearing LC3 ‐ GFP aggregates in the cytoplasm ( LC3 ‐ GFPvac ) are quantified in ( B ) . The insert in ( B ) demonstrates the efficiency of the Beclin ‐ 1 ‐ specific siRNAs , as quantified by immunoblot .

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(A, B) Detection of autophagic vacuoles by LC3‐GFP and their modulation by ABT737 and by Beclin‐1‐specific siRNAs. HeLa cells were transfected with control or Beclin‐1‐specific siRNAs, 24 h later re‐transfected with LC3‐GFP, cultured in complete medium (CM) for 24 h, and finally kept 12 h either in CM or in nutrient‐free (NF) conditions, in the presence or absence of 1 μM ABT737. Representative microphotographs of cells cultured in NF medium are shown in (A) and the percentage (means±s.d., n=3 separate experiments) of LC3‐GFP‐transfected cells bearing LC3‐GFP aggregates in the cytoplasm (LC3‐GFPvac) are quantified in (B). The insert in (B) demonstrates the efficiency of the Beclin‐1‐specific siRNAs, as quantified by immunoblot.


( C , D ) Detection of cytoplasmicvacuoles using chloromethylfluorescein diacetate ( CMFDA ) . Cells were transfected with control or Beclin ‐ 1 ‐ specific siRNAs , cultured for 48 h in CM , washed , cultured in CM ( D ) or NF ( C , D ) for 12 h , stained with CMFDA , and either photographed ( C ) or subjected to the quantification of the cells that bear at least one discernible cytoplasmicvacuole ( arrow head ) ( means ± s . d . , n = 3 separate experiments ) .

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(C, D) Detection of cytoplasmicvacuoles using chloromethylfluorescein diacetate (CMFDA). Cells were transfected with control or Beclin‐1‐specific siRNAs, cultured for 48 h in CM, washed, cultured in CM (D) or NF (C, D) for 12 h, stained with CMFDA, and either photographed (C) or subjected to the quantification of the cells that bear at least one discernible cytoplasmicvacuole (arrow head) (means±s.d., n=3 separate experiments).


( E ) Ultrastructure of autophagic vacuoles induced by ABT737 . Transmission electron microphotographs are shown .

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(E) Ultrastructure of autophagic vacuoles induced by ABT737. Transmission electron microphotographs are shown.


( F ) Detection of dead and dying cells in the cultures . Cells treated as in ( A ) were stained with the ΔΨm ‐ sensitive dye DiOC6 ( 3 ) and the vital dye propidium iodide ( PI ) . The black portions of the columns refer to the DiOC6 ( 3 ) low PI + population ( dead ) and the remaining part of the column corresponds to the DiOC6 ( 3 ) low PI − ( dying ) population . Results are means ± s . d . of three independent experiments .

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(F) Detection of dead and dying cells in the cultures. Cells treated as in (A) were stained with the ΔΨm‐sensitive dye DiOC6(3) and the vital dye propidium iodide (PI). The black portions of the columns refer to the DiOC6(3)low PI+ population (dead) and the remaining part of the column corresponds to the DiOC6(3)low PI− (dying) population. Results are means±s.d. of three independent experiments.


Quantification of autophagic vacuoles induced by ABT737 . HeLa cells transfected with the indicated siRNAs ( specific for Emerin or for Beclin ‐ 1 at 0 h ) were re ‐ transfected with LC3 ‐ GFP finally cultured in nutrient ‐ free ( NF ) conditions ( 60 - 72 h ) , in the presence or absence 1 μM ABT737 and then subjected to electron microscopy detection of immature ( AV1 ) or mature ( AV2 ) autophagic vacuoles . Representative pictures are shown in ( A ) .

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Quantification of autophagic vacuoles induced by ABT737. HeLa cells transfected with the indicated siRNAs (specific for Emerin or for Beclin‐1 at 0 h) were re‐transfected with LC3‐GFP finally cultured in nutrient‐free (NF) conditions (60-72 h), in the presence or absence 1 μM ABT737 and then subjected to electron microscopy detection of immature (AV1) or mature (AV2) autophagic vacuoles. Representative pictures are shown in (A).


The number of AV1 and AV2 was determined for a minimum of 50 cells ( means ± s . e . m . ) ( B ) .

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The number of AV1 and AV2 was determined for a minimum of 50 cells (means±s.e.m.) (B).


( A - D ) Modulation of Beclin ‐ 1 ‐ induced autophagy by BH3 mutants and ABT737 . Cells were transfected with GFP ‐ LC3 together with an empty control vector or Beclin ‐ 1 ( WT , L116A , F123A ) and cultured for 48 h , followed by overnight culture in CM ( B ) or NF ( A , B ) in the absence or presence of 1 μM ABT737 . Representative microphotographs are shown in ( A ) and the LC3 ‐ GFP ‐ positive vacuoles per cell are quantified in ( B ) ( means ± s . d . ; n = 3 separate experiments ) . Alternatively , cells were not transfected and stained with CMFDA to detect vacuoles , as shown in ( C ) and ( D ) , yielding similar results as for the LC3 ‐ GFP method .

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(A-D) Modulation of Beclin‐1‐induced autophagy by BH3 mutants and ABT737. Cells were transfected with GFP‐LC3 together with an empty control vector or Beclin‐1 (WT, L116A, F123A) and cultured for 48 h, followed by overnight culture in CM (B) or NF (A, B) in the absence or presence of 1 μM ABT737. Representative microphotographs are shown in (A) and the LC3‐GFP‐positive vacuoles per cell are quantified in (B) (means±s.d.; n=3 separate experiments). Alternatively, cells were not transfected and stained with CMFDA to detect vacuoles, as shown in (C) and (D), yielding similar results as for the LC3‐GFP method.


( E ) BH3 ‐ dependent modulation of Beclin ‐ 1 ‐ induced autophagy by anti ‐ apoptotic Bcl ‐ 2 proteins . Cells were transfected simultaneously with Beclin ‐ 1 ( or control vector ) and equivalent amounts of plasmids coding for WT Bcl ‐ XL , Bcl ‐ XL G138A , Bcl ‐ 2 or Mcl ‐ 1 ( or control vector ) , as well as GFP ‐ LC3 . The culture in CM or NF in the absence or presence of ABT737 was performed during the last 12 h of the 60 h ‐ long experiment . The percentage of cells exhibiting the accumulation of LC3 ‐ GFP in vacuoles ( LC3 ‐ GFPvac ) is quantified as means ± s . d . ( n = 3 separate experiments ) .

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(E) BH3‐dependent modulation of Beclin‐1‐induced autophagy by anti‐apoptotic Bcl‐2 proteins. Cells were transfected simultaneously with Beclin‐1 (or control vector) and equivalent amounts of plasmids coding for WT Bcl‐XL, Bcl‐XL G138A, Bcl‐2 or Mcl‐1 (or control vector), as well as GFP‐LC3. The culture in CM or NF in the absence or presence of ABT737 was performed during the last 12 h of the 60 h‐long experiment. The percentage of cells exhibiting the accumulation of LC3‐GFP in vacuoles (LC3‐GFPvac) is quantified as means±s.d. (n=3 separate experiments).


( A ) Immunoprecipitation of endogenous Bcl ‐ 2 and Beclin ‐ 1 . Untransfected HeLa or MV4 . 11 cells were treated by culture in nutrient ‐ free EBSS or with ABT737 ( 1 μM ) .

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(A) Immunoprecipitation of endogenous Bcl‐2 and Beclin‐1. Untransfected HeLa or MV4.11 cells were treated by culture in nutrient‐free EBSS or with ABT737 (1 μM).


( B ) Immunoprecipitation of wild ‐ type and ER ‐ targeted Bcl ‐ 2 with Beclin ‐ 1 . Cells stably transfected with wild type , ER ‐ and mitochondrion ‐ targeted Bcl ‐ 2 were subjected to starvation or treated with ABT737 , followed by immunoprecipitation of Bcl ‐ 2 and immunodetection of Beclin ‐ 1 .

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(B) Immunoprecipitation of wild‐type and ER‐targeted Bcl‐2 with Beclin‐1. Cells stably transfected with wild type, ER‐ and mitochondrion‐targeted Bcl‐2 were subjected to starvation or treated with ABT737, followed by immunoprecipitation of Bcl‐2 and immunodetection of Beclin‐1.


( C , D ) Immunoprecipitation of Bcl ‐ 2 and Beclin ‐ 1 in subcellular fractions . Cells treated as in ( B ) were lysed and subjected to subcellular fractionation to enrich either ER vesicles ( microsomes , ( C ) ) or mitochondria ( heavy membrane fraction , ( D ) ) , followed by immunoprecipitation of Bcl ‐ 2 and immunodetection of Beclin ‐ 1 . Calreticulin and Hsp60 were used as internal controls . Results are representative of three independent determinations .

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(C, D) Immunoprecipitation of Bcl‐2 and Beclin‐1 in subcellular fractions. Cells treated as in (B) were lysed and subjected to subcellular fractionation to enrich either ER vesicles (microsomes, (C)) or mitochondria (heavy membrane fraction, (D)), followed by immunoprecipitation of Bcl‐2 and immunodetection of Beclin‐1. Calreticulin and Hsp60 were used as internal controls. Results are representative of three independent determinations.


( A , B ) Interactions between Bcl ‐ XL , Beclin ‐ 1 and Bad in conditions of autophagy induction . Cells were either treated by nutrient depletion ( A ) or addition of 1 μM rapamycin , 1 mM lithium chloride , 100 μM L ‐ 690 , 330 or 50 μM carbamazepine ( B ) , followed by immunprecipitation of Bcl ‐ XL ( as in Figure 2C ) and revealing the immunoblots by antibodies specific for Bcl ‐ XL , Beclin ‐ 1 or Bad .

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(A, B) Interactions between Bcl‐XL, Beclin‐1 and Bad in conditions of autophagy induction. Cells were either treated by nutrient depletion (A) or addition of 1 μM rapamycin, 1 mM lithium chloride, 100 μM L‐690,330 or 50 μM carbamazepine (B), followed by immunprecipitation of Bcl‐XL (as in Figure 2C) and revealing the immunoblots by antibodies specific for Bcl‐XL, Beclin‐1 or Bad.


( C ) Interaction between endogenous Bad and Bcl ‐ 2 . HeLa cells were treated with ABT737 ( 1 μM ) or nutrient ‐ depleted , followed by immunoprecipitation of Bcl ‐ 2 and immunodetection of Bad .

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(C) Interaction between endogenous Bad and Bcl‐2. HeLa cells were treated with ABT737 (1 μM) or nutrient‐depleted, followed by immunoprecipitation of Bcl‐2 and immunodetection of Bad.


( D , E ) Impact of Bad depletion on autophagy . Cells were transfected with LC3 ‐ GFP together with siRNAs specific for Bad , siRNAs specific for emerin or the Beclin ‐ 1 ‐ associated PI3 kinase Vps34 . Forty ‐ eight hours later , when the siRNAs had down ‐ regulated the proteins of interest ( D ) , cells were subjected by nutrient depletion ( NF ) or addition of rapamycin ( E ) and the frequency of cells exhibiting LC3 ‐ GFP aggregation as a marker of autophagy was assessed after 18 h ( means ± s . d . , * P0 . 05 , n = 3 separate 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 B-CONTROLLED_VAR 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

(D, E) Impact of Bad depletion on autophagy. Cells were transfected with LC3‐GFP together with siRNAs specific for Bad, siRNAs specific for emerin or the Beclin‐1‐associated PI3 kinase Vps34. Forty‐eight hours later, when the siRNAs had down‐regulated the proteins of interest (D), cells were subjected by nutrient depletion (NF) or addition of rapamycin (E) and the frequency of cells exhibiting LC3‐GFP aggregation as a marker of autophagy was assessed after 18 h (means±s.d., *P0.05, n=3 separate experiments).


( F ) Effect of the Bad knock ‐ out on autophagy . Wild Type ( WT ) or Bad − / − MEF were transfected with LC3 ‐ GFP and then subjected to nutrient depletion ( NF ) and / or treatment with ABT737 ( 18h ; means ± s . d . , n = 3 ) . The asterisks denotes a significant ( P0 . 05 ) effect of Bad deficiency .

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(F) Effect of the Bad knock‐out on autophagy. Wild Type (WT) or Bad−/− MEF were transfected with LC3‐GFP and then subjected to nutrient depletion (NF) and/or treatment with ABT737 (18h; means±s.d., n=3). The asterisks denotes a significant (P0.05) effect of Bad deficiency.


( G ) Effect of the Bad knockout on the Beclin ‐ 1 / Bcl ‐ 2 interaction . WT or Bad − / − MEF were subjected to the indicated treatments , followed by immunoprecipitation of Bcl ‐ 2 and immunobloting of Beclin ‐ 1 .

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(G) Effect of the Bad knockout on the Beclin‐1/Bcl‐2 interaction. WT or Bad−/− MEF were subjected to the indicated treatments, followed by immunoprecipitation of Bcl‐2 and immunobloting of Beclin‐1.


( H ) Overexpression of Bad triggers autophagy . Cells were transfected with LC3 ‐ GFP alone ( Control ) , together with vector ‐ only or with a vector encoding Bad , and subjected 48 h later to nutrient depletion ( NF ) . Cells were cultured in the continuous presence or absence of the pan ‐ caspase inhibitor Z ‐ VAD ‐ fmk ( 50 μM ) for 16 h , followed by assessment of autophagy as in ( E ) ( means ± s . d . , n = 3 separate experiments , * P0 . 01 ) . Immunoblots are representative of at least three independent experiments .

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(H) Overexpression of Bad triggers autophagy. Cells were transfected with LC3‐GFP alone (Control), together with vector‐only or with a vector encoding Bad, and subjected 48 h later to nutrient depletion (NF). Cells were cultured in the continuous presence or absence of the pan‐caspase inhibitor Z‐VAD‐fmk (50 μM) for 16 h, followed by assessment of autophagy as in (E) (means±s.d., n=3 separate experiments, *P0.01). Immunoblots are representative of at least three independent experiments.


( A ) Representative images of wt and egl ‐ 1 mutant embryos , bearing the plgg −

(A) Representative images of wt and egl‐1 mutant embryos, bearing the plgg−1DsRED


( B ) Quantification of LGG ‐ 1 ∷ DsRED fluorescence as determined in ( A ) . Each point represents the measurement for one individual embryo . Horizontal bars denote means and gray areas denote standard errors .

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(B) Quantification of LGG‐1∷DsRED fluorescence as determined in (A). Each point represents the measurement for one individual embryo. Horizontal bars denote means and gray areas denote standard errors.


Differential expression analysis for ACE2 expression changes in treated vs control samples was performed with the level 3 CMAP data of 24 hours response values using limma ( Ritchie et al , 2015 ; Methods ) . Volcano plots showing the log fold - change ( X - axis ) and uncorrected negative log10P value ( Y - axis ) of ACE2 expression changes are given for ( A ) 48 antihypertensive drugs The enrichment of positive / negative ACE2 expression regulators in different drug classes based on their mechanism of action ( MOA ) was tested with the GSEA method as implemented in the R package fgsea ( Segushichev , 2016 ; Methods ) , and the enrichment significance ( negative log10 P values ) is shown in bar plots in ( B ) , for the analysis on the 48 antihypertensive drugs CCBs , calcium channel blockers ; ARBs , angiotensin II type - I receptor blockers ; ACEIs , angiotensin - converting enzyme

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Differential expression analysis for ACE2 expression changes in treated vs control samples was performed with the level 3 CMAP data of 24 hours response values using limma (Ritchie et al, 2015; Methods). Volcano plots showing the log fold-change (X-axis) and uncorrected negative log10P value (Y-axis) of ACE2 expression changes are given for (A) 48 antihypertensive drugs The enrichment of positive/negative ACE2 expression regulators in different drug classes based on their mechanism of action (MOA) was tested with the GSEA method as implemented in the R package fgsea (Segushichev, 2016; Methods), and the enrichment significance (negative log10 P values) is shown in bar plots in (B) , for the analysis on the 48 antihypertensive drugs CCBs, calcium channel blockers; ARBs, angiotensin II type-I receptor blockers; ACEIs, angiotensin-converting enzyme inhibitors


Differential expression analysis for ACE2 expression changes in treated vs control samples was performed with the level 3 CMAP data of 24 hours response values using limma ( Ritchie et al , 2015 ; Methods ) . ( C ) 672 clinically approved drugs that are each tested on four carcinoma cell lines from CMAP ( Subramanian et al , 2017 ) .

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Differential expression analysis for ACE2 expression changes in treated vs control samples was performed with the level 3 CMAP data of 24 hours response values using limma (Ritchie et al, 2015; Methods). (C) 672 clinically approved drugs that are each tested on four carcinoma cell lines from CMAP (Subramanian et al, 2017).


Differential expression analysis for ACE2 expression changes in treated vs control samples was performed with the level 3 CMAP data of 24 hours response values using limma ( Ritchie et al , 2015 ; Methods ) . The ACE2 expression levels in control and drug - treated groups for the top significant drugs ( vorinostat , panobinostat , and isotretinoin ) among the 672 clinically approved drugs are shown with boxplots in ( D ) , the ACE2 expression log fold - change values and adjusted P values from limma , as well as the number of samples per group are labeled for each drug . In the boxplot of panel ( D ) , the center line , box edges and whiskers denotes the median , interquartile range and the rest of the distribution in respective order , except for points that were determined to be outliers using a method that is a function of the interquartile range , as in standard box plots .

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Differential expression analysis for ACE2 expression changes in treated vs control samples was performed with the level 3 CMAP data of 24 hours response values using limma (Ritchie et al, 2015; Methods). The ACE2 expression levels in control and drug-treated groups for the top significant drugs (vorinostat, panobinostat, and isotretinoin) among the 672 clinically approved drugs are shown with boxplots in (D), the ACE2 expression log fold-change values and adjusted P values from limma, as well as the number of samples per group are labeled for each drug. In the boxplot of panel (D), the center line, box edges and whiskers denotes the median, interquartile range and the rest of the distribution in respective order, except for points that were determined to be outliers using a method that is a function of the interquartile range, as in standard box plots.


Differential expression analysis for ACE2 expression changes in treated vs control samples was performed with the level 3 CMAP data of 24 hours response values using limma ( Ritchie et al , 2015 ; Methods ) . The enrichment of positive / negative ACE2 expression regulators in different drug classes based on their mechanism of action ( MOA ) was tested with the GSEA method as implemented in the R package fgsea ( Segushichev , 2016 ; Methods ) , and the enrichment significance ( negative log10 P values ) is shown in bar plots in ( E ) , for the analysis on the 672 clinically approved drugs We further extended the analysis to a larger set of 989 clinically approved drugs tested on a total of 28 cell lines ( 16 cancer and 12 normal cells ; this list is provided in Table EV2A with details including primary site , subtype and donor demographics ) where each drug may have been tested on a different subset of cells ( Methods ) . and performed enrichment analysis using the WHO ATC drug classification data ( World Health Organization , 2006 ) , and top enriched drug classes are shown in ( F ) .

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Differential expression analysis for ACE2 expression changes in treated vs control samples was performed with the level 3 CMAP data of 24 hours response values using limma (Ritchie et al, 2015; Methods). The enrichment of positive/negative ACE2 expression regulators in different drug classes based on their mechanism of action (MOA) was tested with the GSEA method as implemented in the R package fgsea (Segushichev, 2016; Methods), and the enrichment significance (negative log10 P values) is shown in bar plots in (E), for the analysis on the 672 clinically approved drugs We further extended the analysis to a larger set of 989 clinically approved drugs tested on a total of 28 cell lines (16 cancer and 12 normal cells; this list is provided in Table EV2A with details including primary site, subtype and donor demographics) where each drug may have been tested on a different subset of cells (Methods). and performed enrichment analysis using the WHO ATC drug classification data (World Health Organization, 2006), and top enriched drug classes are shown in (F).


( A - D ) This Fig summarizes the differential expression analysis results for ACE2 upon treatment by clinically approved drugs in samples of lung and kidney datasets mined from the GEO database , spanning both cancer and non - cancer datasets ( Methods ) . Volcano plots showing the log fold - change ( X - axis ) and uncorrected negative log10P value ( Y - axis ) of ACE2 expression changes are displayed in ( A ) for 74 datasets of cells or tissue samples from lung , involving 42 clinically approved drugs , and in ( C ) for 35 kidney datasets involving 29 clinically approved drugs . Among these , we focused on the top significant drugs that modulate ACE2 expression in non - cancerous cells or tissue samples from lung and kidney , which are shown in ( B ) and ( D ) , respectively , where the ACE2 expression ( Y - axis ) difference between control and treated groups ( X - axis ) are shown with boxplots . In the title of each boxplot , the GEO identifier of the respective studies and the corresponding drug name and the sample type are provided . Different datasets involving experiments using the same drug were analyzed and presented separately , since the sample types can be different across datasets . All the P values are computed from differential expression analysis using limma ( Ritchie et al , 2015 ) ( Methods ) . In the boxplots of panels ( B ) and ( D ) , the center line , box edges and whiskers denotes the median , interquartile range and the rest of the distribution in respective order , except for points that were determined to be outliers using a method that is a function of the interquartile range , as in standard box plots . Abbreviations : HBEC , human primary bronchial epithelial cells .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-D) This Fig summarizes the differential expression analysis results for ACE2 upon treatment by clinically approved drugs in samples of lung and kidney datasets mined from the GEO database, spanning both cancer and non-cancer datasets (Methods). Volcano plots showing the log fold-change (X-axis) and uncorrected negative log10P value (Y-axis) of ACE2 expression changes are displayed in (A) for 74 datasets of cells or tissue samples from lung, involving 42 clinically approved drugs, and in (C) for 35 kidney datasets involving 29 clinically approved drugs. Among these, we focused on the top significant drugs that modulate ACE2 expression in non-cancerous cells or tissue samples from lung and kidney, which are shown in (B) and (D), respectively, where the ACE2 expression (Y-axis) difference between control and treated groups (X-axis) are shown with boxplots. In the title of each boxplot, the GEO identifier of the respective studies and the corresponding drug name and the sample type are provided. Different datasets involving experiments using the same drug were analyzed and presented separately, since the sample types can be different across datasets. All the P values are computed from differential expression analysis using limma (Ritchie et al, 2015) (Methods). In the boxplots of panels (B) and (D), the center line, box edges and whiskers denotes the median, interquartile range and the rest of the distribution in respective order, except for points that were determined to be outliers using a method that is a function of the interquartile range, as in standard box plots. Abbreviations: HBEC, human primary bronchial epithelial cells.


( A ) Recombinant c10orf76 directly binds to PI4KB in vitro . His - pulldown assays of baculovirus / Sf9 produced 6xHis - c10orf76 ( 3 μM ) were carried out with untagged PI4KB ( 2 . 5 μM ) .

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

(A) Recombinant c10orf76 directly binds to PI4KB in vitro. His-pulldown assays of baculovirus/Sf9 produced 6xHis-c10orf76 (3 μM) were carried out with untagged PI4KB (2.5 μM).


( B ) PI4KB and c10orf76 form a stable complex . The complex of c10orf76 - PI4KB eluted from a S200 superdex 10 / 300 GL increase gel filtration column ( GE ) at a volume consistent with a heterodimer ( 169 kDa ) , while c10orf76 alone eluted at a volume consistent with a monomer ( 79 kDa ) . Lines with MW values indicate elution of MW standards ( 158 kDa aldolase , 75 kDa conalbumin ) .

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(B) PI4KB and c10orf76 form a stable complex. The complex of c10orf76-PI4KB eluted from a S200 superdex 10/300 GL increase gel filtration column (GE) at a volume consistent with a heterodimer (169 kDa), while c10orf76 alone eluted at a volume consistent with a monomer (79 kDa). Lines with MW values indicate elution of MW standards (158 kDa aldolase, 75 kDa conalbumin).


( C ) PI4KB is potently inhibited by c10orf76 in a dose - dependent manner in vitro . Kinase assays of PI4KB ( 20 nM ) in the presence of varying concentrations of c10orf76 ( 1 . 6 nM - 1 μM ) were carried out on pure PI lipid vesicles ( 0 . 5 mg / L ) in the presence of 100 μM ATP . The data was normalized to the kinase activity of PI4KB alone . IC50 values were determined by one binding site , nonlinear regression ( curve fit ) using Graphpad . Error bars represent standard deviation of independent technical replicates ( n = 3 ) .

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(C) PI4KB is potently inhibited by c10orf76 in a dose-dependent manner in vitro. Kinase assays of PI4KB (20 nM) in the presence of varying concentrations of c10orf76 (1.6 nM-1 μM) were carried out on pure PI lipid vesicles (0.5 mg/L) in the presence of 100 μM ATP. The data was normalized to the kinase activity of PI4KB alone. IC50 values were determined by one binding site, nonlinear regression (curve fit) using Graphpad. Error bars represent standard deviation of independent technical replicates (n=3).


( D ) PI4KB is potently inhibited by c10orf76 on pure PI vesicles and vesicles mimicking Golgi composition . Kinase assays of PI4KB and c10orf76 were carried out on lipid substrate composed of pure PI vesicles ( 0 . 5 mg / mL ) with 100 μM ATP , and Golgi mimic vesicles ( 0 . 5 mg / ml , 10 % PS , 20 % PI , 25 % PE , 45 % PC ) with 10 μM ATP . PI4KB was present at 20 and 300 nM in the PI and Golgi substrate assays respectively , with c10orf76 present at 500 nM in both experiments . The data is normalized to the kinase activity of PI4KB alone . Error bars represent standard deviation of independent technical replicates ( n = 3 ) .

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(D) PI4KB is potently inhibited by c10orf76 on pure PI vesicles and vesicles mimicking Golgi composition. Kinase assays of PI4KB and c10orf76 were carried out on lipid substrate composed of pure PI vesicles (0.5 mg/mL) with 100 μM ATP, and Golgi mimic vesicles (0.5 mg/ml, 10% PS, 20% PI, 25% PE, 45% PC) with 10 μM ATP. PI4KB was present at 20 and 300 nM in the PI and Golgi substrate assays respectively, with c10orf76 present at 500 nM in both experiments. The data is normalized to the kinase activity of PI4KB alone. Error bars represent standard deviation of independent technical replicates (n=3).


( E ) Changes in deuterium incorporation PI4KB in the presence of c10orf76 showed a profound ordering of the kinase domain N - lobe linker and smaller changes in the helical domain and C - lobe of the kinase domain . The sum of the difference mapped as the difference in number of deuterons incorporated for PI4KB ( 400 nM ) in the presence and absence of c10orf76 ( 400 nM ) over all time points ( 3s at 1 oC ; 3s , 30s , and 300s at 23 oC ) . Each dot represents a peptide graphed on the x - axis according to the central residue . The red boxes highlight key regions that showed significant changes ( > 7 % decrease in exchange , > 0 . 5 Da difference , and unpaired two - tailed student t - test p < 0 . 05 ) . Error bars represent standard deviation of independent technical replicates ( n = 3 ) .

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(E) Changes in deuterium incorporation PI4KB in the presence of c10orf76 showed a profound ordering of the kinase domain N-lobe linker and smaller changes in the helical domain and C-lobe of the kinase domain. The sum of the difference mapped as the difference in number of deuterons incorporated for PI4KB (400 nM) in the presence and absence of c10orf76 (400 nM) over all time points (3s at 1 oC; 3s, 30s, and 300s at 23 oC). Each dot represents a peptide graphed on the x-axis according to the central residue. The red boxes highlight key regions that showed significant changes (>7% decrease in exchange, >0.5 Da difference, and unpaired two-tailed student t-test p<0.05). Error bars represent standard deviation of independent technical replicates (n=3).


( G ) Changes in the deuterium incorporation of c10orf76 in the presence of PI4KB . H / D exchange reactions displayed as the sum of the difference in HDX in the number of deuterons for c10orf76 ( 400 nM ) in the presence of PI4KB ( 400 nM ) at all time points ( 3s at 1 oC ; 3s , 30s , and 300s at 23 oC ) analyzed . Red boxes highlight regions that showed significant changes ( > 7 % decrease in exchange , > 0 . 5 Da difference , and unpaired two - tailed student t - test p < 0 . 05 ) . Error bars represent standard deviation of independent technical replicates ( n = 3 ) .

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(G) Changes in the deuterium incorporation of c10orf76 in the presence of PI4KB. H/D exchange reactions displayed as the sum of the difference in HDX in the number of deuterons for c10orf76 (400 nM) in the presence of PI4KB (400 nM) at all time points (3s at 1 oC; 3s, 30s, and 300s at 23 oC) analyzed. Red boxes highlight regions that showed significant changes (>7% decrease in exchange, >0.5 Da difference, and unpaired two-tailed student t-test p<0.05). Error bars represent standard deviation of independent technical replicates (n=3).


( H ) The PI4KB N - lobe linker undergoes a disorder - to - order transition upon binding c10orf76 . Selected peptides ( including the sequence , domain information , and numbering ) of both PI4KB and c10orf76 displayed as the % deuteration incorporation over time . Error bars represent standard deviation of independent technical replicates ( n = 3 ) , and are typically smaller than the size of the point on the graph .

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(H) The PI4KB N-lobe linker undergoes a disorder-to-order transition upon binding c10orf76. Selected peptides (including the sequence, domain information, and numbering) of both PI4KB and c10orf76 displayed as the % deuteration incorporation over time. Error bars represent standard deviation of independent technical replicates (n=3), and are typically smaller than the size of the point on the graph.


( B ) The N - lobe kinase linker of PI4KB can be efficiently phosphorylated by PKA . Recombinant PKA at different concentrations ( 0 , 7 , 34 , 168 , or 840 ng ) was incubated with recombinant ( E . coli ) wild - type PI4KB ( 20 μg ) for 1 hour with 200 μM ATP and the amount of phosphorylation was followed using mass spectrometry . Relative abundance of Ser496 phosphorylated PI4KB was calculated using the relative intensity ( total area ) of the phosphorylated vs non - phosphorylated peptide ( 486 - 506 ) .

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(B) The N-lobe kinase linker of PI4KB can be efficiently phosphorylated by PKA. Recombinant PKA at different concentrations (0, 7, 34, 168, or 840 ng) was incubated with recombinant (E. coli) wild-type PI4KB (20 μg) for 1 hour with 200 μM ATP and the amount of phosphorylation was followed using mass spectrometry. Relative abundance of Ser496 phosphorylated PI4KB was calculated using the relative intensity (total area) of the phosphorylated vs non-phosphorylated peptide (486-506).


( C ) PI4KB phosphorylation by PKA alters the affinity for c10orf76 . The kinase activity of different variants of PI4KB ( 15 nM ) was measured in the presence of varying amounts of c10orf76 ( 23 nM - 1 . 5μM ) with 100 % PI lipid substrate ( 0 . 5 mg / L ) and 100 μM ATP . The data was normalized to the kinase activity of PI4KB alone Error bars represent standard deviation of independent technical replicates ( n = 3 ) .

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(C) PI4KB phosphorylation by PKA alters the affinity for c10orf76. The kinase activity of different variants of PI4KB (15 nM) was measured in the presence of varying amounts of c10orf76 (23 nM-1.5μM) with 100% PI lipid substrate (0.5 mg/L) and 100 μM ATP. The data was normalized to the kinase activity of PI4KB alone Error bars represent standard deviation of independent technical replicates (n=3).


( D ) PI4KB has the same kinase activity when Ser496 is phosphorylated or mutated to alanine . Kinase assay of PI4KB non - phosphorylated , phos - Ser496 or S496A ( 15 nM ) on pure PI lipid vesicles ( 0 . 5 mg / L ) with 100 μM ATP . The data was normalized to the kinase activity of WT PI4KB . Error bars represent standard deviation of independent technical replicates ( n = 3 ) .

(D) PI4KB has the same kinase activity when Ser496 is phosphorylated or mutated to alanine. Kinase assay of PI4KB non-phosphorylated, phos-Ser496 or S496A (15 nM) on pure PI lipid vesicles (0.5 mg/L) with 100 μM ATP. The data was normalized to the kinase activity of WT PI4KB. Error bars represent standard deviation of independent technical replicates (n=3).


( E ) Engineered RL494EA PI4KB mutant shows decreased binding to c10orf76 . His - pulldown assays of 6xHis - c10orf76 ( 3 μM ) with wild - type or RL494EA PI4KB ( 1 - 2 μM ) .

(E) Engineered RL494EA PI4KB mutant shows decreased binding to c10orf76. His-pulldown assays of 6xHis-c10orf76 (3 μM) with wild-type or RL494EA PI4KB (1-2 μM).


( F ) RL494EA PI4KB activity is not inhibited by c10orf76 . Kinase assays of either wild type or mutant RL494EA PI4KB ( 40 nM ) were carried out with varying concentrations of c10orf76 ( 3 . 9 nM - 2μM ) with 100 % PI lipid vesicles ( 0 . 5 mg / L ) and 100 μM ATP . The data was normalized to the kinase activity of PI4KB alone . Error bars represent standard deviation of independent technical replicates ( n = 3 ) .

(F) RL494EA PI4KB activity is not inhibited by c10orf76. Kinase assays of either wild type or mutant RL494EA PI4KB (40 nM) were carried out with varying concentrations of c10orf76 (3.9 nM-2μM) with 100% PI lipid vesicles (0.5 mg/L) and 100 μM ATP. The data was normalized to the kinase activity of PI4KB alone. Error bars represent standard deviation of independent technical replicates (n=3).


( G ) Wild - type PI4KB and RL494EA PI4KB mutant have the same lipid kinase activity . Kinase assays of either wild - type and mutant PI4KB ( 10 nM ) were carried out with 100 % PI lipid vesicles ( 0 . 5 mg / L ) , 100 μM ATP , and PI4KB ( 300 nM ) on Golgi - mimic vesicles ( 0 . 5 mg / mL ) with 10 μM ATP . The data was normalized to the kinase activity of WT PI4KB . Error bars represent standard deviation of independent technical replicates ( n = 3 ) .

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(G) Wild-type PI4KB and RL494EA PI4KB mutant have the same lipid kinase activity. Kinase assays of either wild-type and mutant PI4KB (10 nM) were carried out with 100% PI lipid vesicles (0.5 mg/L), 100 μM ATP, and PI4KB (300 nM) on Golgi-mimic vesicles (0.5 mg/mL) with 10 μM ATP. The data was normalized to the kinase activity of WT PI4KB. Error bars represent standard deviation of independent technical replicates (n=3).


( H ) FLH409AAA - c10orf76 mutant shows decreased affinity for PI4KB . His - pulldown assays of 6xHis - c10orf76 ( 1 μM ) with wild - type PI4KB ( 1 μM ) . Samples washed a total of 4 times .

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(H) FLH409AAA-c10orf76 mutant shows decreased affinity for PI4KB. His-pulldown assays of 6xHis-c10orf76 (1 μM) with wild-type PI4KB (1 μM). Samples washed a total of 4 times.


( I ) Kinase assay shows FLH409AAA c10orf76 inhibition of PI4KB is greatly reduced . Kinase assay of PI4KB ( 40 nM ) and a concentration curve of c10orf76 ( 3 . 9 nM - 2μM ) on pure PI lipid vesicles ( 0 . 5 mg / L ) with 100 μM ATP . The data was normalized to the kinase activity of PI4KB alone . Error bars represent standard deviation of independent technical replicates ( n = 3 ) .

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(I) Kinase assay shows FLH409AAA c10orf76 inhibition of PI4KB is greatly reduced. Kinase assay of PI4KB (40 nM) and a concentration curve of c10orf76 (3.9 nM-2μM) on pure PI lipid vesicles (0.5 mg/L) with 100 μM ATP. The data was normalized to the kinase activity of PI4KB alone. Error bars represent standard deviation of independent technical replicates (n=3).


( A ) Transfections of HEK293 cells revealed that both wild - type GFP - PI4KB and RL494EA GFP - PI4KB localize to the Golgi .

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(A) Transfections of HEK293 cells revealed that both wild-type GFP-PI4KB and RL494EA GFP-PI4KB localize to the Golgi.


( B ) WT c10orf76 also localized to the Golgi , however , the PI4KB binding deficient mutant of c10orf76 ( FLH409AAA ) predominantly localized to the cytosol .

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(B) WT c10orf76 also localized to the Golgi, however, the PI4KB binding deficient mutant of c10orf76 (FLH409AAA) predominantly localized to the cytosol.


( D ) Mitochondria recruitment experiment with wild - type PI4KB and c10orf76 . Left : AKAP1 - FRB - CFP is localized to the mitochondria before ( top ) and 5 minutes after rapamycin ( 100 nM ) treatment ( bottom ) . Middle : mRFP - FKBP12 - PI4KB at high expression level is located in the cytosol before rapamycin ( top ) and translocates to the mitochondria after rapamycin induction ( bottom ) . Right : eGFP - c10orf76 at a high expression level is located in the cytosol before rapamycin ( top ) and translocates to the mitochondria after rapamycin induction ( bottom ) .

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(D) Mitochondria recruitment experiment with wild-type PI4KB and c10orf76. Left: AKAP1-FRB-CFP is localized to the mitochondria before (top) and 5 minutes after rapamycin (100 nM) treatment (bottom). Middle: mRFP-FKBP12-PI4KB at high expression level is located in the cytosol before rapamycin (top) and translocates to the mitochondria after rapamycin induction (bottom). Right: eGFP-c10orf76 at a high expression level is located in the cytosol before rapamycin (top) and translocates to the mitochondria after rapamycin induction (bottom).


( F ) Mitochondria recruitment experiment with mutant PI4KB and WT c10orf76 . Left : AKAP1 - FRB - CFP is localized to the mitochondria before ( top ) and 5 minutes after ( bottom ) rapamycin treatment . Middle : mRFP - FKBP12 - PI4KB ( RL494EA ) is located in the cytosol before rapamycin ( top ) and translocates to the mitochondria after rapamycin induction ( bottom ) . Right : eGFP - c10orf76 is located in the cytosol before ( top ) and after ( bottom ) rapamycin induction .

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(F) Mitochondria recruitment experiment with mutant PI4KB and WT c10orf76. Left: AKAP1-FRB-CFP is localized to the mitochondria before (top) and 5 minutes after (bottom) rapamycin treatment. Middle: mRFP-FKBP12-PI4KB(RL494EA) is located in the cytosol before rapamycin (top) and translocates to the mitochondria after rapamycin induction (bottom). Right: eGFP-c10orf76 is located in the cytosol before (top) and after (bottom) rapamycin induction.


( A HAP1 cells were fixed and stained with antibodies examining PI4P and PI4KB Data information : Nuclei were stained with DAPI ( blue ) . Scale bars represent 10 μm .

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(A HAP1 cells were fixed and stained with antibodies examining PI4P and PI4KB Data information: Nuclei were stained with DAPI (blue). Scale bars represent 10 μm.


B ) HAP1 cells were fixed and stained with antibodies examining Golgi morphology markers ( B ) . ( C ) Western blot of GBF1 , PI4KB and B - Actin levels in both wild - type and c10orf76 KO HAP1 cells . Data information : Nuclei were stained with DAPI ( blue ) . Scale bars represent 10 μm .

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B) HAP1 cells were fixed and stained with antibodies examining Golgi morphology markers (B). (C) Western blot of GBF1, PI4KB and B-Actin levels in both wild-type and c10orf76 KO HAP1 cells. Data information: Nuclei were stained with DAPI (blue). Scale bars represent 10 μm.


( D ) Markers of Arf1 activation - the coatomer proteins COPIα / γ and βCOP act as a readout for GTP - bound Arf1 , while the native coatomer was detected with the CM1 antibody . Data information : Nuclei were stained with DAPI ( blue ) . Scale bars represent 10 μm .

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(D) Markers of Arf1 activation - the coatomer proteins COPIα/γ and βCOP act as a readout for GTP-bound Arf1, while the native coatomer was detected with the CM1 antibody. Data information: Nuclei were stained with DAPI (blue). Scale bars represent 10 μm.


( A ) Viral infection assays determining viral titers by end - point titration at 0 hours and 8 hours in HAP1 wild - type or c10orf76 knockout cells . Left : Coxsackievirus A10 infection . Middle : Poliovirus - 1 infection . Right : Coxsackievirus B3 infection . Titre values for c10orf76 knockout cells at 8 hours were statistically evaluated compared to c10orf76 knockout titres at 0 hours and wild - type titres at 8 hours using a one - way ANOVA . * * , P < 0 . 01 ; N . S . , P > 0 . 05 .

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(A) Viral infection assays determining viral titers by end-point titration at 0 hours and 8 hours in HAP1 wild-type or c10orf76 knockout cells. Left: Coxsackievirus A10 infection. Middle: Poliovirus-1 infection. Right: Coxsackievirus B3 infection. Titre values for c10orf76 knockout cells at 8 hours were statistically evaluated compared to c10orf76 knockout titres at 0 hours and wild-type titres at 8 hours using a one-way ANOVA. **, P<0.01; N.S., P>0.05.


( B ) Viral infection assays determining virus titers by end point titration at 8 hours in HeLa wild - type and PI4KB knockout cells upon transfection of wild - type PI4KB , the complex - disrupting RL494EA PI4KB mutant or the kinase dead D674A PI4KB mutant . Left : Coxsackievirus A10 infection . Middle : Poliovirus - 1 infection . Right : Coxsackievirus B3 infection . Values were statistically evaluated compared to the GalT control using a one - way ANOVA . * * , P < 0 . 01 ; * , P < 0 . 05 ; N . S . , P > 0 . 05 .

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(B) Viral infection assays determining virus titers by end point titration at 8 hours in HeLa wild-type and PI4KB knockout cells upon transfection of wild-type PI4KB, the complex-disrupting RL494EA PI4KB mutant or the kinase dead D674A PI4KB mutant. Left: Coxsackievirus A10 infection. Middle: Poliovirus-1 infection. Right: Coxsackievirus B3 infection. Values were statistically evaluated compared to the GalT control using a one-way ANOVA. **, P<0.01; *, P<0.05; N.S., P>0.05.


( B ) Western blot of RNAi efficiency and specificity for oligos used in ( C - G ) . HeLa Kyoto cells were incubated for 72 h with control oligos or RNAi - oligos depleting CUL4A , CUL4B or DDB1 . Specific downregulation of the indicated proteins was analyzed by immunoblotting . GAPDH controls for equal loading . ( C ) Quantification of western blot bands and the ratio between neddylated vs unneddylated CUL4 with the paralog downregulated is calculated . Graphs displays mean with SEM of N = 3 biological replicates . * * P = 0 . 001 .

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(B) Western blot of RNAi efficiency and specificity for oligos used in (C-G). HeLa Kyoto cells were incubated for 72 h with control oligos or RNAi-oligos depleting CUL4A, CUL4B or DDB1. Specific downregulation of the indicated proteins was analyzed by immunoblotting. GAPDH controls for equal loading. (C) Quantification of western blot bands and the ratio between neddylated vs unneddylated CUL4 with the paralog downregulated is calculated. Graphs displays mean with SEM of N=3 biological replicates. **P = 0.001.


( E ) Flow cytometry analysis of Hela cells treated with the indicated RNAi for 72h and immune - stained for γH2A . X , DNA staining with propidium iodide . One of three biological replicates is represented .

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(E) Flow cytometry analysis of Hela cells treated with the indicated RNAi for 72h and immune-stained for γH2A.X, DNA staining with propidium iodide. One of three biological replicates is represented.


( G ) Live - cell imaging of mitosis in RNAi depleted HeLa Kyoto cells . Brightfield images were taken every 3 min , and mitotic duration was quantified by measuring the time from cell rounding to telophase . Mean with SEM is depicted from N = 3 biological experiments analyzing a total of 30 single cells . nsP = 0 . 9995 ; * * P = 0 . 0040 ; * * P = 0 . 0044 . Red symbols mark incomplete mitotic events during the movie . White arrows mark cells undergoing mitosis . Scale bar : 10 μm .

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(G) Live-cell imaging of mitosis in RNAi depleted HeLa Kyoto cells. Brightfield images were taken every 3 min, and mitotic duration was quantified by measuring the time from cell rounding to telophase. Mean with SEM is depicted from N=3 biological experiments analyzing a total of 30 single cells. nsP= 0.9995; **P=0.0040; **P= 0.0044. Red symbols mark incomplete mitotic events during the movie. White arrows mark cells undergoing mitosis. Scale bar: 10 μm.


( A ) CUL4A and CUL4B were analyzed by immunoblotting of extracts prepared from HeLa Kyoto cells arrested in S - phase ( S = thymidine ) , G2 ( R0 - 3306 ) , or mitosis ( M = nocodazole or taxol ) . One of three biological replicates is shown . H3 pS10 ( phosphorylated histone H3 at serine 10 ) was used as a mitotic marker . Same samples but separate blot for H3pS10 staining due to size differences .

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(A) CUL4A and CUL4B were analyzed by immunoblotting of extracts prepared from HeLa Kyoto cells arrested in S-phase (S= thymidine), G2 (R0-3306), or mitosis (M= nocodazole or taxol). One of three biological replicates is shown. H3 pS10 (phosphorylated histone H3 at serine 10) was used as a mitotic marker. Same samples but separate blot for H3pS10 staining due to size differences.


( C ) Mitotic population of unperturbed cells was harvested by shake - off . Three subsequent harvests are depicted ( R1 - R3 ) . The interphase population are cells that remained attached to the culture dish .

(C) Mitotic population of unperturbed cells was harvested by shake-off. Three subsequent harvests are depicted (R1-R3). The interphase population are cells that remained attached to the culture dish.


( G ) In vitro kinase assays with purified CDK1 - cyclinB and Sf9 - purified CUL4B and mutant proteins in the presence of [ γ - 32P ] ATP . The autoradiogram ( top panel ) shows CUL4B phosphorylation , while coomassie staining ( lower panel ) controls equal loading of CUL4B and mutants ( N = 3 technical replicates for WT , P50L ; NP1 ; N = 2 for NP1 - A49T and NP1 - A49T - P50L ) . Note that the A49T mutation in the NP1 mutant restores phosphorylation of the T49 residue , but only in the presence of the adjacent proline at position 50 .

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(G) In vitro kinase assays with purified CDK1-cyclinB and Sf9-purified CUL4B and mutant proteins in the presence of [γ-32P]ATP. The autoradiogram (top panel) shows CUL4B phosphorylation, while coomassie staining (lower panel) controls equal loading of CUL4B and mutants (N=3 technical replicates for WT, P50L; NP1; N=2 for NP1-A49T and NP1-A49T-P50L). Note that the A49T mutation in the NP1 mutant restores phosphorylation of the T49 residue, but only in the presence of the adjacent proline at position 50.


( A ) Western blot analysis to monitor CUL4A or CUL4B levels in extracts prepared from HeLaFLP control cells or HeLaFLP cells deleted for CUL4B by CRISPR / Cas9 ( ∆ CUL4BFLP ) but stably expressing no CUL4B ( - ) , CUL4B or the indicated NP1 or P50L mutants from the doxycycline - inducible promoter . GAPDH was included to control equal loading . One of three biological replicates is shown .

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(A) Western blot analysis to monitor CUL4A or CUL4B levels in extracts prepared from HeLaFLP control cells or HeLaFLP cells deleted for CUL4B by CRISPR/Cas9 (∆CUL4BFLP) but stably expressing no CUL4B (-), CUL4B or the indicated NP1 or P50L mutants from the doxycycline-inducible promoter. GAPDH was included to control equal loading. One of three biological replicates is shown.


( D ) The expression levels of endogenous HA - tagged WT and the indicated CUL4B mutants were compared by immunoblotting with HA . II antibody . GAPDH controls for equal

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(D) The expression levels of endogenous HA-tagged WT and the indicated CUL4B mutants were compared by immunoblotting with HA.II antibody. GAPDH controls for equal loading


( F ) In vitro E3 ligase activity assays using the indicated reconstituted CRL4BCRBN complexes and the neo - substrate CKlα . At time 0 , lenalidomide was added to start the reaction and CKlα ubiquitination was followed at the times indicated ( in minutes ) by immunoblotting with CKlα antibody . N ≥ 3 technical replicates for CUL4B , CUL4A , P50L , K895R and L864fs * 13 ; N = 1 for NP1 . Note that the in vitro E3 ligase activity of CRL4B and the P50L patient mutation is comparable .

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(F) In vitro E3 ligase activity assays using the indicated reconstituted CRL4BCRBN complexes and the neo-substrate CKlα. At time 0, lenalidomide was added to start the reaction and CKlα ubiquitination was followed at the times indicated (in minutes) by immunoblotting with CKlα antibody. N≥3 technical replicates for CUL4B, CUL4A, P50L, K895R and L864fs*13; N=1 for NP1. Note that the in vitro E3 ligase activity of CRL4B and the P50L patient mutation is comparable.


( C ) To control RNAi efficiency and specificity , HeLa Kyoto cells were incubated for 72 h with control oligos ( control ) or RNAi - oligos depleting CUL4B , LIS1 or WDR1 . Specific downregulation of the indicated proteins was analyzed by immunoblotting . GAPDH controls for equal loading . One of three biological replicates is shown .

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(C) To control RNAi efficiency and specificity, HeLa Kyoto cells were incubated for 72 h with control oligos (control) or RNAi-oligos depleting CUL4B, LIS1 or WDR1. Specific downregulation of the indicated proteins was analyzed by immunoblotting. GAPDH controls for equal loading. One of three biological replicates is shown.


( E ) HA - immunoprecipitation ( IP ) of extracts ( input ) prepared from unedited ( - ) or HA - CUL4B endogenously expressing HeLa Kyoto cell lines arrested in S - phase ( thymidine ) or mitosis ( nocodazole ) were probed with antibodies against LIS1 , WDR1 and DDB1 . The antibody recognizing phospho - H3 ( S19 ) controls mitotic arrest and specificity of the immunoprecipitation .

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(E) HA-immunoprecipitation (IP) of extracts (input) prepared from unedited (-) or HA-CUL4B endogenously expressing HeLa Kyoto cell lines arrested in S-phase (thymidine) or mitosis (nocodazole) were probed with antibodies against LIS1, WDR1 and DDB1. The antibody recognizing phospho-H3(S19) controls mitotic arrest and specificity of the immunoprecipitation.


( F , G ) HeLa Kyoto endogenously expressing HA - CUL4B or the indicated NP1 or P50L mutants were arrested in mitosis with nocodazole . Extracts ( input ) and HA - immunoprecipitants were analyzed for the presence of HA - CUL4B for control and bound DDB1 and LIS1 ( F ) or DDB1 and WDR1 ( G ) , respectively . One of 3 biological replicates is shown .

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(F, G) HeLa Kyoto endogenously expressing HA-CUL4B or the indicated NP1 or P50L mutants were arrested in mitosis with nocodazole. Extracts (input) and HA-immunoprecipitants were analyzed for the presence of HA-CUL4B for control and bound DDB1 and LIS1 (F) or DDB1 and WDR1 (G), respectively. One of 3 biological replicates is shown.


( A ) Coomassie - stained SDS - PAGE of in vitro pull - down assays of FLAG - tagged LIS1 and WDR1 , and His - DDB1 , co - expressed in baculoviral Sf9 cells , F is FLAG and H is His . One of 3 biological replicates is shown . Note that DDB1 specifically interacts with LIS1 and WDR1 .

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(A) Coomassie-stained SDS-PAGE of in vitro pull-down assays of FLAG-tagged LIS1 and WDR1, and His-DDB1, co-expressed in baculoviral Sf9 cells, F is FLAG and H is His. One of 3 biological replicates is shown. Note that DDB1 specifically interacts with LIS1 and WDR1.


( C , D ) Western blot of in vitro pulldown assays of Strep - tagged CUL4B and FLAG - tagged LIS1 ( C ) or WDR1 ( D ) co - expressed in Sf9 cells in the presence ( + ) or absence ( - ) of DDB1 . One of 3 biological replicates is shown . Note that LIS1 and WDR1 specifically bind CUL4B , but this interaction is enhanced by the presence of DDB1 .

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(C, D) Western blot of in vitro pulldown assays of Strep-tagged CUL4B and FLAG-tagged LIS1 (C) or WDR1 (D) co-expressed in Sf9 cells in the presence (+) or absence (-) of DDB1. One of 3 biological replicates is shown. Note that LIS1 and WDR1 specifically bind CUL4B, but this interaction is enhanced by the presence of DDB1.


( G ) FP assay investigating binding of CRL4B and CRL4A to Alexa - labeled

(G) FP assay investigating binding of CRL4B and CRL4A to Alexa-labeled WDR1


Fluorescence polarization assay comparing the binding of CRL4B and λ - PP - treated CRL4B to Alexa - labeled WDR1 . The mean with SD of N = 3 technical replicates is depicted . ( L ) Quantification of in vitro binding assays ( Strep - pull down ) of Strep - LIS1 with purified CRL4B , or the NP1 phospho - mutant ( CRL4BNP1 ) ( N = 2 technical replicates ) , or λ - PP treated CRL4B ( CRL4Bλ - PP ) ( N = 1 ) .

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Fluorescence polarization assay comparing the binding of CRL4B and λ-PP-treated CRL4B to Alexa-labeled WDR1. The mean with SD of N=3 technical replicates is depicted. (L) Quantification of in vitro binding assays (Strep-pull down) of Strep-LIS1 with purified CRL4B, or the NP1 phospho-mutant (CRL4BNP1) (N=2 technical replicates), or λ-PP treated CRL4B (CRL4Bλ-PP) (N=1).


( C ) Extracts ( input ) prepared from S - phase ( S ; thymidine ) or mitosis ( M ; nocodazole ) - arrested HeLa Kyoto cells ( - ) and endogenously expressing HA - tagged WT CUL4B , or the NP1 or P50L mutants were immunoprecipitated with HA . II - antibodies ( IP ) . Bound ACTR3 or DDB1 was detected using specific antibodies . One of three biological replicates is shown . Note that in contrast to DDB1 , ACTR3 binds CUL4B in a phosphorylation - dependent manner .

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(C) Extracts (input) prepared from S-phase (S; thymidine) or mitosis (M; nocodazole)-arrested HeLa Kyoto cells (-) and endogenously expressing HA-tagged WT CUL4B, or the NP1 or P50L mutants were immunoprecipitated with HA.II-antibodies (IP). Bound ACTR3 or DDB1 was detected using specific antibodies. One of three biological replicates is shown. Note that in contrast to DDB1, ACTR3 binds CUL4B in a phosphorylation-dependent manner.


( D ) HA - immunoprecipitants ( IP ) of extracts ( input ) prepared from unedited ( - ) controls or endogenously expressing HA - CUL4B HeLa Kyoto cells arrested in mitosis with nocodazole were probed with antibodies against DDB1 , DCTN1 ( dynactin ) and actin . One of two biological replicates is depicted .

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(D) HA-immunoprecipitants (IP) of extracts (input) prepared from unedited (-) controls or endogenously expressing HA-CUL4B HeLa Kyoto cells arrested in mitosis with nocodazole were probed with antibodies against DDB1, DCTN1 (dynactin) and actin. One of two biological replicates is depicted.


( F ) Live cell imaging of HeLa cells expressing H2B - mcherry and lifeAct - GFP , treated for 72 h with control oligos ( siControl ) or RNAi - oligos depleting CUL4B , LIS1 or WDR1 . Representative cells are shown in time intervals of 6 min during metaphase with identical contrast settings . The unanchored spindle in CUL4B - and LIS1 deleted cells is marked with a white arrow . Scale bar : 10 μm .

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(F) Live cell imaging of HeLa cells expressing H2B-mcherry and lifeAct-GFP, treated for 72 h with control oligos (siControl) or RNAi-oligos depleting CUL4B, LIS1 or WDR1. Representative cells are shown in time intervals of 6 min during metaphase with identical contrast settings. The unanchored spindle in CUL4B- and LIS1 deleted cells is marked with a white arrow. Scale bar: 10 μm.


hESCs were differentiated to generate human forebrain specific regionalised organoids . Sections were stained as indicated with antibodies against SOX2 or CTIP2 to visualize neural stem cell and neuronal cell populations , respectively . ( A ) Example images of day 35 organoids . The stained sections are also shown as merged images at different maginifications , with scale bars of 200 μm and 50 μm , respectively . ( B ) The number of cortical units per mm2 total area was quantified in unedited and ΔCUL4B brain organoids at day 35 . Ventricles were counted and the number divided by the total organoid area on 20 sections from one biological replicate .

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hESCs were differentiated to generate human forebrain specific regionalised organoids. Sections were stained as indicated with antibodies against SOX2 or CTIP2 to visualize neural stem cell and neuronal cell populations, respectively. (A) Example images of day 35 organoids. The stained sections are also shown as merged images at different maginifications, with scale bars of 200 μm and 50 μm, respectively. (B) The number of cortical units per mm2 total area was quantified in unedited and ΔCUL4B brain organoids at day 35. Ventricles were counted and the number divided by the total organoid area on 20 sections from one biological replicate.


C57BL / 6J mice were intraarticularly infected with MRSA ( 4 × 106 CFU ; n = 3 - 4 per group ) and left untreated for 24 hours . H . The amount of infiltrating immune cells in synovial fluid was quantified and detected using MRSA expressive of green fluorescent protein ( GFP ) within immune cells ( Scale bar : 100 μm ) . MRSA bioburden in synovial fluid was quantified in colony - forming units ( CFU ) . Data information : Error bars show means ± SD with individual data points . Two - tailed unpaired t - test analysis was conducted to determine statistical significance ( * p < 0 . 05 or * * p < 0 . 01 ; N . D . = not detected ) .

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C57BL/6J mice were intraarticularly infected with MRSA (4 × 106 CFU; n = 3-4 per group) and left untreated for 24 hours. H. The amount of infiltrating immune cells in synovial fluid was quantified and detected using MRSA expressive of green fluorescent protein (GFP) within immune cells (Scale bar: 100 μm). MRSA bioburden in synovial fluid was quantified in colony-forming units (CFU). Data information: Error bars show means ± SD with individual data points. Two-tailed unpaired t-test analysis was conducted to determine statistical significance (*p<0.05 or **p <0.01; N.D. = not detected).