Figure 6A Expression of KCNA5 ( left ) and KCNJ3 ( right ) in GFP + pools of VM and AM CMs at day 31 , as well as in ventricles and atria of human heart . B , C Typical examples ( left ) and current - voltage relationships ( right ) of ( B ) IKur and ( C ) IK , ACh in VM and AMCMs . Data information : Data are presented as mean ± SEM . In ( A ) , * P < 0 . 05 by unpaired t - test . In ( B , C ) , * P < 0 . 05 by two - way repeated measures ANOVA followed by pairwise comparison using the Student - Newman - Keuls test for ( B ) and Mann - Whitney rank - sum test for ( C ) . In ( B ) , P = 0 . 778 , 0 . 350 , 0 . 03 , 0 . 02 , 0 . 002 , 0 . 001 , < 0 . 001 and < 0 . 001 , respectively , for comparison between VM and AM within membrane potentials of − 20 , − 10 , 0 , 10 , 20 , 30 , 40 and 50 mV . In ( C ) , P = 0 . 01 , 0 . 01 , 0 . 01 , 0 . 01 , 0 . 03 , 0 . 397 , 0 . 397 , 0 . 397 , 0 . 671 , 0 . 207 , 0 . 207 , 0 . 09 , 0 . 01 , 0 . 039 and 0 . 015 , respectively , for comparison between VM and AM within membrane potentials of − 120 , − 110 , − 100 , − 90 , − 80 , − 70 , − 60 , − 50 , − 40 , − 30 , − 20 , − 10 , 0 , 10 and 20 mV . CMs = cardiomyocytes ; hESC - atrial ( AM ) and hESC - ventricular ( VM ) CMs ; IK , ACh = acetylcholine - activated potassium current ; IKur = potassium ultra - rapid delayed rectifier current . 4 - AP = 4 - aminopyridine ; CCh = carbachol . D , E Representative APs of VM and AM at 1 Hz in response to ( D ) IKur block by 4 - AP and ( E ) IK , ACh activation by CCh . AP parameters are shown in Supplementary Table S5 .

O B-PANEL_START O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6A Expression of KCNA5 (left) and KCNJ3 (right) in GFP+ pools of VM and AM CMs at day 31, as well as in ventricles and atria of human heart.B, C Typical examples (left) and current-voltage relationships (right) of (B) IKur and (C) IK,ACh in VM and AMCMs.Data information: Data are presented as mean ± SEM. In (A), *P < 0.05 by unpaired t-test. In (B, C), *P < 0.05 by two-way repeated measures ANOVA followed by pairwise comparison using the Student-Newman-Keuls test for (B) and Mann-Whitney rank-sum test for (C). In (B), P = 0.778, 0.350, 0.03, 0.02, 0.002, 0.001, < 0.001 and < 0.001, respectively, for comparison between VM and AM within membrane potentials of −20, −10, 0, 10, 20, 30, 40 and 50 mV. In (C), P = 0.01, 0.01, 0.01, 0.01, 0.03, 0.397, 0.397, 0.397, 0.671, 0.207, 0.207, 0.09, 0.01, 0.039 and 0.015, respectively, for comparison between VM and AM within membrane potentials of −120, −110, −100, −90, −80, −70, −60, −50, −40, −30, −20, −10, 0, 10 and 20 mV. CMs = cardiomyocytes; hESC-atrial (AM) and hESC-ventricular (VM) CMs; IK,ACh = acetylcholine-activated potassium current; IKur = potassium ultra-rapid delayed rectifier current. 4-AP = 4-aminopyridine; CCh = carbachol.D, E Representative APs of VM and AM at 1 Hz in response to (D) IKur block by 4-AP and (E) IK,ACh activation by CCh. AP parameters are shown in Supplementary Table S5.


Figure 7A Representative APs at 1 Hz of VM and AM CMs in response to vernakalant . Inset shows dV / dtmax . B , C Average APAplat ( B ) and dV / dtmax ( C ) in the absence and presence of vernakalant at 1 - 4 Hz . AP parameters are shown in Supplementary Table S6 . Abbreviations as in Figs3 and 6 . Data information : Data are presented as mean ± SEM . * P < 0 . 05 by Mann - Whitney rank - sum test for ( B ) . P = 0 . 01 , 0 . 008 , 0 . 008 and 0 . 007 , respectively , for comparison of APAplat between VM and AM groups at frequencies of 1 . 0 , 2 . 0 , 3 . 0 and 4 . 0 Hz . Two - way repeated measures ANOVA followed by pairwise comparison using the Student - Newman - Keuls test for ( C ) . P = 0 . 686 between VM and AM groups and hence not statistically significant . For VM , P = 0 . 06 for 1 versus 2 Hz ; P < 0 . 001 for 1 versus 3 Hz ; P < 0 . 001 for 1 versus 4 Hz ; P = 0 . 001 for 2 versus 3 Hz ; P = 0 . 002 for 2 versus 4 Hz ; and P = 0 . 857 for 3 versus 4 Hz . For AM , P = 0 . 03 for 1 versus 2 Hz ; P < 0 . 001 for 1 versus 3 Hz ; P < 0 . 001 for 1 versus 4 Hz ; P = 0 . 02 for 2 versus 3 Hz ; P = 0 . 02 for 2 versus 4 Hz ; and P = 0 . 621 for 3 versus 4 Hz .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7A Representative APs at 1 Hz of VM and AM CMs in response to vernakalant. Inset shows dV/dtmax.B, C Average APAplat (B) and dV/dtmax (C)in the absence and presence of vernakalant at 1-4 Hz. AP parameters are shown in Supplementary Table S6. Abbreviations as in Figs3 and 6.Data information: Data are presented as mean ± SEM. *P < 0.05 by Mann-Whitney rank-sum test for (B). P = 0.01, 0.008, 0.008 and 0.007, respectively, for comparison of APAplat between VM and AM groups at frequencies of 1.0, 2.0, 3.0 and 4.0 Hz. Two-way repeated measures ANOVA followed by pairwise comparison using the Student-Newman-Keuls test for (C). P = 0.686 between VM and AM groups and hence not statistically significant. For VM, P = 0.06 for 1 versus 2 Hz; P < 0.001 for 1 versus 3 Hz; P < 0.001 for 1 versus 4 Hz; P = 0.001 for 2 versus 3 Hz; P = 0.002 for 2 versus 4 Hz; and P = 0.857 for 3 versus 4 Hz. For AM, P = 0.03 for 1 versus 2 Hz; P < 0.001 for 1 versus 3 Hz; P < 0.001 for 1 versus 4 Hz; P = 0.02 for 2 versus 3 Hz; P = 0.02 for 2 versus 4 Hz; and P = 0.621 for 3 versus 4 Hz.


Figure 8A Representative APs of VM and AM CMs in the absence , presence and following washout of 3 μmol / l XEN - D0101 . AP parameters are shown in Supplementary Table S7 . B Representative APs ( 1 Hz ) of VM and AM in the CCh , to activate IK , ACh and subsequent addition of XEN - R0703 . AP parameters are shown in Supplementary Table S9 . C , D Experiments performed in RAP conscious dogs in the presence of vehicle or following 1 , 3 and 10 mg / kg XEN - R0703 show ( C ) mean right AERP values ( left ) , mean Van de Water ' s QTc ( right ) and ( D ) AF inducibility plotted as a function of dose . Data information : For RAP dog experiments , n = 5 ; statistical significance tested with paired t - test . Data are presented as mean ± SEM . AERP = atrial effective refractory period ; AF = atrial fibrillation ; RAP = rapid atrial pacing ; N . S . = not significant . Other abbreviations as in Figs3 , 6 and 7 .

O B-PANEL_START O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8A Representative APs of VM and AM CMs in the absence, presence and following washout of 3 μmol/l XEN-D0101. AP parameters are shown in Supplementary Table S7.B Representative APs (1 Hz) of VM and AM in the CCh, to activate IK,ACh and subsequent addition of XEN-R0703. AP parameters are shown in Supplementary Table S9.C, D Experiments performed in RAP conscious dogs in the presence of vehicle or following 1, 3 and 10 mg/kg XEN-R0703 show (C) mean right AERP values (left), mean Van de Water's QTc (right) and (D) AF inducibility plotted as a function of dose.Data information: For RAP dog experiments, n = 5; statistical significance tested with paired t-test. Data are presented as mean ± SEM. AERP = atrial effective refractory period; AF = atrial fibrillation; RAP = rapid atrial pacing; N.S. = not significant. Other abbreviations as in Figs3, 6 and 7.


Figure 1A Normalized firefly luciferase luminescence ( compared to renilla luciferase ) as a function of external IAA concentration . Arabidopsis protoplast cells were transiently transformed with either AtPIN1 or GFP ( in the case of the control ) both under the control of a constitutive CaMV 35S promoter . Where indicated , 10 µM NPA was added . B Normalized firefly luciferase luminescence ( compared to renilla luciferase ) as a function of external NPA concentration in the presence of 100nM IAA in control GFP - ( blue or red ) or PIN1 - ( green or purple ) transformed protoplasts .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A Normalized firefly luciferase luminescence (compared to renilla luciferase) as a function of external IAA concentration. Arabidopsis protoplast cells were transiently transformed with either AtPIN1 or GFP (in the case of the control) both under the control of a constitutive CaMV 35S promoter. Where indicated, 10 µM NPA was added. B Normalized firefly luciferase luminescence (compared to renilla luciferase) as a function of external NPA concentration in the presence of 100nM IAA in control GFP- (blue or red) or PIN1- (green or purple) transformed protoplasts.


Figure 2A , B Native PAGE separation reveals distinct detergent - sensitive PIN1 - complexes . Plasma membrane preparations from dark grown Arabidopsis cell suspension cultures were solubilized with ( a ) Complexiolyte 27 ( a mixture of ionic and non - ionic detergents ) , and ( b ) Complexiolyte 47 ( lower stringency , non - ionic detergent ) , ( both Logopharm ) ; first dimension BN - PAGE , second dimension SDS - PAGE , blots stained with anti - PIN1 antibody revealing distinct PIN1 complex populations at the indicated positions . Values are given in KDa . c ) Molecular abundance ( abundancenormspec values calculated as summarized peptide PVs divided by the number of MS - accessible protein specific amino acids ) of PIN subunits and ABCB19 in anti - GFP affinity purification of CL47 - solubilized PIN1 - GFP - expressing roots with a GFP - specific DARPin . Note the significant heteromerization of several endogenous PINs with PIN1 - GFP and the absence of other abundant interaction partners ( ABC19B shown as an example ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A, B Native PAGE separation reveals distinct detergent-sensitive PIN1-complexes. Plasma membrane preparations from dark grown Arabidopsis cell suspension cultures were solubilized with (a) Complexiolyte 27 (a mixture of ionic and non-ionic detergents), and (b) Complexiolyte 47 (lower stringency, non-ionic detergent), (both Logopharm); first dimension BN-PAGE, second dimension SDS-PAGE, blots stained with anti-PIN1 antibody revealing distinct PIN1 complex populations at the indicated positions. Values are given in KDa.c) Molecular abundance (abundancenormspec values calculated as summarized peptide PVs divided by the number of MS-accessible protein specific amino acids) of PIN subunits and ABCB19 in anti-GFP affinity purification of CL47-solubilized PIN1-GFP-expressing roots with a GFP-specific DARPin. Note the significant heteromerization of several endogenous PINs with PIN1-GFP and the absence of other abundant interaction partners (ABC19B shown as an example).


Figure 3a ) BN - PAGE was performed with PIN1 - containing microsomes prepared from either PIN1 - expressing HEK293T cells or a dark - grown Arabidopsis cell suspension culture . Prior to solubilization with 1 % dodecyl maltoside , microsomes were incubated with either 10 µM NPA or 10 µM quercetin . b ) PIN1 dimer stability induced by NPA after expression in HEK cells . Relative distribution between monomer and dimer after NPA treatment is given relative to distribution of untreated samples after solubilization with 50 % ( v / v ) CL27 . Each measurement given ( three for each concentration ) represents the mean of three gel lanes for wild type ( circles ) or triple S2523 , S253E , S261E phosphomimetic sequences ( crosses ) ( example images are given in the figure inset ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3a) BN-PAGE was performed with PIN1-containing microsomes prepared from either PIN1-expressing HEK293T cells or a dark-grown Arabidopsis cell suspension culture. Prior to solubilization with 1% dodecyl maltoside, microsomes were incubated with either 10 µM NPA or 10 µM quercetin.b) PIN1 dimer stability induced by NPA after expression in HEK cells. Relative distribution between monomer and dimer after NPA treatment is given relative to distribution of untreated samples after solubilization with 50% (v/v) CL27. Each measurement given (three for each concentration) represents the mean of three gel lanes for wild type (circles) or triple S2523, S253E, S261E phosphomimetic sequences (crosses) (example images are given in the figure inset).


Figure 4a ) Summed 2D projections of PLA interactions ( left ) and co - localisation ( right ) ( n = 3 ) between PIN1 and PIN4 laterally bisected 2D - projected heat map . Treatments as indicated . b ) Quantification of positive PLA interactions . n . t ( no treatment ) and 10µM NPA , n = 4 ; 10µM IAA and 10µM IAA + 10µM NPA ; n = 3 . Error bars indicate standard deviation .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4a) Summed 2D projections of PLA interactions (left) and co-localisation (right) (n=3) between PIN1 and PIN4 laterally bisected 2D-projected heat map. Treatments as indicated. b) Quantification of positive PLA interactions. n.t (no treatment) and 10µM NPA, n=4; 10µM IAA and 10µM IAA+ 10µM NPA; n=3. Error bars indicate standard deviation.


Figure 5a ) Microsomes from HEK293 cells expressing PIN1 - RFP were separated under native conditions in the presence of NPA or an anti - PIN1 Fab fragment . 1 ) PIN1 - RFP , 2 ) PIN1 - RFP + Fab , 3 ) PIN1 - RFP + 10 µM NPA , 4 ) PIN1 - RFP + Fab + 10 µM NPA , 5 ) PIN1 - RFP + 10 µM Quercetin ( Q ) , 6 ) PIN1 - RFP + Fab + 10 µM Quercetin ( Q ) . Protein complexes were solubilized and separated under native conditions as described before . Western blots were performed with an anti - RFP monoclonal antibody . b ) Fourteen - day - old Arabidopsis seedlings ( wild type , brown ; pPIN1 : : Mab9B2 ; blue ) grown on AM containing 0 . 2 µM NPA . Scale bar = 1 cm . c ) NPA - affected lateral root density of nine - day - old Arabidopsis seedlings expressing Mab9B2 scFv fragments ( wild type sample sizes were between 16 and 21 plants ; Mab9B2 sample sizes were between 12 and 31 plants ) . Bars indicate standard error . d ) NPA - affected apical hook angle in dark - grown three - day - old Arabidopsis seedlings expressing Mab9B2 scFv fragments . ( wild type sample sizes were between 38 and 50 plants ; Mab9B2 sample sizes were between 33 and 45 plants ) . e ) Gravitropic curvature in four - day old Arabidopsis seedlings expressing Mab9B2 scFv fragments . ( wild type sample sizes were between 15 and 21 plants ; Mab9B2 sample sizes were between 14 and 20 plants ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5a) Microsomes from HEK293 cells expressing PIN1-RFP were separated under native conditions in the presence of NPA or an anti-PIN1 Fab fragment. 1) PIN1-RFP, 2) PIN1-RFP+Fab, 3) PIN1-RFP+10 µM NPA, 4) PIN1-RFP+Fab+10 µM NPA, 5) PIN1-RFP + 10 µM Quercetin (Q), 6) PIN1-RFP+Fab+10 µM Quercetin (Q). Protein complexes were solubilized and separated under native conditions as described before. Western blots were performed with an anti-RFP monoclonal antibody.b) Fourteen-day-old Arabidopsis seedlings (wild type, brown; pPIN1::Mab9B2; blue) grown on AM containing 0.2 µM NPA. Scale bar = 1 cm.c) NPA-affected lateral root density of nine-day-old Arabidopsis seedlings expressing Mab9B2 scFv fragments (wild type sample sizes were between 16 and 21 plants; Mab9B2 sample sizes were between 12 and 31 plants). Bars indicate standard error.d) NPA-affected apical hook angle in dark-grown three-day-old Arabidopsis seedlings expressing Mab9B2 scFv fragments. (wild type sample sizes were between 38 and 50 plants; Mab9B2 sample sizes were between 33 and 45 plants). e) Gravitropic curvature in four-day old Arabidopsis seedlings expressing Mab9B2 scFv fragments. (wild type sample sizes were between 15 and 21 plants; Mab9B2 sample sizes were between 14 and 20 plants).


Figure 2 B In vitro substrate ubiquitylation assay for Hrd1 with Ubc7 ( left ) and Ubc6 ( right ) . Ubc7 reactions contained equimolar amounts of Cue1 and were performed with Ub ( K48R ) . Top : representative immunoblot using a poly - clonal α - RNase A antibody ; " no E3 " reactions do not contain S - Hrd1 , " no ATP " reactions do not contain ATP , but S - Hrd1 . The RNase - Ub2 band co - migrates with a nonspecific band ( # ) common to all samples . Bottom : Quantification of RNase - Ub signals is shown . Values are reported as means ± standard deviation ( n = 3 ) . Significances for pairwise comparisons were determined by One - way ANOVA Test ; * p < 0 . 05 . For clarity , only significances related to the " no E3 " control of a given E2 are shown . C In vitro Ub nucleophile discharge assays for Hrd1 with U7BR / Ubc7 ( top - yellow ) and Ubc6 ( bottom - green ) with ethanolamine as nucleophile . Representative Coomassie gels are shown . D Quantification of Ub nucleophile discharge assays for Hrd1 with U7BR / Ubc7 ( left ) and Ubc6 ( right ) . Plots of E2 ~ Ub discharge ( dots ) as a function of time with first - order reaction models fitted to the discharge data ( lines ) are shown . Values for each time point are reported as means ± standard deviation ( n = 3 ) . Insets show reaction rates derived from these fits . Significances were determined by student T - test ; * p < 0 . 05 . ( E ) Stimulation of U7BR / Ubc7 and Ubc6 discharge activities by Hrd1 . E2 stimulation is reported as the ratio of rates derived from RING - catalyzed reactions and " no E3 " controls in D . Values are reported as means ± standard deviation ( n = 3 ) . Significance was determined by student T - test ; * p < 0 . 05 .

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

Figure 2 B In vitro substrate ubiquitylation assay for Hrd1 with Ubc7 (left) and Ubc6 (right). Ubc7 reactions contained equimolar amounts of Cue1 and were performed with Ub(K48R). Top: representative immunoblot using a poly-clonal α-RNase A antibody; "no E3" reactions do not contain S-Hrd1, "no ATP" reactions do not contain ATP, but S-Hrd1. The RNase-Ub2 band co-migrates with a nonspecific band (#) common to all samples. Bottom: Quantification of RNase-Ub signals is shown. Values are reported as means ± standard deviation (n = 3). Significances for pairwise comparisons were determined by One-way ANOVA Test; * p < 0.05. For clarity, only significances related to the "no E3" control of a given E2 are shown.  C In vitro Ub nucleophile discharge assays for Hrd1 with U7BR/Ubc7 (top - yellow) and Ubc6 (bottom - green) with ethanolamine as nucleophile. Representative Coomassie gels are shown.  D Quantification of Ub nucleophile discharge assays for Hrd1 with U7BR/Ubc7 (left) and Ubc6 (right). Plots of E2~Ub discharge (dots) as a function of time with first-order reaction models fitted to the discharge data (lines) are shown. Values for each time point are reported as means ± standard deviation (n = 3). Insets show reaction rates derived from these fits. Significances were determined by student T-test; * p < 0.05. (E) Stimulation of U7BR/Ubc7 and Ubc6 discharge activities by Hrd1. E2 stimulation is reported as the ratio of rates derived from RING-catalyzed reactions and "no E3" controls in D. Values are reported as means ± standard deviation (n = 3). Significance was determined by student T-test; * p < 0.05.


Figure 3 A In vitro Ub nucleophile discharge assays for U7BR / Ubc7 with indicated Hrd1 ( left ) and Doa10 ( right ) variants . E2 stimulation is shown as the ratio of rates derived from RING - catalyzed reactions and the " no E3 " control ( see Fig . EV2C - D ) . Values are reported as means ± standard deviation ( n = 3 ) . Significances for pairwise comparisons were determined by One - way ANOVA Test ; * p < 0 . 05 . For clarity only significances related to the " no E3 " control are shown . In addition , reactions for Hrd1 ( 400R ) and Doa10 ( 94R ) are significantly faster than all other reactions and the reaction for Doa10 ( 94H ) is significantly faster than that for Doa10 ( 94E ) . The wild - type E3s are identified by black frames for each set . B In vitro Ub chain formation assay by Ubc7 with indicated Hrd1 ( left ) and Doa10 ( right ) variants in the presence of indicated Cue1 variants . Rates for reactions of mono - Ub to di - Ub and di - Ub to tri - Ub with fluorescently - labeled Ub are shown on a logarithmic scale . Values are reported as means ± standard deviation ( n = 3 ) . Significances for pairwise comparisons were determined by One - way ANOVA Test ; * p < 0 . 05 . For clarity only significances related to the " no E3 " control of given reaction set are shown . D Protein degradation in indicated yeast strains monitored by pulse - chase experiments for the Hrd1 model substrate PrA * - 3xHA ( left ) and by CHX decay assays for the Doa10 model substrate Deg1 - eGFP2 ( right ) . Values for each time point are reported as means ± standard deviation ( n = 4 for PrA * - 3xHA and n = 3 for Deg1 - eGFP2 ) .

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

Figure 3 A In vitro Ub nucleophile discharge assays for U7BR/Ubc7 with indicated Hrd1 (left) and Doa10 (right) variants. E2 stimulation is shown as the ratio of rates derived from RING-catalyzed reactions and the "no E3" control (see Fig. EV2C-D). Values are reported as means ± standard deviation (n = 3). Significances for pairwise comparisons were determined by One-way ANOVA Test; * p < 0.05. For clarity only significances related to the "no E3" control are shown. In addition, reactions for Hrd1(400R) and Doa10(94R) are significantly faster than all other reactions and the reaction for Doa10(94H) is significantly faster than that for Doa10(94E). The wild-type E3s are identified by black frames for each set.  B In vitro Ub chain formation assay by Ubc7 with indicated Hrd1 (left) and Doa10 (right) variants in the presence of indicated Cue1 variants. Rates for reactions of mono-Ub to di-Ub and di-Ub to tri-Ub with fluorescently-labeled Ub are shown on a logarithmic scale. Values are reported as means ± standard deviation (n = 3). Significances for pairwise comparisons were determined by One-way ANOVA Test; * p < 0.05. For clarity only significances related to the "no E3" control of given reaction set are shown.  D Protein degradation in indicated yeast strains monitored by pulse-chase experiments for the Hrd1 model substrate PrA*-3xHA (left) and by CHX decay assays for the Doa10 model substrate Deg1-eGFP2 (right). Values for each time point are reported as means ± standard deviation (n = 4 for PrA*-3xHA and n = 3 for Deg1-eGFP2).


Figure 4 A Protein degradation in indicated yeast strains monitored by pulse - chase experiments for the Hrd1 model substrate PrA * - 3xHA ( left ) and by CHX decay assays for the Doa10 model substrate Deg1 - eGFP2 ( right ) . Values for each time point are reported as means ± standard deviation ( n = 4 for PrA * - 3xHA and n = 3 for Deg1 - eGFP2 ) . Data for control strains ( Hrd1 ( 400R ) wt , Δhrd1 , Doa10 ( 94H ) wt and Δdoa10 ) is the same as in Fig . 3D . B In vitro substrate ubiquitylation assay for Hrd1 variants with Ubc7 . Ubc7 reactions contained equimolar amounts of Cue1 and were performed with Ub ( K48R ) . Top : representative immunoblot using a poly - clonal α - RNase A antibody ; " no E3 " reaction does not contain any S - Hrd1 , " no ATP " reaction does not contain ATP , but wild - type S - Hrd1 ( 400R ) . The RNase - Ub2 band co - migrates with a nonspecific band ( # ) common to all samples . Bottom : Quantification of RNase - Ub signals is shown . Values are reported as means ± standard deviation ( n = 3 ) . Significances for pairwise comparisons were determined by One - way ANOVA Test ; * p < 0 . 05 . For clarity , only significances related to the " no E3 " control are shown . In addition , the reaction containing S - Hrd1 ( 400R ) shows significantly more ubiquitylation than those with other E3 variants . C In vitro Ub nucleophile discharge assays for Ubc6 with indicated Doa10 variants . E2 stimulation is shown as the ratio of rates derived from RING - catalyzed reactions and the " no E3 " control ( see Fig . EV2E ) . Values are reported as means ± standard deviation ( n = 3 ) . Significances for pairwise comparisons were determined by One - way ANOVA Test ; * p < 0 . 05 . For clarity only significances related to the " no E3 " control are shown . In addition , the reaction for Doa10 ( 94R ) is significantly faster than that for Doa10 ( 94H ) and ( 94E ) . The wild - type E3 is identified by a black frame . The scale of the y - axis was set identical to that of plots from other discharge assays D In vitro substrate ubiquitylation assay for Doa10 variants with Ubc7 ( left ) and Ubc6 ( right ) . Ubc7 reactions contained equimolar amounts of Cue1 and were performed with Ub ( K48R ) . Top : representative immunoblot using a poly - clonal α - RNase A antibody ; " no E3 " reaction does not contain any S - Doa10 , " no ATP " reaction does no contain ATP , but wild - type S - Doa10 ( 94H ) . The RNase - Ub2 band co - migrates with a nonspecific band ( # ) common to all samples . Bottom : Quantification of RNase - Ub signals is shown . Values are reported as means ± standard deviation ( n = 3 ) . Significances for pairwise comparisons were determined by One - way ANOVA Test ; * p < 0 . 05 . For clarity , only significances related to the " no E3 " control of a given E2 are shown . In addition , the reaction containing Ubc7 and S - Hrd1 ( 400R ) shows significantly more ubiquitylation than those with Ubc7 and other E3 variants .

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

Figure 4 A Protein degradation in indicated yeast strains monitored by pulse-chase experiments for the Hrd1 model substrate PrA*-3xHA (left) and by CHX decay assays for the Doa10 model substrate Deg1-eGFP2 (right). Values for each time point are reported as means ± standard deviation (n = 4 for PrA*-3xHA and n = 3 for Deg1-eGFP2). Data for control strains (Hrd1(400R) wt, Δhrd1, Doa10(94H) wt and Δdoa10) is the same as in Fig. 3D.  B In vitro substrate ubiquitylation assay for Hrd1 variants with Ubc7. Ubc7 reactions contained equimolar amounts of Cue1 and were performed with Ub(K48R). Top: representative immunoblot using a poly-clonal α-RNase A antibody; "no E3" reaction does not contain any S-Hrd1, "no ATP" reaction does not contain ATP, but wild-type S-Hrd1(400R). The RNase-Ub2 band co-migrates with a nonspecific band (#) common to all samples. Bottom: Quantification of RNase-Ub signals is shown. Values are reported as means ± standard deviation (n = 3). Significances for pairwise comparisons were determined by One-way ANOVA Test; * p < 0.05. For clarity, only significances related to the "no E3" control are shown. In addition, the reaction containing S-Hrd1(400R) shows significantly more ubiquitylation than those with other E3 variants.  C In vitro Ub nucleophile discharge assays for Ubc6 with indicated Doa10 variants. E2 stimulation is shown as the ratio of rates derived from RING-catalyzed reactions and the "no E3" control (see Fig. EV2E). Values are reported as means ± standard deviation (n = 3). Significances for pairwise comparisons were determined by One-way ANOVA Test; * p < 0.05. For clarity only significances related to the "no E3" control are shown. In addition, the reaction for Doa10(94R) is significantly faster than that for Doa10(94H) and (94E). The wild-type E3 is identified by a black frame. The scale of the y-axis was set identical to that of plots from other discharge assays  D In vitro substrate ubiquitylation assay for Doa10 variants with Ubc7 (left) and Ubc6 (right). Ubc7 reactions contained equimolar amounts of Cue1 and were performed with Ub(K48R). Top: representative immunoblot using a poly-clonal α-RNase A antibody; "no E3" reaction does not contain any S-Doa10, "no ATP" reaction does no contain ATP, but wild-type S-Doa10(94H). The RNase-Ub2 band co-migrates with a nonspecific band (#) common to all samples. Bottom: Quantification of RNase-Ub signals is shown. Values are reported as means ± standard deviation (n = 3). Significances for pairwise comparisons were determined by One-way ANOVA Test; * p < 0.05. For clarity, only significances related to the "no E3" control of a given E2 are shown. In addition, the reaction containing Ubc7 and S-Hrd1(400R) shows significantly more ubiquitylation than those with Ubc7 and other E3 variants.


Figure 5 A E2 ~ Ub hydrolysis assays for U7BR / Ubc7 and Ubc6 . E3 - independent Ub discharge assays for both E2s in PBS ( absence of nucleophile = hydrolysis ) were performed at 32 ° C . Left : representative Coomassie gels . Right : Plots of E2 ~ Ub hydrolysis ( dots ) as a function of time with first - order reaction models fitted to the discharge data ( lines ) are shown . Values for each time point are reported as mean ± standard deviation ( n = 3 ) . B HSQC - TROSY experiments comparing free and E2 - conjugated ( 15N ) Ub . Top : CSPs of ( 15N ) Ub ( G76C ) conjugated to indicated E2s via disulfide bond ( 775 µM each ) compared to free ( 15N ) Ub ( G76C ) are shown ; Ub resides known to be involved in the closed conformation interface are underlined in grey ; * = number of resonances with perturbations too large to be assigned confidently ( shown as off - scale in diagram ) . Bottom : Spectra of resonances for selected residues in Ub ' s hydrophobic patch for free Ub ( G76C ) ( gray ) and Ub conjugated to Ubc6 ( green ) and U7BR / Ubc7 ( yellow ) .

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

Figure 5 A E2~Ub hydrolysis assays for U7BR/Ubc7 and Ubc6. E3-independent Ub discharge assays for both E2s in PBS (absence of nucleophile = hydrolysis) were performed at 32°C. Left: representative Coomassie gels. Right: Plots of E2~Ub hydrolysis (dots) as a function of time with first-order reaction models fitted to the discharge data (lines) are shown. Values for each time point are reported as mean ± standard deviation (n = 3).  B HSQC-TROSY experiments comparing free and E2-conjugated (15N)Ub. Top: CSPs of (15N)Ub(G76C) conjugated to indicated E2s via disulfide bond (775 µM each) compared to free (15N)Ub(G76C) are shown; Ub resides known to be involved in the closed conformation interface are underlined in grey; * = number of resonances with perturbations too large to be assigned confidently (shown as off-scale in diagram). Bottom: Spectra of resonances for selected residues in Ub's hydrophobic patch for free Ub(G76C) (gray) and Ub conjugated to Ubc6 (green) and U7BR/Ubc7 (yellow).


Figure 6 Protein degradation in indicated yeast strains monitored by pulse - chase experiments for the Hrd1 model substrates PrA * - 3xHA Values for each time point are reported as means ± standard deviation ( n = 4 for PrA * - 3xHA For A , data for control strains ( Hrd1 ( 400R ) wt , Hrd1 ( 400H ) and Δhrd1 ) are the same as in Fig . 3D and 4A . Protein degradation in indicated yeast strains monitored by CHX decay assays for the Doa10 model substrates Deg1 - eGFP2 Values for each time point are reported as means ± standard deviation n = 3 for Deg1 - eGFP2 Protein degradation in indicated yeast strains monitored by CHX decay assays for the Doa10 model substrates FLAG - Sbh2 Values for each time point are reported as means ± standard deviation n = 3 for FLAG - Sbh2 Protein degradation in indicated yeast strains monitored by pulse - chase experiments for the Hrd1 model substrates PrA * - 3xHA Values for each time point are reported as means ± standard deviation ( n = 4 for PrA * - 3xHA For D , data for control strains ( Doa10 ( 94H ) wt and Δdoa10 ) are the same as in Fig 3D and 4A . Protein degradation in indicated yeast strains monitored by pulse - chase experiments for the lysine - less CPY * - K0 - HA Values for each time point are reported as means ± standard deviation Protein degradation in indicated yeast strains monitored by pulse - chase experiments for FLAG - Sbh2 Values for each time point are reported as means ± standard deviation n = 3 for FLAG - Sbh2

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

Figure 6 Protein degradation in indicated yeast strains monitored by pulse-chase experiments for the Hrd1 model substrates PrA*-3xHA Values for each time point are reported as means ± standard deviation (n = 4 for PrA*-3xHA For A, data for control strains (Hrd1(400R) wt, Hrd1(400H) and Δhrd1) are the same as in Fig. 3D and 4A.  Protein degradation in indicated yeast strains monitored by CHX decay assays for the Doa10 model substrates Deg1-eGFP2 Values for each time point are reported as means ± standard deviation n = 3 for Deg1-eGFP2  Protein degradation in indicated yeast strains monitored by CHX decay assays for the Doa10 model substrates FLAG-Sbh2 Values for each time point are reported as means ± standard deviation n = 3 for FLAG-Sbh2  Protein degradation in indicated yeast strains monitored by pulse-chase experiments for the Hrd1 model substrates PrA*-3xHA Values for each time point are reported as means ± standard deviation (n = 4 for PrA*-3xHA For D, data for control strains (Doa10(94H) wt and Δdoa10) are the same as in Fig 3D and 4A.  Protein degradation in indicated yeast strains monitored by pulse-chase experiments for the lysine-less CPY*-K0-HA Values for each time point are reported as means ± standard deviation  Protein degradation in indicated yeast strains monitored by pulse-chase experiments for FLAG-Sbh2 Values for each time point are reported as means ± standard deviation n = 3 for FLAG-Sbh2


Figure 2A Growth of indicated Y . pestis strains in zinc - limited medium ( cPMH2 ) ( n = 3 ) . B Growth of indicated Y . pestis strains in zinc - replete medium ( cPMH2 + 10μM ZnCl2 ) ( n = 3 ) . Data information : One - way ANOVA with Dunnett test , * p ≤ 0 . 05 , * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001 . For A , B growth of mutants was compared to znuBC . Data represents the mean ± SD of three biological replicates . For some points , error bars are too small to visualize on the graph . C Growth of indicated Y . pestis strains in iron - limited ( cPMH2 + 0μM FeCl2 ) or iron - replete ( cPMH2 + 10μM FeCl2 ) media ( n = 3 ) . D Growth of znuBC y0702 - y0703 , znuBC y0704 , or y0702 - y0703 complemented mutant ( pBCSK + y0702 - 70703 ) in zinc - limited medium ( cPMH2 ) ( n = 3 ) . Data information : One - way ANOVA with Dunnett test , * p ≤ 0 . 05 , * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001 . D , growth of mutants was compared to znuBC . Data represents the mean ± SD of three biological replicates . For some points , error bars are too small to visualize on the graph .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A Growth of indicated Y. pestis strains in zinc-limited medium (cPMH2) (n=3). B Growth of indicated Y. pestis strains in zinc-replete medium (cPMH2 + 10μM ZnCl2) (n=3). Data information: One-way ANOVA with Dunnett test, *p≤0.05, **p≤0.001, ****p≤0.0001. For A, B growth of mutants was compared to znuBC. Data represents the mean ±SD of three biological replicates. For some points, error bars are too small to visualize on the graph.C Growth of indicated Y. pestis strains in iron-limited (cPMH2 + 0μM FeCl2) or iron-replete (cPMH2 + 10μM FeCl2) media (n=3).D Growth of znuBC y0702-y0703, znuBC y0704, or y0702-y0703 complemented mutant (pBCSK+y0702-70703) in zinc-limited medium (cPMH2) (n=3). Data information: One-way ANOVA with Dunnett test, *p≤0.05, **p≤0.001, ****p≤0.0001. D, growth of mutants was compared to znuBC. Data represents the mean ±SD of three biological replicates. For some points, error bars are too small to visualize on the graph.


Figure 3B Growth of znuBC irp2 pGENlux in co - culture with indicated Y . pestis strains in zinc - limited medium ( cPMH2 ) ( n = 3 ) . Data information : For B , Data represents the mean ± SD relative luminescent units ( RLU ) from three biological replicates . For some points , error bars are too small to visualize on the graph . One - way ANOVA with Dunnett test compared to znuBC irp2 + Y . pestis ; * * * * p ≤ 0 . 0001 . C Absorbance of PAR at 500nm incubated with indicated Y . pestis supernatants ( n = 3 ) . D OD600 of bacterial cultures at 8 h for LC - MS ( n = 3 ) . Data information : For One - way ANOVA with Tukey test , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , and * * * * p ≤ 0 . 0001 ; ns = not significant . Mean ± SD of three biological replicates . E LC - MS analysis of Ybt in culture supernatants ( note : both peaks are Ybt ) . F LC - MS quantification ( n = 3 ) . Data information For One - way ANOVA with Tukey test , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , and * * * * p ≤ 0 . 0001 ; ns = not significant . Mean ± SD of three biological replicates . G OD600 of bacterial cultures at 8 h harvested for GFP quantification ( n = 3 ) . Data information : For , One - way ANOVA with Tukey test , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , and * * * * p ≤ 0 . 0001 ; ns = not significant . Mean ± SD of three biological replicates .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3B Growth of znuBC irp2 pGENlux in co-culture with indicated Y. pestis strains in zinc-limited medium (cPMH2) (n=3). Data information: For B, Data represents the mean ±SD relative luminescent units (RLU) from three biological replicates. For some points, error bars are too small to visualize on the graph. One-way ANOVA with Dunnett test compared to znuBC irp2 + Y. pestis; ****p≤0.0001.C Absorbance of PAR at 500nm incubated with indicated Y. pestis supernatants (n=3). D OD600 of bacterial cultures at 8 h for LC-MS (n=3). Data information: For One-way ANOVA with Tukey test, **p≤0.01, ***p≤0.001, and ****p≤0.0001; ns=not significant. Mean ±SD of three biological replicates.E LC-MS analysis of Ybt in culture supernatants (note: both peaks are Ybt). F LC-MS quantification (n=3). Data information For One-way ANOVA with Tukey test, **p≤0.01, ***p≤0.001, and ****p≤0.0001; ns=not significant. Mean ±SD of three biological replicates.G OD600 of bacterial cultures at 8 h harvested for GFP quantification (n=3). Data information: For , One-way ANOVA with Tukey test, **p≤0.01, ***p≤0.001, and ****p≤0.0001; ns=not significant. Mean ±SD of three biological replicates.


Figure 4A IC50 of calprotectin for indicated Y . pestis strains ( n = 3 ) . Data information : For A , mean ± SD of three biological replicates . One - way ANOVA with Tukey ' s comparison : ns = not significant , * * p < 0 . 01 , * * * p = 0 . 001 . B C57BL6 / J mice ( n = 10 ) subcutaneously challenged with 102 CFU of indicated Y . pestis strains . C C57BL6 / J mice ( n = 10 ) intranasally challenged with 104 CFU of indicated Y . pestis strains . Data information : For A , mean ± SD of three biological replicates . One - way ANOVA with Tukey ' s comparison : ns = not significant , * * p < 0 . 01 , * * * p = 0 . 001 . For B and C , differences in survival were calculated by log rank test compared to Y . pestis . For results are combined data from 2 independent experiments ( n = 5 mice per experiment ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A IC50 of calprotectin for indicated Y. pestis strains (n=3). Data information: For A, mean ±SD of three biological replicates. One-way ANOVA with Tukey's comparison: ns= not significant, **p<0.01, ***p=0.001.B C57BL6/J mice (n=10) subcutaneously challenged with 102 CFU of indicated Y. pestis strains. C C57BL6/J mice (n=10) intranasally challenged with 104 CFU of indicated Y. pestis strains. Data information: For A, mean ±SD of three biological replicates. One-way ANOVA with Tukey's comparison: ns= not significant, **p<0.01, ***p=0.001. For B and C, differences in survival were calculated by log rank test compared to Y. pestis. For results are combined data from 2 independent experiments (n=5 mice per experiment).


Figure 5Growth of indicated Y . pestis strains in zinc - limited medium ( cPMH2 ) ( n = 3 ) , One - way ANOVA with Dunnett test compared to znuBC mutant , * * p ≤ 0 . 01 , * * * * p ≤ 0 . 0001 . Data represents the mean ± SD of three biological replicates . For some points , error bars are too small to visualize on the graph .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5Growth of indicated Y. pestis strains in zinc-limited medium (cPMH2) (n=3), One-way ANOVA with Dunnett test compared to znuBC mutant, **p≤0.01, ****p≤0.0001. Data represents the mean ±SD of three biological replicates. For some points, error bars are too small to visualize on the graph.


Figure 1Immunoblot of lysates prepared from the indicated SAMHD1 - proficient ( + / + ) , deficient ( - / - ) and rescue ( WT , H233A ) cell line pairs with the indicated antibodies . Representative of 2 independent experiments . Proliferation inhibition analysis of ara - C and RNRi combination treatment in SAMHD1 + / + or - / - THP - 1 cells . Error bars indicate s . e . m . of two ( HU and dF - dC ) or three ( 3 - AP ) independent experiments , each performed in duplicate . Ara - C EC50 values plotted as a function of RNRi concentration in SAMHD1 + / + , - / - and rescue ( WT , H233A ) THP - 1 cell line pairs . EC50 values in the absence of RNRi are indicated by the black and red dotted line . Error bars indicate s . e . m . of two ( HU and dF - dC ) or three ( 3 - AP ) independent experiments , each performed in duplicate . Drug synergy plots for ara - C and the indicated RNRi in SAMHD1 + / + , - / - and rescue ( WT , H233A ) cell line pairs . Each data point indicates an average delta score from a single dose - response matrix experiment performed in duplicate . Zero , > 0 , or < 0 corresponds to additive , synergy , or antagonism , respectively , whilst > 5 indicates strong synergy . The horizontal line and the error bars indicate the mean and s . d . , respectively , statistical significance was determined using a two - tailed unpaired t - test : ns , not significant , P ≥ 0 . 05 ; * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 ; * * * * , P < 0 . 0001 . Spearman correlation of relative SAMHD1 protein abundance and synergy delta scores for ara - C versus HU or dF - dC in a panel ( n = 9 ) of haematological cancer cell lines . Error bars indicate s . e . m . Each data point corresponds to SAMHD1 protein levels determined by immunoblot analysis ( n = 4 for each cell line , representative blot shown in Appendix Fig S2 ) and an average delta score from repeated dose - response matrix experiments each performed in triplicate : THP - 1 , n = 4 ; HuT - 78 , n = 2 ; HL - 60 / iva , n = 1 ; KBM - 7 , n = 2 ( HU ) and 3 ( dF - dC ) ; K562 , n = 3 ( HU ) and 4 ( dF - dC ) ; CCRF - CEM , n = 3 ( HU ) and 4 ( dF - dC ) ; MV - 4 - 11 , n = 2 ( HU ) and 3 ( dF - dC ) ; Jurkat , n = 2 ( HU ) and 3 ( dF - dC ) ; MOLT - 4 , n = 2 ( HU ) an 3 ( dF - dC ) . Immunoblot analysis of lysates prepared from SAMHD1 + / + or - / - THP - 1 cells treated for 24 h with ara - C and HU , as indicated . Representative of 3 independent experiments .

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Figure 1Immunoblot of lysates prepared from the indicated SAMHD1-proficient (+/+), deficient (-/-) and rescue (WT, H233A) cell line pairs with the indicated antibodies. Representative of 2 independent experiments.Proliferation inhibition analysis of ara-C and RNRi combination treatment in SAMHD1+/+ or -/- THP-1 cells. Error bars indicate s.e.m. of two (HU and dF-dC) or three (3-AP) independent experiments, each performed in duplicate.Ara-C EC50 values plotted as a function of RNRi concentration in SAMHD1+/+, -/- and rescue (WT, H233A) THP-1 cell line pairs. EC50 values in the absence of RNRi are indicated by the black and red dotted line. Error bars indicate s.e.m. of two (HU and dF-dC) or three (3-AP) independent experiments, each performed in duplicate.Drug synergy plots for ara-C and the indicated RNRi in SAMHD1+/+, -/- and rescue (WT, H233A) cell line pairs. Each data point indicates an average delta score from a single dose-response matrix experiment performed in duplicate. Zero, >0, or <0 corresponds to additive, synergy, or antagonism, respectively, whilst >5 indicates strong synergy. The horizontal line and the error bars indicate the mean and s.d., respectively, statistical significance was determined using a two-tailed unpaired t-test: ns, not significant, P ≥ 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.Spearman correlation of relative SAMHD1 protein abundance and synergy delta scores for ara-C versus HU or dF-dC in a panel (n = 9) of haematological cancer cell lines. Error bars indicate s.e.m. Each data point corresponds to SAMHD1 protein levels determined by immunoblot analysis (n = 4 for each cell line, representative blot shown in Appendix Fig S2) and an average delta score from repeated dose-response matrix experiments each performed in triplicate: THP-1, n =4; HuT-78, n = 2; HL-60/iva, n = 1; KBM-7, n = 2 (HU) and 3 (dF-dC); K562, n = 3 (HU) and 4 (dF-dC); CCRF-CEM, n =3 (HU) and 4 (dF-dC); MV-4-11, n = 2 (HU) and 3 (dF-dC); Jurkat, n = 2 (HU) and 3 (dF-dC); MOLT-4, n = 2 (HU) an 3 (dF-dC).Immunoblot analysis of lysates prepared from SAMHD1+/+ or -/- THP-1 cells treated for 24 h with ara-C and HU, as indicated. Representative of 3 independent experiments.


Figure 2Kaplan - Meier analysis of NOD / SCID mice injected i . v . with luciferase - expressing SAMHD1 + / + or - / - THP - 1 cell clones ( day 0 ) and treated with ara - C and / or HU as indicated ( day 6 ) . n = 6 per treatment group . Kaplan - Meier analysis of NOD / SCID mice injected i . v . with luciferase - expressing SAMHD1 + / + or - / - HL - 60 / iva cell clones ( day 0 ) and treated with ara - C and / or HU as indicated ( day 6 ) . n = 6 per treatment group . Kaplan - Meier analysis of NOD / SCID mice injected i . v . with luciferase - expressing SAMHD1 + / + THP - 1 cell clone ( day 0 ) and treated with ara - C and / or dF - dC as indicated ( day 6 ) . n = 7 per treatment group . Kaplan - Meier analysis of CD45 . 2 C57BL / 6J mice injected i . v . with murine MLL - AF9 - transformed AML blasts ( day 0 ) and treated with ara - C and / or HU days 20 - 24 . n = 5 per treatment group , except for vehicle ( n = 4 ) .

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Figure 2Kaplan-Meier analysis of NOD/SCID mice injected i.v. with luciferase-expressing SAMHD1+/+ or -/- THP-1 cell clones (day 0) and treated with ara-C and/or HU as indicated (day 6). n = 6 per treatment group.Kaplan-Meier analysis of NOD/SCID mice injected i.v. with luciferase-expressing SAMHD1+/+ or -/- HL-60/iva cell clones (day 0) and treated with ara-C and/or HU as indicated (day 6). n = 6 per treatment group.Kaplan-Meier analysis of NOD/SCID mice injected i.v. with luciferase-expressing SAMHD1+/+ THP-1 cell clone (day 0) and treated with ara-C and/or dF-dC as indicated (day 6). n = 7 per treatment group.Kaplan-Meier analysis of CD45.2 C57BL/6J mice injected i.v. with murine MLL-AF9-transformed AML blasts (day 0) and treated with ara-C and/or HU days 20-24. n = 5 per treatment group, except for vehicle (n = 4).


Figure 3Drug synergy plots for ara - C and HU or dF - dC in primary patient - derived AML blasts . Each data point indicates an average delta score from a single patient sample subjected to a dose - response matrix experiment performed in triplicate , n = 16 for HU and n = 9 for dF - dC . Zero , > 0 , or < 0 corresponds to additive , synergy , or antagonism , respectively , whilst > 5 indicates strong synergy . Median , upper and lower quartiles , and range of delta scores are indicated by box - and - whisker plots . Pearson correlation of relative SAMHD1 protein abundance and synergy delta scores for ara - C and HU or dF - dC in primary patient - derived AML blasts ( n = 23 ) . Immunoblot of primary patient - derived AML blasts treated with control ( dX ) or Vpx - containing ( X ) virus - like particles ( VLPs ) : patient A2953 ( C ) , ALG17 _ 001 ( E ) . Accompanying proliferation inhibition analysis of ara - C and indicated RNRi combination in these samples : patient A2953 ( D ) , ALG17 _ 001 ( D ) . Error bars indicate s . d . of single experiment performed in triplicate . Paired drug synergy plot for ara - C and RNRi ( HU , n = 7 ; dF - dC , n = 5 ) in primary patient - derived AML blasts pre - treated with control ( dX ) or Vpx - containing ( X ) VLPs . Zero , > 0 , or < 0 corresponds to additive , synergy , or antagonism , respectively , whilst > 5 indicates strong synergy and < 5 indicates strong antagonism . Each data point indicates an average delta score from a single patient sample subjected to a dose - response matrix experiment performed in triplicate . Statistical testing was performed using two - way ANOVA .

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Figure 3Drug synergy plots for ara-C and HU or dF-dC in primary patient-derived AML blasts. Each data point indicates an average delta score from a single patient sample subjected to a dose-response matrix experiment performed in triplicate, n = 16 for HU and n = 9 for dF-dC. Zero, >0, or <0 corresponds to additive, synergy, or antagonism, respectively, whilst >5 indicates strong synergy. Median, upper and lower quartiles, and range of delta scores are indicated by box-and-whisker plots.Pearson correlation of relative SAMHD1 protein abundance and synergy delta scores for ara-C and HU or dF-dC in primary patient-derived AML blasts (n = 23).Immunoblot of primary patient-derived AML blasts treated with control (dX) or Vpx-containing (X) virus-like particles (VLPs): patient A2953 (C), ALG17_001 (E). Accompanying proliferation inhibition analysis of ara-C and indicated RNRi combination in these samples: patient A2953 (D), ALG17_001 (D). Error bars indicate s.d. of single experiment performed in triplicate.Paired drug synergy plot for ara-C and RNRi (HU, n = 7; dF-dC, n = 5) in primary patient-derived AML blasts pre-treated with control (dX) or Vpx-containing (X) VLPs. Zero, >0, or <0 corresponds to additive, synergy, or antagonism, respectively, whilst >5 indicates strong synergy and <5 indicates strong antagonism. Each data point indicates an average delta score from a single patient sample subjected to a dose-response matrix experiment performed in triplicate. Statistical testing was performed using two-way ANOVA.


Figure 4Intracellular dNTP measurements using a primer - extension assay in SAMHD1 + / + THP - 1 cells treated for 4 or 24 h with either 50 µM HU ( middle panel ) or 2 . 5 nM 3 - AP ( right panel ) , ratios of dCTP - to - dATP were calculated . Bars indicate mean values of three independent experiments , error bars indicate s . e . m . Statistical analyses were done using unpaired two - tailed t - tests : * * , P < 0 . 01 . Intracellular ara - CTP ( upper panel ) and dCTP : dATP ratio ( lower panel ) in the indicated cell lines following the indicated treatment determined using HPLC - MS / MS . SAMHD1 - / - THP - 1 cells were treated with 500 nM ara - C and SAMHD1 + / + THP - 1 cells were treated with either solvent , 500 nM ara - C or a combination of 500 nM ara - C and an RNRi ( HU , 50 µM ; dF - dC , 10 nM ; 3 - AP , 150 nM ) for 24 h prior . Values relative to mean ara - CTP amounts in ara - C treated SAMHD1 + / + THP - 1 cells shown ( indicated by dashed line ) . Circles , columns and error bars correspond to individual values , means and s . e . m . of at least three experiments performed independently . Quantificaiton of dCK phosphorylation at serine - 74 ( S74 ) with respect to total dCK in SAMHD1 + / + THP - 1 cells treated with either solvent , 500 nM ara - C or a combination of 500 nM ara - C and an RNRi ( HU , 50 µM ; dF - dC , 10 nM ; 3 - AP , 150 nM ) for 24 h . Circles and squares , columns and error bars correspond to individual measurements , means and s . e . m . of one representative out of two independent experiments performed in triplicates . Measurement of released inorganic triphosphate ( PPPi ) from hydrolysis of ara - CTP ( 200 µM ) by recombinant SAMHD1 ( 0 . 35 µM ) in the presence of GTP ( 200 µM ) and a titration of different non - hydrolysable dNTP analogues ( dNTPαS ) in the enzyme - coupled malachite green assay . Error bars indicate s . e . m . of two independent experiments performed in triplicate and quadruplet .

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Figure 4Intracellular dNTP measurements using a primer-extension assay in SAMHD1+/+ THP-1 cells treated for 4 or 24 h with either 50 µM HU (middle panel) or 2.5 nM 3-AP (right panel), ratios of dCTP-to-dATP were calculated. Bars indicate mean values of three independent experiments, error bars indicate s.e.m. Statistical analyses were done using unpaired two-tailed t-tests: **, P < 0.01.Intracellular ara-CTP (upper panel) and dCTP:dATP ratio (lower panel) in the indicated cell lines following the indicated treatment determined using HPLC-MS/MS. SAMHD1-/- THP-1 cells were treated with 500 nM ara-C and SAMHD1+/+ THP-1 cells were treated with either solvent, 500 nM ara-C or a combination of 500 nM ara-C and an RNRi (HU, 50 µM; dF-dC, 10 nM; 3-AP, 150 nM) for 24 h prior. Values relative to mean ara-CTP amounts in ara-C treated SAMHD1+/+ THP-1 cells shown (indicated by dashed line). Circles, columns and error bars correspond to individual values, means and s.e.m. of at least three experiments performed independently.Quantificaiton of dCK phosphorylation at serine-74 (S74) with respect to total dCK in SAMHD1+/+ THP-1 cells treated with either solvent, 500 nM ara-C or a combination of 500 nM ara-C and an RNRi (HU, 50 µM; dF-dC, 10 nM; 3-AP, 150 nM) for 24 h. Circles and squares, columns and error bars correspond to individual measurements, means and s.e.m. of one representative out of two independent experiments performed in triplicates.Measurement of released inorganic triphosphate (PPPi) from hydrolysis of ara-CTP (200 µM) by recombinant SAMHD1 (0.35 µM) in the presence of GTP (200 µM) and a titration of different non-hydrolysable dNTP analogues (dNTPαS) in the enzyme-coupled malachite green assay. Error bars indicate s.e.m. of two independent experiments performed in triplicate and quadruplet.


Figure 1A . NIH3T3 cells transfected with KV10 . 1 * EGFP did not show primary cilia . Cells were transiently transfected , after 24 h serum was removed for additional 24 hours to induce ciliogenesis , and finally cells were stained with anti - acetylated α - tubulin . While most cells were ciliated , those showing green fluorescence were devoid of cilia . Scale bar : 10µm . B . NIH3T3 cells transfected with KV10 . 1 ( red bars ) showed markedly less cilia than control cells ( empty vector , white bars ) . Subconfluent cultures grown in the presence of FCS were serum starved for 24 hours to induce ciliogenesis . Cilia were stained with anti acetylated α - tubulin antibody . To determine ciliary disassembly , cells were starved for 24 hours and then incubated for 4h in FCS to induce cell cycle re - entry and ciliary resorption . C . Similarly , hTERT - RPE1 cells transfected with KV10 . 1 also showed less cilia . Ciliogenesis and ciliary disassembly were induced as in B and cilia were stained using anti acetylated α - tubulin and quantified as in A . The Inset shows the equivalent experiment using the structurally related potassium channel KV10 . 2 , which did not alter the frequency of expression of cilia . D . Examples of fields of view of hTERT - RPE1 cells transfected with KV10 . 1 , serum starved for 24 hours and cilia were revealed with anti Arl13B antibody ( arrows ) . A majority of control - transfected cells showed cilia , while KV10 . 1 transfected did not . Scale bar : 10µm .

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Figure 1A. NIH3T3 cells transfected with KV10.1*EGFP did not show primary cilia. Cells were transiently transfected, after 24 h serum was removed for additional 24 hours to induce ciliogenesis, and finally cells were stained with anti-acetylated α-tubulin. While most cells were ciliated, those showing green fluorescence were devoid of cilia. Scale bar: 10µm.B. NIH3T3 cells transfected with KV10.1 (red bars) showed markedly less cilia than control cells (empty vector, white bars). Subconfluent cultures grown in the presence of FCS were serum starved for 24 hours to induce ciliogenesis. Cilia were stained with anti acetylated α-tubulin antibody. To determine ciliary disassembly, cells were starved for 24 hours and then incubated for 4h in FCS to induce cell cycle re-entry and ciliary resorption.C. Similarly, hTERT-RPE1 cells transfected with KV10.1 also showed less cilia. Ciliogenesis and ciliary disassembly were induced as in B and cilia were stained using anti acetylated α-tubulin and quantified as in A. The Inset shows the equivalent experiment using the structurally related potassium channel KV10.2, which did not alter the frequency of expression of cilia.D. Examples of fields of view of hTERT-RPE1 cells transfected with KV10.1, serum starved for 24 hours and cilia were revealed with anti Arl13B antibody (arrows). A majority of control-transfected cells showed cilia, while KV10.1 transfected did not. Scale bar: 10µm.


Figure 2A . Representative images showing hTERT - RPE1 grown in the presence of serum and stained with anti acetylated α - tubulin antibody . There were more ciliated cells after treatment with siRNA against KV10 . 1 ( KV10 . 1 KD ) , as compared to scrambled siRNA - transfected cells , which were devoid of cilia ( control ) . Scale bar : 10 µm . B . Quantification of the effect depicted in A for the number of images indicated at the base of the columns . C . Time course of ciliary disassembly . hTERT - RPE1 cells were serum starved for 24 hours , fixed at the times indicated after serum reintroduction , and stained with anti acetylated α - tubulin to reveal cilia . While in the presence of scrambled siRNA the abundance of cilia decreased rapidly ( black line ) , it remained virtually unchanged in cells where KV10 . 1 was knocked down ( red ) . N = 8 - 13 . D . Time course of ciliary shortening after serum reintroduction . In the same experiments as in C , the length of the remaining cilia was measured automatically using ImageJ ( FIJI ) . While ciliary length decreased with a similar time course in the siRNA - treated cells as in controls , cilia were longer in KV10 . 1 - knockdown cells . A re - elongation of cilia was evident in both populations after 6 hours . Values are mean ± SEM for a number of cilia between 62 ( control , 4 . 5 h ) and 308 ( both groups , 1 . 5 h ) . E . Blockade of permeation through the K + channel altered the speed of deciliation . After serum starvation , medium containing serum and Astemizole ( 10 µM ) was added , and cells were fixed and stained at he indicated time points ( N = 12 ) .

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Figure 2A. Representative images showing hTERT-RPE1 grown in the presence of serum and stained with anti acetylated α-tubulin antibody. There were more ciliated cells after treatment with siRNA against KV10.1 (KV10.1 KD), as compared to scrambled siRNA-transfected cells, which were devoid of cilia (control). Scale bar: 10 µm.B. Quantification of the effect depicted in A for the number of images indicated at the base of the columns.C. Time course of ciliary disassembly. hTERT-RPE1 cells were serum starved for 24 hours, fixed at the times indicated after serum reintroduction, and stained with anti acetylated α-tubulin to reveal cilia. While in the presence of scrambled siRNA the abundance of cilia decreased rapidly (black line), it remained virtually unchanged in cells where KV10.1 was knocked down (red). N=8-13.D. Time course of ciliary shortening after serum reintroduction. In the same experiments as in C, the length of the remaining cilia was measured automatically using ImageJ (FIJI). While ciliary length decreased with a similar time course in the siRNA-treated cells as in controls, cilia were longer in KV10.1-knockdown cells. A re-elongation of cilia was evident in both populations after 6 hours. Values are mean ± SEM for a number of cilia between 62 (control, 4.5 h) and 308 (both groups, 1.5 h).E. Blockade of permeation through the K+ channel altered the speed of deciliation. After serum starvation, medium containing serum and Astemizole (10 µM) was added, and cells were fixed and stained at he indicated time points (N=12).


Figure 3A . Quantification of the amount of ciliated cells in primary embryonic fibroblasts from KV10 . 1 knockout mice . Actively growing cultures , cultures after 24 h serum starvation , and four hours after subsequent serum reintroduction were stained for the presence of cilia using anti - acetylated α - tubulin . An increase in frequency of cilia in the knockout cells was evident under all conditions , but especially marked in the ciliary resorption test ( 4h after serum reintroduction ) , indicating an influence of KV10 . 1 expression on deciliation . B . Exogenous expression of human KV10 . 1 reverted the phenotype in MEFs . Cells obtained from three animals were transfected with KV10 . 1 or empty vector ( mock ) , serum starved for 48 h and cilia were revealed by immunocytochemistry using anti - acetylated α - tubulin in the indicated number of images . KV10 . 1 expression decreased the frequency of ciliated cells . C . MEFs from knockout mice did not show primary cilia when transfected with KV10 . 1 . Cells were co - transfected with human KV10 . 1 and mVenus , serum starved for 48 hours and then fixed and stained for cilia ( AcTub ) . Venus - positive cells did not show cilia . Scale bar : 25 µm . D . In wild type MEFs , transfection with KV10 . 1 reduced the presence of primary cilia . When a gain - of - function mutant ( L352V ) that originates developmental defects in human patients was transfected , the frequency of cells with cilia was further reduced . E . Sensitivity to SHH signaling was significantly increased in primary fibroblasts from KV10 . 1 knockout mice , in agreement with the higher abundance of primary cilia . Cells were serum starved and SHH was stimulated after 24 hours by addition of culture supernatant of SHH - expressing HEK293 cells . Expression of mRNA for Gli1 was used as reporter of SHH activation by qRT - PCR .

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Figure 3A. Quantification of the amount of ciliated cells in primary embryonic fibroblasts from KV10.1 knockout mice. Actively growing cultures, cultures after 24 h serum starvation, and four hours after subsequent serum reintroduction were stained for the presence of cilia using anti-acetylated α-tubulin. An increase in frequency of cilia in the knockout cells was evident under all conditions, but especially marked in the ciliary resorption test (4h after serum reintroduction), indicating an influence of KV10.1 expression on deciliation.B. Exogenous expression of human KV10.1 reverted the phenotype in MEFs. Cells obtained from three animals were transfected with KV10.1 or empty vector (mock), serum starved for 48 h and cilia were revealed by immunocytochemistry using anti-acetylated α-tubulin in the indicated number of images. KV10.1 expression decreased the frequency of ciliated cells.C. MEFs from knockout mice did not show primary cilia when transfected with KV10.1. Cells were co-transfected with human KV10.1 and mVenus, serum starved for 48 hours and then fixed and stained for cilia (AcTub). Venus-positive cells did not show cilia. Scale bar: 25 µm.D. In wild type MEFs, transfection with KV10.1 reduced the presence of primary cilia. When a gain-of-function mutant (L352V) that originates developmental defects in human patients was transfected, the frequency of cells with cilia was further reduced.E. Sensitivity to SHH signaling was significantly increased in primary fibroblasts from KV10.1 knockout mice, in agreement with the higher abundance of primary cilia. Cells were serum starved and SHH was stimulated after 24 hours by addition of culture supernatant of SHH-expressing HEK293 cells. Expression of mRNA for Gli1 was used as reporter of SHH activation by qRT-PCR.


Figure 4A . Actively proliferating hTERT - RPE1 cells were transfected with scrambled or KV10 . 1 siRNA , and cell cycle distribution was determined using acridine orange staining . Knockdown of KV10 . 1 induces an increase in cells with DNA / RNA content compatible with G0 ( rectangle ) . However , the quantitative increase ( to approximately 10 % ) was insufficient to explain the abundance of ciliated cells ( over 40 % ) B . The proliferation marker Ki67 frequently coexists with primary cilia in KV10 . 1 knockdown cells . Exponentially growing cells were stained for acetylated α - tubulin ( red , AvTub ) and Ki67 ( green ) . Nuclei were counterstained with Draq5 ( blue ) . Scale bar : 10 µm . C . Quantification of the effects observed in B . Although the positivity for Ki67 ( proliferating cells ) and the presence of cilia were not always excluding each other even in cells transfected with control siRNA , KV10 . 1 knockdown increased the frequency of ciliated cells , while it reduced the amount of Ki67 positive , actively proliferating cells . Of those cells presenting a cilium , the fraction of simultaneously Ki - 67 - positive cells was much larger upon KV10 . 1 knockdown . Scale bar , 10 µm . D . Primary cilia were present in cells actively synthesizing DNA ( measured by EdU incorporation ) . Cells were transfected with siRNA against KV10 . 1 , and 24 hours later were incubated in the presence of EdU for 4h before fixation . KV10 . 1 knockdown resulted in the presence of abundant EdU - positive and simultaneously ciliated cells , suggesting a decoupling between deciliation and cell cycle progression . Scale bar :

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Figure 4A. Actively proliferating hTERT-RPE1 cells were transfected with scrambled or KV10.1 siRNA, and cell cycle distribution was determined using acridine orange staining. Knockdown of KV10.1 induces an increase in cells with DNA/RNA content compatible with G0 (rectangle). However, the quantitative increase (to approximately 10%) was insufficient to explain the abundance of ciliated cells (over 40%)B. The proliferation marker Ki67 frequently coexists with primary cilia in KV10.1 knockdown cells. Exponentially growing cells were stained for acetylated α-tubulin (red, AvTub) and Ki67 (green). Nuclei were counterstained with Draq5 (blue). Scale bar: 10 µm.C. Quantification of the effects observed in B. Although the positivity for Ki67 (proliferating cells) and the presence of cilia were not always excluding each other even in cells transfected with control siRNA, KV10.1 knockdown increased the frequency of ciliated cells, while it reduced the amount of Ki67 positive, actively proliferating cells. Of those cells presenting a cilium, the fraction of simultaneously Ki-67-positive cells was much larger upon KV10.1 knockdown. Scale bar, 10 µm.D. Primary cilia were present in cells actively synthesizing DNA (measured by EdU incorporation). Cells were transfected with siRNA against KV10.1, and 24 hours later were incubated in the presence of EdU for 4h before fixation. KV10.1 knockdown resulted in the presence of abundant EdU-positive and simultaneously ciliated cells, suggesting a decoupling between deciliation and cell cycle progression. Scale bar: 10µm


Figure 5A . An anti - KV10 . 1 antibody directed against an extracellular epitope labels a structure related to the centrosome ( pericentrin - positive ) and positive for Arl13b with an appearance suggestive of the primary cilium in HFF ( human foreskin fibroblast ) cells . Cells were serum starved for 24 or 48h , fixed , blocked ( 10 % BSA ) and then stained without permeabilization with anti - KV10 . 1 antibody recognizing an extracellular epitope . Immunostaining for pericentrin was performed after subsequent permeabilization and additional block . Scale bars : 10 µm ( pericentrin ) , 20 µm ( Arl13b ) . B . Anti - KV10 . 1 antibodies recognize ciliary structures in hTERT - RPE1 cells . After serum starvation for 48h , serum was reintroduced for 4h to induce KV10 . 1 expression and ciliary resorption . Cells were then fixed and stained with a mixture of anti - KV10 . 1 mouse monoclonals and a rabbit monoclonal anti acetylated α - tubulin . Scale bar : 2 µm . C . Specificity of the monoclonal anti KV10 . 1 ( mAb 62 ) as revealed by staining MEFs from wild type ( left ) or knockout animals ( right ) Scale bar : 50 µm : D . Images from primary cilia of MEFs from KV10 . 1 knockout mice obtained using mAb62 and a rabbit polyclonal anti acetylated a - tubulin antibody . No recognizable structures were stained by mAb62 . Scale bar : 1 µm . E . In contrast , mAb62 did stain ciliary - related structures ( acetylated - tubulin - positive ) in wild type mice . Scale bar : 1 µm .

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Figure 5A. An anti-KV10.1 antibody directed against an extracellular epitope labels a structure related to the centrosome (pericentrin-positive) and positive for Arl13b with an appearance suggestive of the primary cilium in HFF (human foreskin fibroblast) cells. Cells were serum starved for 24 or 48h, fixed, blocked (10% BSA) and then stained without permeabilization with anti-KV10.1 antibody recognizing an extracellular epitope. Immunostaining for pericentrin was performed after subsequent permeabilization and additional block. Scale bars: 10 µm (pericentrin), 20 µm (Arl13b).B. Anti-KV10.1 antibodies recognize ciliary structures in hTERT-RPE1 cells. After serum starvation for 48h, serum was reintroduced for 4h to induce KV10.1 expression and ciliary resorption. Cells were then fixed and stained with a mixture of anti-KV10.1 mouse monoclonals and a rabbit monoclonal anti acetylated α-tubulin. Scale bar: 2 µm.C. Specificity of the monoclonal anti KV10.1 (mAb 62) as revealed by staining MEFs from wild type (left) or knockout animals (right) Scale bar: 50 µm:D. Images from primary cilia of MEFs from KV10.1 knockout mice obtained using mAb62 and a rabbit polyclonal anti acetylated a-tubulin antibody. No recognizable structures were stained by mAb62. Scale bar: 1 µm.E. In contrast, mAb62 did stain ciliary-related structures (acetylated-tubulin-positive) in wild type mice. Scale bar: 1 µm.


Figure 6A . KV10 . 1 localizes to pericentrin - positive structures during ciliary resorption . After short ( 4h ) reintroduction of FCS in serum starved hTERT RPE1 cells , the signal of anti - KV10 . 1 antibody co - localized with pericentrin . Scale bar : 25µm . B . Cells expressing a KV10 . 1 * mVenus fusion under the control of the endogenous promoter to render a mild overexpression were fixed and immunostained for pericentrin . Venus fluorescence colocalized with pericentrin - positive structures . Scale bar : 5µm .

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Figure 6A. KV10.1 localizes to pericentrin-positive structures during ciliary resorption. After short (4h) reintroduction of FCS in serum starved hTERT RPE1 cells, the signal of anti-KV10.1 antibody co-localized with pericentrin. Scale bar: 25µm.B. Cells expressing a KV10.1*mVenus fusion under the control of the endogenous promoter to render a mild overexpression were fixed and immunostained for pericentrin. Venus fluorescence colocalized with pericentrin-positive structures. Scale bar: 5µm.


Figure 7A . Density centrifugation of hTERT - RPE1 cell extracts followed by immunoblot revealed the presence of KV10 . 1 in the same fractions as Aurora A , phospho - aurora A and a - tubulin ( centrosomal fractions ) . B . KV10 . 1 is also present in microsomal fractions . Cytoplasmic fractions obtained as in A were ultracentrifuged to sediment membranes . In those preparations , immunoprecipitation revealed the presence of full - length KV10 . 1 in both the centrosomal and the microsomal fractions ( obtained by ultracentrifugation ) .

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Figure 7A. Density centrifugation of hTERT-RPE1 cell extracts followed by immunoblot revealed the presence of KV10.1 in the same fractions as Aurora A, phospho-aurora A and a-tubulin (centrosomal fractions).B. KV10.1 is also present in microsomal fractions. Cytoplasmic fractions obtained as in A were ultracentrifuged to sediment membranes. In those preparations, immunoprecipitation revealed the presence of full-length KV10.1 in both the centrosomal and the microsomal fractions (obtained by ultracentrifugation).


Figure 8B . Deletion of the CLS does not preclude functional expression at the plasma membrane . Two electrode - voltage clamp recordings from Xenopus oocytes injected with cRNA for a KV10 . 1 channel lacking the CLS at the C - terminus revealed voltage - gated currents after stimulation using the potential depicted in the inset . C . The Conductance / voltage relationship of the deleted channel was similar to that of wild - type ( shown for comparison as a solid line ) . D . In the absence of serum , the C - terminus of KV10 . 1 reduced the number of cilia ( maintained cells deciliated ) also after deletion of the CLS ( data for controls is reproduced from Figure 5A ) The CLS is required to induce deciliation . When hTERT - RPE1 and 3T3 cells transfected with KV10 . 1 or KV10 . 1ΔCLS were serum starved , and serum was reintroduced after 24 hours for 4 h , KV10 . 1ΔCLS failed to reduce the fraction of ciliated cells . E . The CLS is required for KV10 . 1 - induced tumorigenesis . CHO cells were transfected with the indicated constructs and implanted into the flank of nude mice . KV10 . 1ΔCLS transfected cells did not induce larger tumors than the control .

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Figure 8B. Deletion of the CLS does not preclude functional expression at the plasma membrane. Two electrode-voltage clamp recordings from Xenopus oocytes injected with cRNA for a KV10.1 channel lacking the CLS at the C-terminus revealed voltage-gated currents after stimulation using the potential depicted in the inset.C. The Conductance/voltage relationship of the deleted channel was similar to that of wild-type (shown for comparison as a solid line).D. In the absence of serum, the C-terminus of KV10.1 reduced the number of cilia (maintained cells deciliated) also after deletion of the CLS (data for controls is reproduced from Figure 5A)The CLS is required to induce deciliation. When hTERT-RPE1 and 3T3 cells transfected with KV10.1 or KV10.1ΔCLS were serum starved, and serum was reintroduced after 24 hours for 4 h, KV10.1ΔCLS failed to reduce the fraction of ciliated cells.E. The CLS is required for KV10.1-induced tumorigenesis. CHO cells were transfected with the indicated constructs and implanted into the flank of nude mice. KV10.1ΔCLS transfected cells did not induce larger tumors than the control.


Figure 9A . Overexpression of cortactin compensated for the knockdown of KV10 . 1 in ciliary disassembly ( 4 h serum reintroduction after starvation ) . B . Deletion of the domain of KV10 . 1 responsible for interaction with cortactin abolished the effects of KV10 . 1 overexpression on the abundance of cilia . C . In mouse embryonic fibroblasts , active ( phosphorylated ) cortactin is increased as compared to wild type , possibly compensating the lack of KV10 . 1 .

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Figure 9A. Overexpression of cortactin compensated for the knockdown of KV10.1 in ciliary disassembly (4 h serum reintroduction after starvation).B. Deletion of the domain of KV10.1 responsible for interaction with cortactin abolished the effects of KV10.1 overexpression on the abundance of cilia.C. In mouse embryonic fibroblasts, active (phosphorylated) cortactin is increased as compared to wild type, possibly compensating the lack of KV10.1.


Figure 1A . Representative confocal micrographs of HeLa cells infected or not with B . abortus 544 for the indicated times , then fixed and immunostained for TOMM20 ( Alexa Fluor 488 - Green ) and B . abortus LPS ( Alexa Fluor 568 - Magenta ) . DNA was stained with Hoechst 33258 ( Blue ) . Scale bars : 20 µm .

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Figure 1A. Representative confocal micrographs of HeLa cells infected or not with B. abortus 544 for the indicated times, then fixed and immunostained for TOMM20 (Alexa Fluor 488 - Green) and B. abortus LPS (Alexa Fluor 568 - Magenta). DNA was stained with Hoechst 33258 (Blue). Scale bars: 20 µm.


Figure 2A . Representative confocal micrographs of HeLa cells infected or not with B . abortus 544 GFP for 48 h , then fixed and immunostained for the β - subunit of the ATP synthase ( Alexa Fluor 633 - Magenta ) and LC3 ( Alexa Fluor 568 - Green ) . DNA was stained with Hoechst 33258 ( Blue ) . Arrows indicate LC3 - ATP synthase β - positive punctae . Scale bars : 20 µm . B . Quantification of the number of LC3 - β - subunit of the ATP synthase - positive punctae per HeLa cell infected or not ( NI ) with B . abortus 544 GFP for 24 , 48 and 72 h from micrographs shown in ( A ) Data are presented as means ± SD from n = 3 ( biological replicates independent experiments ( the numbers indicated in the columns represent the number of cells analysed per condition ) . Statistical analyses were performed using a two - way ANOVA followed by a Šidàk ' s multiple comparisons test ; asterisks indicate significant differences compared to the control ( NI ) ; ns : not significant ; * * * : p < 0 . 001 ; * * * * : p < 0 , 0001 ; hashtags indicate significant differences between infected conditions throughout time ; # : p < 0 . 05 ; # # : p < 0 . 01 . C . Representative confocal micrographs of HeLa cells transfected with a FIS1 - GFP ( Green ) - mCherry ( Magenta ) expression construct , infected or not with B . abortus 544 for 48 h , then fixed and immunostained for B . abortus LPS ( Alexa Fluor 633 - Red ) . DNA was stained with Hoechst 33258 ( Blue ) . Arrows indicate FIS1 - mCherry - positive - GFP - negative punctae . Scale bars : 20 µm . D . Quantification of the number of FIS1 - mCherry - positive - GFP - negative punctae per HeLa cell infected or not ( NI ) with B . abortus 544 for 48 h from micrographs shown in ( C ) . Data are presented as means ± SD from n = 3 ( biological replicates ) independent experiments ( the numbers indicated in the columns represent the number of cells analysed per condition ) . Statistical analyses were performed using an unpaired two - tailed Student ' s t - test ; * * * : p < 0 . 001 ( p = 0 . 0008 ) .

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Figure 2A. Representative confocal micrographs of HeLa cells infected or not with B. abortus 544 GFP for 48 h, then fixed and immunostained for the β-subunit of the ATP synthase (Alexa Fluor 633 - Magenta) and LC3 (Alexa Fluor 568 - Green). DNA was stained with Hoechst 33258 (Blue). Arrows indicate LC3-ATP synthase β-positive punctae. Scale bars: 20 µm. B. Quantification of the number of LC3-β-subunit of the ATP synthase-positive punctae per HeLa cell infected or not (NI) with B. abortus 544 GFP for 24, 48 and 72 h from micrographs shown in (A) Data are presented as means ± SD from n=3 (biological replicates independent experiments (the numbers indicated in the columns represent the number of cells analysed per condition). Statistical analyses were performed using a two-way ANOVA followed by a Šidàk's multiple comparisons test; asterisks indicate significant differences compared to the control (NI); ns: not significant; ***: p <0.001; ****: p <0,0001; hashtags indicate significant differences between infected conditions throughout time; #: p <0.05; ##: p <0.01.C. Representative confocal micrographs of HeLa cells transfected with a FIS1-GFP(Green)-mCherry(Magenta) expression construct, infected or not with B. abortus 544 for 48 h, then fixed and immunostained for B. abortus LPS (Alexa Fluor 633 - Red). DNA was stained with Hoechst 33258 (Blue). Arrows indicate FIS1-mCherry-positive-GFP-negative punctae. Scale bars: 20 µm. D. Quantification of the number of FIS1-mCherry-positive-GFP-negative punctae per HeLa cell infected or not (NI) with B. abortus 544 for 48 h from micrographs shown in (C). Data are presented as means ± SD from n=3 (biological replicates) independent experiments (the numbers indicated in the columns represent the number of cells analysed per condition). Statistical analyses were performed using an unpaired two-tailed Student's t-test; ***: p <0.001 (p = 0.0008).