D Primary hippocampal neurons expressing EGFP - TDP - 43 Wt , 12D or 12A with additional NLS mutation . Bar , 80 µm . Right : Zoomed images of white squares ( TDP - 43 signal ) . Bar , 10 µm .

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

D Primary hippocampal neurons expressing EGFP-TDP-43 Wt, 12D or 12A with additional NLS mutation. Bar, 80 µm. Right: Zoomed images of white squares (TDP-43 signal). Bar, 10 µm.


E SG recruitment of EGFP - TDP - 43 NLS mutant variants ( Wt , 12D , 12A ) in primary hippocampal neurons . SG formation was induced by 1h heat shock at 42 ° C . SGs and TDP - 43 were monitored by G3BP1 antibody staining and EGFP fluorescence , respectively . For clarity , signals were converted to grey values in the individual channels ( first two columns ) . In the merge ( third column ) , EGFP - TDP - 43 shown in green , G3BP1 in red and nuclei ( DAPI staining ) in turquoise . Bar , 20 µm .

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

E SG recruitment of EGFP-TDP-43 NLS mutant variants (Wt, 12D, 12A) in primary hippocampal neurons. SG formation was induced by 1h heat shock at 42°C. SGs and TDP-43 were monitored by G3BP1 antibody staining and EGFP fluorescence, respectively. For clarity, signals were converted to grey values in the individual channels (first two columns). In the merge (third column), EGFP-TDP-43 shown in green, G3BP1 in red and nuclei (DAPI staining) in turquoise. Bar, 20 µm.


Effects of ActA and BMP4 on the expression of selected pluripotency , PGC , and mesodermal genes during PGC ‐ precursor induction . DM : dorsomorphin , SB : SB431542 . Samples were calibrated with iPSC values , and iPSC values depict 1 . Y ‐ axes are in log2 scale .

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

Effects of ActA and BMP4 on the expression of selected pluripotency, PGC, and mesodermal genes during PGC‐precursor induction. DM: dorsomorphin, SB: SB431542. Samples were calibrated with iPSC values, and iPSC values depict 1. Y‐axes are in log2 scale.


Gene expression dynamics of selected pluripotency , PGC , and mesodermal genes during prolonged PGC ‐ precursor induction of up to day 7 . Y ‐ axes are in log2 scale .

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

Gene expression dynamics of selected pluripotency, PGC, and mesodermal genes during prolonged PGC‐precursor induction of up to day 7. Y‐axes are in log2 scale.


Immunofluorescence analysis for OCT4 ( red ) , BLIMP1 ( green ) , and T ( white ) on days 0 , 1 , and 2 . Nuclei were stained with Hoechst ( blue ) . The culture contained BMP4 ( 5 ng / ml ) , Activin A ( 50 ng / ml ) , and bFGF ( 20 ng / ml ) . Scale bar : 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 O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O

Immunofluorescence analysis for OCT4 (red), BLIMP1 (green), and T (white) on days 0, 1, and 2. Nuclei were stained with Hoechst (blue). The culture contained BMP4 (5 ng/ml), Activin A (50 ng/ml), and bFGF (20 ng/ml). Scale bar: 100 μm.


FACS analysis of the concentration ‐ dependent effect of BMP4 on TRA ‐ 1 ‐ 81 + / cKIT + PGCLCs on day 4 . B25 : 25 ng / ml BMP4 ; B50 : 50 ng / ml BMP4 ; B100 : 100 ng / ml BMP4 .

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

FACS analysis of the concentration‐dependent effect of BMP4 on TRA‐1‐81+/cKIT+ PGCLCs on day 4. B25: 25 ng/ml BMP4; B50: 50 ng/ml BMP4; B100: 100 ng/ml BMP4.


FACS analysis of TRA ‐ 1 ‐ 81 and cKIT during PGC induction of up to day 8 .

FACS analysis of TRA‐1‐81 and cKIT during PGC induction of up to day 8.


Gene expression analysis of selected PGC markers in TRA ‐ 1 ‐ 81 + / cKIT + and SSEA1 + / cKIT + FACS fractions of d4 PGCLCs . Samples were calibrated with iPSC values , and iPSC values depict 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

Gene expression analysis of selected PGC markers in TRA‐1‐81+/cKIT+ and SSEA1+/cKIT+ FACS fractions of d4 PGCLCs. Samples were calibrated with iPSC values, and iPSC values depict 1.


Gene expression dynamics of selected pluripotency and PGC genes in FACS ‐ sorted , specified cells during PGC induction . The value for iPSCs is set as 1 , and values are on log2 scale .

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

Gene expression dynamics of selected pluripotency and PGC genes in FACS‐sorted, specified cells during PGC induction. The value for iPSCs is set as 1, and values are on log2 scale.


Immunofluorescence analysis for OCT4 ( green ) , BLIMP1 ( red ) , and STELLA ( yellow ) in TRA ‐ 1 ‐ 81 + / cKIT + PGCLCs on day 6 . Nuclei were stained with Hoechst ( blue ) . Scale bar : 15 μm .

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

Immunofluorescence analysis for OCT4 (green), BLIMP1 (red), and STELLA (yellow) in TRA‐1‐81+/cKIT+ PGCLCs on day 6. Nuclei were stained with Hoechst (blue). Scale bar: 15 μm.


Bisulfite sequence analysis of DMRs of the imprinted genes ( PEG1 , KvDMR1 , PEG10 , and NESP55 ) in iPSCs ( top ) and d6 TRA ‐ 1 ‐ 81 + / cKIT + PGCLCs ( bottom ) . White and black circles represent unmethylated and methylated CpG sequences , respectively .

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

Bisulfite sequence analysis of DMRs of the imprinted genes (PEG1, KvDMR1, PEG10, and NESP55) in iPSCs (top) and d6 TRA‐1‐81+/cKIT+ PGCLCs (bottom). White and black circles represent unmethylated and methylated CpG sequences, respectively.


Immunofluorescence analysis for 5mC in d6 PGCLCs . Nuclei were stained with Hoechst ( blue ) . Scale bar : 50 μm .

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

Immunofluorescence analysis for 5mC in d6 PGCLCs.Nuclei were stained with Hoechst (blue). Scale bar: 50 μm.


Transplantation of human iPSCs and PGCLCs into ovaries of recipient mice . ( i ) left ovary : control ovary without transplantation ; right ovary : teratoma from iPSC ‐ reconstituted ovary ; ( ii ) PGCLC ‐ reconstituted ovaries . Note that no teratomas had formed . Bottom panels : Immunohistochemical analysis for NUMA in sections of iPSC ‐ and PGCLC ‐ reconstituted ovaries . ( iii ) iPSC ‐ induced teratoma shown in ( i ) ; ( iv ) ovary containing NUMA + cells shown in ( ii ) . Insert : higher magnification of selected area . Scale bars : ( i ) and ( ii ) , 0 . 5 cm ; ( iii ) and ( iv ) , 50 μm .

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

Transplantation of human iPSCs and PGCLCs into ovaries of recipient mice. (i) left ovary: control ovary without transplantation; right ovary: teratoma from iPSC‐reconstituted ovary; (ii) PGCLC‐reconstituted ovaries. Note that no teratomas had formed. Bottom panels: Immunohistochemical analysis for NUMA in sections of iPSC‐ and PGCLC‐reconstituted ovaries. (iii) iPSC‐induced teratoma shown in (i); (iv) ovary containing NUMA+ cells shown in (ii). Insert: higher magnification of selected area. Scale bars: (i) and (ii), 0.5 cm; (iii) and (iv), 50 μm.


Effects of KSR and WNT3A during PGC ‐ precursor induction on the expression of selected pluripotency , PGC , and a mesodermal gene of d2 cultures . The value for iPSCs is set as 1 , and values are on log2 scale . Data are presented as means ± SD ( n = 3 ) .

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

Effects of KSR and WNT3A during PGC‐precursor induction on the expression of selected pluripotency, PGC, and a mesodermal gene of d2 cultures. The value for iPSCs is set as 1, and values are on log2 scale. Data are presented as means ± SD (n = 3).


Effects of KSR and WNT3A on the induction of d6 PGCLCs as analyzed by FACS gated for TRA ‐ 1 ‐ 81 and cKIT .

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

Effects of KSR and WNT3A on the induction of d6 PGCLCs as analyzed by FACS gated for TRA‐1‐81 and cKIT.


Gene expression analysis of selected PGC and hematopoietic markers in d6 PGCLCs that were cultured in 0 % ( control ) or 20 % KSR ( KSR ) condition during PGC ‐ precursor induction . Samples were calibrated with iPSC values , and iPSC values depict 1 . Data are presented as means ± SD ( n = 3 ) .

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

Gene expression analysis of selected PGC and hematopoietic markers in d6 PGCLCs that were cultured in 0% (control) or 20% KSR (KSR) condition during PGC‐precursor induction. Samples were calibrated with iPSC values, and iPSC values depict 1. Data are presented as means ± SD (n = 3).


Immunofluorescence analysis for OCT4 ( red ) , BLIMP1 ( green ) , and T ( yellow ) in d2 precursors cultured in increasing KSR concentrations . Nuclei were stained with Hoechst ( blue ) . Scale bar : 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 B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O

Immunofluorescence analysis for OCT4 (red), BLIMP1 (green), and T (yellow) in d2 precursors cultured in increasing KSR concentrations. Nuclei were stained with Hoechst (blue). Scale bar: 100 μm.


Effect of knockdown of BLIMP1 and PRDM14 on PGCLC induction . Knockdown of BLIMP1 ( F ) and PRDM14 ( G ) was done using the lentiviral system with two individual shRNA vectors for each gene . The knockdown efficiencies were assessed by qPCR ( left panel , each ) . The induction of d4 PGCLCs analyzed by FACS , gated for TRA ‐ 1 ‐ 81 and cKIT ( right panel , each ) . Data are presented as means ± SD ( n = 3 ) .

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

Effect of knockdown of BLIMP1 and PRDM14 on PGCLC induction. Knockdown of BLIMP1 (F) and PRDM14 (G) was done using the lentiviral system with two individual shRNA vectors for each gene. The knockdown efficiencies were assessed by qPCR (left panel, each). The induction of d4 PGCLCs analyzed by FACS, gated for TRA‐1‐81 and cKIT (right panel, each). Data are presented as means ± SD (n = 3).


( A ) Oxygen consumption ratio , , in the indicated VSMC types .

(A) Oxygen consumption ratio, , in the indicated VSMC types.


( B ) mitochondrial ATP synthesis in the indicated VSMC types .

(B) mitochondrial ATP synthesis in the indicated VSMC types.


( C ) mitochondrial membrane potential ( MP ) , in the indicated VSMC types .

(C) mitochondrial membrane potential (MP), in the indicated VSMC types.


( A ) Cytosolic ATP synthesis in the indicated VSMC types , measured by incorporation of phosphate - 32 ( 32Pi ) into ADP . [ 32Pi ] - ATP was separated from 32Pi using the molybdate method , as explained in the Methods section .

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

(A) Cytosolic ATP synthesis in the indicated VSMC types, measured by incorporation of phosphate-32 (32Pi) into ADP. [32Pi]-ATP was separated from 32Pi using the molybdate method, as explained in the Methods section.


(B) Intracellular lactate concentration


( C ) external acidification in the indicated VSMC types .

(C) external acidification in the indicated VSMC types.


( D ) Calcium accumulation in mitochondria after 24 h of incubation in MEM media containing 10 µCi / mL calcium - 45 ( 45Ca2 + ) as a radiotracer .

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

(D) Calcium accumulation in mitochondria after 24 h of incubation in MEM media containing 10 µCi/mL calcium-45 (45Ca2+) as a radiotracer.


( E ) Magnesium concentration in isolated mitochondria .

(E) Magnesium concentration in isolated mitochondria.


( B ) Representative time - course of 2 mmol / L phosphate on calcification of LmnaG609G / + VSMCs ( up ) . Calcification was visualized with Alizarin red . Representative microscopic images ( 10x ; scale bar : 100 µm ) showing calcification of treated and untreated LmnaG609G / + VSMCs ( down ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative time-course of 2 mmol/L phosphate on calcification of LmnaG609G/+ VSMCs (up). Calcification was visualized with Alizarin red. Representative microscopic images (10x; scale bar: 100 µm) showing calcification of treated and untreated LmnaG609G/+ VSMCs (down).


( C - F ) Measures of calcium in treated living LmnaG609G / + VSMCs ( C ) , treated fixed LmnaG609G / + VSMCs ( D ) , untreated living LmnaG609G / + VSMCs ( E ) , and untreated fixed LmnaG609G / + VSMCs ( F ) .

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

(C-F) Measures of calcium in treated living LmnaG609G/+ VSMCs (C), treated fixed LmnaG609G/+ VSMCs (D), untreated living LmnaG609G/+ VSMCs (E), and untreated fixed LmnaG609G/+ VSMCs (F).


( G ) The calcification inhibitory capacity was calculated as the difference in calcium deposition between living and fixed cells ( ΔCa2 + ) .

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

(G) The calcification inhibitory capacity was calculated as the difference in calcium deposition between living and fixed cells (ΔCa2+).


( A ) Magnesium intake in 34 - week - old untreated and treated LmnaG609G / + mice ( n = 16 ) .

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

(A) Magnesium intake in 34-week-old untreated and treated LmnaG609G/+ mice (n=16).


( B ) Plasma magnesium concentration in 34 - week - old untreated and treated LmnaG609G / + mice ( n = 16 ) .

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

(B) Plasma magnesium concentration in 34-week-old untreated and treated LmnaG609G/+ mice (n=16).


( C ) Calcium content of aortas obtained from 34 - week - old wild - type mice and untreated and treated LmnaG609G / + mice ( n = 20 ) .

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

(C) Calcium content of aortas obtained from 34-week-old wild-type mice and untreated and treated LmnaG609G/+ mice (n=20).


( D ) Body masses of 34 - week - old untreated and treated LmnaG609G / + mice .

(D) Body masses of 34-week-old untreated and treated LmnaG609G/+ mice.


( E ) Kaplan - Meier graph for untreated and treated LmnaG609G / + mice ( n = 16 ) .

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

(E) Kaplan-Meier graph for untreated and treated LmnaG609G/+ mice (n=16).


( F ) Representative photographs of 40 - wk - old wild - type , untreated and treated LmnaG609G / + mice .

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

(F) Representative photographs of 40-wk-old wild-type, untreated and treated LmnaG609G/+ mice.


( A ) ATP concentration in liver homogenates obtained from 34 - week - old wild - type , untreated or treated LmnaG609G / + mice .

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

(A) ATP concentration in liver homogenates obtained from 34-week-old wild-type, untreated or treated LmnaG609G/+ mice.


( B ) Mitochondrial calcium measured in liver mitochondria isolated from the indicated experimental mouse groups .

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

(B) Mitochondrial calcium measured in liver mitochondria isolated from the indicated experimental mouse groups.


( C ) Magnesium concentration in isolated mitochondria .

(C) Magnesium concentration in isolated mitochondria.


( D ) Activities of the indicated mitochondrial complexes ( I , II , III , IV , and V ) in the absence or presence of rotenone ( 2 µmol / L ) , 2 - thenoyltrifluoroacetone ( TTFA ; 1 mmol / L ) , antimycin A ( 10 µmol / L ) , potassium cyanide ( KCN ; 1 mmol / L ) , or oligomycin ( 10 µmol / L ) .

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

(D) Activities of the indicated mitochondrial complexes (I, II, III, IV, and V) in the absence or presence of rotenone (2 µmol/L), 2-thenoyltrifluoroacetone (TTFA; 1 mmol/L), antimycin A (10 µmol/L), potassium cyanide (KCN; 1 mmol/L), or oligomycin (10 µmol/L).


A ) Infection susceptibility ( top ) to VSVG and Spike - typed lentiviruses of a panel of lung cancer cell lines with or without ( Empty vector ) overexpression of ACE2 . ACE2 protein levels were determined by Western - blot ( bottom ) .

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

A) Infection susceptibility (top) to VSVG and Spike-typed lentiviruses of a panel of lung cancer cell lines with or without (Empty vector) overexpression of ACE2. ACE2 protein levels were determined by Western-blot (bottom).


C ) Gene - level scores obtained by robust ranking algorithm analysis of the sgRNA relative abundances between cells infected with Spike - typed lentiviruses and uninfected cells . Genes were ordered alphabetically in the x - axis and the top hits ( log10 ( enrichment - score ) < - 5 ) were colored and labelled . Note that CTSL and CTSL1 are different gene isoforms .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Gene-level scores obtained by robust ranking algorithm analysis of the sgRNA relative abundances between cells infected with Spike-typed lentiviruses and uninfected cells. Genes were ordered alphabetically in the x-axis and the top hits (log10(enrichment-score) < -5) were colored and labelled. Note that CTSL and CTSL1 are different gene isoforms.


A ) Scheme depicting the screen validation strategy ( top ) and representative density plots ( bottom ) of flow cytometry data from cells infected with VSVG - or Spike - typed lentivirus . The infected population is labeled with a darker color .

O O O O O O O O O O O O O O O O O O O O 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) Scheme depicting the screen validation strategy (top) and representative density plots (bottom) of flow cytometry data from cells infected with VSVG- or Spike-typed lentivirus. The infected population is labeled with a darker color.


B ) Susceptibility to infection with Spike - typed and VSVG - typed lentiviruses in Calu1ACE2 CRISPR KO cell lines with loss - of - function of selected screen candidates . NT596 and NT764 are two non - targeting CRISPR controls . ACE2 are Calu1ACE2 cells transduced with a CRISPR vector against ACE2 . Bars represent the average and standard error of the mean ( SEM ) of the percentage of infected cells in each condition ( three biological replicates ) , normalized to the non - targeting control cell line NT596 . The t - test p - value between the relative percentage of infection with Spike - typed and VSVG - typed for each cell line is indicated .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Susceptibility to infection with Spike-typed and VSVG-typed lentiviruses in Calu1ACE2 CRISPR KO cell lines with loss-of-function of selected screen candidates. NT596 and NT764 are two non-targeting CRISPR controls. ACE2 are Calu1ACE2 cells transduced with a CRISPR vector against ACE2. Bars represent the average and standard error of the mean (SEM) of the percentage of infected cells in each condition (three biological replicates), normalized to the non-targeting control cell line NT596. The t-test p-value between the relative percentage of infection with Spike-typed and VSVG-typed for each cell line is indicated.


C ) Confocal microscopy images of SPNS1 - KO1 and PLAC8 - KO1 Calu1ACE2 cells overexpressing CRISPR - resistant SPNS1 - or GFP - PLAC8 , respectively .

O O O O 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) Confocal microscopy images of SPNS1-KO1 and PLAC8-KO1 Calu1ACE2 cells overexpressing CRISPR-resistant SPNS1- or GFP-PLAC8, respectively.


D ) Rescue experiments in PLAC8 and SPNS1 Calu1ACE2 KO cell lines : bar plot showing the average ( three biological replicates ) and SEM percentage of infection ( normalized to NT596 with GFP overexpression ) of Spike - typed and VSVG - typed lentiviruses in PLAC8 - KO1 and SPNS1 - KO1 cell lines that overexpress CRISPR - resistant PLAC8 - or GFP - SPNS1 constructs . The significance above each bar represents the t - test p - value between each condition and the control cell line ( NT596 with GFP overexpression )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Rescue experiments in PLAC8 and SPNS1 Calu1ACE2 KO cell lines: bar plot showing the average (three biological replicates) and SEM percentage of infection (normalized to NT596 with GFP overexpression) of Spike-typed and VSVG-typed lentiviruses in PLAC8-KO1 and SPNS1-KO1 cell lines that overexpress CRISPR-resistant PLAC8- or GFP-SPNS1 constructs. The significance above each bar represents the t-test p-value between each condition and the control cell line (NT596 with GFP overexpression)


A , B ) Susceptibility to infection with SARS - CoV - 2 viruses in Calu1ACE2 ( A ) and H1299ACE2 ( B ) cell lines with loss - of - function of the indicated genes . Left : representative immunofluorescence images of SARS - CoV - 2 nucleocapsid protein ( green ) and cell nuclei ( red ) in the indicated cell lines . Scale bar : 20 µm . Right : immunofluorescence quantification of the number of infected cells at 24 h post - infection in the different loss - of - function cell lines infected with SARS - CoV - 2 . Bars represent the average and SEM of the percentage of infected cells relative to CRISPR non - targeting transduced ( NT596 ) cells in three biological replicates ( three different CRISPR KO cell lines infected independently ) . The significance above each bar represents the one - tail t - test p - value between each condition and CRISPR non - targeting transduced ( NT596 ) 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

A, B) Susceptibility to infection with SARS-CoV-2 viruses in Calu1ACE2 (A) and H1299ACE2 (B) cell lines with loss-of-function of the indicated genes. Left: representative immunofluorescence images of SARS-CoV-2 nucleocapsid protein (green) and cell nuclei (red) in the indicated cell lines. Scale bar: 20 µm. Right: immunofluorescence quantification of the number of infected cells at 24 h post-infection in the different loss-of-function cell lines infected with SARS-CoV-2. Bars represent the average and SEM of the percentage of infected cells relative to CRISPR non-targeting transduced (NT596) cells in three biological replicates (three different CRISPR KO cell lines infected independently). The significance above each bar represents the one-tail t-test p-value between each condition and CRISPR non-targeting transduced (NT596) cells.


C ) Western - blot analyses of SPNS1 and PLAC8 protein levels in the different lung cancer cell lines tested in Fig 1 with or without ACE2 overexpression . Note that PLAC8 and SPNS1 immunodetection was performed on the same blots than in Figure 1A , and that ACE2 and Actin panels from Figure 1A were included here to allow comparison .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Western-blot analyses of SPNS1 and PLAC8 protein levels in the different lung cancer cell lines tested in Fig 1 with or without ACE2 overexpression. Note that PLAC8 and SPNS1 immunodetection was performed on the same blots than in Figure 1A, and that ACE2 and Actin panels from Figure 1A were included here to allow comparison.


D ) Susceptibility to Spike - typed lentiviruses in the indicated ACE2 overexpressing cancer cell lines that were transduced with GFP , GFP - PLAC8 or GFP - SPNS1 . Bars represent the average and SEM of the percentage of infected cells in three technical replicates ( independent infections of the same cell line ) relative to Calu1ACE2 - GFP cells . The significance above each bar represents the t - test between GFP - PLAC8 or GFP - SPNS1 and GFP cells for each cell line .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Susceptibility to Spike-typed lentiviruses in the indicated ACE2 overexpressing cancer cell lines that were transduced with GFP, GFP-PLAC8 or GFP-SPNS1. Bars represent the average and SEM of the percentage of infected cells in three technical replicates (independent infections of the same cell line) relative to Calu1ACE2-GFP cells. The significance above each bar represents the t-test between GFP-PLAC8 or GFP-SPNS1 and GFP cells for each cell line.


E ) Susceptibility to infection with SARS - CoV - 2 viruses in cell lines that overexpress ACE2 and GFP - SPNS1 or GFP - PLAC8 . Bars represent the immunofluorescence quantification of the number of SARS - CoV - 2 infected cells at 24 h post - infection . Representative images of SARS - CoV - 2 nucleocapsid immunofluorescence ( green ) and cell nuclei ( red ) in GFP and GFP - PLAC8 cells are shown right to 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

E) Susceptibility to infection with SARS-CoV-2 viruses in cell lines that overexpress ACE2 and GFP-SPNS1 or GFP-PLAC8. Bars represent the immunofluorescence quantification of the number of SARS-CoV-2 infected cells at 24 h post-infection. Representative images of SARS-CoV-2 nucleocapsid immunofluorescence (green) and cell nuclei (red) in GFP and GFP-PLAC8 cells are shown right to each plot.


A ) Binding and endocytosis studies with Alexa Fluor 647 - conjugated S - RBD or transferrin in Calu1 - ACE2 - RFP cells deficient in PLAC8 and SPNS1 . Left : scatterplots of flow cytometry data showing the cutoffs used to define ACE2 - RFP and Alexa Fluor 647 positive cells . Right : bar plots showing the ratio of cells positive for Alexa Fluor 647 in either ACE2 - RFP positive ( red ) or negative ( blue ) cells in binding and endocytosis experiments with S - RBD or transferrin . Bars represent the average and SEM of three biological replicates ( three different CRISPR KO cell lines ) . The significance above each bar represents the t - test p - value between each condition and CRISPR non - targeting transduced ( NT596 ) 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

A) Binding and endocytosis studies with Alexa Fluor 647-conjugated S-RBD or transferrin in Calu1-ACE2-RFP cells deficient in PLAC8 and SPNS1. Left: scatterplots of flow cytometry data showing the cutoffs used to define ACE2-RFP and Alexa Fluor 647 positive cells. Right: bar plots showing the ratio of cells positive for Alexa Fluor 647 in either ACE2-RFP positive (red) or negative (blue) cells in binding and endocytosis experiments with S-RBD or transferrin. Bars represent the average and SEM of three biological replicates (three different CRISPR KO cell lines). The significance above each bar represents the t-test p-value between each condition and CRISPR non-targeting transduced (NT596) cells.


B - E ) Endocytosis experiments with S - RBD in PLAC8 - or SPNS1 - KO Calu1 - ACE2 - RFP cells with overexpression of GFP - PLAC8 or GFP - SPNS1 , respectively . Cells were incubated with S - RBD for 1 h at 37 ºC and fixed with 4 % p - formaldehyde after an acid wash to remove non - internalized S - RBD . Representative wide - field fluorescence images ( B ) and quantification of mean fluorescence intensities of each fluorophore ( C ) in each cell show that S - RBD signal correlates with ACE2 - RFP expression but not with GFP - PLAC8 or GFP - SPNS1 . The Pearson correlation coefficient ( r ) and correlation test p - value ( p ) is indicated within each scatterplot . Each dot represents a cell measurement from different microphotographs of a single replicate . Scale bar : 20 µm . Representative fluorescence confocal microphotographs ( D ) and spot colocalization analyses using ComDet plugin ( E ) demonstrate colocalization of S - RBD endosomes with ACE2 - RFP and GFP - PLAC8 or GFP - SPNS1 . ( D ) Red ovals defining the detected S - RBD dots are overlaid in each channel to assess the colocalization with ACE2 - RFP , GFP - PLAC8 or GFP - SPNS1 . Inlets show a magnification of the region defined by the white squares . Scale bar : 20 µm . ( E ) Boxplots representing the percentage of internalized S - RBD that colocalize with ACE2 - RFP , GFP - PLAC8 , GFP - SPNS1 or their combination ( ACE2 - RFP and GFP - PLAC8 or ACE2 - RFP and GFP - SPNS1 ) . The central band represents the median , the box represents the interquartile range ( IQR ) between the 25th and 75th percentile , and the whiskers extend from the box to 1 . 5 * IQR . Each dot represents a cell measurement from different microphotographs of a single replicate .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-E) Endocytosis experiments with S-RBD in PLAC8- or SPNS1-KO Calu1-ACE2-RFP cells with overexpression of GFP-PLAC8 or GFP-SPNS1, respectively. Cells were incubated with S-RBD for 1 h at 37 ºC and fixed with 4% p-formaldehyde after an acid wash to remove non-internalized S-RBD. Representative wide-field fluorescence images (B) and quantification of mean fluorescence intensities of each fluorophore (C) in each cell show that S-RBD signal correlates with ACE2-RFP expression but not with GFP-PLAC8 or GFP-SPNS1. The Pearson correlation coefficient (r) and correlation test p-value (p) is indicated within each scatterplot. Each dot represents a cell measurement from different microphotographs of a single replicate. Scale bar: 20 µm. Representative fluorescence confocal microphotographs (D) and spot colocalization analyses using ComDet plugin (E) demonstrate colocalization of S-RBD endosomes with ACE2-RFP and GFP-PLAC8 or GFP-SPNS1. (D) Red ovals defining the detected S-RBD dots are overlaid in each channel to assess the colocalization with ACE2-RFP, GFP-PLAC8 or GFP-SPNS1. Inlets show a magnification of the region defined by the white squares. Scale bar: 20 µm. (E) Boxplots representing the percentage of internalized S-RBD that colocalize with ACE2-RFP, GFP-PLAC8, GFP-SPNS1 or their combination (ACE2-RFP and GFP-PLAC8 or ACE2-RFP and GFP-SPNS1). The central band represents the median, the box represents the interquartile range (IQR) between the 25th and 75th percentile, and the whiskers extend from the box to 1.5*IQR. Each dot represents a cell measurement from different microphotographs of a single replicate.


C ) Representative images ( left ) and quantification ( right ) of the number of acidic particles in PLAC8 - and SPNS1 - KO Calu1ACE2 cells compared to CRISPR non - targeting control cells ( NT596 ) using live - cell lysotracker staining . The two samples Wilcoxon Test p - value of the pooled observations compared to NT596 cells is indicated above each condition . The central band represents the median , the box represents the interquartile range ( IQR ) between the 25th and 75th percentile , and the whiskers extend from the box to 1 . 5 * IQR . Each dot represents a cell measurement from different microphotographs of a single replicate . Three biological replicates per condition were used .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative images (left) and quantification (right) of the number of acidic particles in PLAC8- and SPNS1-KO Calu1ACE2 cells compared to CRISPR non-targeting control cells (NT596) using live-cell lysotracker staining. The two samples Wilcoxon Test p-value of the pooled observations compared to NT596 cells is indicated above each condition. The central band represents the median, the box represents the interquartile range (IQR) between the 25th and 75th percentile, and the whiskers extend from the box to 1.5*IQR. Each dot represents a cell measurement from different microphotographs of a single replicate. Three biological replicates per condition were used.


D ) Lysosomal degradation studies in Calu1ACE2 cells using the fluorogenic substrate DQ - Red - BSA . Left : density plots representing the shift in fluorescence intensity in NT596 cells untreated or treated with DQ - Red - BSA under growth or serum starvation conditions . Right : bar plot representing the mean and SEM of the median fluorescence intensity values ( MFI ) of three biological replicates of PLAC8 - and SPNS1 - KO and CRISPR non - targeting cells in growth and serum starvation conditions .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Lysosomal degradation studies in Calu1ACE2 cells using the fluorogenic substrate DQ-Red-BSA. Left: density plots representing the shift in fluorescence intensity in NT596 cells untreated or treated with DQ-Red-BSA under growth or serum starvation conditions. Right: bar plot representing the mean and SEM of the median fluorescence intensity values (MFI) of three biological replicates of PLAC8- and SPNS1-KO and CRISPR non-targeting cells in growth and serum starvation conditions.


E ) Representative images ( left ) and quantification ( right ) of the number of LC3B particles in PLAC8 - and SPNS1 - KO Calu1ACE2 cells compared to CRISPR non - targeting control cells ( NT596 ) using LC3B immunofluorescence staining . The two samples Wilcoxon Test p - value of the pooled observations compared to NT596 cells is indicated above each condition . The central band represents the median , the box represents the interquartile range ( IQR ) between the 25th and 75th percentile , and the whiskers extend from the box to 1 . 5 * IQR . Each dot represents a cell measurement from different microphotographs of a single replicate . Three biological replicates per condition were used .

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

E) Representative images (left) and quantification (right) of the number of LC3B particles in PLAC8- and SPNS1-KO Calu1ACE2 cells compared to CRISPR non-targeting control cells (NT596) using LC3B immunofluorescence staining. The two samples Wilcoxon Test p-value of the pooled observations compared to NT596 cells is indicated above each condition. The central band represents the median, the box represents the interquartile range (IQR) between the 25th and 75th percentile, and the whiskers extend from the box to 1.5*IQR. Each dot represents a cell measurement from different microphotographs of a single replicate. Three biological replicates per condition were used.


F ) Autophagic flux analyses using the fluorescence reporter GFP - LC3 - RFP - LC3ΔG in PLAC8 - and SPNS1 - KO Calu1ACE2 cells compared to CRISPR non - targeting control cells ( NT596 ) . Left : density plots showing the shift in GFP intensity under starvation conditions compared to control growth conditions in NT596 Calu1ACE2 cells . Right : bar plots showing the mean and SEM of the median GFP / RFP intensity ratio of three biological replicates in the different cell lines and conditions .

F) Autophagic flux analyses using the fluorescence reporter GFP-LC3-RFP-LC3ΔG in PLAC8- and SPNS1-KO Calu1ACE2 cells compared to CRISPR non-targeting control cells (NT596). Left: density plots showing the shift in GFP intensity under starvation conditions compared to control growth conditions in NT596 Calu1ACE2 cells. Right: bar plots showing the mean and SEM of the median GFP/RFP intensity ratio of three biological replicates in the different cell lines and conditions.


Induction of UAS constructs and EPs by the ap promoter directed Gal4 expression within the dorsal compartment of the wing imaginal disc : ( A ) ap - Gal4 control ; ( B , E , H , J , K ) UAS - dS6K ; ( C , F , I ) UAS - S6K1dE / D3E ; ( D , E , F ) DHR3 - EP ; ( G , H , I , K ) UAS - DHR3 - RNAi and ( J ) unidirectional DHR3 - EP . The bending down of the wing indicates a slight overgrowth of the dorsal compartment , whereas a bending up reveals a slight growth deficit of this compartment .

Induction of UAS constructs and EPs by the ap promoter directed Gal4 expression within the dorsal compartment of the wing imaginal disc: (A) ap-Gal4 control; (B, E, H, J, K) UAS-dS6K; (C, F, I) UAS-S6K1dE/D3E; (D, E, F) DHR3-EP; (G, H, I, K) UAS-DHR3-RNAi and (J) unidirectional DHR3-EP. The bending down of the wing indicates a slight overgrowth of the dorsal compartment, whereas a bending up reveals a slight growth deficit of this compartment.


Induction of UAS constructs and DHR3 - EP by the eyeless promoter - directed Gal4 expression in the developing eye : ( A ) eye - Gal4 control ; ( B , D , E ) DHR3 - EP ; ( C , D ) UAS - dTsc1 and UAS - dTsc2 ; ( E , F ) UAS - dPTEN .

Induction of UAS constructs and DHR3-EP by the eyeless promoter-directed Gal4 expression in the developing eye: (A) eye-Gal4 control; (B, D, E) DHR3-EP; (C, D) UAS-dTsc1 and UAS-dTsc2; (E, F) UAS-dPTEN.


( B - G ) Adult wings in which the ap - Gal4 induces expression of the following UAS constructs : ( B ) ap - Gal4 control ; ( C ) UAS - dS6K ; ( D ) UAS - DHR3 - RS ; ( E ) both UAS - dS6K and UAS - DHR3 - RS ; ( F ) UAS - DHR3 - RS in a dS6Kl - 1 mutant escaper ; ( G ) UAS - dS6K , UAS - DHR3 - RS , and UAS - DHR3 - RNAi together .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-G) Adult wings in which the ap-Gal4 induces expression of the following UAS constructs: (B) ap-Gal4 control; (C) UAS-dS6K; (D) UAS-DHR3-RS; (E) both UAS-dS6K and UAS-DHR3-RS; (F) UAS-DHR3-RS in a dS6Kl-1 mutant escaper; (G) UAS-dS6K, UAS-DHR3-RS, and UAS-DHR3-RNAi together.


( H ) Upper panel , H2B substrate phosphorylation by dS6K from larval protein extracts . Ubiquitous induction of UAS - DHR3 RNAi , using either a daughterless - Gal4 ( da > Ri ) or an actin - Gal4 ( ac > Ri ) driver , provoked a drop in dS6K activity , as compared with control drivers alone ( da , ac ) . Middle and lower panels are western blots detecting dS6K and α - tubulin , respectively .

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

(H) Upper panel, H2B substrate phosphorylation by dS6K from larval protein extracts. Ubiquitous induction of UAS-DHR3 RNAi, using either a daughterless-Gal4 (da>Ri) or an actin-Gal4 (ac>Ri) driver, provoked a drop in dS6K activity, as compared with control drivers alone (da, ac). Middle and lower panels are western blots detecting dS6K and α-tubulin, respectively.


( A - F ) Wing imaginal discs stained with : ( A , C , D , F ) antibody to DHR3 , and ( B , E ) antibody to GFP . ( A , B ) Overexpression of DHR3 - RS and USA - GFP in the posterior compartment was induced using an engrailed - Gal4 driver . ( C ) and ( D ) ap - Gal4 drives overexpression of DHR3 - RS and DHR3 - EP , respectively . ( E , F ) Flip - out clone ( induced 3 days prior to dissection ) in a prepupal wing imaginal disc expressing UAS - DHR3 - RNAi and UAS - GFP . ( E ) Decrease in DHR3 staining in a flip - out clone labeled by GFP ( F ) , indicating that DHR3 is expressed in this tissue .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-F) Wing imaginal discs stained with: (A, C, D, F) antibody to DHR3, and (B, E) antibody to GFP. (A, B) Overexpression of DHR3-RS and USA-GFP in the posterior compartment was induced using an engrailed-Gal4 driver. (C) and (D) ap-Gal4 drives overexpression of DHR3-RS and DHR3-EP, respectively. (E, F) Flip-out clone (induced 3 days prior to dissection) in a prepupal wing imaginal disc expressing UAS-DHR3-RNAi and UAS-GFP. (E) Decrease in DHR3 staining in a flip-out clone labeled by GFP (F), indicating that DHR3 is expressed in this tissue.


( G ) Western blot analysis of DHR3 in larval protein extracts from control ( Co ) and heat shock - induced UAS lines expressing DHR3 - RA ( RA ) , DHR3 - RB ( RB ) , and DHR3 - RS ( RS ) transcripts , or the DHR3 - EPs ( E1 and E2 ) .

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

(G) Western blot analysis of DHR3 in larval protein extracts from control (Co) and heat shock-induced UAS lines expressing DHR3-RA (RA), DHR3-RB (RB), and DHR3-RS (RS) transcripts, or the DHR3-EPs (E1 and E2).


( H ) Western blot of the endogenous DHR3 protein in late third - instar larvae ( L3 ) , prepupae ( pp ) , and prepupae expressing an RNAi to DHR3 ( RNAipp ) . In ( G , H ) , the arrows at the left indicate the position of the DBD - containing ( high ) and DBD - lacking ( low ) DHR3 proteins .

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

(H) Western blot of the endogenous DHR3 protein in late third-instar larvae (L3), prepupae (pp), and prepupae expressing an RNAi to DHR3 (RNAipp). In (G, H), the arrows at the left indicate the position of the DBD-containing (high) and DBD-lacking (low) DHR3 proteins.


( A , B ) The ap - Gal4 driver induces dS6K in combination with either ( A ) the DHR3 - EP transgene or ( B ) the EP with the EMS DHR3

O O O O O O O O O O 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, B) The ap-Gal4 driver induces dS6K in combination with either (A) the DHR3-EP transgene or (B) the EP with the EMS DHR3 mutation


( D ) dS6K activity in larval protein extracts measured by H2B target phosphorylation and ( E ) corresponding western blot of dS6K in the trans - heterozygote LBD - mutant combination ( LBD ) , as compared to heterozygous larvae ( Co ) .

O O O O O O O O O O O O O O O O O O O O O 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) dS6K activity in larval protein extracts measured by H2B target phosphorylation and (E) corresponding western blot of dS6K in the trans-heterozygote LBD-mutant combination (LBD), as compared to heterozygous larvae (Co).


( F , G ) The eyeless promoter directs the flipase recombinase during eye development . Flipase allows recombination on the right arm of the second chromosome and eventually leads to an adult eye ( F ) that is homozygous for the wild - type allele of DHR3 . ( G ) Homozygosity for a DHR3K243X mutation results in a decrease in the size of the adult eye . A dotted yellow line surrounding the control eye in ( F ) has been copied and pasted on the mutant eyes ( G ) ; insets are higher magnification images of ommatidia showing misorientation of bristles .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, G) The eyeless promoter directs the flipase recombinase during eye development. Flipase allows recombination on the right arm of the second chromosome and eventually leads to an adult eye (F) that is homozygous for the wild-type allele of DHR3. (G) Homozygosity for a DHR3K243X mutation results in a decrease in the size of the adult eye. A dotted yellow line surrounding the control eye in (F) has been copied and pasted on the mutant eyes (G); insets are higher magnification images of ommatidia showing misorientation of bristles.


( H ) At the scutellum , a y - marked homozygous DHR3K243X mutant bristle ( arrow ) , recognizable by its light color , is smaller than the symmetrical neighboring bristles .

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

(H) At the scutellum, a y-marked homozygous DHR3K243X mutant bristle (arrow), recognizable by its light color, is smaller than the symmetrical neighboring bristles.


( A ) Normalized eye size ( mm2 ) of control ( black bar ; n = 20 ) , DHR3W284X ( grey bar ; n = 18 ) and DHR3K243X ( white bar ; n = 18 ) mutants . As compared to control recombined eyes ( Co ) , DHR3W284X and DHR3K243X mutant eyes exhibit a highly significant size reduction ( * * * P < 0 . 001 ) . ( B ) Ommatidia size of control ( black bar ) , DHR3W284X ( grey bar ) , and DHR3K243X ( white bar ) mutants . As compared to control recombined eyes ( Co ) , DHR3W284X and DHR3K243X mutant ommatidia exhibit a significant size reduction ( * * P < 0 . 01 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Normalized eye size (mm2) of control (black bar; n = 20), DHR3W284X (grey bar; n = 18) and DHR3K243X (white bar; n = 18) mutants. As compared to control recombined eyes (Co), DHR3W284X and DHR3K243X mutant eyes exhibit a highly significant size reduction (***P<0.001). (B) Ommatidia size of control (black bar), DHR3W284X (grey bar), and DHR3K243X (white bar) mutants. As compared to control recombined eyes (Co), DHR3W284X and DHR3K243X mutant ommatidia exhibit a significant size reduction (**P<0.01).


( C - E ) Comparison of y - marked homozygous clonal bristles ( black arrows ) to their neighboring heterozygous bristles ( white arrows ) at the wing margin . Note that control clonal bristles are of normal size and orientation ( C ) , whereas those of DHR3W284X ( D ) and DHR3K243X ( E ) are reduced in size and are misoriented . ( F ) Size measurements ( µm ) of the length of homozygous clones ( y − ) and their neighboring heterozygous ( y + ) bristles ( n = 51 for each bar ) . The size reduction of clonal versus neighbor bristles is highly significant for DHR3K243X ( * * * P < 0 . 001 ) and DHR3W284X ( * * * P < 0 . 001 ) mutants , but not for the control ( Co ) bristles ( P = 0 . 028 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-E) Comparison of y-marked homozygous clonal bristles (black arrows) to their neighboring heterozygous bristles (white arrows) at the wing margin. Note that control clonal bristles are of normal size and orientation (C), whereas those of DHR3W284X (D) and DHR3K243X (E) are reduced in size and are misoriented. (F) Size measurements (µm) of the length of homozygous clones (y−) and their neighboring heterozygous (y+) bristles (n = 51 for each bar). The size reduction of clonal versus neighbor bristles is highly significant for DHR3K243X (***P<0.001) and DHR3W284X (***P<0.001) mutants, but not for the control (Co) bristles (P = 0.028).


Exponential growth of E . coli K - 12 , measured using optical density ( OD600 ) , in lysogenic broth ( LB , white circles ) or minimal medium supplemented with different carbon sources , listed in the legend on the bottom right in a greyscale . All the cultures are grown in batch mode , see Methods .

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

Exponential growth of E. coli K-12, measured using optical density (OD600), in lysogenic broth (LB, white circles) or minimal medium supplemented with different carbon sources, listed in the legend on the bottom right in a greyscale. All the cultures are grown in batch mode, see Methods.


Bacterial viability in colony forming units ( CFU ) per ml of cultures from panel A , grown until OD600 0 . 5 , washed and re - suspended in carbon - free minimal medium . Death rates ( slopes of the exponential fits ) are listed in Table EV1 . Initial density for fast growth ( lighter shades ) is lower than for slow growth ( darker shades ) , due larger cell sizes that lead to lower cell densities per OD600 .

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

Bacterial viability in colony forming units (CFU) per ml of cultures from panel A, grown until OD600 0.5, washed and re-suspended in carbon-free minimal medium. Death rates (slopes of the exponential fits) are listed in Table EV1. Initial density for fast growth (lighter shades) is lower than for slow growth (darker shades), due larger cell sizes that lead to lower cell densities per OD600.


Death rates of panel B plotted against growth rates of panel A shown as circles in a grey - scale . Squares show data obtained using eight other carbon substrates , also listed in Table EV1 . Color circles show data obtained by varying growth rate in a chemostat . The black line shows a linear weighted least square fit to log - transformed data , that takes uncertainty in data points into account ( see Methods ) . Data shown as mean ± ( standard deviation ) SD . Two or three replicates per condition .

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

Death rates of panel B plotted against growth rates of panel A shown as circles in a grey-scale. Squares show data obtained using eight other carbon substrates, also listed in Table EV1. Color circles show data obtained by varying growth rate in a chemostat. The black line shows a linear weighted least square fit to log-transformed data, that takes uncertainty in data points into account (see Methods). Data shown as mean ± (standard deviation) SD. Two or three replicates per condition.


Death rates of wild - type and GlpK22 mutants plotted versus growth rates . Wild type is grown in glycerol minimal medium in batch cultures ( black circle ) and in ' chemostat ' continuous cultures with growth rates coded in colors , see legend on the center right . GlpK22 mutants ( white circle ) are grown in glycerol minimal medium in batch culture . Data shown as mean ± ( standard deviation ) SD . Two replicates per condition .

Death rates of wild-type and GlpK22 mutants plotted versus growth rates. Wild type is grown in glycerol minimal medium in batch cultures (black circle) and in 'chemostat' continuous cultures with growth rates coded in colors, see legend on the center right. GlpK22 mutants (white circle) are grown in glycerol minimal medium in batch culture. Data shown as mean ± (standard deviation) SD. Two replicates per condition.


Example experiment of the assay sketched in panel C . Cells previously grown at 0 . 3 h − 1 in continuous culture are starved ( green circles ) . After 2 days of starvation , a sample is ( 1 ) extracted and ( 2 ) UV - sterilized , see dashed line , followed by ( 3 ) inoculation of 1 % of starved and ( 4 ) growth of the culture . The difference between the maximum viability after growth , and the initial viability at point ( 3 ) is the absolute yield .

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

Example experiment of the assay sketched in panel C. Cells previously grown at 0.3 h − 1 in continuous culture are starved (green circles). After 2 days of starvation, a sample is (1) extracted and (2) UV-sterilized, see dashed line, followed by (3) inoculation of 1% of starved and (4) growth of the culture. The difference between the maximum viability after growth, and the initial viability at point (3) is the absolute yield.


The absolute yield measured at three different time points for each of the growth conditions shown in panel A is plotted against the viability of the starvation culture at the extraction time . The recycling yield is extracted as the slope of the linear fits . Recycling yield from panel E plotted versus growth rate . Data shown as mean ± SD . Two replicates per condition .

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

The absolute yield measured at three different time points for each of the growth conditions shown in panel A is plotted against the viability of the starvation culture at the extraction time. The recycling yield is extracted as the slope of the linear fits. Recycling yield from panel E plotted versus growth rate. Data shown as mean ± SD. Two replicates per condition.


Example experiment from a starved culture previously grown at a rate of 0 . 3 h − 1 with a viability after 5 days of starvation . After ( 1 ) extraction and ( 2 ) addition of 40 µM of glycerol the decay of viability is delayed ( black circles ) compared to a control without glycerol ( green symbols ) . Per viable cell , the glycerol addition in this experiment is 0 . 33 fmol CFU − 1 . After an initial period of survival , the culture with added glycerol ( black ) dies at the same rate as the control ( green ) . The ' lag time ' fitted to this data is 1 day .

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

Example experiment from a starved culture previously grown at a rate of 0.3 h − 1 with a viability after 5 days of starvation. After (1) extraction and (2) addition of 40 µM of glycerol the decay of viability is delayed (black circles) compared to a control without glycerol (green symbols). Per viable cell, the glycerol addition in this experiment is 0.33 fmol CFU − 1. After an initial period of survival, the culture with added glycerol (black) dies at the same rate as the control (green). The 'lag time' fitted to this data is 1 day.


Lag time of the experiments of panel B for different glycerol concentrations . Lag increases linearly with glycerol concentration and maintenance rate is extracted as the inverse of the slope of the linear fits .

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

Lag time of the experiments of panel B for different glycerol concentrations. Lag increases linearly with glycerol concentration and maintenance rate is extracted as the inverse of the slope of the linear fits.


Maintenance rate values extracted from panel I plotted versus growth rate . Data shown as mean ± SD . Two replicates per condition .

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

Maintenance rate values extracted from panel I plotted versus growth rate. Data shown as mean ± SD. Two replicates per condition.


( K ) Consistency check of the equation in panel A . Fold changes of maintenance rate , FC ( β ) , divided by the fold changes of the recycling yield , FC ( α ) , plotted versus the fold changes of death rate , FC ( γ ) . All fold changes are relative to wild - type in batch , growing at 0 . 7 h − 1 . The dashed line shows the unity line FC ( γ ) = FC ( β ) / FC ( α ) .

(K) Consistency check of the equation in panel A. Fold changes of maintenance rate, FC(β), divided by the fold changes of the recycling yield, FC(α), plotted versus the fold changes of death rate, FC(γ). All fold changes are relative to wild-type in batch, growing at 0.7 h − 1. The dashed line shows the unity line FC(γ)= FC(β)/FC(α).


Cell size during starvation . Length and width of the cells are measured with phase - contrast microscopy and the volume is computed considering cell shape as a cylinder with two semi - spheres , as described in the graphical synopsis at the top ( see also Table EV3 and Methods ) . Each measurement is an average of 200 cells . ( A ) Measured length and width of wild type cells starved in batch cultures and previously grown in the chemostat at different steady - state growth rates and of GlpK22 cells starved and previously grown in batch culture at μ = 0 . 9 h − 1 ( see upper color legend ) . Note that , as shown in Schink et al . ( 2019 ) , cell widths do not change from steady state growth to starvation , while lengths decrease . Both length and width increase exponentially with growth rate . ( B ) Starvation volume of the cells described in panel A , computed as explained in the graphical synopsis ( see also Methods ) . In agreement with literature ( Schaechter et al . , 1958 ) , it increases exponentially with growth rate ( black line ) .

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

Cell size during starvation. Length and width of the cells are measured with phase-contrast microscopy and the volume is computed considering cell shape as a cylinder with two semi-spheres, as described in the graphical synopsis at the top (see also Table EV3 and Methods). Each measurement is an average of 200 cells. (A) Measured length and width of wild type cells starved in batch cultures and previously grown in the chemostat at different steady-state growth rates and of GlpK22 cells starved and previously grown in batch culture at μ = 0.9 h − 1 (see upper color legend). Note that, as shown in Schink et al. (2019), cell widths do not change from steady state growth to starvation, while lengths decrease. Both length and width increase exponentially with growth rate. (B) Starvation volume of the cells described in panel A, computed as explained in the graphical synopsis (see also Methods). In agreement with literature (Schaechter et al., 1958), it increases exponentially with growth rate (black line).


Recycling yield and maintenance rate per cell volume . ( C ) Recycling yields and ( D ) maintenance rate measured as described in Fig . 2 are normalized per cell volume and plotted versus the previous growth rate of the cultures they refer to ( see color legend at the top and Table EV3 ) . The normalized yield is constant within the uncertainty . Maintenance rate increases significantly with a slope of ( 0 . 99 ± 0 . 55 ) h , matching the increase of the death rate , ( 1 . 1 ± 0 . 4 ) h , shown in Fig 2B . Solid black lines show linear fits on log transformed data , using weighted least square fits .

Recycling yield and maintenance rate per cell volume. (C) Recycling yields and (D) maintenance rate measured as described in Fig. 2 are normalized per cell volume and plotted versus the previous growth rate of the cultures they refer to (see color legend at the top and Table EV3). The normalized yield is constant within the uncertainty. Maintenance rate increases significantly with a slope of (0.99±0.55) h, matching the increase of the death rate, (1.1±0.4) h, shown in Fig 2B. Solid black lines show linear fits on log transformed data, using weighted least square fits.


Death rates of ∆ rpoS mutants versus the growth rate ( grey symbols ) , compared to wild - type E . coli K - 12 ( white symbols , data from Fig . 1 ) . Death rate increases with growth rate with a smaller slope ( ∆ rpoS : 0 . 57 ± 0 . 13 h ) compared to wild - type ( WT : 0 . 88 ± 0 . 13 h ) . Black and grey lines show linear weighted least square fits to log - transformed data , which take uncertainty in data points into account ( see Methods ) . Data shown as mean ± SD . Two or three replicates per condition .

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

Death rates of ∆rpoS mutants versus the growth rate (grey symbols), compared to wild-type E. coli K-12 (white symbols, data from Fig. 1). Death rate increases with growth rate with a smaller slope (∆rpoS: 0.57±0.13 h) compared to wild-type (WT: 0.88±0.13 h). Black and grey lines show linear weighted least square fits to log-transformed data, which take uncertainty in data points into account (see Methods). Data shown as mean ± SD. Two or three replicates per condition.


The optimal growth rate , i . e . the one that maximizes fitness over a cycle of famine and feast , depends on the ratio of feast and famine periods . For long periods of feast interrupted by short periods of famine , E . coli the optimal strategy is to grow fast . For long periods of starvation intermitted by short periods of growth , the optimal strategy is long survival and slow growth . For the scenario of panel A and B , $ \ frac { T _ { - } } { T _ { + } } = 48 $ , the optimum is μ = 0 . 86 h − 1 and γ = 0 . 5 d − 1 , see dashed line .

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

The optimal growth rate, i.e. the one that maximizes fitness over a cycle of famine and feast, depends on the ratio of feast and famine periods. For long periods of feast interrupted by short periods of famine, E. coli the optimal strategy is to grow fast. For long periods of starvation intermitted by short periods of growth, the optimal strategy is long survival and slow growth. For the scenario of panel A and B, $\frac{T_{-}}{T_{+}} = 48$, the optimum is μ = 0.86 h − 1 and γ = 0.5 d − 1, see dashed line.


Adaptation at the end of growth . When the nutrient concentration decreases , the nutrient uptake rate slows down with a Michaelis - Menten type kinetics ( top ) . Depending on the uptake affinity , bacteria will experience either a sharp or smooth decrease of growth rate ( bottom left , bright orange : 10 µM , dark orange : 1 mM ) . During this slow down , the proteome adapts ( bottom center ) , with lower affinities ( dark orange ) showing a slight improvement of proteome adaptation , which results in a slightly lower death rate ( bottom right ) .

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

Adaptation at the end of growth. When the nutrient concentration decreases, the nutrient uptake rate slows down with a Michaelis-Menten type kinetics (top). Depending on the uptake affinity, bacteria will experience either a sharp or smooth decrease of growth rate (bottom left, bright orange: 10 µM, dark orange: 1 mM). During this slow down, the proteome adapts (bottom center), with lower affinities (dark orange) showing a slight improvement of proteome adaptation, which results in a slightly lower death rate (bottom right).


Adaptation in complex media . A medium supplemented with multiple nutrients ( one - red , two - green , ten - blue ) leads to a step - wise decrease of growth rate during nutrient depletion ( bottom left ) . Due to the extended periods of slow growth after exhaustion of primary nutrients , bacteria can adapt their proteome substantially ( bottom center ) , and decrease their death rate ( bottom right ) .

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

Adaptation in complex media. A medium supplemented with multiple nutrients (one - red, two - green, ten - blue) leads to a step-wise decrease of growth rate during nutrient depletion (bottom left). Due to the extended periods of slow growth after exhaustion of primary nutrients, bacteria can adapt their proteome substantially (bottom center), and decrease their death rate (bottom right).


A . In vivo genetic complementation of the E . coli MC4100 wild - type ( WT ) and secB or / and tig knock - out derivatives by either an empty vector or one carrying the secB or tig ( encoding TF ) genes or derivatives ( see " Plasmid " table in Appendix ) , as indicated . Serial dilutions of a culture ( OD600 = 0 . 5 ) were spotted ( 12μl ) on LB - Ampicillin plates containing or not anhydrotetracycline ( AHT ; 5 ng / ml ) and grown at 16 ° C or 30 ° C ( as indicated ) . n = 3 - 7 biological replicates .

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

A. In vivo genetic complementation of the E. coli MC4100 wild-type (WT) and secB or/and tig knock-out derivatives by either an empty vector or one carrying the secB or tig (encoding TF) genes or derivatives (see "Plasmid" table in Appendix), as indicated. Serial dilutions of a culture (OD600= 0.5) were spotted (12μl) on LB-Ampicillin plates containing or not anhydrotetracycline (AHT; 5 ng/ml) and grown at 16°C or 30°C (as indicated). n= 3-7 biological replicates.


C . SecA ATPase activity determination in solution ( basal ; B ) , or plus SecYEG IMVs ; ( membrane ; M ) or plus proOmpA ( pre - treated with 10 mM DTT ; 5 mM EDTA ; 20 min ; 4 ° C ; translocation ; T ) . n = 21 biological replicates . Mean values ( ± SEM ) shown .

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

C. SecA ATPase activity determination in solution (basal; B), or plus SecYEG IMVs; (membrane; M) or plus proOmpA (pre-treated with 10 mM DTT; 5 mM EDTA; 20 min; 4°C; translocation; T). n= 21 biological replicates. Mean values (± SEM) shown.


D . proOmpA translocation ATPase activity stimulation of SecA ( as in C ) in the absence or presence of TF2 or / and SecB4 , added at the indicated molar excess over proOmpA1 . The T / M ratios were calculated ; the one in the absence of chaperones was considered as 100 % , all other values were expressed as % of this . n = 2 - 4 biological replicates . Mean values ( ± SEM ) shown . Unpaired parametric t - test , 95 % confidence interval : ns : not significant ( p = 0 . 1032 ) ; * * * * : p < 0 . 0001 ; * * : p = 0 . 0037 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. proOmpA translocation ATPase activity stimulation of SecA (as in C) in the absence or presence of TF2 or/and SecB4, added at the indicated molar excess over proOmpA1. The T/M ratios were calculated; the one in the absence of chaperones was considered as 100%, all other values were expressed as % of this. n= 2-4 biological replicates. Mean values (± SEM) shown. Unpaired parametric t-test, 95% confidence interval: ns: not significant (p = 0.1032); ****: p < 0.0001; **: p = 0.0037.


E . Titrated TF - mediated inhibition ( 0 - 6 µM range ) of proOmpA - stimulated translocation ATPase activity ( as in C ) . Values were normalized ( effectmax = 100 % ; effectmin = 0 % ) and plotted ( n = 1 - 4 biological replicates ; mean values ± SEM ) versus the log10 [ TF2 ] . The Ki was determined using a variable slope fit ( log [ inhibitor ] versus normalized response ; GraphPad Prism ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Titrated TF-mediated inhibition (0-6 µM range) of proOmpA-stimulated translocation ATPase activity (as in C). Values were normalized (effectmax=100%; effectmin=0%) and plotted (n= 1-4 biological replicates; mean values ± SEM) versus the log10[TF2]. The Ki was determined using a variable slope fit (log[inhibitor] versus normalized response; GraphPad Prism).


F . SecB - mediated relief of TF - mediated inhibition of proOmpA translocation ATPase activity ( as in C ) . TF2 ( 1μM ) and SecB4 ( 0 - 6 µM ) were used . The T / M ratios were calculated , normalized ( as in D ) and plotted ( n = 2 - 3 biological replicates ; mean values ± SEM ) versus the log10 [ SecB4 ] . An apparent relief constant ( Krel ) was determined ( as in E ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. SecB-mediated relief of TF-mediated inhibition of proOmpA translocation ATPase activity(as in C). TF2 (1μM) and SecB4 (0-6 µM) were used. The T/M ratios were calculated, normalized (as in D) and plotted (n= 2-3 biological replicates; mean values ± SEM) versus the log10[SecB4]. An apparent relief constant (Krel) was determined (as in E).


A . TF - proOmpA physical interaction in solution . proOmpA1 ( 5 μM ) was incubated ( 50 μl ; Buffer C ; < 0 . 2 M urea ; 37 ° C ; 60 min ) with the indicated molar excess of TF2 . Soluble proteins were analyzed on 10 % native - PAGE ( 4 mA ; 16h ; 4 ° C ) and Coomassie - blue stained . A representative experiment is shown ; n = 4 biological replicates

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

A. TF-proOmpA physical interaction in solution. proOmpA1 (5 μM) was incubated (50 μl; Buffer C; < 0.2 M urea; 37°C; 60 min) with the indicated molar excess of TF2. Soluble proteins were analyzed on 10% native-PAGE (4 mA; 16h; 4°C) and Coomassie-blue stained. A representative experiment is shown; n= 4 biological replicates


C . TF solubilizes proOmpA * . proOmpA * 1 ( 5 μM ) was incubated ( 50 μl ; Buffer C ; < 0 . 2 M urea ; 37 ° C ; 60 min ) alone or in the presence of TF . Proteins in the soluble fraction were analyzed on 15 % SDS - PAGE and Coomassie - blue stained . Lane 2 : 2 . 84 μg proOmpA * . Representative experiment shown ; n = 5 biological replicates

C. TF solubilizes proOmpA*. proOmpA*1 (5 μM) was incubated (50 μl; Buffer C; < 0.2 M urea; 37°C; 60 min) alone or in the presence of TF. Proteins in the soluble fraction were analyzed on 15% SDS-PAGE and Coomassie-blue stained. Lane 2: 2.84 μg proOmpA*. Representative experiment shown; n= 5 biological replicates


D . proOmpA preincubated at 37oC fails to stimulate SecA ATPase activity . proOmpA ( 0 . 5 μM ) was either diluted directly ( lane 1 ) or after preincubation alone ( 10 min ; buffer D ; < 0 . 2 M urea ; either at 4 ° C or 37 ° C ) into reactions containing SecYEG - SecA alone or together with TF2 or SecB4 ( 2 and 4 x molar excess over proOmpA , respectively ) . T / M ratios ( as in Fig . 1D ) are shown ( n = 8 biological replicates ; mean values ± SEM ; Unpaired parametric t - test , 95 % confidence interval : * * : p = 0 . 0017 ; * * * * : p < 0 . 0001 )

D. proOmpA preincubated at 37oC fails to stimulate SecA ATPase activity. proOmpA (0.5 μM) was either diluted directly (lane 1) or after preincubation alone (10 min; buffer D; < 0.2 M urea; either at 4°C or 37°C) into reactions containing SecYEG-SecA alone or together with TF2 or SecB4 (2 and 4 x molar excess over proOmpA, respectively). T/M ratios (as in Fig. 1D) are shown (n= 8 biological replicates; mean values ± SEM; Unpaired parametric t-test, 95% confidence interval: **: p = 0.0017; ****: p < 0.0001)


E . proOmpA pre - incubated at 37oC does not interact with TF . proOmpA1 ( 5 μM ) was preincubated ( Buffer D ; < 0 . 2 M urea ; 37 ° C ; 10 min ) or not ( lane 3 ) , prior to addition of the indicated molar excess of TF2 ( analyzed as in A ) . Representative experiment shown ; n = 4 biological replicates

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

E. proOmpA pre-incubated at 37oC does not interact with TF. proOmpA1 (5 μM) was preincubated (Buffer D; < 0.2 M urea; 37°C; 10 min) or not (lane 3), prior to addition of the indicated molar excess of TF2 (analyzed as in A). Representative experiment shown; n= 4 biological replicates


B . SecB protects proOmpA * from aggregation . proOmpA * 1 ( 5 μM ) was incubated ( 50 μl ; Buffer C ; 37 ° C ; 60 min ) alone or in the presence of the indicated molar excess of SecB4 , and proOmpA * in the soluble fraction ( after centrifugation ) was analyzed by SDS - PAGE ( as in Fig . 2C ) . Lane 2 : 2 . 84 μg proOmpA * . Representative experiment shown ; n = 4 biological

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. SecB protects proOmpA* from aggregation. proOmpA*1 (5 μM) was incubated (50 μl; Buffer C; 37°C; 60 min) alone or in the presence of the indicated molar excess of SecB4, and proOmpA* in the soluble fraction (after centrifugation) was analyzed by SDS-PAGE (as in Fig. 2C). Lane 2: 2.84 μg proOmpA*. Representative experiment shown; n= 4 biological replicates


C . SecB physically interacts with proOmpA . proOmpA1 ( 5 μM ) was incubated ( 50 μl ; Buffer C ; < 0 . 2 M urea ; 4 ° C ; 60 min ) , with the indicated molar excess of SecB4 ( filled arrow ) , analyzed by native - PAGE as in Fig . 2A , and stained as indicated . brace : higher order proOmpA species . Representative experiment shown ; n = 5 biological replicates

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

C. SecB physically interacts with proOmpA. proOmpA1 (5 μM) was incubated (50 μl; Buffer C; < 0.2 M urea; 4°C; 60 min), with the indicated molar excess of SecB4 (filled arrow), analyzed by native-PAGE as in Fig. 2A, and stained as indicated. brace: higher order proOmpA species. Representative experiment shown; n= 5 biological replicates


B . Blow up of the ESI - mass spectrum of the TF : proOmpA : SecB4 super - complex from A ( complete dataset in Fig . EV3 ) . Left : charge state distributions , at the indicated m / z range . Right : masses deconvoluted from the mass spectra ( as in Fig . 2B ) . Inset cartoon depicts stoichiometry . Representative spectrum shown ; n = 2 biological replicates .

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

B. Blow up of the ESI-mass spectrum of the TF:proOmpA:SecB4 super-complex from A (complete dataset in Fig. EV3). Left: charge state distributions, at the indicated m/z range. Right: masses deconvoluted from the mass spectra (as in Fig. 2B). Inset cartoon depicts stoichiometry. Representative spectrum shown; n=2 biological replicates.


C . TF : proOmpA : SecB supercomplex analysis using GPC - MALS in Buffer A . TF1 : proOmpA1 ( 10 µM each ; green ) , SecB4 ( 40 µM ; blue ) and TF1 : proOmpA1 : SecB4 ( 10 , 10 and 40 µM , respectively ; black ) . n = 5 biological replicates ; mean mass : 137 kDa ( ± 6 kDa ) representative experiment shown ; red circles : determined mass ( kDa ) ; AU : arbitrary units .

C. TF:proOmpA:SecB supercomplex analysis using GPC-MALS in Buffer A. TF1:proOmpA1 (10 µM each; green), SecB4 (40 µM; blue) and TF1:proOmpA1:SecB4 (10, 10 and 40 µM, respectively; black). n= 5 biological replicates; mean mass: 137 kDa (± 6 kDa) representative experiment shown; red circles: determined mass (kDa); AU: arbitrary units.


A . SecB fails to restore proOmpA translocation ATPase activity stimulation of SecA ( noC - tail ) in the presence of TF . proOmpA translocation ATPase activity stimulation ( as in Fig . 1C ; SecB molar excess over proOmpA indicated ) . T / M ratio of SecA ( noC - tail ) in the absence of TF or SecB = 100 % ; lane 1 ; all other values were expressed as a % of this value . n = 4 biological replicates ; mean values ± SEM . Unpaired parametric t - test , 95 % confidence interval : * * : p = 0 . 0048 ; * * * * : p < 0 . 0001 .

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

A. SecB fails to restore proOmpA translocation ATPase activity stimulation of SecA(noC-tail) in the presence of TF. proOmpA translocation ATPase activity stimulation (as in Fig. 1C; SecB molar excess over proOmpA indicated). T/M ratio of SecA(noC-tail) in the absence of TF or SecB= 100%; lane 1; all other values were expressed as a % of this value. n= 4 biological replicates; mean values ± SEM. Unpaired parametric t-test, 95% confidence interval: **: p= 0.0048; ****: p < 0.0001.


B . Excess of SecB inhibits the proOmpA - stimulated translocation ATPase activity of SecA ( noCtail ) . ATPase measured as in Fig . 1C ; SecB concentration range : 0 - 6 µM . Values were normalized ( effectmax = 100 % ; effectmin = 0 % ) and plotted ( n = 3 biological replicates ; mean values ± SEM ) versus the log10 [ SecB4 ] . The Ki was determined as in Fig . 1E .

B. Excess of SecB inhibits the proOmpA-stimulated translocation ATPase activity of SecA(noCtail). ATPase measured as in Fig. 1C; SecB concentration range: 0-6 µM. Values were normalized (effectmax=100%; effectmin=0%) and plotted (n= 3 biological replicates; mean values ± SEM) versus the log10[SecB4]. The Ki was determined as in Fig. 1E.


C . TF and SecB - regulated , SecA - dependent in vitro translocation of proOmpA into SecYEG / A - IMVs , analyzed after proteolytic treatment . Translocated , protease - resistant proOmpA was visualized using immunostaining ( α - His ) in the absence or presence of TF2 or / and SecB4 ( as indicated ) . Translocation in the absence of TF or SecB was considered 100 % ( lane 3 ) ; all other values were expressed as a percentage of this . A summary of all repeats is presented ( mean ± SEM ; n = 6 biological replicates ; ns : not significant ( p = 0 . 1568 for lane 3 vs 4 ; p = 0 . 4835 for lane 3 vs 6 ) ; Unpaired parametric t - test , 95 % confidence interval : * * * * : p < 0 . 0001 ) with a representative western blot ( bottom ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. TF and SecB-regulated, SecA-dependent in vitro translocation of proOmpA into SecYEG/A-IMVs, analyzed after proteolytic treatment. Translocated, protease-resistant proOmpA was visualized using immunostaining (α-His) in the absence or presence of TF2 or/and SecB4 (as indicated). Translocation in the absence of TF or SecB was considered 100% (lane 3); all other values were expressed as a percentage of this. A summary of all repeats is presented (mean ± SEM; n= 6 biological replicates; ns: not significant (p = 0.1568 for lane 3 vs 4; p = 0.4835 for lane 3 vs 6); Unpaired parametric t-test, 95% confidence interval: ****: p < 0.0001) with a representative western blot (bottom).


pH - dependent dissociation of E and M . TBEV preparations treated at pH 8 ( left ) and pH 6 ( right ) were solubilized with Triton X - 100 and subjected to sucrose gradient centrifugation . The fractions were analyzed by SDS - PAGE and densitometry . Representative examples of at least two independent experiments are shown . Positions of E dimers ( D ) and E trimers ( T ) in the gradients are indicated .

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

pH-dependent dissociation of E and M. TBEV preparations treated at pH 8 (left) and pH 6 (right) were solubilized with Triton X-100 and subjected to sucrose gradient centrifugation. The fractions were analyzed by SDS-PAGE and densitometry. Representative examples of at least two independent experiments are shown. Positions of E dimers (D) and E trimers (T) in the gradients are indicated.


Trimer stability expressed as percentage of the fraction detected in the trimer peak and normalized with respect to the corresponding Ev fraction in sedimentation analyses after incubation at 70 ° C relative to 37 ° C ( 100 % ) . Data are from at least three independent experiments .

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

Trimer stability expressed as percentage of the fraction detected in the trimer peak and normalized with respect to the corresponding Ev fraction in sedimentation analyses after incubation at 70°C relative to 37°C (100%). Data are from at least three independent experiments.


Difference in deuterium uptake by sE404r and sE419r trimers ( D404 - D419 ) . Left panel , deuteration difference for each peptide reported against the 1 - p value obtained via Student ' s t - test between the two different constructs . In the right panel , peptides are reported as short horizontal lines , with length and position corresponding to the peptide length and location along the protein sequence ( x - axis ) and with their deuteration delta on the y - axis . In both panels , red ( positive ) and blue ( negative ) indicate changes above the Student ' s t - test threshold , and in grey those not reaching this threshold . Four peptides , termed HDX - a , HDX - b , HDX - c and HDX - d , were identified as showing a strong increase in deuteration in sE404r compared to sE419r . The same pattern was observed in the comparison between sE404r and sE428r , whereas no difference deemed " significant " by the same test was seen when comparing sE419r with sE428r ( see Fig EV3 ) .

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

Difference in deuterium uptake by sE404r and sE419r trimers (D404-D419). Left panel, deuteration difference for each peptide reported against the 1-p value obtained via Student's t-test between the two different constructs. In the right panel, peptides are reported as short horizontal lines, with length and position corresponding to the peptide length and location along the protein sequence (x-axis) and with their deuteration delta on the y-axis. In both panels, red (positive) and blue (negative) indicate changes above the Student's t-test threshold, and in grey those not reaching this threshold. Four peptides, termed HDX-a, HDX-b, HDX-c and HDX-d, were identified as showing a strong increase in deuteration in sE404r compared to sE419r. The same pattern was observed in the comparison between sE404r and sE428r, whereas no difference deemed "significant" by the same test was seen when comparing sE419r with sE428r (see Fig EV3).


Blocking ELISA with mabs A1 , A2 , A3 and B4 . Serial dilutions of the trimers were pre - incubated with a predetermined concentration of the respective mabs . The fraction of mab not blocked by the antigen was detected in ELISA with TBEV - coated plates ( Materials and Methods ) . The bar charts show the results expressed relative to the reactivity with the sE401v trimer . Data are from at least three independent experiments .

Blocking ELISA with mabs A1, A2, A3 and B4. Serial dilutions of the trimers were pre-incubated with a predetermined concentration of the respective mabs. The fraction of mab not blocked by the antigen was detected in ELISA with TBEV-coated plates (Materials and Methods). The bar charts show the results expressed relative to the reactivity with the sE401v trimer. Data are from at least three independent experiments.


( A - B ) Primary cilia are enriched at branch points and the greater curvature of the aorta of wild type mice , schematized in ( A ) and quantified in ( B ) . The color in ( A ) indicates which side of the aorta is quantified in ( B ) . Numbers indicate the region of the aorta ( A ) whose proportion of ciliated cells is quantified in ( B ) . Error bars are + / - 1 S . E . M and n = 7 mice . ( C ) Aortic endothelial cilia are ablated in Tek - Cre Ift88C / - mice . Endothelial cells and cilia are labeled by staining for Pecam1 and Arl13b , respectively . Scale bars are 10μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-B) Primary cilia are enriched at branch points and the greater curvature of the aorta of wild type mice, schematized in (A) and quantified in (B). The color in (A) indicates which side of the aorta is quantified in (B). Numbers indicate the region of the aorta (A) whose proportion of ciliated cells is quantified in (B). Error bars are +/- 1 S.E.M and n=7 mice. (C) Aortic endothelial cilia are ablated in Tek-Cre Ift88C/- mice. Endothelial cells and cilia are labeled by staining for Pecam1 and Arl13b, respectively. Scale bars are 10μm.


( A ) The vasculature in P5 retinas from ( A ) mTmG reporter mice with and without EC cilia was visualized by EGFP fluorescence . ( B ) The fractional distance migrated to the edge of the retina was quantified . There was no statistically significant difference ( p = 0 . 305 , Student ' s 2 - tailed t - test , n = 6 mice for each group ) . The average value of 8 measurements ( 4 retina leaflets measured per eye on both eyes ) was used for each mouse . ( C ) The number of vascular branchpoints was counted . There was no statistically significant difference ( p = 0 . 25 , Student ' s 2 - tailed t - test , n = 6 for control and n = 5 for mutant ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) The vasculature in P5 retinas from (A) mTmG reporter mice with and without EC cilia was visualized by EGFP fluorescence.(B) The fractional distance migrated to the edge of the retina was quantified. There was no statistically significant difference (p=0.305, Student's 2-tailed t-test, n=6 mice for each group). The average value of 8 measurements (4 retina leaflets measured per eye on both eyes) was used for each mouse.(C) The number of vascular branchpoints was counted. There was no statistically significant difference (p=0.25, Student's 2-tailed t-test, n=6 for control and n=5 for mutant).


( D ) Control and mutant adult mouse aortas were stained for GM130 ( green ) to mark the Golgi , CD144 ( red ) to mark ECs and DAPI ( blue ) to label DNA . ( E ) The angle between the Golgi , nucleus and tissue axis was measured and binned as ' left ' , ' right ' , ' upstream ' or ' downstream ' . ( F ) Six areas in the aorta were analyzed . ( G ) There was no difference in Golgi distribution at any single region or overall , nor was there a difference in the variance of the individual un - binned angles . Error bars are + 1 S . E . M . Scale bars are 500μm . n = 3 control and mutant mice for each region except region 2 where n = 3 control and n = 2 mutant mice .

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

(D) Control and mutant adult mouse aortas were stained for GM130 (green) to mark the Golgi, CD144 (red) to mark ECs and DAPI (blue) to label DNA.(E) The angle between the Golgi, nucleus and tissue axis was measured and binned as 'left', 'right', 'upstream' or 'downstream'.(F) Six areas in the aorta were analyzed.(G) There was no difference in Golgi distribution at any single region or overall, nor was there a difference in the variance of the individual un-binned angles. Error bars are +1 S.E.M. Scale bars are 500μm. n=3 control and mutant mice for each region except region 2 where n=3 control and n=2 mutant mice.


( B ) Loss of EC cilia by Tek - Cre increases atherosclerosis in the aortas of Apoe - / - mice on a high - fat high - cholesterol diet as visualized by Oil Red O ( ORO ) staining ( left panels ) whereas loss of cilia genes in blood by Mx1 - Cre has no effect ( right panels ) . ( C ) Cropped views of the boxed areas in ( B ) showing increased atherosclerosis in mice with Tek - Cre but not Mx1 - Cre mediated loss of Ift88 in the aortic arch . ( D - G ) Scatter plots of the percent ORO - positive area of the thoracic aorta in ( D - E ) Tek - Cre and ( F - G ) Mx1 - Cre mice , separated by ( D , F ) females and ( E , G ) males . Tek - Cre Apoe - / - Ift88C / - mice had a 58 . 9 % ( p = 0 . 0135 , Student ' s 2 - tailed t - test , n = 13 control and n = 10 mutant mice ) and 67 % ( p = 0 . 0106 , Student ' s 2 - tailed t - test , n = 17 control and n = 14 mutant ) increase in ORO - positive area versus control littermates in females and males , respectively . Mx1 - Cre Apoe - / - Ift88C / - mice had no significant difference in ORO - positive area in females ( p = 0 . 6643 , Student ' s 2 - tailed t - test , n = 7 control and mutant mice ) or males ( p = 0 . 9381 , Student ' s 2 - tailed t - test , n = 9 control and n = 7 mutant mice ) . Black bars represent the mean and error bars are + / - 1 S . E . M . Aortas depicted in ( B - C ) are red in the scatter plots .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Loss of EC cilia by Tek-Cre increases atherosclerosis in the aortas of Apoe-/- mice on a high-fat high-cholesterol diet as visualized by Oil Red O (ORO) staining (left panels) whereas loss of cilia genes in blood by Mx1-Cre has no effect (right panels).(C) Cropped views of the boxed areas in (B) showing increased atherosclerosis in mice with Tek-Cre but not Mx1-Cre mediated loss of Ift88 in the aortic arch.(D-G) Scatter plots of the percent ORO-positive area of the thoracic aorta in (D-E) Tek-Cre and (F-G) Mx1-Cre mice, separated by (D, F) females and (E, G) males. Tek-Cre Apoe-/- Ift88C/- mice had a 58.9% (p=0.0135, Student's 2-tailed t-test, n=13 control and n=10 mutant mice) and 67% (p=0.0106, Student's 2-tailed t-test, n=17 control and n=14 mutant) increase in ORO-positive area versus control littermates in females and males, respectively. Mx1-Cre Apoe-/- Ift88C/- mice had no significant difference in ORO-positive area in females (p=0.6643, Student's 2-tailed t-test, n=7 control and mutant mice) or males (p=0.9381, Student's 2-tailed t-test, n=9 control and n=7 mutant mice). Black bars represent the mean and error bars are +/- 1 S.E.M. Aortas depicted in (B-C) are red in the scatter plots.


( A ) Transcripts for the inflammatory cytokines Il1b , Il6 , Tnf1 and Tgfb1 , the adhesion molecules Vcam1 and Sele , the lymphocyte marker Nos2 and macrophage marker Cd68 , and the NFκB responsive genes Tnfaip3 and Hmox1 were upregulated in aortas from Apoe - / - mice lacking EC cilia by RT - qPCR , while transcripts of the Hh target genes Gli1 , Ptc1 , and Ptc2 were not . Error bars are + 1 S . E . M . n = 8 control and n = 10 mutant mice of mixed sex . P - values calculated by Student ' s 2 - tailed t - test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Transcripts for the inflammatory cytokines Il1b, Il6, Tnf1 and Tgfb1, the adhesion molecules Vcam1 and Sele, the lymphocyte marker Nos2 and macrophage marker Cd68, and the NFκB responsive genes Tnfaip3 and Hmox1 were upregulated in aortas from Apoe-/- mice lacking EC cilia by RT-qPCR, while transcripts of the Hh target genes Gli1, Ptc1, and Ptc2 were not. Error bars are +1 S.E.M. n=8 control and n=10 mutant mice of mixed sex. P-values calculated by Student's 2-tailed t-test.


( B ) Pkd2 localizes to EC cilia in vivo . Adult mouse aortas were stained for Pkd2 , acetylated α - Tubulin , and Hoechst to label DNA . Pkd2 localized strongly to primary cilia but also to other membranous structures in endothelial cells . Arrowheads point out cilia . Example cilium highlighted in inset .

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

(B) Pkd2 localizes to EC cilia in vivo. Adult mouse aortas were stained for Pkd2, acetylated α-Tubulin, and Hoechst to label DNA. Pkd2 localized strongly to primary cilia but also to other membranous structures in endothelial cells. Arrowheads point out cilia. Example cilium highlighted in inset.


( C ) Phosphorylation at Serine 1176 of eNOS was reduced in the aortas of mice lacking EC cilia . Aortas from control and mutant mice were stained for eNOS , p - eNOS - S1176 , acetylated α - Tubulin , and Hoechst to label DNA .

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

(C) Phosphorylation at Serine 1176 of eNOS was reduced in the aortas of mice lacking EC cilia. Aortas from control and mutant mice were stained for eNOS, p-eNOS-S1176, acetylated α-Tubulin, and Hoechst to label DNA.


( D ) The ratio of the total fluorescence p - eNOS - S1176 to total eNOS in the upper thoracic aorta ( near the first intercostal artery ) was calculated for control and mutant mice . The relative p - eNOS - S1176 to eNOS ratio was reduced in the mutant to 72 . 7 % the level of control siblings ( p = 0 . 009 , paired 2 - tailed ratio t - test , n = 7 sets of mice ) . Error bar is + 1 S . E . M . Four sets consisted of one control mouse and one mutant mouse . Three sets consisted of two control or mutant mice and the average value of the two was used . Scale bars are 10μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) The ratio of the total fluorescence p-eNOS-S1176 to total eNOS in the upper thoracic aorta (near the first intercostal artery) was calculated for control and mutant mice. The relative p-eNOS-S1176 to eNOS ratio was reduced in the mutant to 72.7% the level of control siblings (p=0.009, paired 2-tailed ratio t-test, n=7 sets of mice). Error bar is +1 S.E.M. Four sets consisted of one control mouse and one mutant mouse. Three sets consisted of two control or mutant mice and the average value of the two was used. Scale bars are 10μm.