( D ) Boxplots representing the expression level of INHBB , PMEPA1 , MYC and NRL in the different MB subtypes , as defined in Cavalli et al . ( Cavalli et al . , 2017 ) . Only p - values corresponding to comparisons between Group 3 subtypes are indicated . Patient samples are colored by subtypes as indicated . ( E ) Scatter plot of INHBB and PMEPA1 , expression levels in Group 3 patient samples . Colored dots represent each patient sample and colors represent the group 3 MB subtypes ( α in yellow , β in brown and δ in orange ) . Wilcoxon rank - sum tests were performed for panel D . Spearman ' s rank correlation coefficient ρ and p - value are indicated on panel E . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 and * * * * p < 0 . 0001 . Bars represent the mean ± SD . Number of replicates is 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Boxplots representing the expression level of INHBB, PMEPA1, MYC and NRL in the different MB subtypes, as defined in Cavalli et al. (Cavalli et al., 2017). Only p-values corresponding to comparisons between Group 3 subtypes are indicated. Patient samples are colored by subtypes as indicated. (E) Scatter plot of INHBB and PMEPA1, expression levels in Group 3 patient samples. Colored dots represent each patient sample and colors represent the group 3 MB subtypes (α in yellow, β in brown and δ in orange). Wilcoxon rank-sum tests were performed for panel D. Spearman's rank correlation coefficient ρ and p-value are indicated on panel E. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001. Bars represent the mean ± SD. Number of replicates is n≥3.


B Representative adhesion frequency ( Pa ) vs . contact duration ( tc ) curves forNKG2D expressing NK cells ( n ≥ 3 ) in contact with RBCs ( n ≥ 3 ) coated with a ligand ( MICA in red , MICB in orange , ULBP1 in green , or ULBP3 in blue ) at different contact durations , fitted by non - linear in - situ binding kinetics model Site densities of NKG2D ( mr ) and its ligands ( ml ) are indicated . Data information : Error bars are mean ± SEM for at least three independent biological experiments where * P < 0 . 05 , * * P < 0 . 01 ( two - tailed unpaired 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 O O O O O O O O O O O O O O

B Representative adhesion frequency (Pa) vs. contact duration (tc) curves forNKG2D expressing NK cells (n ≥ 3) in contact with RBCs (n ≥ 3) coated with a ligand (MICA in red, MICB in orange, ULBP1 in green, or ULBP3 in blue) at different contact durations, fitted by non-linear in-situ binding kinetics model Site densities of NKG2D (mr) and its ligands (ml) are indicated. Data information: Error bars are mean ± SEM for at least three independent biological experiments where *P < 0.05, **P < 0.01 (two-tailed unpaired t-test).


In - situ force - free affinities ( C ) , on - rates ( D ) of NKG2D binding with indicated ligands from mammalian cells . The in - situ force - free kinetics were obtained from fittings with in - situ binding kinetics modeling in ( B ) . Data information : Every dot represents one independent binding experiment . Error bars are mean ± SEM for at least three independent biological experiments where * P < 0 . 05 , * * P < 0 . 01 ( two - tailed unpaired 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

In-situ force-free affinities (C), on-rates (D) of NKG2D binding with indicated ligands from mammalian cells. The in-situ force-free kinetics were obtained from fittings with in-situ binding kinetics modeling in (B). Data information: Every dot represents one independent binding experiment. Error bars are mean ± SEM for at least three independent biological experiments where *P < 0.05, **P < 0.01 (two-tailed unpaired t-test).


In - situ force - free off - rates ( E ) of NKG2D binding with indicated ligands from mammalian cells . The in - situ force - free kinetics were obtained from fittings with in - situ binding kinetics modeling in ( B ) . Data information : Every dot represents one independent binding experiment . Error bars are mean ± SEM for at least three independent biological experiments where * P < 0 . 05 , * * P < 0 . 01 ( two - tailed unpaired 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

In-situ force-free off-rates (E) of NKG2D binding with indicated ligands from mammalian cells. The in-situ force-free kinetics were obtained from fittings with in-situ binding kinetics modeling in (B). Data information: Every dot represents one independent binding experiment. Error bars are mean ± SEM for at least three independent biological experiments where *P < 0.05, **P < 0.01 (two-tailed unpaired t-test).


F Detection range comparison in affinity measurement of NKG2D and indicated ligands between in - situ and in - solution assay . Bars in different colors are the ratios of the affinities of indicated ligands divided by that of ULBP1 . Data information : In - solution affinities of proteins purified from E . coli In - solution affinities of proteins purified from mammalian cells were measured by

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Detection range comparison in affinity measurement of NKG2D and indicated ligands between in-situ and in-solution assay. Bars in different colors are the ratios of the affinities of indicated ligands divided by that of ULBP1. Data information: In-solution affinities of proteins purified from E. coli In-solution affinities of proteins purified from mammalian cells were measured by BLI


IFN - γ release assay and IFN - γ production ( B ) by human peripheral NK cells stimulated with plate - coated NKG2D ligands ( MICA in red , MICB in green and ULBP3 in blue ) assessed by Cytometric Bead Array , of which are their 50 % effective concentration ( EC50 ) ( C ) . Data information : The IFN - γ release assay in ( B ) was one representative experiment of three total independent experiments . Data points in ( B ) and bars in ( C ) represent mean values . Error bars in ( B ) and ( C ) represent mean ± SEM . * * * P < 0 . 001 , * * * * P < 0 . 0001 ( two - tailed unpaired 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O

IFN-γ release assay and IFN-γ production (B) by human peripheral NK cells stimulated with plate-coated NKG2D ligands (MICA in red, MICB in green and ULBP3 in blue) assessed by Cytometric Bead Array, of which are their 50% effective concentration (EC50) (C). Data information: The IFN-γ release assay in (B) was one representative experiment of three total independent experiments. Data points in (B) and bars in (C) represent mean values. Error bars in (B) and (C) represent mean ± SEM. ***P < 0.001, **** P <0.0001 (two-tailed unpaired t-test).


D - F Plots of reciprocals of EC50 vs . the effective in - situ affinities ( AcKa ) ( D ) , the effective in - situ on - rate ( AcKon ) ( E ) and in - situ off - rate ( koff ) ( F ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-F Plots of reciprocals of EC50 vs. the effective in-situ affinities (AcKa) (D), the effective in-situ on-rate (AcKon) (E) and in-situ off-rate (koff) (F).


C Verification of the binding specificity of NKG2D with different ligands . Data information : Every dot represents the adhesion frequency of one cell - bead pair and the experimental data came from at least two to three repeated trials . Horizontal lines represent mean values . Error bars represent mean ± SEM .

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

C Verification of the binding specificity of NKG2D with different ligands. Data information: Every dot represents the adhesion frequency of one cell-bead pair and the experimental data came from at least two to three repeated trials. Horizontal lines represent mean values. Error bars represent mean ± SEM.


E , F Force - dependent bond lifetimes of NKG2D and various ligands at 5 pN , 10 pN and 15 pN ( E ) and full force spectrum ( F ) . Data information : Bond lifetimes in ( E ) and ( F ) ( in which n = 1505 for MICA , n = 1852 for MICB , n = 531 for ULBP1 , n = 1241 for ULBP3 ) of all ligands with NKG2D came from at least 19 NK cell - bead pairs of at least four independent biological experiments . bars in ( E ) , and data points in ( F ) represent mean values . Error bars represent mean ± SEM .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, F Force-dependent bond lifetimes of NKG2D and various ligands at 5 pN, 10 pN and 15 pN (E) and full force spectrum (F). Data information: Bond lifetimes in (E) and (F) (in which n = 1505 for MICA, n = 1852 for MICB, n = 531 for ULBP1, n = 1241 for ULBP3) of all ligands with NKG2D came from at least 19 NK cell-bead pairs of at least four independent biological experiments. bars in (E), and data points in (F) represent mean values. Error bars represent mean ± SEM.


C The force vs . extension trajectories for the simulations are shown in ( A ) and ( B ) . Occurrence of the sudden extension changes are indicated in the shaded area and time points correspond with the snapshots in ( A ) and ( B ) are marked .

C The force vs. extension trajectories for the simulations are shown in (A) and (B). Occurrence of the sudden extension changes are indicated in the shaded area and time points correspond with the snapshots in (A) and (B) are marked.


F Distance vs . time curves for force - induced H - bond formation between indicated residues within NKG2D - MICA binding interfaces . The dashed red lines represent H - bonds whose distances are less than 3 . 5 Å .

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

F Distance vs. time curves for force-induced H-bond formation between indicated residues within NKG2D-MICA binding interfaces. The dashed red lines represent H-bonds whose distances are less than 3.5 Å.


A , B Abolishments of force - induced binding residues by 3A mutations ( MICA 3A , n = 526 ) and TAT mutations ( MICA TAT , n = 580 ) impairs NKG2D ' s catch bond with MICA WT ( n = 1505 ) ( A ) and IFN - γ release ( B ) . Data information : Bond lifetimes in ( A ) of MICA WT and mutants with NKG2D came from at least 21 pairs of cells and beads of at least three repeated experiments . IFN - γ release of NK cells in ( B ) was one representative experiment of three total independent experiments . Data points represent mean values . Error bars represent mean ± SEM for biological triplicate experiments . * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 ( two - tailed unpaired 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Abolishments of force-induced binding residues by 3A mutations (MICA 3A, n = 526) and TAT mutations (MICA TAT, n= 580) impairs NKG2D's catch bond with MICA WT (n = 1505) (A) and IFN-γ release (B). Data information: Bond lifetimes in (A) of MICA WT and mutants with NKG2D came from at least 21 pairs of cells and beads of at least three repeated experiments. IFN-γ release of NK cells in (B) was one representative experiment of three total independent experiments. Data points represent mean values. Error bars represent mean ± SEM for biological triplicate experiments. **P < 0.01, ***P < 0.001, **** P <0.0001 (two-tailed unpaired t-test).


50 % effective concentration ( EC50 ) of NK cells stimulated by indicated MICA mutants Data information : horizontal lines represent mean values . Error bars represent mean ± SEM for biological triplicate experiments . * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 ( two - tailed unpaired 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

50% effective concentration (EC50) of NK cells stimulated by indicated MICA mutants Data information: horizontal lines represent mean values. Error bars represent mean ± SEM for biological triplicate experiments. **P < 0.01, ***P < 0.001, **** P <0.0001 (two-tailed unpaired t-test).


IFN - γ release of NK cells stimulated by indicated MICA mutants at a concentration of 11nM ( D ) and 22nM ( E ) . Data information : bars in ( E ) , and data point in ( F ) represent mean values . Error bars represent mean ± SEM for biological triplicate experiments . * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 ( two - tailed unpaired 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

IFN-γ release of NK cells stimulated by indicated MICA mutants at a concentration of 11nM (D) and 22nM (E). Data information: bars in (E), and data point in (F) represent mean values. Error bars represent mean ± SEM for biological triplicate experiments. **P < 0.01, ***P < 0.001, **** P <0.0001 (two-tailed unpaired t-test).


A - C Plots and Pearson correlation analysis of reciprocals of EC50 vs . the force - dependent in - situ koff at 5 pN ( A ) , 10 pN ( B ) 15pN ( C ) .

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

A-C Plots and Pearson correlation analysis of reciprocals of EC50 vs. the force-dependent in-situ koff at 5 pN (A), 10 pN (B) 15pN (C).


I Detection range comparison in force - dependent affinity of NKG2D and indicated ligands under difference force . Bars in different colors are the ratios of the affinities of indicated ligands divided by that of ULBP1 .

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

I Detection range comparison in force-dependent affinity of NKG2D and indicated ligands under difference force. Bars in different colors are the ratios of the affinities of indicated ligands divided by that of ULBP1.


D Contour plots showed probabilities of output signals at 300s produced by continuously variable initial on - rates and force - dependent off - rates .

O O 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 Contour plots showed probabilities of output signals at 300s produced by continuously variable initial on-rates and force-dependent off-rates.


A : Snapshots of LimE - GFP , GFP - myo , and corresponding kymographs for type 1 cells . Corresponding speeds are 10 . 8µm / min and 9 . 4µm / min . White dots in the fluorescent kymographs indicate the location of the two poles of force at each timepoint , as extracted from the corresponding stress kymographs . C : Snapshots of LimE - GFP , GFP - myo , and corresponding kymographs for type 2 cells . Corresponding speeds are 5 . 6µm / min and 8 . 7µm / min . White dots in the fluorescent kymographs indicate the location of the two poles of force at each timepoint .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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: Snapshots of LimE-GFP, GFP-myo, and corresponding kymographs for type 1 cells. Corresponding speeds are 10.8µm/min and 9.4µm/min. White dots in the fluorescent kymographs indicate the location of the two poles of force at each timepoint, as extracted from the corresponding stress kymographs. C: Snapshots of LimE-GFP, GFP-myo, and corresponding kymographs for type 2 cells. Corresponding speeds are 5.6µm/min and 8.7µm/min. White dots in the fluorescent kymographs indicate the location of the two poles of force at each timepoint.


B : Stress maps quantifying the magnitude of the force per area using a color scale with blue / red corresponding to small / large stresses and the direction of forces using vectors . Fan - shaped cells were rotated so that the vertical ( x ) axis is the direction of motion ( see Methods ) . Shown are the overall stress T , the stress in the direction of motion Tx and the stress perpendicular to motion Ty for the type 1 cell with the LimE marker , and the corresponding Tx and Ty kymographs along the cells outlines . D : Stress maps of T , Tx and Ty for the type 2 cell with the LimE marker , and corresponding Tx and Ty kymographs along the cells outlines .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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: Stress maps quantifying the magnitude of the force per area using a color scale with blue/red corresponding to small/large stresses and the direction of forces using vectors. Fan-shaped cells were rotated so that the vertical (x) axis is the direction of motion (see Methods). Shown are the overall stress T, the stress in the direction of motion Tx and the stress perpendicular to motion Ty for the type 1 cell with the LimE marker, and the corresponding Tx and Ty kymographs along the cells outlines. D: Stress maps of T, Tx and Ty for the type 2 cell with the LimE marker, and corresponding Tx and Ty kymographs along the cells outlines.


A : Sequence of snapshots of the LimE - GFP distribution and stress T for an oscillatory cell together with snapshots of the GFP - myo distribution for a separate oscillatory cell . Corresponding speeds are 7 . 0µm / min and 4 . 6µm / min . The sequences show an entire cycle of oscillation ( spreading and contraction ) . White arrows represent the direction of motion before the cell spreads and contracts during which no net motion exists .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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: Sequence of snapshots of the LimE-GFP distribution and stress T for an oscillatory cell together with snapshots of the GFP-myo distribution for a separate oscillatory cell. Corresponding speeds are 7.0µm/min and 4.6µm/min. The sequences show an entire cycle of oscillation (spreading and contraction). White arrows represent the direction of motion before the cell spreads and contracts during which no net motion exists.


A : Sequence of snapshots of the LimE - GFP distribution and stress T for an amoeboid cell and the GFP - myo distribution for a separate amoeboid cell . Corresponding speeds are 7 . 4µm / min and 4 . 5µm / min . The sequences show both the phases of protrusion and retraction . White arrows represent the direction of motion .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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: Sequence of snapshots of the LimE-GFP distribution and stress T for an amoeboid cell and the GFP-myo distribution for a separate amoeboid cell. Corresponding speeds are 7.4µm/min and 4.5µm/min. The sequences show both the phases of protrusion and retraction. White arrows represent the direction of motion.


A : Protrusion and retraction speed for amoeboid , oscillatory and fan - shaped cells , defined as the average of the pixels with the 20 % lowest and highest membrane speed .

O O O O O O O O O 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: Protrusion and retraction speed for amoeboid, oscillatory and fan-shaped cells, defined as the average of the pixels with the 20% lowest and highest membrane speed.


C : Average edge velocity in regions of low and high LimE - GFP and GFP - myo fluorescence . High fluorescence was defined as the 20 % brightest LimE - GFP and GFP - myo pixels in the kymographs while low fluorescence consisted of the remaining 80 % pixels .

C: Average edge velocity in regions of low and high LimE-GFP and GFP-myo fluorescence. High fluorescence was defined as the 20% brightest LimE-GFP and GFP-myo pixels in the kymographs while low fluorescence consisted of the remaining 80% pixels.


( a ) Lipid phosphatase activity of Paladin , wild - type and phosphatase - dead C / S mutant , towards PI ( 4 , 5 ) P2 and PI ( 3 , 4 , 5 ) P3 substrates . Positive control ; wild - type phosphatase and tensin homolog ( PTEN ) ; negative control ; lipid phosphatase - dead C124S PTEN . Mean ± SEM , Paired t - test , n = 3 biological replicates . Data information : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * 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

(a) Lipid phosphatase activity of Paladin, wild-type and phosphatase-dead C/S mutant, towards PI(4,5)P2 and PI(3,4,5)P3 substrates. Positive control; wild-type phosphatase and tensin homolog (PTEN); negative control; lipid phosphatase-dead C124S PTEN. Mean±SEM, Paired t-test, n=3 biological replicates. Data information: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.


( b ) Representative confocal image of HDMEC stained for Paladin ( green ) , Vascular Endothelial Cadherin ( VEC , red ) and nuclei ( DAPI , blue ) , scale bar : 10 μm . White boxes in the upper image are showed at higher magnification below , scale bar : 3 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

(b) Representative confocal image of HDMEC stained for Paladin (green), Vascular Endothelial Cadherin (VEC, red) and nuclei (DAPI, blue), scale bar: 10 μm. White boxes in the upper image are showed at higher magnification below, scale bar: 3 µm.


( c ) HDMEC stained for VEGFR2 ( green ) , Paladin ( red ) and nuclei ( DAPI , blue ) and with or without stimulation of 50 ng / ml VEGF - A for 0 , 2 or 10 min . Scale bar : 10 µm . ( d ) Quantification of VEGFR2 - Paladin co - staining before and after VEGF - A stimulation as shown in ( C ) . Mean ± SEM , One - way ANOVA . n = 3 biological replicates . Data information : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * 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 O O O

(c) HDMEC stained for VEGFR2 (green), Paladin (red) and nuclei (DAPI, blue) and with or without stimulation of 50 ng/ml VEGF-A for 0, 2 or 10 min. Scale bar: 10 µm. (d) Quantification of VEGFR2-Paladin co-staining before and after VEGF-A stimulation as shown in (C). Mean±SEM, One-way ANOVA. n=3 biological replicates. Data information: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.


( e ) HDMEC analysed using Proximity Ligand Assay ( PLA ) for Paladin and VEGFR2 . Green dots indicate complex formation , VE - cadherin ( red ) and nuclei ( DAPI , blue ) . Cells stimulated with 50 ng / ml VEGF - A for 0 , 2 or 10 min . Scale bar : 10 µm . ( f ) Quantification of ( e ) , the number of PLA Paldin - VEGFR2 complexes per cell at 0 , 2 and 10 minutes after VEGF - A stimulation ( top ) . Quantification of ( e ) , average distance for the PLA complexes to the nearest VE - cadherin positive junction at 0 , 2 and 10 minutes after VEGF - A stimulation ( bottom ) . Mean ± SEM , One - way ANOVA . n = 3 biological replicates . Data information : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HDMEC analysed using Proximity Ligand Assay (PLA) for Paladin and VEGFR2. Green dots indicate complex formation, VE-cadherin (red) and nuclei (DAPI, blue). Cells stimulated with 50 ng/ml VEGF-A for 0, 2 or 10 min. Scale bar: 10 µm. (f) Quantification of (e), the number of PLA Paldin-VEGFR2 complexes per cell at 0, 2 and 10 minutes after VEGF-A stimulation (top). Quantification of (e), average distance for the PLA complexes to the nearest VE-cadherin positive junction at 0, 2 and 10 minutes after VEGF-A stimulation (bottom). Mean±SEM, One-way ANOVA. n=3 biological replicates. Data information: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.


( g ) HDMEC stained for Paladin ( green ) and EEA1 ( red ) , colocalization in yellow , and nuclei ( DAPI , blue ) after VEGF - A stimulation for 0 , 2 or 10 minutes , a representative image from a single confocal plane is shown , scale bar : 10 µm . White boxes in the upper image are showed at higher magnification below , scale bar 3 µm . ( h ) Quantification of ( g ) , EEA1 / Paladin double positive particles per field of view at 0 , 2 and 10 minutes after VEGF - A stimulation . Mean ± SEM , One - way ANOVA . n = 3 biological replicates . Data information : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * 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 O O O O O O O O O O O O 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) HDMEC stained for Paladin (green) and EEA1 (red), colocalization in yellow, and nuclei (DAPI, blue) after VEGF-A stimulation for 0, 2 or 10 minutes, a representative image from a single confocal plane is shown, scale bar: 10 µm. White boxes in the upper image are showed at higher magnification below, scale bar 3 µm. (h) Quantification of (g), EEA1/Paladin double positive particles per field of view at 0, 2 and 10 minutes after VEGF-A stimulation. Mean±SEM, One-way ANOVA. n= 3 biological replicates. Data information: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.


( a ) Cell surface VEGFR2 levels detected by cell surface biotinylation , using thiol - cleavable sulfo - NHS - SS - biotin , of HDMEC transfected with PALD1 siRNA ( # 1 and # 2 ) or non - targeting control ( " c " ) siRNA , followed by VEGF - A stimulation ( 50 ng / ml ) for indicated time periods . Total lysates ( input ) and streptavidin ( SA ) pull down , immunoblotted for VEGFR2 , Paladin , and actin . ' No biotin ctrl ' , cells not treated with sulfo - NHS - SS - biotin . ( b ) Quantification of data in ( a ) . VEGFR2 surface levels ( data pooled for the indicated time points ) normalized to total VEGFR2 levels and compared between control and siRNA treated HDMEC . n = 4 for each time point , biological replicates , Mean ± SEM . Data information : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Cell surface VEGFR2 levels detected by cell surface biotinylation, using thiol-cleavable sulfo-NHS-SS-biotin, of HDMEC transfected with PALD1 siRNA (#1 and #2) or non-targeting control ("c") siRNA, followed by VEGF-A stimulation (50 ng/ml) for indicated time periods. Total lysates (input) and streptavidin (SA) pull down, immunoblotted for VEGFR2, Paladin, and actin. 'No biotin ctrl', cells not treated with sulfo-NHS-SS-biotin. (b) Quantification of data in (a). VEGFR2 surface levels (data pooled for the indicated time points) normalized to total VEGFR2 levels and compared between control and siRNA treated HDMEC. n=4 for each time point, biological replicates, Mean±SEM. Data information: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.


( c ) Internalized pool of VEGFR2 after VEGF - A treatment ( 50 ng / ml ) of non - transfected HDMEC ( " NT " ) or HDMEC transfected with PALD1 siRNA ( # 1 and # 2 ) or non - targeting control siRNA ( " c " ) . Cell surface biotinylation was performed prior to VEGF - A stimulation and at indicated time points , remaining cell surface biotin was stripped and the internalized pool of VEGFR2 was collected by SA pull down . Immunoblotting of the total lysate ( input ) and SA pull down fraction for VEGFR2 , Paladin and actin . ( d ) Quantification of data in ( c ) . Data were normalized to total VEGFR2 levels in the lysate after subtraction of signals in biotinylated and stripped samples . Mean ± SEM , unpaired t - test for indicated time points , normalized to cntrl siRNA sample . n = 3 for each time point , biological replicates . Data information : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Internalized pool of VEGFR2 after VEGF-A treatment (50 ng/ml) of non-transfected HDMEC ("NT") or HDMEC transfected with PALD1 siRNA (#1 and #2) or non-targeting control siRNA ("c"). Cell surface biotinylation was performed prior to VEGF-A stimulation and at indicated time points, remaining cell surface biotin was stripped and the internalized pool of VEGFR2 was collected by SA pull down. Immunoblotting of the total lysate (input) and SA pull down fraction for VEGFR2, Paladin and actin. (d) Quantification of data in (c). Data were normalized to total VEGFR2 levels in the lysate after subtraction of signals in biotinylated and stripped samples. Mean±SEM, unpaired t-test for indicated time points, normalized to cntrl siRNA sample. n=3 for each time point, biological replicates. Data information: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.


( e ) Analysis of EEA1 and VEGFR2 vesicles following PALD1 knockdown . Representative images of VEGFR2 ( green ) / EEA1 ( red ) double - positive ( yellow ) vesicles in negative control siRNA , and PALD1 KD # 2 siRNA - silenced HDMEC at 0 , 2 and 10 min of VEGF - A stimulation ( 50 ng / ml ) . DAPI in blue , scale bar : 10 µm . Inset shows only EEA1 channel , scale bar : 10 µm . ( f ) Quantification of ( e ) , number of EEA1 positive ( top ) or VEGFR2 - EEA1 double - positive vesicles ( bottom ) per field of view . Mean ± SEM , Two - way ANOVA , n = 3 biological replicates . Data information : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * 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 O O O O O O O O O O O O O O O O O O O O O O O O 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) Analysis of EEA1 and VEGFR2 vesicles following PALD1 knockdown. Representative images of VEGFR2 (green)/EEA1 (red) double-positive (yellow) vesicles in negative control siRNA, and PALD1 KD#2 siRNA-silenced HDMEC at 0, 2 and 10 min of VEGF-A stimulation (50 ng/ml). DAPI in blue, scale bar: 10 µm. Inset shows only EEA1 channel, scale bar: 10 µm. (f) Quantification of (e), number of EEA1 positive (top) or VEGFR2-EEA1 double-positive vesicles (bottom) per field of view. Mean±SEM, Two-way ANOVA, n=3 biological replicates. Data information: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.


( g ) HDMEC stained for PI ( 4 , 5 ) P2 ( cyan ) , VE - cadherin ( red ) and Paladin ( yellow in inset ) following treatment using negative control or PALD1 ( KD # 2 ) siRNA . VEGF - A stimulation for 0 , 2 or 10 minutes ( 50 ng / ml ) . DAPI in blue . Scale bar : 10 µm ( h ) Quantification of ( g ) , Total PI ( 4 , 5 ) P2 signal ( top ) or intracellular PI ( 4 , 5 ) P2 not overlapping with VE - cadherin ( bottom ) . Mean ± SEM , two - way ANOVA , n = 3 biological replicates . Data information : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

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

(g) HDMEC stained for PI(4,5)P2 (cyan), VE-cadherin (red) and Paladin (yellow in inset) following treatment using negative control or PALD1 (KD#2) siRNA. VEGF-A stimulation for 0, 2 or 10 minutes (50 ng/ml). DAPI in blue. Scale bar: 10 µm (h) Quantification of (g), Total PI(4,5)P2 signal (top) or intracellular PI(4,5)P2 not overlapping with VE-cadherin (bottom). Mean±SEM, two-way ANOVA, n=3 biological replicates. Data information: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.


Signalling downstream of VEGF - A / VEGFR2 assessed in HDMEC , untreated ( NT , non - transfected ) , transfected with non - targeting siRNA ( c / cntrl ) , or with two different PALD1 - targeting siRNAs ( KD # 1 and KD # 2 ) , and treated with VEGF - A for 0 - 10 and 0 - 20 min , respectively . Immunoblotting of cell lysates for phosphorylated ( p ) VEGFR2 ( pY1175 ) , total VEGFR2 Actin served as loading control ( a PALD1 knockdown was verified by blotting for Paladin . Quantification of pVEGFR2 ( normalized to total VEGFR2 ) ( b ) Mean ± SEM , two - way ANOVA , n = 3 biological replicates . Data information : * p < 0 . 05 , * * 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 O O O O O O O O O O O

Signalling downstream of VEGF-A/VEGFR2 assessed in HDMEC, untreated (NT, non-transfected), transfected with non-targeting siRNA (c/cntrl), or with two different PALD1-targeting siRNAs (KD#1 and KD#2), and treated with VEGF-A for 0-10 and 0-20 min, respectively. Immunoblotting of cell lysates for phosphorylated (p) VEGFR2 (pY1175), total VEGFR2 Actin served as loading control (a PALD1 knockdown was verified by blotting for Paladin. Quantification of pVEGFR2 (normalized to total VEGFR2) (b) Mean±SEM, two-way ANOVA, n=3 biological replicates. Data information: *p < 0.05, **p < 0.01.


Signalling downstream of VEGF - A / VEGFR2 assessed in HDMEC , untreated ( NT , non - transfected ) , transfected with non - targeting siRNA ( c / cntrl ) , or with two different PALD1 - targeting siRNAs ( KD # 1 and KD # 2 ) , and treated with VEGF - A for 0 - 10 and 0 - 20 min , respectively . Immunoblotting of cell lysates for phosphorylated Erk1 / 2 ( pT202 and pY204 ) , and total Erk . Actin served as loading control c ) . PALD1 knockdown was verified by blotting for Paladin . Quantification of pErk1 / 2 ( normalized to total Erk1 / 2 ) ( d ) . Mean ± SEM , two - way ANOVA , n = 3 biological replicates . Data information : * p < 0 . 05 , * * 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 O O O O O O O O O O O O O O O O O O O O

Signalling downstream of VEGF-A/VEGFR2 assessed in HDMEC, untreated (NT, non-transfected), transfected with non-targeting siRNA (c/cntrl), or with two different PALD1-targeting siRNAs (KD#1 and KD#2), and treated with VEGF-A for 0-10 and 0-20 min, respectively. Immunoblotting of cell lysates for phosphorylated Erk1/2 (pT202 and pY204), and total Erk. Actin served as loading control c). PALD1 knockdown was verified by blotting for Paladin. Quantification of pErk1/2 (normalized to total Erk1/2) (d). Mean±SEM, two-way ANOVA, n=3 biological replicates. Data information: *p < 0.05, **p < 0.01.


( e - f ) Immunoblotting on total heart lysates from adult Pald1 + / + and Pald1 - / - mice , tail - vein injected with VEGF - A ( 0 . 25µg / g body weight ) or PBS for the indicated time points , for Paladin , phosphorylated and total levels of VEGFR2 , Erk1 / 2 and β2 - microglobulin ( β 2 - MG , loading control ) . ( f ) Quantification of pVEGFR pY1173 normalized to total VEGFR2 ( n = 3 ) , total VEGFR2 levels normalized to total loading control ( n = 4 ) , pT202 / pY204 Erk1 / 2 normalized to Erk1 / 2 ( n = 5 ) . Mean ± SEM , multiple t - test with Holm - Sidak correction ( total VEGFR2 ) , two - way ANOVA ( others ) . Data information : * p < 0 . 05 , * * 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 O O O O O O O O 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-f) Immunoblotting on total heart lysates from adult Pald1+/+ and Pald1-/- mice, tail-vein injected with VEGF-A (0.25µg/g body weight) or PBS for the indicated time points, for Paladin, phosphorylated and total levels of VEGFR2, Erk1/2 and β2-microglobulin (β 2-MG, loading control). (f) Quantification of pVEGFR pY1173 normalized to total VEGFR2 (n=3), total VEGFR2 levels normalized to total loading control (n=4), pT202/pY204 Erk1/2 normalized to Erk1/2 (n=5). Mean±SEM, multiple t-test with Holm-Sidak correction (total VEGFR2), two-way ANOVA (others). Data information: *p < 0.05, **p < 0.01.


( a , b ) Delayed vascular outgrowth and hyperdense vascular front in isolectinB4 - stained P5 retina from Pald1 - / - mouse compared with Pald1 + / + ( a ) . Orange arrow indicates radial expansion of the vascular plexus in the Pald1 + / + retina . Scale bar : 1 mm . Quantification of radial expansion ( b ) as shown in ( a ) normalized to wild type litter mates . Mean ± SEM , unpaired t - test . n = 14 litters , 18 wild type and 23 knock - out pups . Data information : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Delayed vascular outgrowth and hyperdense vascular front in isolectinB4-stained P5 retina from Pald1-/- mouse compared with Pald1+/+ (a). Orange arrow indicates radial expansion of the vascular plexus in the Pald1+/+ retina. Scale bar: 1 mm. Quantification of radial expansion (b) as shown in (a) normalized to wild type litter mates. Mean±SEM, unpaired t-test. n=14 litters, 18 wild type and 23 knock-out pups. Data information: *p < 0.05, **p < 0.01, ***p < 0.001.


( c , d ) Increased filopodia number in Pald1 - / - mouse retina at vascular front , visualized by isolectinB4 staining ( c ) . Scale bar : 50 μm . Quantification of filopodia per 100 µm at the vascular front ( d ) . Mean ± SEM , unpaired t - test . n = 3 litters , 7 wild type and 5 knock - out pups . Data information : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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

(c, d) Increased filopodia number in Pald1-/- mouse retina at vascular front, visualized by isolectinB4 staining (c). Scale bar: 50 μm. Quantification of filopodia per 100 µm at the vascular front (d). Mean±SEM, unpaired t-test. n=3 litters, 7 wild type and 5 knock-out pups. Data information: *p < 0.05, **p < 0.01, ***p < 0.001.


( e , f ) P5 retina vascular front ( isolectinB4 , green ) and endothelial nuclei ( Erg , red ) ( e ) . Scale bar : 100 μm . Magenta stippled square area quantified in ( f ) . Vascular density was determined in the capillary bed ( left ) ( 11 wild type , 12 knock - out ) and in area around veins ( right ) ( 10 wild type , 9 knock - out , biological replicates ) . Mean ± SEM , unpaired t - test . Data information : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, f) P5 retina vascular front (isolectinB4, green) and endothelial nuclei (Erg, red) (e). Scale bar: 100 μm. Magenta stippled square area quantified in (f). Vascular density was determined in the capillary bed (left) (11 wild type, 12 knock-out) and in area around veins (right) (10 wild type, 9 knock-out, biological replicates). Mean±SEM, unpaired t-test. Data information: *p < 0.05, **p < 0.01, ***p < 0.001.


( g , h ) pT202 / pY204 Erk1 / 2 immunostaining ( black ) in Pald1 + / + and Pald1 - / - P5 pups . IsolectinB4 ( white ) visualizes the entire vasculature . Scale bar : 100 μm . Quantification of pT202 / pY204 Erk1 / 2 area ( h ) as in the black stippled square in ( g ) ( 400 µm from the retina rim ) , normalized to isolectinB4 area . Mean ± SEM , unpaired t - test . n = 5 litters , 14 wild type and 10 knock - out retinas per genotype . Data information : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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

(g, h) pT202/pY204 Erk1/2 immunostaining (black) in Pald1+/+ and Pald1-/- P5 pups. IsolectinB4 (white) visualizes the entire vasculature. Scale bar: 100 μm. Quantification of pT202/pY204 Erk1/2 area (h) as in the black stippled square in (g) (400 µm from the retina rim), normalized to isolectinB4 area. Mean±SEM, unpaired t-test. n=5 litters, 14 wild type and 10 knock-out retinas per genotype. Data information: *p < 0.05, **p < 0.01, ***p < 0.001.


( i ) Quantitative real - time PCR analysis of P4 - P5 retinas from Pald1 + / + and Pald1 - / - pups . Ccnd1 transcript levels , normalized to Gapdh . Mean ± SEM , unpaired t - test . n = 10 Pald1 + / + and 14 Pald1 - / - pups . Data information : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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

(i) Quantitative real-time PCR analysis of P4-P5 retinas from Pald1+/+ and Pald1-/- pups. Ccnd1 transcript levels, normalized to Gapdh. Mean±SEM, unpaired t-test. n=10 Pald1+/+ and 14 Pald1-/- pups. Data information: *p < 0.05, **p < 0.01, ***p < 0.001.


( j ) Cyclin D1 ( black ) and isolectinB4 ( white ) staining of P5 retinas from Pald1 + / + and Pald1 - / - pups . 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

(j) Cyclin D1 (black) and isolectinB4 (white) staining of P5 retinas from Pald1+/+ and Pald1-/-pups. Scale bar: 100 µm.


( k ) Quantification of number of Cyclin D1 positive nuclei normalized for isolectinB4 area in retinas from Pald1 + / + and Pald1 - / P5 pups . Mean ± SEM , unpaired t - test . n = 7 Pald1 + / + and 6 Pald1 - / - pups . Data information : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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

(k) Quantification of number of Cyclin D1 positive nuclei normalized for isolectinB4 area in retinas from Pald1+/+ and Pald1-/ P5 pups. Mean±SEM, unpaired t-test. n=7 Pald1+/+ and 6 Pald1-/- pups. Data information: *p < 0.05, **p < 0.01, ***p < 0.001.


( l ) Quantification of relative radial expansion in vehicle ( n = 2 litters , 5 Pald1 + / + and 4 Pald1 - / - pups ) and MEK inhibitor ( U0126 ) - treated pups ( n = 4 litters , 7 Pald1 + / + and 5 Pald1 - / - pups ) . MEK inhibitor / vehicle was administered twice at 12h interval at P4 and eyes collected at P5 . Each dot is one mouse . Mean ± SEM , one - way ANOVA , n = 4 - 7 . Data information : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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

(l) Quantification of relative radial expansion in vehicle (n=2 litters, 5 Pald1+/+ and 4 Pald1-/- pups) and MEK inhibitor (U0126)-treated pups (n=4 litters, 7 Pald1+/+ and 5 Pald1-/- pups). MEK inhibitor/vehicle was administered twice at 12h interval at P4 and eyes collected at P5. Each dot is one mouse. Mean±SEM, one-way ANOVA, n=4-7. Data information: *p < 0.05, **p < 0.01, ***p < 0.001.


( m ) Quantification of the tip cell number in vehicle - ( n = 3 litters , 5 Pald1 + / + and 5 Pald1 - / - pups ) and MEK inhibitor ( U0126 ) - treated pups ( n = 3 litters , 5 Pald1 + / + and 4 Pald1 - / - pups ) . MEK inhibitor / vehicle administered twice at P5 at 2 - h intervals , and eyes collected 2 h after the second injection . Each dot is one mouse . Mean ± SEM , one - way ANOVA . Data information : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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

(m) Quantification of the tip cell number in vehicle- (n=3 litters, 5 Pald1+/+ and 5 Pald1-/- pups) and MEK inhibitor (U0126)-treated pups (n=3 litters, 5 Pald1+/+ and 4 Pald1-/- pups). MEK inhibitor/vehicle administered twice at P5 at 2-h intervals, and eyes collected 2 h after the second injection. Each dot is one mouse. Mean±SEM, one-way ANOVA. Data information: *p < 0.05, **p < 0.01, ***p < 0.001.


( a ) Eyes from Pald1 + / LacZ mice collected at P17 from untreated animals or animals with oxygen - induced retinopathy ( OIR ) . Pald1 - promoter driven LacZ expression and X - gal staining generated signals in capillaries , veins and arteries in the OIR retina at P17 compared to the normoxia control with predominantly arterial LacZ expression . Scale bar : 1 mm .

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

(a) Eyes from Pald1+/LacZ mice collected at P17 from untreated animals or animals with oxygen-induced retinopathy (OIR). Pald1-promoter driven LacZ expression and X-gal staining generated signals in capillaries, veins and arteries in the OIR retina at P17 compared to the normoxia control with predominantly arterial LacZ expression. Scale bar: 1 mm.


( b ) Paladin levels in primary human umbilical vein endothelial cells ( HUVEC ) untreated or treated for 24 h with VEGF - A ( 50 ng / ml ) , FGF2 ( 50 ng / ml ) , or SDF1α ( 30 ng / ml ) , quantified by immunoblotting for Paladin and β - actin ( loading control ) . Mean ± SEM , one way - ANOVA , n = 5 biological replicates . Data information : * p < 0 . 05 , * * 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

(b) Paladin levels in primary human umbilical vein endothelial cells (HUVEC) untreated or treated for 24 h with VEGF-A (50 ng/ml), FGF2 (50 ng/ml), or SDF1α (30 ng/ml), quantified by immunoblotting for Paladin and β-actin (loading control). Mean±SEM, one way-ANOVA, n=5 biological replicates. Data information: *p < 0.05, **p < 0.01.


( c ) Eyes from adult Pald1LacZ / + mice collected at 72 h after single - bolus intravitreal injection of 1 μg VEGF - A , and PBS in the contralateral eye , followed by X - gal staining . Arrow indicates Pald1 promoter activity in veins ( outlined by dashed line ) specifically after VEGF - A treatment . A , artery ; V , vein . 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 O O O O 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) Eyes from adult Pald1LacZ/+ mice collected at 72 h after single-bolus intravitreal injection of 1 μg VEGF-A, and PBS in the contralateral eye, followed by X-gal staining. Arrow indicates Pald1 promoter activity in veins (outlined by dashed line) specifically after VEGF-A treatment. A, artery; V, vein. Scale bar: 100 μm.


( d - f ) Representative images of isolectinB4 - stained P17 retinas from OIR - challenged Pald1 + / + and Pald1 - / - mice ( detailed view in the insets ) ( d ) . Scale bar : 1 mm ( inset 250 µm ) . Quantification of neovascular tuft area ( e ) and avascular area ( f ) . Each dot represents the mean of both retinas per mouse . Mean ± SEM , unpaired t - test , n = 3 litters , 4 Pald1 + / + and 3 Pald1 - / - pups . Data information : * p < 0 . 05 , * * 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

(d-f) Representative images of isolectinB4-stained P17 retinas from OIR-challenged Pald1+/+ and Pald1-/- mice (detailed view in the insets) (d). Scale bar: 1 mm (inset 250 µm). Quantification of neovascular tuft area (e) and avascular area (f). Each dot represents the mean of both retinas per mouse. Mean±SEM, unpaired t-test, n=3 litters, 4 Pald1+/+ and 3 Pald1-/- pups. Data information: *p < 0.05, **p < 0.01.


g ) Representative images of retina vasculature immunostained for isolectinB4 and pT202 / pY204 Erk1 / 2 ( pErk1 / 2 ) . pErk1 / 2 staining within the vessels is also visualized using a 16 - color heatmap to display staining intensity . Scale bar : 30 µm ( inset 10µm ) . ( h ) Quantification of pErk1 / 2 immunostaining as shown in ( g ) within isolectinB4 - positive vessels , as fold - change of pErk1 / 2 stained area . Each dot represents the mean of both retinas per mouse . Mean ± SEM , unpaired t - test , n = 3 litters , 5 Pald1 + / + and 5 Pald1 - / - pups . Data information : * p < 0 . 05 , * * 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 O O O O O O O

g) Representative images of retina vasculature immunostained for isolectinB4 and pT202/pY204 Erk1/2 (pErk1/2). pErk1/2 staining within the vessels is also visualized using a 16-color heatmap to display staining intensity. Scale bar: 30 µm (inset 10µm). (h) Quantification of pErk1/2 immunostaining as shown in (g) within isolectinB4-positive vessels, as fold-change of pErk1/2 stained area. Each dot represents the mean of both retinas per mouse. Mean±SEM, unpaired t-test, n=3 litters, 5 Pald1+/+ and 5 Pald1-/- pups. Data information: *p < 0.05, **p < 0.01.


A NHF , Lowe 1676 and Lowe 3265 cells were grown in complete media or 5 h serum starvation , then immunostained with LAMP1 antibody . Dashed lines in images outline cell boundaries . Scale bar , 10 μm . C Quantitative analyses of lysosome perinuclear distribution in the experiments shown in A . The intracellular distribution of LAMP1 positive vesicles was quantified as described in the Methods . n = 3 per group in which 30 cells were counted per condition per experiment . Data information : data are presented as mean ± SEM . * ≤ 0 . 05 , * * ≤ 0 . 01 , * * * ≤ 0 . 001 , * * * * P ≤ 0 . 0001 , n . s . , not significant , calculated by two - tailed Student ' s 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 O O O O O O O O O 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 NHF, Lowe 1676 and Lowe 3265 cells were grown in complete media or 5 h serum starvation, then immunostained with LAMP1 antibody. Dashed lines in images outline cell boundaries. Scale bar, 10 μm. C Quantitative analyses of lysosome perinuclear distribution in the experiments shown in A. The intracellular distribution of LAMP1 positive vesicles was quantified as described in the Methods. n = 3 per group in which 30 cells were counted per condition per experiment. Data information: data are presented as mean ± SEM. * ≤ 0.05, ** ≤ 0.01, ***≤ 0.001, ****P ≤ 0.0001, n.s., not significant, calculated by two-tailed Student's t-test.


B Wildtype ( Ocrl + / y ) and Ocrl - / - MEF cells were grown in complete media or 5 h serum starvation , then immunostained with indicated antibodies . Dashed lines in images outline cell boundaries . Scale 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 O O

B Wildtype (Ocrl+/y) and Ocrl-/- MEF cells were grown in complete media or 5 h serum starvation, then immunostained with indicated antibodies. Dashed lines in images outline cell boundaries. Scale bar, 10 μm.


D , E Quantitative analyses of lysosome and mTOR perinuclear distribution in the experiments shown in B . The intracellular distribution of mTOR positive vesicles was quantified as described in the Methods . n = 3 per group in which 30 cells were counted per condition per experiment . Data information : , data are presented as mean ± SEM . * ≤ 0 . 05 , * * ≤ 0 . 01 , * * * ≤ 0 . 001 , * * * * P ≤ 0 . 0001 , n . s . , not significant , calculated by two - tailed Student ' s 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

D, E Quantitative analyses of lysosome and mTOR perinuclear distribution in the experiments shown in B. The intracellular distribution of mTOR positive vesicles was quantified as described in the Methods. n = 3 per group in which 30 cells were counted per condition per experiment. Data information: , data are presented as mean ± SEM. * ≤ 0.05, ** ≤ 0.01, ***≤ 0.001, ****P ≤ 0.0001, n.s., not significant, calculated by two-tailed Student's t-test.


I Snap images of WT ( Ocrl + / y ) , OCRL knockout ( Ocrl - / - ) and IOB MEFs transfected with lysosome - GFP . After transfection , WT , OCRL knockout and IOB MEFs were left in serum starvation for 8 h . Scale bar , 5 μm . J Quantitative analyses of tubulated lysosomes in the experiments shown in H . n = 3 per group in which 10 cells were counted per condition per experiment . D Data information : data are presented as mean ± SEM . * ≤ 0 . 05 , * * ≤ 0 . 01 , * * * ≤ 0 . 001 , * * * * P ≤ 0 . 0001 , n . s . , not significant , calculated by two - tailed Student ' s 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

I Snap images of WT (Ocrl+/y), OCRL knockout (Ocrl-/-) and IOB MEFs transfected with lysosome-GFP. After transfection, WT, OCRL knockout and IOB MEFs were left in serum starvation for 8 h. Scale bar, 5 μm. J Quantitative analyses of tubulated lysosomes in the experiments shown in H. n = 3 per group in which 10 cells were counted per condition per experiment. D Data information: data are presented as mean ± SEM. * ≤ 0.05, ** ≤ 0.01, ***≤ 0.001, ****P ≤ 0.0001, n.s., not significant, calculated by two-tailed Student's t-test.


A Immunoblot analysis of endogenous p - mTOR ( Ser2448 ) , total mTOR , p - p70 S6K ( Thr389 ) and total p70 S6K in NHF , Lowe 1676 and Lowe 3265 cells grown in complete media or 5 h serum starvation . p - p70 S6K ( Thr389 ) over total p70 S6K ratios normalized to NHF cells , three independent experiments . B Immunoblot analysis of endogenous p - mTOR ( Ser2448 ) , total mTOR , p - p70 S6K ( Thr389 ) and total p70 S6K in WT ( Ocrl + / y ) and IOB MEFs grown in complete medium or 5 h serum starvation . p - p70 S6K ( Thr389 ) over total p70 S6K ratios normalized to NHF cells , three independent experiments . C Data information : data are presented as mean ± SEM . * ≤ 0 . 05 , * * ≤ 0 . 01 , * * * ≤ 0 . 001 , * * * * P ≤ 0 . 0001 , n . s . , not significant , calculated by two - tailed Student ' s t - test unless otherwise stated .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Immunoblot analysis of endogenous p-mTOR (Ser2448), total mTOR, p-p70 S6K (Thr389) and total p70 S6K in NHF, Lowe 1676 and Lowe 3265 cells grown in complete media or 5 h serum starvation. p-p70 S6K (Thr389) over total p70 S6K ratios normalized to NHF cells, three independent experiments. B Immunoblot analysis of endogenous p-mTOR (Ser2448), total mTOR , p-p70 S6K (Thr389) and total p70 S6K in WT (Ocrl+/y) and IOB MEFs grown in complete medium or 5 h serum starvation. p-p70 S6K (Thr389) over total p70 S6K ratios normalized to NHF cells, three independent experiments. C Data information: data are presented as mean ± SEM. * ≤ 0.05, ** ≤ 0.01, ***≤ 0.001, ****P ≤ 0.0001, n.s., not significant, calculated by two-tailed Student's t-test unless otherwise stated.


D NHF and Lowe 1676 cells were grown in complete media , amino - acid ( aa ) / FBS starved for 5 h , or starved and then recovered in amino - acid / FBS - containing medium , then immunoblotted using antibodies against endogenous p - mTOR ( Ser2448 ) , total mTOR , p - p70 S6K ( Thr389 ) and total p70 S6K . Data information : data are presented as mean ± SEM . * ≤ 0 . 05 , * * ≤ 0 . 01 , * * * ≤ 0 . 001 , * * * * P ≤ 0 . 0001 , n . s . , not significant , calculated by two - tailed Student ' s t - test unless otherwise stated .

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

D NHF and Lowe 1676 cells were grown in complete media, amino-acid (aa)/FBS starved for 5 h, or starved and then recovered in amino-acid/FBS-containing medium, then immunoblotted using antibodies against endogenous p-mTOR (Ser2448), total mTOR, p-p70 S6K (Thr389) and total p70 S6K. Data information: data are presented as mean ± SEM. * ≤ 0.05, ** ≤ 0.01, ***≤ 0.001, ****P ≤ 0.0001, n.s., not significant, calculated by two-tailed Student's t-test unless otherwise stated.


A Representative images of NHF , Lowe 1676 and Lowe 3265 cells immunostained with anti - α - tubulin ( red ) and anti - γ - tubulin ( green ) antibodies . Scale bar , 10 μm . B Quantitative analyses of focused microtubule arrays numbers at γ - tubulin ( microtubule organizing center MTOC ) measured in the experiments shown in A . n = 3 cells per group , three independent experiments . C Data information : data are presented as mean ± SEM . * ≤ 0 . 05 , * * ≤ 0 . 01 , calculated by two - way ANOVA , multiple comparisons .

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

A Representative images of NHF, Lowe 1676 and Lowe 3265 cells immunostained with anti-α-tubulin (red) and anti-γ-tubulin (green) antibodies. Scale bar, 10 μm. B Quantitative analyses of focused microtubule arrays numbers at γ-tubulin (microtubule organizing center MTOC) measured in the experiments shown in A. n = 3 cells per group, three independent experiments. C Data information: data are presented as mean ± SEM. * ≤ 0.05, ** ≤ 0.01, calculated by two-way ANOVA, multiple comparisons.


C WT ( Ocrl + / y ) , OCRL knockout ( Ocrl - / - ) and IOB MEFs derived from the corresponding mice were costained with anti - α - tubulin and anti - γ - tubulin antibodies . Nuclei were stained with DAPI ( blue ) . Scale bar , 10 μm . D Quantitative analyses of focused microtubule arrays numbers at γ - tubulin ( microtubule organizing center MTOC ) measured in the experiments shown in C . n = 3 per group in which 30 cells were counted per condition per experiment . E Data information : data are presented as mean ± SEM . * ≤ 0 . 05 , * * ≤ 0 . 01 , calculated by two - way ANOVA , multiple comparisons .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 WT (Ocrl+/y), OCRL knockout (Ocrl-/-) and IOB MEFs derived from the corresponding mice were costained with anti-α-tubulin and anti- γ-tubulin antibodies. Nuclei were stained with DAPI (blue). Scale bar, 10 μm. D Quantitative analyses of focused microtubule arrays numbers at γ-tubulin (microtubule organizing center MTOC) measured in the experiments shown in C. n = 3 per group in which 30 cells were counted per condition per experiment. E Data information: data are presented as mean ± SEM. * ≤ 0.05, ** ≤ 0.01, calculated by two-way ANOVA, multiple comparisons.


E Microtubule regrowth assay was performed on NHF , Lowe 1676 and Lowe 3265 cells with 20 μM nocodazole for 2 h on ice , followed by rinse with PBS , and addition of pre - warmed medium to induce microtubule regrowth . Cells were then immunostained with anti - α - tubulin and anti - γ - tubulin antibodies at the indicated time point after addition of pre - warmed medium . The magnified view shows the cytoplasmic microtubules organization center . Scale bar , 10 μm . F Quantitative analyses of focused microtubule arrays numbers at γ - tubulin ( microtubule organizing center , MTOC ) measured in the experiments shown in E . n = 3 per group in which 30 cells were counted per condition per experiment . D Data information : data are presented as mean ± SEM . * ≤ 0 . 05 , * * ≤ 0 . 01 , calculated by two - way ANOVA , multiple comparisons .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Microtubule regrowth assay was performed on NHF, Lowe 1676 and Lowe 3265 cells with 20 μM nocodazole for 2 h on ice, followed by rinse with PBS, and addition of pre-warmed medium to induce microtubule regrowth. Cells were then immunostained with anti-α-tubulin and anti-γ-tubulin antibodies at the indicated time point after addition of pre-warmed medium. The magnified view shows the cytoplasmic microtubules organization center. Scale bar, 10 μm. F Quantitative analyses of focused microtubule arrays numbers at γ-tubulin (microtubule organizing center, MTOC) measured in the experiments shown in E. n = 3 per group in which 30 cells were counted per condition per experiment. D Data information: data are presented as mean ± SEM. * ≤ 0.05, ** ≤ 0.01, calculated by two-way ANOVA, multiple comparisons.


E Super - resolution images of centrosome stained with anti - CEP164 ( red , labeling distal appendages of mother centriole ) and anti - OCRL ( blue ) were acquired using Centrin2 - GFP - RPE - 1 cells by DeltaVision OMX V4 BLAZE system . Scale bar , 0 . 5 μm . F Super - resolution images of centrosome stained with anti - CNAP1 ( red , labeling proximal ends of centriole ) and anti - OCRL ( blue ) were acquired using Centrin2 - GFP - RPE - 1 cells by DeltaVision OMX V4 BLAZE system . Scale bar , 0 . 5 μm . G

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Super-resolution images of centrosome stained with anti-CEP164 (red, labeling distal appendages of mother centriole) and anti-OCRL (blue) were acquired using Centrin2-GFP-RPE-1 cells by DeltaVision OMX V4 BLAZE system. Scale bar, 0.5 μm. F Super-resolution images of centrosome stained with anti-CNAP1 (red, labeling proximal ends of centriole) and anti-OCRL (blue) were acquired using Centrin2-GFP-RPE-1 cells by DeltaVision OMX V4 BLAZE system. Scale bar, 0.5 μm. G


H Representative images of the co - localization of OCRL - WT , GFP - OCRL - △ RhoGAP and GFP - OCRL - △ ASH + RhoGAP with γ - tubulin . Insets , enlargements of the outlined areas . Scale bars , 10 μm . I Quantitative analysis of cells with and without centrosomal OCRL ( centrosomal GFP signal ) in the experiments shown in A . n = 3 per group in which 10 cells were counted per condition per experiment . Means ± SEM , * * * * P < 0 . 0001 , n . s . , not significant , calculated by two - way ANOVA , multiple comparisons .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Representative images of the co-localization of OCRL-WT, GFP-OCRL-△RhoGAP and GFP-OCRL-△ASH+RhoGAP with γ-tubulin. Insets, enlargements of the outlined areas. Scale bars, 10 μm. I Quantitative analysis of cells with and without centrosomal OCRL (centrosomal GFP signal) in the experiments shown in A. n = 3 per group in which 10 cells were counted per condition per experiment. Means ±SEM, ****P < 0.0001, n.s., not significant, calculated by two-way ANOVA, multiple comparisons.


A RPE - 1 cells transfected with nontargeting control siRNA or OCRL siRNA , were left for 48 h , then subjected to IF microscopy to visualize PI ( 4 , 5 ) P2 ( green ) , γ - tubulin ( red ) and nuclei ( blue , DAPI staining ) . Scale bar , 10 μm . B Quantitative analyses of centrosomal PI ( 4 , 5 ) P2 fluorescence intensity in the experiments shown in A . n = 3 per group in which 50 cells were counted per condition per experiment . C Data information : data are presented as mean ± SEM . n . s . , not significant , calculated by calculated by two - tailed Student ' s 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 B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A RPE-1 cells transfected with nontargeting control siRNA or OCRL siRNA, were left for 48 h, then subjected to IF microscopy to visualize PI(4,5)P2 (green), γ-tubulin (red) and nuclei (blue, DAPI staining). Scale bar, 10 μm. B Quantitative analyses of centrosomal PI(4,5)P2 fluorescence intensity in the experiments shown in A. n = 3 per group in which 50 cells were counted per condition per experiment. C Data information: data are presented as mean ± SEM. n.s., not significant, calculated by calculated by two-tailed Student's t-test.


C NHF , Lowe 1676 and Lowe 3265 cells were subjected to IF microscopy to visualize PI ( 4 , 5 ) P2 ( green ) , gamma - tubulin ( red ) and nuclei ( blue , DAPI staining ) . Scale bar , 10 μm . D The fluorescence intensity of PI ( 4 , 5 ) P2 on centrosomes in C was measured . n = 3 per group in which 50 cells were counted per condition per experiment . D Data information : data are presented as mean ± SEM . n . s . , not significant , calculated by calculated by two - tailed Student ' s t - test .

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

C NHF, Lowe 1676 and Lowe 3265 cells were subjected to IF microscopy to visualize PI(4,5)P2 (green), gamma-tubulin (red) and nuclei (blue, DAPI staining). Scale bar, 10 μm. D The fluorescence intensity of PI (4,5)P2 on centrosomes in C was measured. n = 3 per group in which 50 cells were counted per condition per experiment. D Data information: data are presented as mean ± SEM. n.s., not significant, calculated by calculated by two-tailed Student's t-test.


A SSX2IP interacts with OCRL . Plasmids carrying GFP - OCRL - WT , GFP - OCRL - △ RhoGAP and GFP - OCRL - △ ASH + RhoGAP were transfected into RPE - 1 cells . Immunoprecipitation was performed with GFP - Trap , followed by blotting with the indicated antibodies .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 SSX2IP interacts with OCRL. Plasmids carrying GFP-OCRL-WT, GFP-OCRL-△RhoGAP and GFP-OCRL-△ASH+RhoGAP were transfected into RPE-1 cells. Immunoprecipitation was performed with GFP-Trap, followed by blotting with the indicated antibodies.


D NHF , Lowe 1676 and Lowe 3265 cells were transfected with wildtype GFP - SSX2IP or GFP - PACT - SSX2IP for 48 h , then immunostained with anti - β - tubulin ( red ) antibody . Nuclei ( blue ) were stained with DAPI . The centrosomal microtubule nucleation in GFP - PACT - SSX2IP transfected cells was indicated by white arrowhead . Scale bar , 5 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 NHF, Lowe 1676 and Lowe 3265 cells were transfected with wildtype GFP-SSX2IP or GFP-PACT-SSX2IP for 48 h, then immunostained with anti-β-tubulin (red) antibody. Nuclei (blue) were stained with DAPI. The centrosomal microtubule nucleation in GFP-PACT-SSX2IP transfected cells was indicated by white arrowhead. Scale bar, 5 μm.


F Western blot analysis of GFP , endogenous phospho - mTOR , total mTOR , in NHF and Lowe 1676 cells . NHF and Lowe 1676 cells were left untransfected , transfected with GFP - SSX2IP - WT or GFP - PACT - SSX2IP , then immunoblotted with indicated antibodies .

O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Western blot analysis of GFP, endogenous phospho-mTOR, total mTOR, in NHF and Lowe 1676 cells. NHF and Lowe 1676 cells were left untransfected, transfected with GFP-SSX2IP-WT or GFP-PACT-SSX2IP, then immunoblotted with indicated antibodies.


( A ) HeLa cells expressing GFP ‐ tagged UBQLN1 or UBQLN2 ( or GFP for controls ) were transferred to starvation medium ( PBS ) for the indicated time . Where specified , only serum was removed from the culture medium . Viability of transfected cells was assessed by PI staining and analysed by flow cytometry . The graph represents the mean of three experiments ± s . 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

(A) HeLa cells expressing GFP‐tagged UBQLN1 or UBQLN2 (or GFP for controls) were transferred to starvation medium (PBS) for the indicated time. Where specified, only serum was removed from the culture medium. Viability of transfected cells was assessed by PI staining and analysed by flow cytometry. The graph represents the mean of three experiments±s.e.


( B ) HeLa cells were depleted of UBQLN1 or UBQLN2 by siRNA transfection ; depletion was verified by Western blot . Cells were starved for increasing periods of time , and their viability was analysed as in ( A ) . The graph represents the mean of three experiments ± s . e ; * P0 . 05 between control and UBQLN2 ‐ KD at 6 h ( paired Student ' s t ‐ test ) . GAPDH , glyceraldehyde ‐ 3 ‐ phosphate dehydrogenase ; GFP , green fluorescent protein ; KD , knockdown ; PI , propidium iodide ; siRNA , short interfering RNA ; UBQLN , ubiquilin .

(B) HeLa cells were depleted of UBQLN1 or UBQLN2 by siRNA transfection; depletion was verified by Western blot. Cells were starved for increasing periods of time, and their viability was analysed as in (A). The graph represents the mean of three experiments±s.e; *P0.05 between control and UBQLN2‐KD at 6 h (paired Student's t‐test). GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase; GFP, green fluorescent protein; KD, knockdown; PI, propidium iodide; siRNA, short interfering RNA; UBQLN, ubiquilin.


( A ) HeLa cells were transfected with siRNAs for autophagy - related gene ATG5 ( siATG5 ) or with a non - silencing siRNA ( siC ) . Depletion of ATG5 , which decreased the levels of the ATG5 - ATG12 covalent complex , was confirmed by Western blot . Loading control : transferrin receptor ( TfR ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HeLa cells were transfected with siRNAs for autophagy-related gene ATG5 (siATG5) or with a non-silencing siRNA (siC). Depletion of ATG5, which decreased the levels of the ATG5-ATG12 covalent complex, was confirmed by Western blot. Loading control: transferrin receptor (TfR).


( B ) Control or ATG5 ‐ KD cells transfected with UBQLN1 were starved , and the cell viability was assessed as in Fig 1 ; the graph represents the mean of three experiments ± s . 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

(B) Control or ATG5‐KD cells transfected with UBQLN1 were starved, and the cell viability was assessed as in Fig 1; the graph represents the mean of three experiments±s.e.


( C ) HeLa cells were stably transfected with control ( shC ) or ATG7 ( shATG7 ) shRNA ; depletion was verified by Western blot ( loading control : GAPDH ) .

O O O O O O O O O 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) HeLa cells were stably transfected with control (shC) or ATG7 (shATG7) shRNA; depletion was verified by Western blot (loading control: GAPDH).


( D ) Control or ATG7 ‐ KD cells transfected with UBQLN1 were starved for 3 or 6 h and assessed for their viability . The graph represents the mean of three experiments ± s . e . GAPDH , glyceraldehyde ‐ 3 ‐ phosphate dehydrogenase ; KD , knockdown ; Pl , propidium iodide ; shRNA , short hairpin RNA ; siRNA , short interfering RNA ; UBQLN , ubiquilin .

(D) Control or ATG7‐KD cells transfected with UBQLN1 were starved for 3 or 6 h and assessed for their viability. The graph represents the mean of three experiments±s.e. GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase; KD, knockdown; Pl, propidium iodide; shRNA, short hairpin RNA; siRNA, short interfering RNA; UBQLN, ubiquilin.


( A ) Immunolocalization of transfected Myc ‐ UBQLN ( 1 or 2 ) and GFP ‐ tagged microtubule ‐ associated protein 1 light chain 3 ( GFP ‐ LC3 ) was analysed by microscopy . Inset : magnification of UBQLN1 vesicular staining ( arrow ) , which , after acquisition of Z ‐ stacks , was found in all UBQLN structures analysed .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunolocalization of transfected Myc‐UBQLN (1 or 2) and GFP‐tagged microtubule‐associated protein 1 light chain 3 (GFP‐LC3) was analysed by microscopy. Inset: magnification of UBQLN1 vesicular staining (arrow), which, after acquisition of Z‐stacks, was found in all UBQLN structures analysed.


( B ) Cells were starved for 2 h , with ( top row ) or without BAF A1 ( bottom row ) . Cells were stained for endogenous UBQLN2 ( magenta ) and LC3 ( green ) .

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

(B) Cells were starved for 2 h, with (top row) or without BAF A1 (bottom row). Cells were stained for endogenous UBQLN2 (magenta) and LC3 (green).


( C ) Control cells ( untransfected , right panel ) or cells overexpressing Myc ‐ UBQLN2 and GFP ‐ LC3 ( left panel ) were transferred into starvation medium for 2 h . Localization of Myc ‐ UBQLN2 and GFP ‐ LC3 was analysed by immunoelectron microscopy using Myc ( arrow ) and GFP ( arrowhead ) antibodies coupled to 10 and 15 nm gold particles , respectively . Double ‐ membrane vacuoles ( autophagosomes ) in transfected cells ( left panel ) show increased staining by both antibodies compared with autophagosomes in untransfected cells ( right panel ) . Scale bar , 0 . 5 μm . BAF A , bafilomycin A1 ; GFP , green fluorescent protein ; UBQLN , ubiquilin .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Control cells (untransfected, right panel) or cells overexpressing Myc‐UBQLN2 and GFP‐LC3 (left panel) were transferred into starvation medium for 2 h. Localization of Myc‐UBQLN2 and GFP‐LC3 was analysed by immunoelectron microscopy using Myc (arrow) and GFP (arrowhead) antibodies coupled to 10 and 15 nm gold particles, respectively. Double‐membrane vacuoles (autophagosomes) in transfected cells (left panel) show increased staining by both antibodies compared with autophagosomes in untransfected cells (right panel). Scale bar, 0.5 μm. BAF A, bafilomycin A1; GFP, green fluorescent protein; UBQLN, ubiquilin.


( B ) Each mutant was co ‐ transfected with GFP ‐ tagged microtubule ‐ associated protein 1 light chain 3 ( GFP ‐ LC3 ) and the localization of each protein was analysed by microscopy .

O O O O O O O O O O O O O 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) Each mutant was co‐transfected with GFP‐tagged microtubule‐associated protein 1 light chain 3 (GFP‐LC3) and the localization of each protein was analysed by microscopy.


( C ) Starvation ‐ induced cell death was assessed in cells expressing UBQLN1 or UBQLN1ΔUBA . The graph represents the mean of three experiments ± s . e . , with * P ⩽ 0 . 05 ( P = 0 . 0017 by one ‐ way ANOVA test , comparing control , UBQLN1 and UBQLN1ΔUBA ) . No difference was found between control and UBQLN1ΔUBA at any time point . ANOVA , analysis of variance ; GFP , green fluorescent protein ; Pl , propidium iodide ; UBA , ubiquitin ‐ associated ; UBQLN , ubiquilin .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Starvation‐induced cell death was assessed in cells expressing UBQLN1 or UBQLN1ΔUBA. The graph represents the mean of three experiments±s.e., with *P⩽0.05 (P=0.0017 by one‐way ANOVA test, comparing control, UBQLN1 and UBQLN1ΔUBA). No difference was found between control and UBQLN1ΔUBA at any time point. ANOVA, analysis of variance; GFP, green fluorescent protein; Pl, propidium iodide; UBA, ubiquitin‐associated; UBQLN, ubiquilin.


( A ) Autophagosome subcellular localization during starvation was analysed by microscopy in UBQLN ‐ or autophagy ‐ related gene ATG5 ‐ KD cells expressing GFP ‐ tagged microtubule associated protein 1 light chain 3 ( GFP ‐ LC3 ) .

O O O O O O O O O O O O O O O O O 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) Autophagosome subcellular localization during starvation was analysed by microscopy in UBQLN‐ or autophagy‐related gene ATG5‐KD cells expressing GFP‐tagged microtubule associated protein 1 light chain 3 (GFP‐LC3).


( B ) Autophagosome spatial distribution was quantified by measuring the distance of each LC3 vesicle from the nucleus ( in pixels ) and by analysing the frequency distribution .

O O O O O O 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) Autophagosome spatial distribution was quantified by measuring the distance of each LC3 vesicle from the nucleus (in pixels) and by analysing the frequency distribution.


( C ) Control or UBQLN ‐ KD cells expressing GFP ‐ LC3 were starved and stained with the mitochondria dye Mitotracker ™ . Dual staining of LC3 ( green ) with Mitotracker ( magenta ) was analysed by microscopy .

(C) Control or UBQLN‐KD cells expressing GFP‐LC3 were starved and stained with the mitochondria dye Mitotracker™. Dual staining of LC3 (green) with Mitotracker (magenta) was analysed by microscopy.


( D ) Control and UBQLN ‐ KD cells stained with Mitotracker were starved for 6 h ; BAF A was added where indicated . Mitotracker fluorescence was measured by flow cytometry . Unless specified otherwise , values for UBQLN ‐ KD and control + BAF A were compared with that of controls at the same time points , with * * * P0 . 001 , * * P0 . 01 and * P0 . 05 ( paired Student ' s t ‐ test ) .

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

(D) Control and UBQLN‐KD cells stained with Mitotracker were starved for 6 h; BAF A was added where indicated. Mitotracker fluorescence was measured by flow cytometry. Unless specified otherwise, values for UBQLN‐KD and control+BAF A were compared with that of controls at the same time points, with ***P0.001, **P0.01 and *P0.05 (paired Student's t‐test).


( E ) Cells were transfected with pShooter ™ together with control or UBQLN ( 1 + 2 ) siRNA . Expression of pShooter was comparable in control and UBQLN ‐ KD cells ( top ) . Cells were starved for 3 or 6 h , and the intensity of GFP was quantified by flow cytometry ( bottom ) . The fluorescence value obtained with controls ( no starvation ) was considered as 100 % , and the value obtained for the 3 ‐ and 6 ‐ h time points was normalized to controls . The graph represents the mean ± s . e . of four assessments .

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

(E) Cells were transfected with pShooter™ together with control or UBQLN (1+2) siRNA. Expression of pShooter was comparable in control and UBQLN‐KD cells (top). Cells were starved for 3 or 6 h, and the intensity of GFP was quantified by flow cytometry (bottom). The fluorescence value obtained with controls (no starvation) was considered as 100%, and the value obtained for the 3‐ and 6‐h time points was normalized to controls. The graph represents the mean±s.e. of four assessments.


( F ) Cells expressing a chimaeric LC3 fused to both GFP and mCherry were transfected with control or UBQLN siRNA , and starvation was induced for the indicated times . Puncta from 25 cells were scored and analysed by microscopy for their fluorescence colour . The graph represents the mean of three experiments ± s . e . The number of mCherry single ‐ positive puncta in UBQLN ‐ KD cells was compared with that of controls for each time point ; * * P0 . 01 and * P0 . 05 ( paired Student ' s 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

(F) Cells expressing a chimaeric LC3 fused to both GFP and mCherry were transfected with control or UBQLN siRNA, and starvation was induced for the indicated times. Puncta from 25 cells were scored and analysed by microscopy for their fluorescence colour. The graph represents the mean of three experiments±s.e. The number of mCherry single‐positive puncta in UBQLN‐KD cells was compared with that of controls for each time point; **P0.01 and *P0.05 (paired Student's t‐test).


( G ) Control or UBQLN ‐ KD cells were starved for increasing periods . Where indicated , BAF A was added to the starvation medium ( + B ) . Cell lysates were analysed by Western blot for their content in LC3 ‐ II . Loading control : transferrin receptor ( TfR ) . BAF A , bafilomycin A1 ; GFP , green fluorescent protein ; KD , knockdown ; siRNA , short interfering RNA ; UBQLN , ubiquilin .

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 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 O O

(G) Control or UBQLN‐KD cells were starved for increasing periods. Where indicated, BAF A was added to the starvation medium (+B). Cell lysates were analysed by Western blot for their content in LC3‐II. Loading control: transferrin receptor (TfR). BAF A, bafilomycin A1; GFP, green fluorescent protein; KD, knockdown; siRNA, short interfering RNA; UBQLN, ubiquilin.


( A , B ) BMDCs were infected with Pru parasites ( at a MOI of 4 parasites / BMDC ) or treated with tunicamycin ( TN ) for 16h and the expression of UPR target genes were analyzed by RT - qPCR . The results are expressed as ( Log2 ) Fold change ( A ) or relative mRNA level ( B ) compared to non - infected ( NI ) conditions . IRE1α / XBP1s pathway : Ern1 ( IRE1α ) , Xbp1s , Dnajb9 ( ERdj4 ) , Sec24D , Bloc1s1 and Sec61a1 ; PERK / ATF4 pathway : Atf4 , Ddit3 ( CHOP ) and Ppp1r15a ( GADD34 ) ; ATF6 pathway : Atf6 and Herpud1 ; classical ER chaperons : Hspa5 ( BIP ) and Hsp90b1 ( GRP94 ) . Results are normalized to the housekeeping gene Gapdh . Unpaired Student T - test , ns : p > 0 . 05 ; * * : p < 0 . 01 ; * * * : p < 0 . 001 ; mean ± S . E . M ( n = 2 - 5 independent experiments depending of the studied gene , 3 mice / independent experiment ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) BMDCs were infected with Pru parasites (at a MOI of 4 parasites/BMDC) or treated with tunicamycin (TN) for 16h and the expression of UPR target genes were analyzed by RT-qPCR. The results are expressed as (Log2) Fold change (A) or relative mRNA level (B) compared to non-infected (NI) conditions. IRE1α/XBP1s pathway: Ern1 (IRE1α), Xbp1s, Dnajb9 (ERdj4), Sec24D, Bloc1s1 and Sec61a1; PERK/ATF4 pathway: Atf4, Ddit3 (CHOP) and Ppp1r15a (GADD34); ATF6 pathway: Atf6 and Herpud1; classical ER chaperons: Hspa5 (BIP) and Hsp90b1 (GRP94). Results are normalized to the housekeeping gene Gapdh. Unpaired Student T-test, ns: p>0.05; **: p<0.01; ***: p<0.001; mean ± S.E.M (n=2-5 independent experiments depending of the studied gene, 3 mice / independent experiment).


( C , D ) Immunoblot analysis of IRE1α , PERK , Phospho - PERK ( detected by the presence of a second upper band after migration on a 6 % acrylamide gel ) , Phospho - eIF2α , CHOP and XBP1s protein expression in non - infected BMDCs , BMDCs treated with TN and Pru infected BMDCs ( C ) and in non - infected and Pru infected Flt3 / Notch - DCs ( D ) for the indicated times . GAPDH and tubulin were used as loading controls . The same blots are shown in Figure EV3B - C .

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

(C, D) Immunoblot analysis of IRE1α, PERK, Phospho-PERK (detected by the presence of a second upper band after migration on a 6% acrylamide gel), Phospho-eIF2α, CHOP and XBP1s protein expression in non-infected BMDCs, BMDCs treated with TN and Pru infected BMDCs (C) and in non-infected and Pru infected Flt3/Notch-DCs (D) for the indicated times. GAPDH and tubulin were used as loading controls. The same blots are shown in Figure EV3B-C.


( D ) mRNA relative expression level of Xbp1s , Dnajb9 ( ERdj4 ) , Ddit3 ( CHOP ) and Ppp1r15a ( GADD34 ) from WT and MyD88KO BMDCs infected with Pru parasites for 16h compared to non - infected ( NI ) conditions . Results are normalized to the housekeeping gene Gapdh . Two - way ANOVA Tukey ' s multiple comparisons , ns : p > 0 . 05 ; * : p < 0 . 05 ; * * : p < 0 . 01 ; * * * : p < 0 . 001 , * * * * : p < 0 . 0001 ; mean ± S . E . M ( n = 4 - 5 mice pooled from 2 independent experiments ) .

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

(D) mRNA relative expression level of Xbp1s, Dnajb9 (ERdj4), Ddit3 (CHOP) and Ppp1r15a (GADD34) from WT and MyD88KO BMDCs infected with Pru parasites for 16h compared to non-infected (NI) conditions. Results are normalized to the housekeeping gene Gapdh. Two-way ANOVA Tukey's multiple comparisons, ns: p>0.05; *: p<0.05; **: p<0.01; ***: p<0.001, ****: p<0.0001; mean ± S.E.M (n=4-5 mice pooled from 2 independent experiments).


( A ) BMDCs ( CD11c + ) were incubated with Pru - Tomato parasites for 16h . Infected cells ( red gating ) and non - infected bystander cells ( green gating ) were sorted - purified and further analyzed . ( B ) The expression of UPR target genes were analyzed by RT - qPCR in infected and non - infected bystander cells compared to non - infected BMDCs ( NI ) . The results are expressed as relative mRNA level compared to non - infected conditions . Results are normalized to the housekeeping gene Gapdh . Unpaired Student T - test , ns : p > 0 . 05 ; * : p < 0 . 05 ; * * : p < 0 . 01 ; * * * : p < 0 . 001 ; mean ± S . E . M ( n = 6 mice pooled from 2 independent experiments ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) BMDCs (CD11c+) were incubated with Pru-Tomato parasites for 16h. Infected cells (red gating) and non-infected bystander cells (green gating) were sorted-purified and further analyzed. (B) The expression of UPR target genes were analyzed by RT-qPCR in infected and non-infected bystander cells compared to non-infected BMDCs (NI). The results are expressed as relative mRNA level compared to non-infected conditions. Results are normalized to the housekeeping gene Gapdh. Unpaired Student T-test, ns: p>0.05; *: p<0.05; **: p<0.01; ***: p<0.001; mean ± S.E.M (n=6 mice pooled from 2 independent experiments).


( E ) mRNA relative expression level of Il - 6 , Il - 12 and Il - 23 from XBP1fl / flIRE1fl / fl , XBP1ΔDC IRE1truncDC and XBP1ΔDC BMDCs infected with Pru parasites for 16 h compared to non - infected ( NI ) conditions . Results are normalized to the housekeeping gene Gapdh . Two - way ANOVA Tukey ' s multiple comparisons , ns : p > 0 . 05 ; * : p < 0 . 05 ; * * : p < 0 . 01 ; * * * : p < 0 . 001 ; * * * * : p < 0 . 0001 ; mean ± S . E . M ( n = 3 - 5 mice pooled from 2 independent experiments ) .

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

(E) mRNA relative expression level of Il-6, Il-12 and Il-23 from XBP1fl/flIRE1fl/fl, XBP1ΔDC IRE1truncDC and XBP1ΔDC BMDCs infected with Pru parasites for 16 h compared to non-infected (NI) conditions. Results are normalized to the housekeeping gene Gapdh. Two-way ANOVA Tukey's multiple comparisons, ns: p>0.05; *: p<0.05; **: p<0.01; ***: p<0.001; ****: p<0.0001; mean ± S.E.M (n=3-5 mice pooled from 2 independent experiments).


( I ) Graph depicts the expression ( indicated as the Mean Fluorescence Intensity ) of H - 2Kb by Pru Tomato SAG1 ‐ OVA infected XBP1fl / fl IRE1fl / fl treated or not with 4μ8C , XBP1ΔDC IRE1truncDC and XBP1ΔDC BMDCs . Bar graphs depict mean ± S . E . M ( n = 3 mice , one representative experiment from 2 independent experiments ) . Tukey ' s ANOVA one - way test , ns : p > 0 . 05 .

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

(I) Graph depicts the expression (indicated as the Mean Fluorescence Intensity) of H-2Kb by Pru Tomato SAG1‐OVA infected XBP1fl/fl IRE1fl/fl treated or not with 4μ8C, XBP1ΔDC IRE1truncDC and XBP1ΔDC BMDCs. Bar graphs depict mean ± S.E.M (n=3 mice, one representative experiment from 2 independent experiments). Tukey's ANOVA one-way test, ns: p>0.05.


( A , B ) Expression of VenusFP by splenic cDC1 ( CD26 + CD11c + MHCII + XCR1 + CD172a - , upper panels ) and cDC2 ( CD26 + CD11c + MHCII + XCR1 - CD172a + , lower panel ) from WT ( A ) and ERAI ( B ) mice non - infected ( NI ) or infected with Pru ( 6 days post - infection ( dpi ) ) reporting on IRE1α pathway activation .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Expression of VenusFP by splenic cDC1 (CD26+ CD11c+ MHCII+ XCR1+ CD172a-, upper panels) and cDC2 (CD26+ CD11c+ MHCII+ XCR1- CD172a+, lower panel) from WT (A) and ERAI (B) mice non-infected (NI) or infected with Pru (6 days post-infection (dpi)) reporting on IRE1α pathway activation.


( E , F ) RT - qPCR analysis of the indicated UPR genes among total RNA prepared from splenic cDC1 ( E ) and cDC2 ( F ) sorted by flow cytometry 6 dpi from wild - type mice , non - infected ( NI ) or infected with Pru parasites . Results were normalized to the housekeeping gene Gapdh and are expressed as relative mRNA level compared to the NI condition . Bar graphs depict mean ± S . E . M . Unpaired Student T - test , ns : p > 0 . 05 ; * : p < 0 . 05 ; * * : p < 0 . 01 ; mean ± S . E . M ( n = 3 - 5 mice pooled from 2 independent experiments ) .

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

(E, F) RT-qPCR analysis of the indicated UPR genes among total RNA prepared from splenic cDC1 (E) and cDC2 (F) sorted by flow cytometry 6 dpi from wild-type mice, non-infected (NI) or infected with Pru parasites. Results were normalized to the housekeeping gene Gapdh and are expressed as relative mRNA level compared to the NI condition. Bar graphs depict mean ± S.E.M. Unpaired Student T-test, ns: p>0.05; *: p<0.05; **: p<0.01; mean ± S.E.M (n=3-5 mice pooled from 2 independent experiments).


( A ) Survival curves of XBP1fl / flIRE1fl / fl and XBP1ΔDC IRE1truncDC mice infected intraperitoneally with 500 Pru parasites . Log - rank ( Mantel - Cox ) test , p = 0 , 0004 ; n = 10 mice per genotype .

O O O O O O O O O O O O O O O O O O O O O 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) Survival curves of XBP1fl/flIRE1fl/fl and XBP1ΔDC IRE1truncDC mice infected intraperitoneally with 500 Pru parasites. Log-rank (Mantel-Cox) test, p=0,0004; n=10 mice per genotype.


( D , E ) Quantification of the number of total ( left histogram ) and activated ( right histogram ) ( IFNγ - positive ) CD4 + T cells ( D ) and CD8 + T cells ( E ) in the spleen of control mice ( XBP1fl / flIRE1fl / fl ) and XBP1ΔDC IRE1truncDC infected for 6 days . Two - way ANOVA Tukey ' s multiple compare , ns : p > 0 . 05 ; * : p < 0 . 05 ; * * : p < 0 . 01 ; * * * : p < 0 . 001 ; * * * * : p < 0 . 0001 ; mean ± S . E . M ( n = 2 independent experiments combined ) .

(D, E) Quantification of the number of total (left histogram) and activated (right histogram) (IFNγ-positive) CD4+ T cells (D) and CD8+ T cells (E) in the spleen of control mice (XBP1fl/flIRE1fl/fl) and XBP1ΔDC IRE1truncDC infected for 6 days. Two-way ANOVA Tukey's multiple compare, ns: p> 0.05; *: p <0.05; **: p <0.01; ***: p <0.001; ****: p <0.0001; mean ± S.E.M (n=2 independent experiments combined).