IIA1 . 6 B lymphoma cells were stimulated with BCR - ligand - ( IgM ) or BCR - ligand + ( IgG ) beads for 60 min , fixed and stained for F - actin ( top ) and α - tubulin ( bottom ) . Scale bar : 3 µm .

B-CELL_LINE I-CELL_LINE I-CELL_LINE B-DISEASE I-DISEASE O O O O B-SUBCELLULAR O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O B-EXP_ASSAY O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O

IIA1.6 B lymphoma cells were stimulated with BCR-ligand- (IgM) or BCR-ligand+ (IgG) beads for 60 min, fixed and stained for F-actin (top) and α-tubulin (bottom). Scale bar: 3 µm.


Histograms show the quantifications of the polymerized tubulin and filamentous F - actin at the centrosome ( dashed outline on the image , values correspond to the fraction of fluorescence in a 2 - micron - wide area around the centrosome relative to the total fluorescence in the cell ) and the total amount of polymerized fluorescent tubulin ( bottom right , values were normalized with respect to the mean of control condition ) . Measurements were pooled from 3 independent experiments ; IgM ( BCR - ligand - ) : n = 88 ; IgG ( BCR - ligand + ) : n = 93 . Error bars correspond to standard deviations . P values were calculated with Mann - Whitney test . Scale bar : 2 µm .

O O O O O O O B-GENEPROD O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O B-SUBCELLULAR 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-SUBCELLULAR O O O O O O O O O O O O O O O B-EXP_ASSAY B-GENEPROD O O O 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-SUBCELLULAR O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Histograms show the quantifications of the polymerized tubulin and filamentous F-actin at the centrosome (dashed outline on the image, values correspond to the fraction of fluorescence in a 2-micron-wide area around the centrosome relative to the total fluorescence in the cell) and the total amount of polymerized fluorescent tubulin (bottom right, values were normalized with respect to the mean of control condition). Measurements were pooled from 3 independent experiments; IgM (BCR-ligand-): n= 88; IgG (BCR-ligand+): n= 93. Error bars correspond to standard deviations. P values were calculated with Mann-Whitney test. Scale bar: 2 µm.


Percentage differences of centrosomal F - actin and centrosomal microtubule fluorescence intensities in cells stimulated with BCR - ligand + beads with respect to cells stimulated with BCR - ligand - beads . The data set is identical to panel ( B ) . Measurements were pooled from 3 independent experiments ; IgM ( BCR - ligand - ) : n = 88 ; IgG ( BCR - ligand + ) : n = 93 . Error bars correspond to standard deviations . P values were calculated with one - sample t - test ( i . e . comparison to a theoretical mean of " 0 " ) .

O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR B-EXP_ASSAY I-EXP_ASSAY O O O O B-SUBCELLULAR O O O O O O O O O O B-SUBCELLULAR O O 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-SUBCELLULAR O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Percentage differences of centrosomal F-actin and centrosomal microtubule fluorescence intensities in cells stimulated with BCR-ligand+ beads with respect to cells stimulated with BCR-ligand- beads. The data set is identical to panel (B). Measurements were pooled from 3 independent experiments; IgM (BCR-ligand-): n= 88; IgG (BCR-ligand+): n= 93. Error bars correspond to standard deviations. P values were calculated with one-sample t-test (i.e. comparison to a theoretical mean of "0").


The graph shows the variations of the total amount of tubulin per cell with respect to the content of actin at the centrosome in an XY representation of individual measurements . The two lines correspond to linear regressions of the two sets of data relative to cells stimulated with BCR - ligand + ( activated cells ) or BCR - ligand - ( resting cells ) beads .

O O O O O O O O O O B-GENEPROD O O O O O O O O B-SUBCELLULAR O O B-SUBCELLULAR O O 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-SUBCELLULAR O O O O O O O O B-SUBCELLULAR O O O O O O O O O

The graph shows the variations of the total amount of tubulin per cell with respect to the content of actin at the centrosome in an XY representation of individual measurements. The two lines correspond to linear regressions of the two sets of data relative to cells stimulated with BCR-ligand+ (activated cells) or BCR-ligand- (resting cells) beads.


IIA1 . 6 B lymphoma cells were fixed and immuno - stained for F - actin ( red ) and microtubules ( green ) . Images show the projection of maximal intensity of three confocal slices spaced by 0 . 5 µm apart from the centrosome . Scale bars : 2 µm ( 0 . 5 µm in the zoomed insets ) .

B-CELL_LINE I-CELL_LINE I-CELL_LINE B-DISEASE I-DISEASE O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O B-SUBCELLULAR O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O

IIA1.6 B lymphoma cells were fixed and immuno-stained for F-actin (red) and microtubules (green). Images show the projection of maximal intensity of three confocal slices spaced by 0.5 µm apart from the centrosome. Scale bars: 2 µm (0.5 µm in the zoomed insets).


IIA1 . 6 B lymphoma cells were treated 45 min with indicated inhibitors ( CK666 at 25 µM , SMIFH2 at 25µM ) or DMSO as control prior to being fixed and stained for α - tubulin ( left column ) and F - actin ( right column ) . Scale bar : 3 µm .

B-CELL_LINE I-CELL_LINE I-CELL_LINE B-DISEASE I-DISEASE O O O O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O

IIA1.6 B lymphoma cells were treated 45 min with indicated inhibitors (CK666 at 25 µM, SMIFH2 at 25µM) or DMSO as control prior to being fixed and stained for α-tubulin (left column) and F-actin (right column). Scale bar: 3 µm.


Histograms show the quantifications of the amount of polymerized fluorescent tubulin ( right , values were normalized with respect to the mean of control condition ) and filamentous actin at the centrosome ( left , values correspond to the fraction of fluorescence in a 2 - micron - wide area around the centrosome relative to the total fluorescence in the cell ) . Measurements were pooled from 3 independent experiments ; DMSO : n = 91 , CK666 : n = 82 , SMIFH2 : n = 74 , latrunculinA : n = 96 . Error bars correspond to standard deviations . P values were calculated with Mann - Whitney test .

O O O O O O O O O B-EXP_ASSAY B-GENEPROD O O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O

Histograms show the quantifications of the amount of polymerized fluorescent tubulin (right, values were normalized with respect to the mean of control condition) and filamentous actin at the centrosome (left, values correspond to the fraction of fluorescence in a 2-micron-wide area around the centrosome relative to the total fluorescence in the cell). Measurements were pooled from 3 independent experiments; DMSO: n= 91, CK666: n= 82, SMIFH2: n= 74, latrunculinA: n= 96. Error bars correspond to standard deviations. P values were calculated with Mann-Whitney test.


Percentage differences of centrosomal F - actin and microtubule fluorescence intensities in cells treated with cytoskeleton inhibitors in comparison with the respective densities in cells treated with DMSO . Errors bars represent standard deviations . P values were calculated with one - sample t - test ( i . e . comparison to a theoretical mean of " 0 " ) .

O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR B-EXP_ASSAY I-EXP_ASSAY O O O O B-SUBCELLULAR O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Percentage differences of centrosomal F-actin and microtubule fluorescence intensities in cells treated with cytoskeleton inhibitors in comparison with the respective densities in cells treated with DMSO. Errors bars represent standard deviations. P values were calculated with one-sample t-test (i.e. comparison to a theoretical mean of "0").


The graph shows the same measurements as in panel ( B ) in an XY representation of individual measurements . The three lines correspond to linear regressions of the three sets of data relative to cells stimulated with each actin drug .

O O O O O O O O O O O O O O O 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-SUBCELLULAR O O

The graph shows the same measurements as in panel (B) in an XY representation of individual measurements. The three lines correspond to linear regressions of the three sets of data relative to cells stimulated with each actin drug.


IIA1 . 6 B lymphoma cells were transfected to transiently express centrin1 - GFP ( red ) and EB3 - mCherry ( green ) and video - recorded at the contact site with the glass coverslip ( left ) and at the centrosome ( right ) . Scale bar : 3 µm .

B-CELL_LINE I-CELL_LINE I-CELL_LINE B-DISEASE I-DISEASE O O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O

IIA1.6 B lymphoma cells were transfected to transiently express centrin1-GFP (red) and EB3-mCherry (green) and video-recorded at the contact site with the glass coverslip (left) and at the centrosome (right). Scale bar: 3 µm.


The duration of EB3 - positive comets presence in the bottom plane was measured in DMSO and CK666 treated cells ( left ) . Error bars correspond to standard deviations . The number of EB3 - positive comets exiting the a 2 - µm wide centrosomal area was also compared between the two conditions ( right ) . In both cases P values were calculated with Mann - Whitney test .

O O O B-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O

The duration of EB3-positive comets presence in the bottom plane was measured in DMSO and CK666 treated cells (left). Error bars correspond to standard deviations. The number of EB3-positive comets exiting the a 2-µm wide centrosomal area was also compared between the two conditions (right). In both cases P values were calculated with Mann-Whitney test.


IIA1 . 6 B lymphoma cells were transfected to transiently express centrin1 - VCA - GFP ( bottom ) or centrin1 - GFP ( top ) as control prior to be fixed and stained for α - tubulin ( left column ) and F - actin ( middle column ) . The GFP signal of centrin1 or centrin1 - VCA is shown in the right column to illustrate the proper centrosome targeting . Scale bar : 3 µm .

B-CELL_LINE I-CELL_LINE I-CELL_LINE B-DISEASE I-DISEASE O O O O O O B-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O

IIA1.6 B lymphoma cells were transfected to transiently express centrin1-VCA-GFP (bottom) or centrin1-GFP (top) as control prior to be fixed and stained for α-tubulin (left column) and F-actin (middle column). The GFP signal of centrin1 or centrin1-VCA is shown in the right column to illustrate the proper centrosome targeting. Scale bar: 3 µm.


Histograms show the quantifications of the amount of polymerized fluorescent tubulin ( right ) and filamentous actin at the centrosome ( left ) . Values correspond to the fraction of fluorescence in a 2 - micron - wide area around the centrosome relative to the total fluorescence in the cell . Measurements were pooled from 3 independent experiments ; centrin1 - GFP : n = 88 , centrin1 - VCA - GFP : n = 87 . Errors bars represent standard deviations . P values were calculated with Mann - Whitney test .

O O O O O O O O O B-EXP_ASSAY B-GENEPROD O O O O B-SUBCELLULAR I-SUBCELLULAR O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O

Histograms show the quantifications of the amount of polymerized fluorescent tubulin (right) and filamentous actin at the centrosome (left). Values correspond to the fraction of fluorescence in a 2-micron-wide area around the centrosome relative to the total fluorescence in the cell. Measurements were pooled from 3 independent experiments; centrin1-GFP: n= 88, centrin1-VCA-GFP: n= 87. Errors bars represent standard deviations. P values were calculated with Mann-Whitney test.


Percentage differences of F - actin and microtubule fluorescence intensities at the centrosome were compared in cells transfected either with centrin1 - VCA - GFP or with centrin1 - GFP . Errors bars represent standard deviations . P values were calculated with one - sample t - test ( i . e . comparison to a theoretical mean of " 0 " ) .

O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR B-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR O O O O O O O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Percentage differences of F-actin and microtubule fluorescence intensities at the centrosome were compared in cells transfected either with centrin1-VCA-GFP or with centrin1-GFP. Errors bars represent standard deviations. P values were calculated with one-sample t-test (i.e. comparison to a theoretical mean of "0").


The graph shows the variations of the total amount of tubulin per cell with respect to the content of actin at the centrosome . The two lines correspond to linear regressions of the two sets of data relative to cells transfected with centrin1 - VCA - GFP or centrin1 - GFP .

O O O O O O O O O O B-GENEPROD O O O O O O O O B-SUBCELLULAR O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O

The graph shows the variations of the total amount of tubulin per cell with respect to the content of actin at the centrosome. The two lines correspond to linear regressions of the two sets of data relative to cells transfected with centrin1-VCA-GFP or centrin1-GFP.


Two sets of representative images showing fluorescent microtubules and actin filaments assembled from isolated centrosomes . Centrosomes were isolated from Jurkat cells expressing centrin1 - GFP . Upper and lower lines show actin filaments and microtubules radiating from two distinct centrosomes with low ( top ) and high ( bottom ) densities of actin filaments . Scale bars : 10 μm .

O O O O O O B-EXP_ASSAY B-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR O O O B-SUBCELLULAR O B-SUBCELLULAR O O O B-CELL_LINE O O B-GENEPROD O B-GENEPROD I-GENEPROD O O O O O B-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O

Two sets of representative images showing fluorescent microtubules and actin filaments assembled from isolated centrosomes. Centrosomes were isolated from Jurkat cells expressing centrin1-GFP. Upper and lower lines show actin filaments and microtubules radiating from two distinct centrosomes with low (top) and high (bottom) densities of actin filaments. Scale bars: 10 μm.


The graph shows the number of microtubules per centrosome relative to the density of actin filaments . Inset shows actin filaments at the centrosome with a FIRE look - up table and a 20 µm - wide circle in which actin fluorescence intensity is measured . Measurements were pooled from 5 independent experiments ; n = 50 .

O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O B-SUBCELLULAR I-SUBCELLULAR O O B-SUBCELLULAR O O O O O O O O O O O O O O O O B-SUBCELLULAR B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O

The graph shows the number of microtubules per centrosome relative to the density of actin filaments. Inset shows actin filaments at the centrosome with a FIRE look-up table and a 20 µm-wide circle in which actin fluorescence intensity is measured. Measurements were pooled from 5 independent experiments; n= 50.


Microtubules ( top line ) and actin filaments ( bottom line ) assembly from isolated centrosomes in the presence of increasing concentration of monomeric actin ( from left to right ) . Scale bar : 20 μm .

B-SUBCELLULAR O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O B-SUBCELLULAR O O O O O O O O B-GENEPROD O O O O O O O O O O O O O

Microtubules (top line) and actin filaments (bottom line) assembly from isolated centrosomes in the presence of increasing concentration of monomeric actin (from left to right). Scale bar: 20 μm.


The graph shows the number of microtubules per centrosome in response to increasing concentrations of monomeric actin . Data were pooled from 2 independent experiments ; 0 µM : n = 21 ; 0 . 3 µM : n = 17 ; 0 . 5 µM : n = 17 ; 1 . 0 µM : n = 17 . * * * * : P < 0 . 001 Mann - Whitney test .

O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

The graph shows the number of microtubules per centrosome in response to increasing concentrations of monomeric actin. Data were pooled from 2 independent experiments; 0 µM: n=21; 0.3 µM: n=17; 0.5 µM: n=17; 1.0 µM: n=17. ****:P<0.001 Mann-Whitney test.


The image shows the density of actin filaments ( in the presence of 1µM of actin monomers ) at the centrosome color - coded with the FIRE look - up table and the definition of central , peripheral and distal regions corresponding to decreasing concentrations of actin filaments . Scale bar : 20 μm . The graph shows the measurements of microtubule growth rate in each region . Data were pooled from 3 independent experiments : central zone : n = 58 , peripheral : n = 104 , outside : n = 61 ; n . s . means no statistical difference between the data set according to Mann - Whitney test .

O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O B-GENEPROD O O O O B-SUBCELLULAR O 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-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O B-SUBCELLULAR B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

The image shows the density of actin filaments (in the presence of 1µM of actin monomers) at the centrosome color-coded with the FIRE look-up table and the definition of central, peripheral and distal regions corresponding to decreasing concentrations of actin filaments. Scale bar: 20 μm. The graph shows the measurements of microtubule growth rate in each region. Data were pooled from 3 independent experiments: central zone: n=58, peripheral: n=104, outside: n=61; n. s. means no statistical difference between the data set according to Mann-Whitney test.


The graphs show the various intensities of centrosome immuno - staining with antibodies against gamma - tubulin on the same coverslip depending on the presence / absence of actin filaments ( left ) or on the amount of actin filaments ( right ) . Data were pooled from 3 independent experiments . Left graph : without actin n = 69 , with actin n = 26 , right graph n = 26 . N . s . means no statistical difference between the data set according to Mann - Whitney test .

O O O O O O O B-SUBCELLULAR B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

The graphs show the various intensities of centrosome immuno-staining with antibodies against gamma-tubulin on the same coverslip depending on the presence/absence of actin filaments (left) or on the amount of actin filaments (right). Data were pooled from 3 independent experiments. Left graph: without actin n=69, with actin n=26, right graph n=26. N.s. means no statistical difference between the data set according to Mann-Whitney test.


Schematic illustration of the first dynamic assay : sequential addition of tubulin followed by tubulin and actin on isolated centrosomes . Representative images showing microtubules ( top line ) and the merged images of actin and microtubules ( bottom line ) for the two steps of the assay ; in the presence of tubulin only ( left column ) and in the presence of tubulin and actin ( right column ) . Scale bar : 10 µm . Quantification of the differences in the number of microtubules per centrosome between the two stages of the experiment described above on centrosomes capable ( first condition ) , or not ( second condition ) , to grow actin filaments . Data were collected from a single experiment ; asters without actin filaments : n = 29 ; asters with actin filaments : n = 13 . Data were analyzed using Mann - Whitney test .

O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O

Schematic illustration of the first dynamic assay: sequential addition of tubulin followed by tubulin and actin on isolated centrosomes. Representative images showing microtubules (top line) and the merged images of actin and microtubules (bottom line) for the two steps of the assay; in the presence of tubulin only (left column) and in the presence of tubulin and actin (right column). Scale bar: 10 µm. Quantification of the differences in the number of microtubules per centrosome between the two stages of the experiment described above on centrosomes capable (first condition), or not (second condition), to grow actin filaments. Data were collected from a single experiment; asters without actin filaments: n= 29; asters with actin filaments: n= 13. Data were analyzed using Mann-Whitney test.


Schematic illustration of the second dynamic assay : tubulin is added to measure centrosome nucleation capacity and washed out . Then actin is added followed by actin and tubulin . Representative images showing microtubules ( top line ) and the merged images of actin and microtubule ( bottom line ) during the three steps of the assay ; in the presence of tubulin only ( left column ) , in the absence of tubulin and presence of actin ( middle column ) and in the presence of tubulin and actin ( right column ) . Scale bar : 10 µm . Arrowheads indicate microtubules unable to re - grow after assembly of actin filaments . Quantification of the differences in the number of microtubules per centrosome between the first and last steps of the experiment described above ( panels D and E ) on centrosomes capable ( first condition ) , or not ( second condition ) , to grow actin filaments . Data were pooled from 2 independent experiments ; asters without actin filaments : n = 24 ; asters with actin filaments : n = 13 . * * * * : P < 0 . 001 Student t - test .

O O O O O O O O B-GENEPROD O O O O B-SUBCELLULAR B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O B-SUBCELLULAR O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O

Schematic illustration of the second dynamic assay: tubulin is added to measure centrosome nucleation capacity and washed out. Then actin is added followed by actin and tubulin. Representative images showing microtubules (top line) and the merged images of actin and microtubule (bottom line) during the three steps of the assay; in the presence of tubulin only (left column), in the absence of tubulin and presence of actin (middle column) and in the presence of tubulin and actin (right column). Scale bar:10 µm. Arrowheads indicate microtubules unable to re-grow after assembly of actin filaments. Quantification of the differences in the number of microtubules per centrosome between the first and last steps of the experiment described above (panels D and E) on centrosomes capable (first condition), or not (second condition), to grow actin filaments. Data were pooled from 2 independent experiments; asters without actin filaments: n= 24; asters with actin filaments: n= 13. ****:P<0.001 Student t-test.


Representative images of microtubules ( top ) and actin filaments ( bottom ) growth from micropatterns . Scale bars : 20 μm .

O O O B-SUBCELLULAR O O O O B-SUBCELLULAR I-SUBCELLULAR O O O B-EXP_ASSAY O O O O O O O O O

Representative images of microtubules (top) and actin filaments (bottom) growth from micropatterns. Scale bars: 20 μm.


Schematic illustration of the assay on micropatterned substrate . Tubulin was first added alone to measure the nucleation capacity of each micropattern , and then washed out . Later on , actin was added followed by actin and tubulin . Finally , actin was rinsed out and gelsolin was added to fully disassemble actin filaments . Representative images showing microtubules ( top line ) and the merged images of actin and microtubules ( bottom line ) during the four steps of the assay ; in the presence of tubulin only , in the absence of tubulin and presence of actin , in the presence of tubulin and actin , and finally in the presence of tubulin and gelsolin but in the absence of actin filaments ( from left to right ) . Scale bar : 10 μm .

O O O O O O O O O B-GENEPROD O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O B-SUBCELLULAR O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O

Schematic illustration of the assay on micropatterned substrate. Tubulin was first added alone to measure the nucleation capacity of each micropattern, and then washed out. Later on, actin was added followed by actin and tubulin. Finally, actin was rinsed out and gelsolin was added to fully disassemble actin filaments. Representative images showing microtubules (top line) and the merged images of actin and microtubules (bottom line) during the four steps of the assay; in the presence of tubulin only, in the absence of tubulin and presence of actin, in the presence of tubulin and actin, and finally in the presence of tubulin and gelsolin but in the absence of actin filaments (from left to right). Scale bar: 10 μm.


Quantification of the number of microtubules per micropattern in the presence of tubulin only ( left ) , actin and tubulin ( middle ) and tubulin only after actin filaments disassembly ( right ) . Data were pooled from 2 independent experiments ; n = 133 . * * * * : P < 0 . 001 Student t - test . The graph shows the same measurements as in panel ( E ) in an XY representation of individual measurements . It illustrates the differences in the number of microtubules per micropattern between the first to the second step ( tubulin only versus actin and tubulin together ) with respect to the density of actin filaments per micropattern .

O O O O O B-SUBCELLULAR O O O O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SUBCELLULAR O O O O O O O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O

Quantification of the number of microtubules per micropattern in the presence of tubulin only (left), actin and tubulin (middle) and tubulin only after actin filaments disassembly (right). Data were pooled from 2 independent experiments; n= 133. ****:P<0.001 Student t-test. The graph shows the same measurements as in panel (E) in an XY representation of individual measurements. It illustrates the differences in the number of microtubules per micropattern between the first to the second step (tubulin only versus actin and tubulin together) with respect to the density of actin filaments per micropattern.


RPE1 cells stably expressing centrin1 - GFP were plated for 3 h on coverslips coated with different ratios ( 100 : 0 ; 50 : 50 or 1 : 99 ) of fibronectin and PLL - PEG prior to fixation and staining for F - actin ( top line and magnified views around centrosome below . Scale bars : 10 µm and 2 µm , respectively ) and α - tubulin ( bottom line . Scale bar : 10 µm ) .

B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O 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-GENEPROD O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-EXP_ASSAY O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O

RPE1 cells stably expressing centrin1-GFP were plated for 3 h on coverslips coated with different ratios (100:0; 50:50 or 1:99) of fibronectin and PLL-PEG prior to fixation and staining for F-actin (top line and magnified views around centrosome below. Scale bars: 10 µm and 2 µm, respectively) and α-tubulin (bottom line. Scale bar: 10 µm).


IIA1 . 6 B lymphoma cells were plated for 60 min on poly - L - lysine , fibronectin or ICAM - 1 coated cover slides prior to be fixed and stained for F - actin ( top line ) and α - tubulin ( bottom line ) . Scale bar : 3 µm .

B-CELL_LINE I-CELL_LINE I-CELL_LINE B-DISEASE I-DISEASE O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-EXP_ASSAY O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O

IIA1.6 B lymphoma cells were plated for 60 min on poly-L-lysine, fibronectin or ICAM-1 coated cover slides prior to be fixed and stained for F-actin (top line) and α-tubulin (bottom line). Scale bar: 3 µm.


Quantification of the area occupied by RPE1 cells on the substrate ( top left ) , F - actin content at the centrosome ( top right ) polymerized tubulin at the centrosome ( bottom left ) and in the entire cell ( bottom right ) for the three conditions of cell adhesion described in ( B ) . Measurements came from 3 independent experiments with more than 60 analyzed cells in each . Errors bars represent standard deviations . F - actin and microtubule contents were compared using Mann - Whitney test , and variations of the cell area were compared using unpaired t - test .

O O O O O O B-CELL_LINE O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O B-SUBCELLULAR O O O O O B-GENEPROD O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O 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-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O

Quantification of the area occupied by RPE1 cells on the substrate (top left), F-actin content at the centrosome (top right) polymerized tubulin at the centrosome (bottom left) and in the entire cell (bottom right) for the three conditions of cell adhesion described in (B). Measurements came from 3 independent experiments with more than 60 analyzed cells in each. Errors bars represent standard deviations. F-actin and microtubule contents were compared using Mann-Whitney test, and variations of the cell area were compared using unpaired t-test.


Quantification of the area occupied by B lymphoma cells on the substrate ( top left ) , F - actin content at the centrosome ( top right ) polymerized tubulin at the centrosome ( bottom left ) and in the entire cell ( bottom right ) for the three conditions of cell adhesion described in ( D ) . Measurements came from 3 independent experiments with more than 80 analyzed cells in each . Errors bars represent standard deviations . F - actin and microtubule contents were compared using Mann - Whitney test , and variations of the cell area were compared using unpaired t - test .

O O O O O O B-DISEASE I-DISEASE O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O B-SUBCELLULAR O O O O O B-GENEPROD O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O 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-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O

Quantification of the area occupied by B lymphoma cells on the substrate (top left), F-actin content at the centrosome (top right) polymerized tubulin at the centrosome (bottom left) and in the entire cell (bottom right) for the three conditions of cell adhesion described in (D). Measurements came from 3 independent experiments with more than 80 analyzed cells in each. Errors bars represent standard deviations. F-actin and microtubule contents were compared using Mann-Whitney test, and variations of the cell area were compared using unpaired t-test.


B HeLa cells expressing GFP - parkin ( green ) , OCT - DsRed2 ( red ) and CFP - Drp1K38E were left untreated ( untreated ) or treated for 2 h with 10 µM CCCP ( CCCP ) , then fixed and stained for TOM20 ( blue ) . Scale bars , 30 µm .

O B-CELL_LINE O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O O O O O O O O O O

B HeLa cells expressing GFP-parkin (green), OCT-DsRed2 (red) and CFP-Drp1K38E were left untreated (untreated) or treated for 2 h with 10 µM CCCP (CCCP), then fixed and stained for TOM20 (blue). Scale bars, 30 µm.


C Cells prepared as in ( B ) were treated with 50 µM antimycin A for 2 h . Arrows indicate OCT - DsRed2 - positive / TOM20 - negative MDVs that colocalize with GFP - parkin , while circles indicate MDVs that are parkin - negative . Arrowheads indicate nascent vesicles , adjacent to mitochondria , exhibiting cargo selectivity and parkin recruitment . Open arrowheads indicate parkin - negative MDVs containing the reciprocal cargo ( TOM20 - positive / OCT - DsRed2 - negative ) . Cell boundaries are delineated in the GFP - parkin single - channel image . Scale bars , 30 µm .

O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O B-SUBCELLULAR O O O B-GENEPROD O B-GENEPROD O O O O B-SUBCELLULAR O O B-GENEPROD O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O B-SUBCELLULAR O O O O O B-GENEPROD O O O O O B-GENEPROD O O B-SUBCELLULAR O O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O

C Cells prepared as in (B) were treated with 50 µM antimycin A for 2 h. Arrows indicate OCT-DsRed2-positive/TOM20-negative MDVs that colocalize with GFP-parkin, while circles indicate MDVs that are parkin-negative. Arrowheads indicate nascent vesicles, adjacent to mitochondria, exhibiting cargo selectivity and parkin recruitment. Open arrowheads indicate parkin-negative MDVs containing the reciprocal cargo (TOM20-positive/OCT-DsRed2-negative). Cell boundaries are delineated in the GFP-parkin single-channel image. Scale bars, 30 µm.


A Quantification of the number of OCTμDsRed2μpositive / TOM20μnegative vesicles in cells expressing GFP , GFPμparkinWT , GFPμparkinR42P , GFPμparkinK211N , or GFPμparkinC431F , treated with or without antimycin A ( anti A ) as in ( C ) ; both the total number ( white bars ) and the number colocalizing with GFPμparkin ( gray bars ) are indicated . Bars represent the mean ± s . e . m . Pμvalues are given first for GFPμ / GFPμparkinμpositive vesicles , then for total vesicle number ( n = 49 - 68 cells in 2 - 3 experiments ) ; ns , not significant ; * * P < 0 . 01 ; * * * P < 0 . 001 ) .

O O O O O O B-GENEPROD O B-GENEPROD B-SUBCELLULAR O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O O O O O O O O O O O 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 Quantification of the number of OCTμDsRed2μpositive/TOM20μnegative vesicles in cells expressing GFP, GFPμparkinWT, GFPμparkinR42P, GFPμparkinK211N, or GFPμparkinC431F, treated with or without antimycin A (anti A) as in (C); both the total number (white bars) and the number colocalizing with GFPμparkin (gray bars) are indicated. Bars represent the mean ± s.e.m. Pμvalues are given first for GFPμ/GFPμparkinμpositive vesicles, then for total vesicle number (n = 49-68 cells in 2-3 experiments); ns, not significant; **P < 0.01; ***P < 0.001).


C HeLa cells expressing various GFP - parkin mutant contructs ( green ) , pOCT - DsRed2 ( red ) and CFP - Drp1K38E were treated with 50 µM antimycin A for 2 h , then fixed and immunostained against TOM20 . Arrows indicate matrix - positive / TOM20 - negative structures colocalizing with GFP - parkin . Scale bars , 30 µm .

O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O O O

C HeLa cells expressing various GFP-parkin mutant contructs (green), pOCT-DsRed2 (red) and CFP-Drp1K38E were treated with 50 µM antimycin A for 2 h, then fixed and immunostained against TOM20. Arrows indicate matrix-positive/TOM20-negative structures colocalizing with GFP-parkin. Scale bars, 30 µm.


D Immunoblot of whole - cell lysates of HeLa cells expressing various GFP - parkin constructs , treated with 50 µM antimycin A ( anti A , + ) or DMSO ( - ) for 2 h .

O B-EXP_ASSAY O O O O O O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O

D Immunoblot of whole-cell lysates of HeLa cells expressing various GFP-parkin constructs, treated with 50 µM antimycin A (anti A, +) or DMSO (-) for 2 h.


A Representative immunoblot of whole ‐ cell lysates from U2OS : GFP and U2OS : GFP ‐ parkin cells transfected with non ‐ targeting siRNA or siRNA targeting Drp1 ( siDrp1 ) .

O O B-EXP_ASSAY O O O O O O B-CELL_LINE O B-GENEPROD O B-CELL_LINE O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O

A Representative immunoblot of whole‐cell lysates from U2OS:GFP and U2OS:GFP‐parkin cells transfected with non‐targeting siRNA or siRNA targeting Drp1 (siDrp1).


B U2OS : GFP − parkin cells transfected with siRNA targeting Drp1 were treated with DMSO ( upper panels ) or 25 μM antimycin A ( anti A , lower panels ) for 90 min , then fixed and immunostained against PDH ( red ) and TOM20 ( blue ) . PDHE2 / E3 bp − positive / TOM20 − negative MDVs colocalizing with GFP − parkin ( arrows ) or not ( circles ) are indicated . Scale bars , 20 μm ( first panels on left ) and 2 μm .

O B-CELL_LINE O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O O B-SUBCELLULAR O O B-GENEPROD O B-GENEPROD O O 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 U2OS:GFP−parkin cells transfected with siRNA targeting Drp1 were treated with DMSO (upper panels) or 25 μM antimycin A (anti A, lower panels) for 90 min, then fixed and immunostained against PDH (red) and TOM20 (blue). PDHE2/E3 bp−positive/TOM20−negative MDVs colocalizing with GFP−parkin (arrows) or not (circles) are indicated. Scale bars, 20 μm (first panels on left) and 2 μm.


C Quantification of PDH − positive / TOM20 − negative vesicles in U2OS : GFP and U2OS : GFP − parkin cells transfected with the indicated siRNA , treated with DMSO or antimycin A ( anti A ) as in ( B ) ; both the total number ( white bars ) and the number colocalizing with GFP − parkin ( gray bars ) are indicated . Bars represent the mean ± s . e . m . P − values are given first for GFP − / GFP − parkin − positive vesicles , then for total vesicle number ( n = 48 - 85 cells in 2 - 3 experiments ) ; ns , not significant ; * * * P < 0 . 001 ) .

O O O O O O O B-GENEPROD O O B-SUBCELLULAR O B-CELL_LINE O B-GENEPROD O B-CELL_LINE O B-GENEPROD O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O 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-GENEPROD O O B-GENEPROD O B-GENEPROD O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O O O 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 Quantification of PDH−positive/TOM20−negative vesicles in U2OS:GFP and U2OS:GFP−parkin cells transfected with the indicated siRNA, treated with DMSO or antimycin A (anti A) as in (B); both the total number (white bars) and the number colocalizing with GFP−parkin (gray bars) are indicated. Bars represent the mean ± s.e.m. P−values are given first for GFP−/GFP−parkin−positive vesicles, then for total vesicle number (n = 48-85 cells in 2-3 experiments); ns, not significant; ***P < 0.001).


D 5 μm ‐ long profile of the parkin ‐ positive vesicle and adjacent mitochondrial tubule depicted in the antimycin A ‐ treated cell from ( B ) ( left ) . Increasing position values on the x ‐ axis of the fluorescence intensity plot ( right ) correspond to moving from the top to the bottom of the profile . * MDV indicated by an arrow in ( B ) .

O O O O O O O O B-GENEPROD O O B-SUBCELLULAR O O B-SUBCELLULAR I-SUBCELLULAR O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O 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-SUBCELLULAR O O O O O O O O O

D 5 μm‐long profile of the parkin‐positive vesicle and adjacent mitochondrial tubule depicted in the antimycin A‐treated cell from (B) (left). Increasing position values on the x‐axis of the fluorescence intensity plot (right) correspond to moving from the top to the bottom of the profile. *MDV indicated by an arrow in (B).


E Quantification of the distance between the centres of GFP − parkin − negative ( black bar ) or − positive ( gray bar ) vesicles and the edge of the nearest mitochondrial tubule for the vesicles quantified in antimycin A − treated U2OS : GFP − parkin cells transfected with siDrp1 in ( C ) . Error bars represent the mean ± s . e . m . ; * P < 0 . 05 ( obtained by Student ' s t − test ) .

O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O B-SUBCELLULAR O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O B-CELL_LINE O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O 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 Quantification of the distance between the centres of GFP−parkin−negative (black bar) or −positive (gray bar) vesicles and the edge of the nearest mitochondrial tubule for the vesicles quantified in antimycin A−treated U2OS:GFP−parkin cells transfected with siDrp1 in (C). Error bars represent the mean ± s.e.m.; *P < 0.05 (obtained by Student's t−test).


F U2OS : GFP ‐ parkin cells transfected with siRNA targeting Drp1 ( siDrp1 ) were treated with 25 μM antimycin A for 90 min prior to fixation . Samples were immunostained against TOM20 ( blue ) and the indicated mitochondrial marker ( red ) . TOM20 ‐ negative MDVs containing the specified cargo and colocalizing with GFP ‐ parkin ( arrows ) or not ( circles ) are indicated . Arrowheads show lack of colocalization between GFP ‐ parkin and the indicated mitochondrial marker . Scale bars , 20 μm ( first two columns of panels ) and 2 μm ( remaining columns of panels ) .

O B-CELL_LINE O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O B-SUBCELLULAR O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O 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 U2OS:GFP‐parkin cells transfected with siRNA targeting Drp1 (siDrp1) were treated with 25 μM antimycin A for 90 min prior to fixation. Samples were immunostained against TOM20 (blue) and the indicated mitochondrial marker (red). TOM20‐negative MDVs containing the specified cargo and colocalizing with GFP‐parkin (arrows) or not (circles) are indicated. Arrowheads show lack of colocalization between GFP‐parkin and the indicated mitochondrial marker. Scale bars, 20 μm (first two columns of panels) and 2 μm (remaining columns of panels).


G Quantification of TOM20 − negative structures that stained positively for the indicated cargo in U2OS : GFP ( GFP ) and U2OS : GFP − parkin ( GFP − parkin ) cells , transfected with siRNA targeting Drp1 , treated with antimycin A ( anti A ) ; both the total number ( white bars ) and the number colocalizing with GFP − parkin ( gray bars ) are indicated . Error bars represent the mean ± s . e . m . P − values are given first for GFP − / GFP − parkin − positive vesicles , then for total vesicle number ( n = 24 - 59 cells in two experiments ; ns , not significant ; * * * P < 0 . 001 ) .

O O O B-GENEPROD O O O O O O O O O O O B-CELL_LINE O B-GENEPROD O B-GENEPROD O O B-CELL_LINE O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O 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-GENEPROD O O B-GENEPROD O B-GENEPROD O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O

G Quantification of TOM20−negative structures that stained positively for the indicated cargo in U2OS:GFP (GFP) and U2OS:GFP−parkin (GFP−parkin) cells, transfected with siRNA targeting Drp1, treated with antimycin A (anti A); both the total number (white bars) and the number colocalizing with GFP−parkin (gray bars) are indicated. Error bars represent the mean ± s.e.m. P−values are given first for GFP−/GFP−parkin−positive vesicles, then for total vesicle number (n = 24-59 cells in two experiments; ns, not significant; ***P < 0.001).


AHeLa cells , transfected with siRNA targeting Drp1 and GFP ‐ parkin ( green ) , were treated with 25 μM antimycin A for 90 min following a 30 ‐ min pretreatment with 50 nM bafilomycin A1 , then fixed and immunostained against PDH E2 / E3 bp ( red ) and TOM20 ( blue ) . PDH E2 / E3 bp ‐ positive / TOM20 ‐ negative MDVs colocalizing with GFP ‐ parkin ( arrows ) or not ( circles ) are indicated . Scale bar , 30 μm .

O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O O B-SUBCELLULAR O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O

AHeLa cells, transfected with siRNA targeting Drp1 and GFP‐parkin (green), were treated with 25 μM antimycin A for 90 min following a 30‐min pretreatment with 50 nM bafilomycin A1, then fixed and immunostained against PDH E2/E3 bp (red) and TOM20 (blue). PDH E2/E3 bp‐positive/TOM20‐negative MDVs colocalizing with GFP‐parkin (arrows) or not (circles) are indicated. Scale bar, 30 μm.


B Quantification of PDH E2 / E3bp ‐ positive / TOM20 ‐ negative vesicles in HeLa cells treated with antimycin A in the presence of the lysosomal inhibitors bafilomycin A1 or pepstatin A and E ‐ 64d ; both total number ( white bars ) and the number colocalizing with GFP ‐ parkin ( gray bars ) are indicated . Bars represent the mean ± s . e . m . P ‐ values are given for total vesicle number ( n = 19 to 120 cells in 2 - 3 experiments ; * * P < 0 . 01 ; * * * P < 0 . 001 ) .

O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O O B-SUBCELLULAR O B-CELL_LINE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O 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-SUBCELLULAR O O O O O O O O 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 Quantification of PDH E2/E3bp‐positive/TOM20‐negative vesicles in HeLa cells treated with antimycin A in the presence of the lysosomal inhibitors bafilomycin A1 or pepstatin A and E‐64d; both total number (white bars) and the number colocalizing with GFP‐parkin (gray bars) are indicated. Bars represent the mean ± s.e.m. P‐values are given for total vesicle number (n = 19 to 120 cells in 2-3 experiments; **P < 0.01; ***P < 0.001).


CHeLa cells , transfected with GFP ‐ parkin ( green ) and siRNA targeting Drp1 and Atg5 , were treated with 25 μM antimycin A for 90 min , then fixed , immunostained for PDH ( red ) and TOM20 ( blue ) , and counterstained for Hoescht ( gray ) . PDH E2 / E3bp bp ‐ positive / TOM20 ‐ negative MDVs colocalizing with GFP ‐ parkin ( arrows ) or not ( circles ) are indicated . Scale bars , 20 μm .

O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O O O O O B-EXP_ASSAY O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O O B-SUBCELLULAR O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O

CHeLa cells, transfected with GFP‐parkin (green) and siRNA targeting Drp1 and Atg5, were treated with 25 μM antimycin A for 90 min, then fixed, immunostained for PDH (red) and TOM20 (blue), and counterstained for Hoescht (gray). PDH E2/E3bp bp‐positive/TOM20‐negative MDVs colocalizing with GFP‐parkin (arrows) or not (circles) are indicated. Scale bars, 20 μm.


D Quantification of PDH E2 / E3 bp ‐ positive / TOM20 ‐ negative vesicles from HeLa cells transfected with the indicated siRNA ; both the total number ( white bars ) and the number colocalizing with GFP ‐ parkin ( gray bars ) are indicated . Error bars represent the mean ± s . e . m . P ‐ values are given for the total vesicle number ( n = 42 - 64 cells in 2 - 3 experiments ) ; ns , not significant .

O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O O B-SUBCELLULAR O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O 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-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O

D Quantification of PDH E2/E3 bp‐positive/TOM20‐negative vesicles from HeLa cells transfected with the indicated siRNA; both the total number (white bars) and the number colocalizing with GFP‐parkin (gray bars) are indicated. Error bars represent the mean ± s.e.m. P‐values are given for the total vesicle number (n = 42-64 cells in 2-3 experiments); ns, not significant.


A Representative immunoblot of whole ‐ cell lysates from HeLa cells transfected with non ‐ targeting siRNA or siRNA targeting PINK1 ( siPINK1 ) , treated with 10 μM CCCP for 6 h in order to stabilize the PINK1 full ‐ length band .

O O B-EXP_ASSAY O O O O O O B-CELL_LINE O O O O O O O O O O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-GENEPROD O O O O O

A Representative immunoblot of whole‐cell lysates from HeLa cells transfected with non‐targeting siRNA or siRNA targeting PINK1 (siPINK1), treated with 10 μM CCCP for 6 h in order to stabilize the PINK1 full‐length band.


B HeLa cells transfected with GFP − parkin ( green ) and siRNA targeting PINK1 ( siPINK1 ) or non − targeting control ( ctrl siRNA ) , were treated with 25 μM antimycin A for 90 min , then fixed and immunostained for PDH E2 / E3 bp ( red ) and TOM20 ( blue ) . PDH E2 / E3 bp − positive / TOM20 − negative MDVs colocalizing with GFP − parkin ( arrows ) or not ( circles ) are indicated . Arrowheads indicate parkin − positive MDVs adjacent to mitochondria , possibly budding . Scale bar , 20 μm .

O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O O B-SUBCELLULAR O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O B-SUBCELLULAR O O B-SUBCELLULAR O O O O O O O O O O

B HeLa cells transfected with GFP−parkin (green) and siRNA targeting PINK1 (siPINK1) or non−targeting control (ctrl siRNA), were treated with 25 μM antimycin A for 90 min, then fixed and immunostained for PDH E2/E3 bp (red) and TOM20 (blue). PDH E2/E3 bp−positive/TOM20−negative MDVs colocalizing with GFP−parkin (arrows) or not (circles) are indicated. Arrowheads indicate parkin−positive MDVs adjacent to mitochondria, possibly budding. Scale bar, 20 μm.


C Quantification of PDHE2 / E3 bp − positive / TOM20 − negative structures from ( B ) ; both the total number ( white bars ) and the number colocalizing with GFP − parkin ( gray bars ) are indicated . Error bars represent the mean ± s . e . m . P − values are given first for GFP − / GFP − parkin − positive vesicles , then for total vesicle number ( n = 42 - 60 cells in three experiments ) ; ns , not significant ; * * * P < 0 . 001 ) .

O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O 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-GENEPROD O O B-GENEPROD O B-GENEPROD O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O 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 Quantification of PDHE2/E3 bp−positive/TOM20−negative structures from (B); both the total number (white bars) and the number colocalizing with GFP−parkin (gray bars) are indicated. Error bars represent the mean ± s.e.m. P−values are given first for GFP−/GFP−parkin−positive vesicles, then for total vesicle number (n = 42-60 cells in three experiments); ns, not significant; ***P < 0.001).


A Quantification of TMRM fluorescence by flow cytometry in U2OS : GFP ‐ parkin cells treated with 25 μM antimycin A ( anti A ) , 25 μM antimycin A and 10 μM oligomycin ( anti A + oligo ) , and 20 μM CCCP ( CCCP ) for the indicated time , represented as a fraction of fluorescence intensity compared to DMSO ‐ treated cells . Error bars represent the mean ± s . e . m . ( n = 3 experiments ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O B-GENEPROD O B-GENEPROD O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O

A Quantification of TMRM fluorescence by flow cytometry in U2OS:GFP‐parkin cells treated with 25 μM antimycin A (anti A), 25 μM antimycin A and 10 μM oligomycin (anti A + oligo), and 20 μM CCCP (CCCP) for the indicated time, represented as a fraction of fluorescence intensity compared to DMSO‐treated cells. Error bars represent the mean ± s.e.m. (n = 3 experiments).


B U2OS : GFP ‐ parkin cells were treated with DMSO ( DMSO ) , 25 μM antimycin A ( anti A ) , 25 μM antimycin A and 10 μM oligomycin ( anti A + oligo ) , or 20 μM CCCP ( CCCP ) for two hours , then fixed and immunostained for TOM20 ( red ) . Scale bars , 50 μm .

O B-CELL_LINE O B-GENEPROD O B-GENEPROD O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O

B U2OS:GFP‐parkin cells were treated with DMSO (DMSO), 25 μM antimycin A (anti A), 25 μM antimycin A and 10 μM oligomycin (anti A + oligo), or 20 μM CCCP (CCCP) for two hours, then fixed and immunostained for TOM20 (red). Scale bars, 50 μm.


C Quantification ofPDHE2 / E3 bp − positive / TOM20 − negative structures from ( B ) ; both the total number ( white bars ) and the number colocalizing with GFP − parkin ( gray bars ) are indicated . Error bars represent the mean ± s . e . m . P − values are given first for GFP − / GFP − parkin − positive vesicles , then for total vesicle number ( n = 42 - 60 cells in three experiments ) ; ns , not significant ; * * * P < 0 . 001 ) .

O O O B-GENEPROD I-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O 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-GENEPROD O O B-GENEPROD O B-GENEPROD O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O 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 Quantification ofPDHE2/E3 bp−positive/TOM20−negative structures from (B); both the total number (white bars) and the number colocalizing with GFP−parkin (gray bars) are indicated. Error bars represent the mean ± s.e.m. P−values are given first for GFP−/GFP−parkin−positive vesicles, then for total vesicle number (n = 42-60 cells in three experiments); ns, not significant; ***P < 0.001).


D U2OS : GFP − parkin cells were treated with DMSO ( DMSO ) , 25 μM antimycin A ( anti A ) , 25 μM antimycin A and 10 μM oligomycin ( anti A + oligo ) , or 20 μM CCCP ( CCCP ) for two hours , then fixed and immunostained for PDH E2 / E3 bp ( red ) and TOM20 ( blue ) . A merged image of both mitochondrial markers ( mito ) is shown at the far right . Arrows indicate PDH E2 / E3 bp − positive / TOM20 − negative structures colocalizing with GFP − parkin ( green ) . Scale bars , 20 μm .

O B-CELL_LINE O B-GENEPROD O B-GENEPROD O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O

D U2OS:GFP−parkin cells were treated with DMSO (DMSO), 25 μM antimycin A (anti A), 25 μM antimycin A and 10 μM oligomycin (anti A + oligo), or 20 μM CCCP (CCCP) for two hours, then fixed and immunostained for PDH E2/E3 bp (red) and TOM20 (blue). A merged image of both mitochondrial markers (mito) is shown at the far right. Arrows indicate PDH E2/E3 bp−positive/TOM20−negative structures colocalizing with GFP−parkin (green). Scale bars, 20 μm.


A Representative immunoblot of whole ‐ cell lysates from U2OS : GFP and GFP ‐ parkin cells treated with DMSO , 25 μM antimycin A ( anti A ) , 25 μM antimycin A with 10 μM oligomycin ( anti A + oligo ) , or 20 μM CCCP for the indicated time period .

O O B-EXP_ASSAY O O O O O O B-CELL_LINE O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O O O O

A Representative immunoblot of whole‐cell lysates from U2OS:GFP and GFP‐parkin cells treated with DMSO, 25 μM antimycin A (anti A), 25 μM antimycin A with 10 μM oligomycin (anti A + oligo), or 20 μM CCCP for the indicated time period.


B Quantification of PDH E1a signal intensity relative to that of actin in immunoblots from ( A ) . Error bars represent the mean ± s . e . m . ( n = 3 experiments ) .

O O O B-GENEPROD I-GENEPROD O O O O O O B-GENEPROD O O 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 Quantification of PDH E1a signal intensity relative to that of actin in immunoblots from (A). Error bars represent the mean ± s.e.m. (n = 3 experiments).


C Quantification of mitochondrial clearance in U2OS : GFP − parkin cells treated as in ( A ) , fixed and immunostained for TOM20 . Data are shown as percentage of cells containing mitochondria , by TOM20 staining , visualized by fluorescence microscopy . Error bars represent the mean ± s . e . m . ( n = 3 experiments , with at least 85 cells quantified per condition , per experiment ) .

O O O B-SUBCELLULAR O O B-CELL_LINE O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O B-SUBCELLULAR O O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O 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 Quantification of mitochondrial clearance in U2OS:GFP−parkin cells treated as in (A), fixed and immunostained for TOM20. Data are shown as percentage of cells containing mitochondria, by TOM20 staining, visualized by fluorescence microscopy. Error bars represent the mean ± s.e.m. (n = 3 experiments, with at least 85 cells quantified per condition, per experiment).


D U2OS : GFP ‐ parkin cells were treated as in ( A ) for the indicated time period , then fixed and immunostained for TOM20 ( red ) . Cell boundaries are delineated in single ‐ channel images . Scale bars , 20 μm .

O B-CELL_LINE O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O O O O O O O

D U2OS:GFP‐parkin cells were treated as in (A) for the indicated time period, then fixed and immunostained for TOM20 (red). Cell boundaries are delineated in single‐channel images. Scale bars, 20 μm.


A . Colony formation assay ( CFA ) with imatinib with representative images of colonies formed by each cell line within the panel . Error bars represent mean ± S . E . M . , n = 9 , * P < 0 . 001 by two - tailed unpaired Student ' s t - test ( H1915 P = 0 . 0004 , H2110 P = 0 . 0001 ) .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O 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-CELL_LINE O O O O O O B-CELL_LINE O O O O O O O

A. Colony formation assay (CFA) with imatinib with representative images of colonies formed by each cell line within the panel. Error bars represent mean ±S.E.M., n=9, *P < 0.001 by two-tailed unpaired Student's t-test (H1915 P= 0.0004, H2110 P=0.0001).


B . Western blot analysis after 1 hour treatment with imatinib .

O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-SMALL_MOLECULE O

B. Western blot analysis after 1 hour treatment with imatinib.


C . MTT assay for NSCLC cell lines and Beas - 2B cells with dasatinib or DMSO . Error bars represent mean ± S . E . M . , n = 9 , * P < 0 . 001 by two - tailed unpaired Student ' s t - test ( H1915 P = 1 . 09 × 10 - 5 , H2110 P = 0 . 009 and A549 P = 6 × 10 - 6 ) .

O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O 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-CELL_LINE O O O O O O O O O O B-CELL_LINE O O O O O O B-CELL_LINE O O O O O O O O O

C. MTT assay for NSCLC cell lines and Beas-2B cells with dasatinib or DMSO. Error bars represent mean ±S.E.M., n=9, *P<0.001 by two-tailed unpaired Student's t-test (H1915 P=1.09×10-5, H2110 P=0.009 and A549 P=6×10-6).


D . Western blot analysis after 1 h treatment with dasatinib .

D. Western blot analysis after 1 h treatment with dasatinib.


A - C . Drug - resistant ABL1 - T315I rescues dasatinib - and imatinib - induced cytotoxicity . Western blot analysis of ( A ) transient and ( B ) tetracycline - inducible overexpression of ABL1 - T315I in H1915 cells treated with dasatinib or imatinib .

O O O O O O O B-GENEPROD O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O O O B-CELL_LINE O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O

A -C. Drug-resistant ABL1-T315I rescues dasatinib- and imatinib-induced cytotoxicity. Western blot analysis of (A) transient and (B) tetracycline-inducible overexpression of ABL1-T315I in H1915 cells treated with dasatinib or imatinib.


C . CFA with dasatinib or imatinib for H1915 cells with or without tetracycline - inducible expression of ABL1 - T315I . Error bars represent mean ± S . E . M . , n = 9 , * P < 0 . 001 by two - tailed unpaired Student ' s t - test ( control , DMSO vs dasatinib P = 4 . 3 × 10 - 12 vs imatinib P = 6 × 10 - 6 ; dasatinib , control vs tetracycline , P = 9 × 10 - 7 ; imatinib , control vs tetracycline P = 9 . 7 × 10 - 5 ) .

O O B-EXP_ASSAY O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-CELL_LINE O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O O O

C. CFA with dasatinib or imatinib for H1915 cells with or without tetracycline-inducible expression of ABL1-T315I. Error bars represent mean ±S.E.M., n=9, *P<0.001 by two-tailed unpaired Student's t-test (control, DMSO vs dasatinib P=4.3×10-12 vs imatinib P=6×10-6; dasatinib, control vs tetracycline, P=9×10-7; imatinib, control vs tetracycline P=9.7×10-5).


D - F . Knockdown of mutated ABL1 reduces viability of lung cancer cells . Top , western blot analysis of ABL1 knockdown in ( D ) Beas - 2B , ( E ) H1915 and ( F ) H2110 cells upon transfection with three ABL1 siRNAs , negative control ( scrambled ) and CyclophilinB positive control . Bottom , MTT assay showing viability of ( D ) Beas - 2B , ( E ) H1915 and ( F ) H2110 cells in response to ABL1 knockdown . Error bars represent mean ± S . E . M , n = 9 , * P < 0 . 005 , * * P < 0 . 001 by two - tailed unpaired Student ' s t - test ( Beas - 2B , scrambled vs siABL1 - 3 P = 0 . 002 ; H1915 , scrambled vs siABL1 - 1 P = 0 . 004 , vs siABL1 - 2 P = 0 . 001 , vs siABL1 - 3 P = 0 . 002 ; H2110 scrambled vs siABL1 - 1 P = 5 × 10 - 6 , vs siABL1 - 2 P = 2 × 10 - 5 , vs siABL1 - 3 P = 4 . 2 × 10 - 6 ) .

O O O O O O O B-GENEPROD O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-CELL_LINE O O O O B-CELL_LINE O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-CELL_LINE O O O O B-CELL_LINE O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O 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-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D-F. Knockdown of mutated ABL1 reduces viability of lung cancer cells. Top, western blot analysis of ABL1 knockdown in (D) Beas-2B, (E) H1915 and (F) H2110 cells upon transfection with three ABL1 siRNAs, negative control (scrambled) and CyclophilinB positive control. Bottom, MTT assay showing viability of (D) Beas-2B, (E) H1915 and (F) H2110 cells in response to ABL1 knockdown. Error bars represent mean ±S.E.M, n=9, *P<0.005, **P<0.001 by two-tailed unpaired Student's t-test (Beas-2B, scrambled vs siABL1-3 P=0.002; H1915, scrambled vs siABL1-1 P=0.004, vs siABL1-2 P=0.001, vs siABL1-3 P=0.002; H2110 scrambled vs siABL1-1 P=5×10-6, vs siABL1-2 P=2×10-5, vs siABL1-3 P=4.2×10-6).


A - C . Average tumor volumes of xenografts derived from H1915 ( A ) , H650 ( B ) and ( C ) H1915 cell line with doxycycline inducible ABL1 gate - keeper mutation ( H1915 - ABL1T315I ) in mice treated with either vehicle or imatinib ( 100mg / kg ) . Error bars represent ± S . D . , n = 6 - 10 mice per group , * P = 0 . 0016 by Mann - Whitney U test .

O O O O O O O O O O O B-CELL_LINE O O O O B-CELL_LINE O O O O O O O B-CELL_LINE O O O B-SMALL_MOLECULE O B-GENEPROD O O O O O B-CELL_LINE O B-GENEPROD O O B-ORGANISM O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O

A-C. Average tumor volumes of xenografts derived from H1915 (A), H650 (B) and (C) H1915 cell line with doxycycline inducible ABL1 gate-keeper mutation (H1915-ABL1T315I) in mice treated with either vehicle or imatinib (100mg/kg). Error bars represent ±S.D., n=6-10 mice per group, *P=0.0016 by Mann-Whitney U test.


A and C . Root mean square deviation ( RMSD ) analysis of the movement of ( A ) αC - helix and ( C ) DFG motif in the active and inactive conformation of ABL1 kinase with R351W mutation ( orange ) compared to the wild type ( WT ) kinase ( black ) . B and D . Time - lapse images show movement within the αC - helix ( B ) and DFG motif ( D ) of the inactive structure of the WT ( black outline ) and R351W ( orange outline ) constructs .

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD 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-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O 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 and C. Root mean square deviation (RMSD) analysis of the movement of (A) αC-helix and (C) DFG motif in the active and inactive conformation of ABL1 kinase with R351W mutation (orange) compared to the wild type (WT) kinase (black). B and D. Time-lapse images show movement within the αC-helix (B) and DFG motif (D) of the inactive structure of the WT (black outline) and R351W (orange outline) constructs.


E . left , western blot analysis of transient overexpression of wild type ABL1 ( WT ) and ABL1 - R351W , compared to empty vector control ( EV ) in HEK293T cells . Right , quantification of p - CRKL signal based on densitometry analysis of blots from 6 independent experiments . Error bars represent ± S . D . , n = 6 , * P = 0 . 0004 by two - tailed unpaired Student ' s t - test .

O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O B-CELL_LINE O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O 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. left, western blot analysis of transient overexpression of wild type ABL1 (WT) and ABL1-R351W, compared to empty vector control (EV) in HEK293T cells. Right, quantification of p-CRKL signal based on densitometry analysis of blots from 6 independent experiments. Error bars represent ±S.D., n=6, *P=0.0004 by two-tailed unpaired Student's t-test.


F . Overexpression of mutant ABL1 constructs with mutations identified in selected primary lung tumors ( Ding et al , 2008 ) in H157 cells . Values indicate quantified band intensity for p - CRKL , relative to α - tubulin and HA - tagged ABL1 .

O O O O O B-GENEPROD O O O O O O O B-TISSUE O O O O O O O O O B-CELL_LINE O O O O O O O O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O B-GENEPROD O

F. Overexpression of mutant ABL1 constructs with mutations identified in selected primary lung tumors (Ding et al, 2008) in H157 cells. Values indicate quantified band intensity for p-CRKL, relative to α-tubulin and HA-tagged ABL1.


A . Immunofluorescence imaging of endogenous ABL1 in the panel of NSCLC cell lines ; scale bars = 20 μm . B and C Quantification of nuclear versus cytoplasmic localization of ( B ) endogenous ABL1 in NSCLC cell lines and ( C ) tetracycline - inducible expression of ABL1 - WT , ABL1 - R351W and ABL1 - G340L in Beas - 2B cells . Error bars represent ± S . D . , n = 6 - 10 images , * P < 0 . 005 by two - tailed unpaired Student ' s t - test ( Beas - 2B vs H1915 P = 6 . 6 × 10 - 5 , vs H2110 P = 0 . 0001 ; WT vs R351W P = 0 . 003 , vs G340L P = 0 . 002 ) .

O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-CELL_LINE O O O O O O O O O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O

A. Immunofluorescence imaging of endogenous ABL1 in the panel of NSCLC cell lines; scale bars=20 μm. B and C Quantification of nuclear versus cytoplasmic localization of (B) endogenous ABL1 in NSCLC cell lines and (C) tetracycline-inducible expression of ABL1-WT, ABL1-R351W and ABL1-G340L in Beas-2B cells. Error bars represent ±S.D., n=6-10 images, *P<0.005 by two-tailed unpaired Student's t-test (Beas-2B vs H1915 P=6.6×10-5, vs H2110 P=0.0001; WT vs R351W P=0.003, vs G340L P=0.002).


IL - 1β concentrations in supernatants of macrophages , unstimulated ( control ) ( B ) or stimulated with SP / N ( C ) or LPS / N ( D ) .

B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-CELL_TYPE O O O O O O O O O O O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O O O

IL-1β concentrations in supernatants of macrophages, unstimulated (control) (B) or stimulated with SP/N (C) or LPS/N (D).


E . SARS - CoV - 2 spike ELISA of the corresponding plasma : ( EC50 titer ) of samples collected at T0 ( n = 35 individuals ) , T1 ( n = 28 individuals ) or T2 ( n = 44 individuals ) .

O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD B-EXP_ASSAY O O O B-TISSUE O O B-EXP_ASSAY O O O O O O 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. SARS-CoV-2 spike ELISA of the corresponding plasma: (EC50 titer) of samples collected at T0 (n = 35 individuals), T1 (n = 28 individuals) or T2 (n = 44 individuals).


F . Immunofluorescence microscopy images of ASC specks ( green ) in T2 macrophages . Nuclei were stained with DAPI ( blue ) . Scale bar indicates 20 µm . Arrow indicates ASC speck formation with magnified illustration in the right corner of the image .

O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD I-GENEPROD O O O O O B-CELL_TYPE O B-SUBCELLULAR O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O

F. Immunofluorescence microscopy images of ASC specks (green) in T2 macrophages. Nuclei were stained with DAPI (blue). Scale bar indicates 20 µm. Arrow indicates ASC speck formation with magnified illustration in the right corner of the image.


G , H . ASC specks were quantified microscopically at T0 ( G ) ( n = 7 individuals ) and T2 ( H ) ( n = 8 individuals ) upon indicated stimuli .

O O O O B-GENEPROD I-GENEPROD O O B-EXP_ASSAY O O 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. ASC specks were quantified microscopically at T0 (G) (n = 7 individuals) and T2 (H) (n = 8 individuals) upon indicated stimuli.


I . Cleaved gasdermin D ( lysate ) and cleaved IL - 1β ( supernatant ) detected by Western Blot in macrophages stimulated with LPS / N or SP / N at T0 and T2 .

O O O B-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE O O B-SMALL_MOLECULE O O O B-GENEPROD O O O O O O O

I. Cleaved gasdermin D (lysate) and cleaved IL-1β (supernatant) detected by Western Blot in macrophages stimulated with LPS/N or SP/N at T0 and T2.


J , K . Cell death quantification of macrophages by flow cytometry in unstimulated or SP / N stimulated cells at T0 ( n = 5 individuals ) ( J ) and T2 ( n = 5 individuals ) ( K ) using the viability dye Zombie UV . LPS / N was used as positive control .

O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-CELL_TYPE O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O O 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-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O

J, K. Cell death quantification of macrophages by flow cytometry in unstimulated or SP/N stimulated cells at T0 (n = 5 individuals) (J) and T2 (n = 5 individuals) (K) using the viability dye Zombie UV. LPS/N was used as positive control.


L . LDH release of macrophages ( n = 6 individuals ) stimulated with SP / N at T2 compared to unstimulated cells . M . IL - 1β concentrations of MCC950 treated T2 macrophage ( n = 44 individuals ) stimulation with SP / N .

O O B-GENEPROD O O B-CELL_TYPE O O O O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-SMALL_MOLECULE O O B-CELL_TYPE O O O O O O O O B-GENEPROD O O O

L. LDH release of macrophages (n = 6 individuals) stimulated with SP/N at T2 compared to unstimulated cells. M. IL-1β concentrations of MCC950 treated T2 macrophage (n = 44 individuals) stimulation with SP/N.


B . Volcano plot showing DEGs in the same samples . Negative log10 padj - values are plotted against the log2 fold - change . Selected genes are labeled .

O O O O O O 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. Volcano plot showing DEGs in the same samples. Negative log10 padj-values are plotted against the log2 fold-change. Selected genes are labeled.


C , D . CLEC4D ( C ) and CLEC4E ( D ) surface expression on patient - derived macrophages prior ( n = 5 individuals ) or post ( n = 5 individuals ) vaccination were quantified by flow cytometry . Graphs indicate the mean fluorescence intensity ( MFI ) .

O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-EXP_ASSAY O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O

C, D. CLEC4D (C) and CLEC4E (D) surface expression on patient-derived macrophages prior (n = 5 individuals) or post (n = 5 individuals) vaccination were quantified by flow cytometry. Graphs indicate the mean fluorescence intensity (MFI).


E , F . SP or LPS as a control PAMP were incubated with affinity purified CLRs ( ratio : 10 vs . 1 for SP ) for 1h and then added to primary T2 macrophages ( n = 5 individuals ) . Subsequently , IL - 1β was quantified in the supernatants . ( E ) shows absolute values for SP ; ( F ) shows the fold change calculated with the corresponding unstimulated control for SP and LPS .

O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O B-CELL_TYPE O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O

E, F. SP or LPS as a control PAMP were incubated with affinity purified CLRs (ratio: 10 vs. 1 for SP) for 1h and then added to primary T2 macrophages (n = 5 individuals). Subsequently, IL-1β was quantified in the supernatants. (E) shows absolute values for SP; (F) shows the fold change calculated with the corresponding unstimulated control for SP and LPS.


G . Monocytes were isolated prior ( n = 5 individuals ) or after vaccination ( n = 5 individuals ) and differentiated to macrophages . IL - 1β concentrations in supernatants of macrophages were measured after stimulation with the SP S1 subunit SP and nigericin . H . Monocytes were isolated prior ( T0 ; n = 35 individuals ) or after vaccination [ 2 weeks after 1st ( T1 ; n = 28 individuals ) , 2 weeks ( T2 ; n = 38 individuals ) and 10 weeks ( T3 ; n = 33 individuals ) after 2nd vaccination ] and differentiated to macrophages . IL - 1β concentrations in supernatants of macrophages , stimulated with the SP S2 subunit SP are shown .

O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-CELL_TYPE O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CELL_TYPE O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-CELL_TYPE O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O

G. Monocytes were isolated prior (n = 5 individuals) or after vaccination (n = 5 individuals) and differentiated to macrophages. IL-1β concentrations in supernatants of macrophages were measured after stimulation with the SP S1 subunit SP and nigericin. H. Monocytes were isolated prior (T0; n = 35 individuals) or after vaccination [2 weeks after 1st (T1; n = 28 individuals), 2 weeks (T2; n = 38 individuals) and 10 weeks (T3; n =33 individuals) after 2nd vaccination] and differentiated to macrophages. IL-1β concentrations in supernatants of macrophages, stimulated with the SP S2 subunit SP are shown.


B . Phospho - SYK Immunofluorescence - microscopy in macrophages before and after vaccination . Nuclei stained with DAPI . Scale bars indicate 20 µm .

O O B-EXP_ASSAY O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE O O O O O B-SUBCELLULAR B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O

B. Phospho-SYK Immunofluorescence-microscopy in macrophages before and after vaccination. Nuclei stained with DAPI. Scale bars indicate 20 µm.


C . Quantification of pSYK positive macrophages by flow cytometry before ( n = 3 individuals ) and 14 d after vaccination ( n = 3 individuals ) . Total SYK was measured as control .

O O O O O O B-CELL_TYPE O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O

C. Quantification of pSYK positive macrophages by flow cytometry before (n = 3 individuals) and 14 d after vaccination (n = 3 individuals). Total SYK was measured as control.


D . Western blot analysis of phospho - NF - κB and NLRP3 in cell lysates of macrophages ( T0 ; T2 ) stimulated with LPS / N or SP / N .

O O B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O B-CELL_TYPE O O O O O O O B-SMALL_MOLECULE O O O B-GENEPROD O O O

D. Western blot analysis of phospho-NF-κB and NLRP3 in cell lysates of macrophages (T0; T2) stimulated with LPS/N or SP/N.


E . Macrophages were incubated with IKK - β - inhibitor KINK - 1 ( n = 10 individuals ) and SYK - inhibitors entospletinib ( n = 20 individuals ) , R406 ( n = 13 individuals ) prior to SP / nigericin stimulation . IL - 1β concentrations measured with ELISA . DMSO - treated , SP / N - stimulated and untreated unstimulated macrophages were used as control ( both n = 44 individuals ) .

O O B-CELL_TYPE O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-GENEPROD O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O B-GENEPROD O O O O O O O B-CELL_TYPE O O O O O O O O O O O O

E. Macrophages were incubated with IKK-β-inhibitor KINK-1 (n = 10 individuals) and SYK-inhibitors entospletinib (n = 20 individuals), R406 (n = 13 individuals) prior to SP/nigericin stimulation. IL-1β concentrations measured with ELISA. DMSO-treated, SP/N-stimulated and untreated unstimulated macrophages were used as control (both n = 44 individuals).


F . IL - 1β quantification in THP - 1 wild - type cells incubated with SYK inhibitors entospletinib ( n = 4 independent experiments ) and R406 ( n = 2 independent experiments ) or in THP - 1 SYKKO cells stimulated with SP / N ( n = 5 independent experiments ) .

O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE B-GENEPROD O O O B-GENEPROD O O O O O O O O O O

F. IL-1β quantification in THP-1 wild-type cells incubated with SYK inhibitors entospletinib (n = 4 independent experiments) and R406 (n = 2 independent experiments) or in THP-1 SYKKO cells stimulated with SP/N (n = 5 independent experiments).


G . ASC formation quantified by immunofluorescence - microscopy in SP / N stimulated primary macrophages after vaccination ( n = 5 individuals ) .

O O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O B-CELL_TYPE O O O O O O O O O

G. ASC formation quantified by immunofluorescence-microscopy in SP/N stimulated primary macrophages after vaccination (n = 5 individuals).


H . Quantification of cell death in T2 macrophages ( n = 4 individuals ) treated with R406 prior to SP / N - stimulation .

O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-CELL_TYPE O O O O O O O O B-SMALL_MOLECULE O O B-GENEPROD O O O O O

H. Quantification of cell death in T2 macrophages (n = 4 individuals) treated with R406 prior to SP/N-stimulation.


I - K . mROS quantified by flow cytometry using MitoSOX red staining at T0 and T2 in SP / N - stimulated macrophages without inhibitor ( I [ exemplary results ] , J [ T0 : n = 5 individuals ; T2 : n = 5 individuals ] )

O O O O B-SMALL_MOLECULE O O B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE B-EXP_ASSAY O O O O O B-GENEPROD O O O O B-CELL_TYPE O O 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-K. mROS quantified by flow cytometry using MitoSOX red staining at T0 and T2 in SP/N-stimulated macrophages without inhibitor (I [exemplary results], J [T0: n = 5 individuals; T2: n = 5 individuals])


mROS quantified MitoSOX red staining at T0 and T2 in SP / N - stimulated macrophages with R406 treatment ( n = 4 individuals ) ( K ) .

B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE B-EXP_ASSAY O O O O O B-GENEPROD O O O O B-CELL_TYPE O B-SMALL_MOLECULE O O O O O O O O O O O

mROS quantified MitoSOX red staining at T0 and T2 in SP/N-stimulated macrophages with R406 treatment (n = 4 individuals) (K).


L . Western blot analysis of SOD2 in macrophages at T0 and T2 treated with SP / N or LPS / N .

O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-CELL_TYPE O O O O O O B-GENEPROD O O O B-SMALL_MOLECULE O O O

L. Western blot analysis of SOD2 in macrophages at T0 and T2 treated with SP/N or LPS/N.


M . Mitochondrial membrane potential determined with flow cytometry of tetramethylrhodamine methyl ester ( TMRM ) stained macrophages at T0 ( n = 5 individuals ) and T2 ( n = 5 individuals ) stimulated with SP / N . N . T2 - macrophages ( n = 4 individuals ) were treated with the SYK - inhibitor R406 . Data information : For statistical analysis , one - or two - way ANOVA with tukey post hoc test was used . Box plots indicate the median and the upper and lower quartile . Outliers are plotted as individual dots ( outside the 10 - 90 percentile ) . Scatter dot plots show mean ± SD . * p < 0 . 05 ; * * p < 0 . 01 , * * * p < 0 . 001 . ( S - protein : SP , lipopolysaccharide : LPS , nigericin : N )

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O B-EXP_ASSAY B-CELL_TYPE O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O B-CELL_TYPE O O O O O O O O O O B-GENEPROD O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O

M. Mitochondrial membrane potential determined with flow cytometry of tetramethylrhodamine methyl ester (TMRM) stained macrophages at T0 (n = 5 individuals) and T2 (n = 5 individuals) stimulated with SP/N. N. T2-macrophages (n = 4 individuals) were treated with the SYK-inhibitor R406. Data information: For statistical analysis, one- or two-way ANOVA with tukey post hoc test was used. Box plots indicate the median and the upper and lower quartile. Outliers are plotted as individual dots (outside the 10-90 percentile). Scatter dot plots show mean ± SD. *p<0.05; **p<0.01, ***p<0.001. (S-protein: SP, lipopolysaccharide: LPS, nigericin: N)


A - H . ( A ) T cells at T0 ( n = 6 individuals ) and T2 ( n = 10 individuals ) were incubated with SP / N - stimulated macrophages . Co - cultures of unstimulated macrophages were used as control ( con ) . CD4 + subpopulation analyses performed with flow cytometry ( Experimental setup : Fig EV3 ) . ( B ) Quantification of fold change of the CD4 + T cell subpopulation upon SP / N stimulation ( T0 ; T2 ) . Dotted line indicates the baseline ( fold change = 1 . 0 ) . ( C ) Fold change of CD4 + naïve T cell and CD4 + effector memory T cell populations ( D ) upon co - culture of macrophages stimulated with SP / N in presence of MCC950 or R406 . Identical analyses performed with CD8 + T cells ( E , F , G , H ) .

O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-CELL_TYPE O O O O O O B-CELL_TYPE O O O O O O O O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O B-GENEPROD O O O O O 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-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O O O O B-CELL_TYPE O O B-GENEPROD O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O O O O O

A-H. (A) T cells at T0 (n = 6 individuals) and T2 (n = 10 individuals) were incubated with SP/N-stimulated macrophages. Co-cultures of unstimulated macrophages were used as control (con). CD4+ subpopulation analyses performed with flow cytometry (Experimental setup: Fig EV3). (B) Quantification of fold change of the CD4+ T cell subpopulation upon SP/N stimulation (T0; T2). Dotted line indicates the baseline (fold change = 1.0). (C) Fold change of CD4+ naïve T cell and CD4+ effector memory T cell populations (D) upon co-culture of macrophages stimulated with SP/N in presence of MCC950 or R406. Identical analyses performed with CD8+ T cells (E, F, G, H).


I , J . PD - 1 expression was analyzed in CD4 + ( I ) and CD8 + T cells ( J ) by flow cytometry upon co - culture with SP / N - stimulated T0 ( n = 6 individuals ) or T2 ( n = 11 individuals ) macrophages . Co - cultures of unstimulated macrophages were used as control .

O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O B-GENEPROD O O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O B-CELL_TYPE O O O O O

I, J. PD-1 expression was analyzed in CD4+ (I) and CD8+ T cells (J) by flow cytometry upon co-culture with SP/N-stimulated T0 (n = 6 individuals) or T2 (n = 11 individuals) macrophages. Co-cultures of unstimulated macrophages were used as control.


K , L . Interferon - γ quantified in supernatants of corresponding co - cultures , untreated ( T0 [ n = 6 individuals ] and T2 [ n = 11 individuals ] ) ( K ) and treated with MCC950 or R406 ( L ) at T2 ( n = 11 individuals ) .

O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O 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-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O

K, L. Interferon-γ quantified in supernatants of corresponding co-cultures, untreated (T0 [n = 6 individuals] and T2 [n = 11 individuals]) (K) and treated with MCC950 or R406 (L) at T2 (n = 11 individuals).


M . IL - 1β concentrations determined in SP / N - stimulated monocyte - derived macrophages at 34 weeks after 2nd vaccination ( first booster ) ( T4 = 7 individuals ) and 2 weeks after the 3rd vaccination ( second booster ) ( T5 = 11 individuals ) .

O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O O O O B-CELL_TYPE O O B-CELL_TYPE O O O O O O O O O O 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. IL-1β concentrations determined in SP/N-stimulated monocyte-derived macrophages at 34 weeks after 2nd vaccination (first booster) (T4 = 7 individuals) and 2 weeks after the 3rd vaccination (second booster) (T5 = 11 individuals).


A . The Armi complex immunoisolated from OSCs using anti - Armi antibodies contains Yb , SoYb , and Vret .

O O O B-GENEPROD O B-EXP_ASSAY O O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O

A. The Armi complex immunoisolated from OSCs using anti-Armi antibodies contains Yb, SoYb, and Vret.


B . The Vret complex immunoisolated from OSCs using anti - Vret antibodies contains Yb , Armi , and SoYb .

B. The Vret complex immunoisolated from OSCs using anti-Vret antibodies contains Yb, Armi, and SoYb.


C . Silver staining of protein components in the Vret complex The lane α - Vret ( left ) contained only the anti - Vret antibodies used in these experiments .

O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O

C. Silver staining of protein components in the Vret complex The lane α-Vret (left) contained only the anti-Vret antibodies used in these experiments.


D . Immunoprecipitation and western blotting were performed in OSCs upon RNAi treatment ( kd ) . Even without the association of Yb , Armi , SoYb , and Vret associated . Yb and Armi remained bound to each other upon SoYb or Vret depletion . The level of SoYb ( input ) decreased upon Vret depletion . Likewise , the level of Vret ( input ) decreased upon SoYb depletion .

O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O

D. Immunoprecipitation and western blotting were performed in OSCs upon RNAi treatment (kd). Even without the association of Yb, Armi, SoYb, and Vret associated. Yb and Armi remained bound to each other upon SoYb or Vret depletion. The level of SoYb (input) decreased upon Vret depletion. Likewise, the level of Vret (input) decreased upon SoYb depletion.


E . Upon Armi depletion , SoYb and Vret were still bound to each other but this heterodimer no longer associated with Yb .

O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O B-EXP_ASSAY O O O O O O O O B-EXP_ASSAY O B-GENEPROD O

E. Upon Armi depletion, SoYb and Vret were still bound to each other but this heterodimer no longer associated with Yb.


F . Immunofluorescence analyses in OSCs . Yb bodies disappeared upon Yb depletion , which led Armi , SoYb and Vret to be localized in the cytosol . Yb bodies were detected in OSCs upon Armi depletion , but SoYb and Vret localized in the cytosol . Armi was detected at Yb bodies upon SoYb or Vret depletion . Yb bodies are indicated by white arrows . Nuclei are shown in blue . Scale bar : 2 μm .

O O B-EXP_ASSAY O O O O B-SUBCELLULAR I-SUBCELLULAR O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-EXP_ASSAY O O B-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR O O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O O B-SUBCELLULAR O B-GENEPROD O O O B-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD O B-GENEPROD O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O O O O O O O O O

F. Immunofluorescence analyses in OSCs. Yb bodies disappeared upon Yb depletion, which led Armi, SoYb and Vret to be localized in the cytosol. Yb bodies were detected in OSCs upon Armi depletion, but SoYb and Vret localized in the cytosol. Armi was detected at Yb bodies upon SoYb or Vret depletion. Yb bodies are indicated by white arrows. Nuclei are shown in blue. Scale bar: 2 μm.


B . In Yb - lacking OSCs ( Yb kd ) , WT Yb and ∆ eTud bound to Armi but ∆ eTud did not . Note that WT Yb and both mutants were engineered to be RNAi - resistant by mutations . β - gal was used as a negative control .

O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O

B. In Yb-lacking OSCs (Yb kd), WT Yb and ∆eTud bound to Armi but ∆eTud did not. Note that WT Yb and both mutants were engineered to be RNAi-resistant by mutations. β-gal was used as a negative control.


C . In normal OSCs , WT Yb and ∆ eTud bound with Yb but ∆ Hel - C did not .

O O O O O O O B-GENEPROD O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O

C. In normal OSCs, WT Yb and ∆eTud bound with Yb but ∆Hel-C did not.


D . Flag - Hel - C copurified with Myc - Hel - C but not with BSD ( negative control ) .

O O B-GENEPROD O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O B-GENEPROD O O O O O

D. Flag-Hel-C copurified with Myc-Hel-C but not with BSD (negative control).


E . Subcellular localization of WT Yb and deletion mutants ∆ Hel - C and ∆ eTud . Myc - Yb is shown in green . Endogenous Armi is shown in red . DAPI shows nuclei ( blue ) . Scale bar : 5 μm . Western blotting ( right ) showing WT Yb and deletion mutants ∆ Hel - C and ∆ eTud expressed in Yb - lacking OSCs .

O O O B-EXP_ASSAY O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O B-SUBCELLULAR O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O

E. Subcellular localization of WT Yb and deletion mutants ∆Hel-C and ∆eTud. Myc-Yb is shown in green. Endogenous Armi is shown in red. DAPI shows nuclei (blue). Scale bar: 5 μm. Western blotting (right) showing WT Yb and deletion mutants ∆Hel-C and ∆eTud expressed in Yb-lacking OSCs.


F . CLIP shows that WT Yb and ∆ Hel - C bind RNA in vivo but ∆ eTud does not .

O O B-EXP_ASSAY O O O B-GENEPROD O O O O O B-EXP_ASSAY B-SMALL_MOLECULE O O O O O O O O

F. CLIP shows that WT Yb and ∆Hel-C bind RNA in vivo but ∆eTud does not.


A . Rescue experiments showing that ∆ Hel - C but not ∆ eTud restored piRNA biogenesis attenuated by endogenous Yb depletion . Flag - Piwi was expressed upon endogenous Yb depletion .

O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-EXP_ASSAY O O B-GENEPROD O O

A. Rescue experiments showing that ∆Hel-C but not ∆eTud restored piRNA biogenesis attenuated by endogenous Yb depletion. Flag-Piwi was expressed upon endogenous Yb depletion.


B . The levels of transposon mdg1 Bars and error bars represent means ± SEM values of three independent experiments . p values were calculated by t - test . * : p < 0 . 05 .

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

B. The levels of transposon mdg1 Bars and error bars represent means ± SEM values of three independent experiments. p values were calculated by t-test. *: p < 0.05.