Reduction of cell wall synthesis gene expression causes a tolerance defect . ( C ) Survival of a ∆ pbp1b ∆ pbp1a Piptg : pbp1a strain after 6 hours of PenG exposure . Pup indicates conditions of high IPTG concentrations ( 100 µM ) , Pdown low IPTG concentration ( 2 µM ) . Survival fraction is cfu / ml of PenG - treated cultures normalized to untreated cultures grown in and plated on medium containing the same IPTG concentration .

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

Reduction of cell wall synthesis gene expression causes a tolerance defect. (C) Survival of a ∆pbp1b ∆pbp1a Piptg:pbp1a strain after 6 hours of PenG exposure. Pup indicates conditions of high IPTG concentrations (100 µM), Pdown low IPTG concentration (2 µM). Survival fraction is cfu/ml of PenG-treated cultures normalized to untreated cultures grown in and plated on medium containing the same IPTG concentration.


( D ) Time - dependent killing experiment in the presence of PenG ( 100 µg / ml , 10 x MIC ) . All data are means ( + / - standard error ) of 3 independent biological replicates .

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

(D) Time-dependent killing experiment in the presence of PenG (100 µg/ml, 10 x MIC). All data are means (+/- standard error) of 3 independent biological replicates.


( C ) Time - course of iron accumulation in PenG - treated WT and ∆ vxrAB cells as measured by ICP - MS . Data are average of 3 independent biological replicates , error bars represent standard deviation . ∗ ∗ P = 0 . 001 ; ∗ ∗ ∗ P < 0 . 0003 ( paired t - test ) .

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

(C) Time-course of iron accumulation in PenG-treated WT and ∆vxrAB cells as measured by ICP-MS. Data are average of 3 independent biological replicates, error bars represent standard deviation. ∗∗P=0.001; ∗∗∗P<0.0003 (paired t-test).


( B ) . To test PenG - mediated induction of Fur - regulated genes , S1 nuclease mapping assays were performed on the small RNA ryhB , the RyhB target sodB , hutA / B , coding for heme transport systems and vc2212 , which encodes a component of a ferric citrate uptake system ) . Cells were grown to mid - exponential phase followed by exposure to H2O2 or PenG for the indicated duration . Numbers represent expression levels normalized to untreated control ( averages from three independent replicates ) . White demarcation line between time points indicates that these data points were not adjacent in the original experiment but were cropped for presentation purposes . Source data is available as supplemental material .

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

(B). To test PenG-mediated induction of Fur-regulated genes, S1 nuclease mapping assays were performed on the small RNA ryhB, the RyhB target sodB, hutA/B, coding for heme transport systems and vc2212, which encodes a component of a ferric citrate uptake system). Cells were grown to mid-exponential phase followed by exposure to H2O2 or PenG for the indicated duration. Numbers represent expression levels normalized to untreated control (averages from three independent replicates). White demarcation line between time points indicates that these data points were not adjacent in the original experiment but were cropped for presentation purposes. Source data is available as supplemental material.


( B ) and ( C ) Iron uptake mutants restore ∆ vxrAB growth on 10 % sucrose . The indicated strains were grown to exponential phase ( OD600 ~ 0 . 5 ) in LB medium and spot - plated on 10 % Sucrose plates with or without additional iron sulfate . An example is shown in ( B ) , quantification of cfu is shown in ( C ) . Data information : Data are average of 3 independent biological replicates , error bars represent standard deviation . ∗ ∗ ∗ P < 0 . 0003 ; ∗ ∗ ∗ ∗ P < 0 . 0001 ( paired t - test ) .

O O O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD B-EXP_ASSAY 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 B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE 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 O O O O O O O O O O O O O O O O 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) and (C) Iron uptake mutants restore ∆vxrAB growth on 10% sucrose. The indicated strains were grown to exponential phase (OD600~0.5) in LB medium and spot-plated on 10% Sucrose plates with or without additional iron sulfate. An example is shown in (B), quantification of cfu is shown in (C). Data information: Data are average of 3 independent biological replicates, error bars represent standard deviation. ∗∗∗ P<0.0003; ∗∗∗∗ P<0.0001 (paired t-test).


( B ) Excretion of V . cholerae . Fecal samples were collected every two hours and assessed for their viable cell content ( Colony Forming Units , CFU ) to monitor the level of V . cholerae excretion . Each symbol represents fecal samples from an individual mouse ( n = 4 ) . The number of bacterial CFU was normalized per milligram of feces ( CFU / mg ) . Error bars represent standard deviation . ∗ ∗ P < 0 . 005 ; ∗ ∗ ∗ P < 0 . 001 ( two - way ANOVA ) .

O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O B-TISSUE O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O B-TISSUE O O O O B-ORGANISM O O O O O O O B-EXP_ASSAY O O B-EXP_ASSAY O O O O O B-TISSUE 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 O O O

(B) Excretion of V. cholerae. Fecal samples were collected every two hours and assessed for their viable cell content (Colony Forming Units, CFU) to monitor the level of V. cholerae excretion. Each symbol represents fecal samples from an individual mouse (n=4). The number of bacterial CFU was normalized per milligram of feces (CFU/mg). Error bars represent standard deviation. ∗∗ P<0.005; ∗∗∗ P<0.001 (two-way ANOVA).


( E ) Time - dependent killing of respiratory chain mutants ( ∆ nqrA , encoding NADH dehydrogenase and ∆ ubiA , encoding an early step of ubiquinone synthesis ) in the presence of PenG ( 100 µg / ml , 10xMIC ) . All strains were grown in LB 0 . 2 % glucose . Data information : Data are mean of three independent biological replicates and error bars represent standard deviation .

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

(E) Time-dependent killing of respiratory chain mutants (∆nqrA, encoding NADH dehydrogenase and ∆ubiA, encoding an early step of ubiquinone synthesis) in the presence of PenG (100 µg/ml, 10xMIC). All strains were grown in LB 0.2 % glucose. Data information: Data are mean of three independent biological replicates and error bars represent standard deviation.


( F ) Growth - phase dependent killing assay of ROS detoxification systems ( ∆ fur , ∆ sodB , and ∆ oxyR1 ∆ katG ∆ katB ) . At designated time - points , a 5 ml aliquot was withdrawn and exposed to 100 µg / ml PenG for 3 h . CFU / mL for each time point was assessed before ( solid lines ) and after addition of PenG ( dotted lines ) . Data information : Data are mean of three independent biological replicates and error bars represent standard deviation .

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

(F) Growth-phase dependent killing assay of ROS detoxification systems (∆fur, ∆sodB, and ∆oxyR1∆katG∆katB). At designated time-points, a 5 ml aliquot was withdrawn and exposed to 100 µg/ml PenG for 3 h. CFU/mL for each time point was assessed before (solid lines) and after addition of PenG (dotted lines). Data information: Data are mean of three independent biological replicates and error bars represent standard deviation.


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 .

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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 B-EXP_ASSAY 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 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 ) .

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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 O 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 O 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 O 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 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 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 B-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 O O O O O O O O O O O O O O O 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 B-EXP_ASSAY 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 B-EXP_ASSAY 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 .

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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 ) .

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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.


( G ) GTT or ITT in 17 - week - old wild - type or JFKTG mice fed on HFD . Error bars represent mean ± SEM ( n = 6 , * p < 0 . 05 , paired two - tailed Student ' s t - test ) .

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(G) GTT or ITT in 17-week-old wild-type or JFKTG mice fed on HFD. Error bars represent mean ± SEM (n = 6, *p < 0.05, paired two-tailed Student's t-test).


( H ) H & E and Oil Red O staining of liver sections from wild - type or JFKTG mice fed on ND or HFD . Scale bar , 100 μm .

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(H) H&E and Oil Red O staining of liver sections from wild-type or JFKTG mice fed on ND or HFD. Scale bar, 100 μm.


( D ) The body weight of male wild - type or JfkKO mice fed on HFD . Error bars represent mean ± SEM ( n = 6 , * p < 0 . 05 , paired two - tailed Student ' s t - test ) .

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(D) The body weight of male wild-type or JfkKO mice fed on HFD. Error bars represent mean ± SEM (n = 6, *p < 0.05, paired two-tailed Student's t-test).


( G ) H & E and Oil Red O staining of liver sections from wild - type or JfkKO mice fed on HFD . Scale bar , 100 μm .

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(G) H&E and Oil Red O staining of liver sections from wild-type or JfkKO mice fed on HFD. Scale bar, 100 μm.


( H ) Wild - type or JfkKO mice were fed with HFD for 11 weeks prior to injection of adenovirally - delivered vector or JFK via tail vein . Seven days after injection , total proteins prepared from liver tissues were subjected to western blotting analysis for the expression of Jfk or measurement for hepatic TG and NEFA content . Error bars represent mean ± SEM ( n = 6 , * p < 0 . 05 , one - way ANOVA with Tukey ' s HSD test ) . H & E and Oil Red O staining of liver sections are shown . Scale bar , 100 μm .

O O O O O O O B-GENEPROD B-ORGANISM O O O O O O O O O O O B-ORGANISM O O O O B-GENEPROD O B-TISSUE I-TISSUE O O O O O O O O O O B-TISSUE O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY O B-GENEPROD O O O B-TISSUE 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 B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-TISSUE O O O O O O O O O O

(H) Wild-type or JfkKO mice were fed with HFD for 11 weeks prior to injection of adenovirally-delivered vector or JFK via tail vein. Seven days after injection, total proteins prepared from liver tissues were subjected to western blotting analysis for the expression of Jfk or measurement for hepatic TG and NEFA content. Error bars represent mean ± SEM (n = 6, *p < 0.05, one-way ANOVA with Tukey's HSD test). H&E and Oil Red O staining of liver sections are shown. Scale bar, 100 μm.


( A ) HepG2 cells were stably transfected with vector or FLAG - JFK . Cellular extracts were immunopurified with anti - FLAG immunoafﬁnity resin and eluted with FLAG peptides . The eluates were resolved on SDS - PAGE and silver - stained . The proteins bands were retrieved and analyzed by mass spectrometry .

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

(A) HepG2 cells were stably transfected with vector or FLAG-JFK. Cellular extracts were immunopurified with anti-FLAG immunoafﬁnity resin and eluted with FLAG peptides. The eluates were resolved on SDS-PAGE and silver-stained. The proteins bands were retrieved and analyzed by mass spectrometry.


( B ) Cellular extracts from HepG2 cells were immunoprecipitated with antibodies against ING5 followed by immunoblotting with the antibodies against the indicated proteins .

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(B) Cellular extracts from HepG2 cells were immunoprecipitated with antibodies against ING5 followed by immunoblotting with the antibodies against the indicated proteins.


( E ) HepG2 cells were transfected with FLAG - JFK and treated with 50 μg / ml CHX for the indicated times in the presence or absence of MG132 for western blotting analysis . Quantitation was done by densitometry and expressed as signals of ING5 / β - actin .

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(E) HepG2 cells were transfected with FLAG-JFK and treated with 50 μg/ml CHX for the indicated times in the presence or absence of MG132 for western blotting analysis. Quantitation was done by densitometry and expressed as signals of ING5/β-actin.


( H ) Bacterially - expressed GST - JFK or JFK mutants was incubated with in vitro transcribed / translated ING5 for in vitro ubiquitination assays .

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(H) Bacterially-expressed GST-JFK or JFK mutants was incubated with in vitro transcribed/translated ING5 for in vitro ubiquitination assays.


( D ) ChIP - seq track at the representative promoter of target genes . The red rectangles indicate the peak regions / binding sites of ING5 on the target genes promoter .

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(D) ChIP-seq track at the representative promoter of target genes. The red rectangles indicate the peak regions/binding sites of ING5 on the target genes promoter.


( C ) HepG2 cells were treated with control siRNAs or siRNAs against JFK or / and ING5 , or transfected with vector , JFK , or / and ING5 . Cellular extracts were prepared for western blotting analysis with the indicated antibodies .

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(C) HepG2 cells were treated with control siRNAs or siRNAs against JFK or/and ING5, or transfected with vector, JFK, or/and ING5. Cellular extracts were prepared for western blotting analysis with the indicated antibodies.


( D ) HepG2 cells were transfected with vector or JFK , AMPKα1 , or / and ING5 , or treated with control siRNAs or siRNAs against JFK , AMPKα1 , or / and ING5 , Cellular extracts were prepared for AMPK activity assays by ELISA . Error bars represent mean ± SD for triplicate experiments ( * p < 0 . 05 , paired two - tailed Student ' s t - test ) .

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(D) HepG2 cells were transfected with vector or JFK, AMPKα1, or/and ING5, or treated with control siRNAs or siRNAs against JFK, AMPKα1, or/and ING5, Cellular extracts were prepared for AMPK activity assays by ELISA. Error bars represent mean ± SD for triplicate experiments (*p < 0.05, paired two-tailed Student's t-test).


( D ) The measurement of AMPK activity and the rate of fatty acid β - oxidation of wild - type or JfkKO mice . Cellular extracts prepared from MEFs or liver tissues were subjected to AMPK activity assays by ELISA . Mitochondria were isolated from MEFs or liver tissues , and the rate of fatty acid β - oxidation was quantified by spectrophotometric detection for ferricyanide - trapped reduced products . Error bars represent mean ± SEM ( n = 6 , * p < 0 . 05 , paired two - tailed Student ' s t - test ) .

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

(D) The measurement of AMPK activity and the rate of fatty acid β-oxidation of wild-type or JfkKO mice. Cellular extracts prepared from MEFs or liver tissues were subjected to AMPK activity assays by ELISA. Mitochondria were isolated from MEFs or liver tissues, and the rate of fatty acid β-oxidation was quantified by spectrophotometric detection for ferricyanide-trapped reduced products. Error bars represent mean ± SEM (n = 6, *p < 0.05, paired two-tailed Student's t-test).


Wild - type or JfkKO mice were fed with HFD for 11 weeks prior to injection of adenovirally - delivered vector control , shIng5 , or shAmpkα1 via tail vein . Seven days after injection , cellular extracts prepared from liver tissues were subjected to western blotting analysis or measurement for hepatic TG and NEFA content ( E ) . Asterisk ( * ) represents significant comparison to wild - type mice , octothorpe ( # ) represents significant comparison to JfkKO mice . Error bars represent mean ± SEM ( n = 6 , * p < 0 . 05 , # p < 0 . 05 , one - way ANOVA with Tukey ' s HSD test ) .

O O O O B-GENEPROD B-ORGANISM O O O O O O O O O O O B-ORGANISM O O O O O O O O O O B-TISSUE I-TISSUE O O O O O O O O O O B-TISSUE O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-TISSUE B-SMALL_MOLECULE O B-SMALL_MOLECULE 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 B-GENEPROD B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Wild-type or JfkKO mice were fed with HFD for 11 weeks prior to injection of adenovirally-delivered vector control, shIng5, or shAmpkα1 via tail vein. Seven days after injection, cellular extracts prepared from liver tissues were subjected to western blotting analysis or measurement for hepatic TG and NEFA content (E). Asterisk (*) represents significant comparison to wild-type mice, octothorpe (#) represents significant comparison to JfkKO mice. Error bars represent mean ± SEM (n = 6, *p < 0.05, #p < 0.05, one-way ANOVA with Tukey's HSD test).


D . Expression distribution of lncORF in both groups by Ribo - seq .

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

D. Expression distribution of lncORF in both groups by Ribo-seq.


H . Scatter diagram of lncRNA - seq in HCT116 / L - OHP and HCT116 cells .

H. Scatter diagram of lncRNA-seq in HCT116/L-OHP and HCT116 cells.


A . Schematic diagram of sucrose density gradient ultracentrifugation for RNC isolation , and PCR verification of the 12 screened lncRNA in both RNC - RNA and total RNA from two groups of CRC cells , among which the translation level of lnc - AP ( marked with a green box ) was the most notably dysregulated in both groups of drug - resistant cells ( n = 3 , biological replicates , mean ± SEM , t test ) . Data information : * for P < 0 . 05 ; * * for P < 0 . 01 ; NS for no significance .

O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR O O O B-EXP_ASSAY O O O O O B-GENEPROD O O B-SUBCELLULAR O O O O O O O O O B-DISEASE 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 O O O O O O O O O O O O O O O O O O O O O O O O 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. Schematic diagram of sucrose density gradient ultracentrifugation for RNC isolation, and PCR verification of the 12 screened lncRNA in both RNC-RNA and total RNA from two groups of CRC cells, among which the translation level of lnc-AP (marked with a green box) was the most notably dysregulated in both groups of drug-resistant cells (n = 3, biological replicates, mean±SEM, t test). Data information: * for P < 0.05; ** for P < 0.01; NS for no significance.


B . PCR analysis of lnc - AP in serum samples from oxaliplatin - resistant ( PD , n = 15 ) and - sensitive ( PR , n = 9 ) CRC patients ( mean ± SEM , t test ) .

O O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O B-TISSUE O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O B-DISEASE O O O O O O O O O O

B. PCR analysis of lnc-AP in serum samples from oxaliplatin-resistant (PD, n = 15) and -sensitive (PR, n = 9) CRC patients (mean±SEM, t test).


C . In the public database lncCAR , high expression level of lnc - AP was correlated with long overall survival in CRC patients .

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C. In the public database lncCAR, high expression level of lnc-AP was correlated with long overall survival in CRC patients.


E . PCR and WB analysis of HEK293T cells transfected with either lnc - AP - Flag OE plasmid ( n = 3 , biological replicates , mean ± SEM , Dunnett - t test ) . F . PCR and WB analysis of HCT116 / L - OHP and SW480 / L - OHP cells transfected with either lnc - AP - Flag OE plasmid ( n = 3 , biological replicates , mean ± SEM , Dunnett - t test ) . Data information : * for P < 0 . 05 ; * * for P < 0 . 01 ; NS for no significance . Control referred to the corresponding plasmid vector .

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

E. PCR and WB analysis of HEK293T cells transfected with either lnc-AP-Flag OE plasmid (n = 3, biological replicates, mean±SEM, Dunnett-t test). F. PCR and WB analysis of HCT116/L-OHP and SW480/L-OHP cells transfected with either lnc-AP-Flag OE plasmid (n = 3, biological replicates, mean±SEM, Dunnett-t test). Data information: * for P < 0.05; ** for P < 0.01; NS for no significance. Control referred to the corresponding plasmid vector.


J . IF detection of pep - AP in HCT116 / L - OHP and SW480 / L - OHP cells transfected with either HA - lnc - AP OE plasmid . Scale bar , 10 µm .

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

J. IF detection of pep-AP in HCT116/L-OHP and SW480/L-OHP cells transfected with either HA-lnc-AP OE plasmid. Scale bar, 10 µm.


B . CCK - 8 assay of inhibition ratio by L - OHP in two groups of CRC cells with down - or up - regulation of lnc - AP ( n = 3 , biological replicates , mean ± SEM ) . Data information : * si - NC was the abbreviation of commercial normal control of siRNA .

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

B. CCK-8 assay of inhibition ratio by L-OHP in two groups of CRC cells with down- or up-regulation of lnc-AP (n = 3, biological replicates, mean±SEM). Data information: * si-NC was the abbreviation of commercial normal control of siRNA.


C . IF detection of H2AFX for DNA damage by L - OHP in two groups of CRC cells with down - or up - regulation of lnc - AP , and nuclei were stained with DAPI ( n = 3 , biological replicates , mean ± SEM , Dunnett - t test ) .

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

C. IF detection of H2AFX for DNA damage by L-OHP in two groups of CRC cells with down- or up-regulation of lnc-AP, and nuclei were stained with DAPI (n = 3, biological replicates, mean±SEM, Dunnett-t test).


G . Verification of the interaction between CEBPA and the upstream region of AP002387 via ChIP assay followed by PCR ( n = 3 , biological replicates , mean ± SEM , t test ) . Data information : * for P < 0 . 05 ; * * for P < 0 .

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

G. Verification of the interaction between CEBPA and the upstream region of AP002387 via ChIP assay followed by PCR (n = 3, biological replicates, mean±SEM, t test). Data information: * for P < 0.05; ** for P < 0.01


H . PCR analysis of lnc - AP attenuation and restoration in HCT116 and SW480 cells ( n = 3 , biological replicates , mean ± SEM , Dunnett - t test ) . Data information : * for P < 0 . 05 ; * * for P < 0 . 01 ; NS for no significance . Control referred to the corresponding plasmid vector and si - NC was the abbreviation of commercial normal control of siRNA .

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

H. PCR analysis of lnc-AP attenuation and restoration in HCT116 and SW480 cells (n = 3, biological replicates, mean±SEM, Dunnett-t test). Data information: * for P < 0.05; ** for P < 0.01; NS for no significance. Control referred to the corresponding plasmid vector and si-NC was the abbreviation of commercial normal control of siRNA.


J . CCK - 8 assay of inhibition ratio by L - OHP in HCT116 and SW480 cells with lnc - AP / pep - AP attenuation and restoration ( n = 3 , biological replicates , mean ± SEM ) . Data information : * si - NC was the abbreviation of commercial normal control of siRNA .

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

J. CCK-8 assay of inhibition ratio by L-OHP in HCT116 and SW480 cells with lnc-AP/pep-AP attenuation and restoration (n = 3, biological replicates, mean±SEM). Data information: * si-NC was the abbreviation of commercial normal control of siRNA.


D . Survival analysis correlated with TALDO1 using TCGA data . Upper : the Kaplan curve of overall survival probability for patients with high or low level of TALDO1 . Lower : the expression graph of TALDO1 in patients who were dead or not .

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

D. Survival analysis correlated with TALDO1 using TCGA data. Upper: the Kaplan curve of overall survival probability for patients with high or low level of TALDO1. Lower: the expression graph of TALDO1 in patients who were dead or not.


F . IF detection of the colocalization of pep - AP - Flag and TALDO1 in HCT116 / L - OHP and SW480 / L - OHP cells . Scale bar , 10 µm .

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

F. IF detection of the colocalization of pep-AP-Flag and TALDO1 in HCT116/L-OHP and SW480/L-OHP cells. Scale bar, 10 µm.


G . WB confirmation of the interaction between HA - pep - AP and TALDO1 in HCT116 / L - OHP and SW480 / L - OHP cells .

G. WB confirmation of the interaction between HA-pep-AP and TALDO1 in HCT116/L-OHP and SW480/L-OHP cells.


A . WB analysis of TALDO1 overexpression or knock down in two groups of CRC cells , which was quantified by gray analysis on the right ( n = 3 , biological replicates , mean ± SEM , t test ) . Data information : * for P < 0 . 05 ; * * for P < 0 . 01 . Control referred to the corresponding plasmid vector and si - NC was the abbreviation of commercial normal control of siRNA .

O O B-EXP_ASSAY O O B-GENEPROD O O O O O O O O B-DISEASE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. WB analysis of TALDO1 overexpression or knock down in two groups of CRC cells, which was quantified by gray analysis on the right (n = 3, biological replicates, mean±SEM, t test). Data information: * for P < 0.05; ** for P < 0.01. Control referred to the corresponding plasmid vector and si-NC was the abbreviation of commercial normal control of siRNA.


D . Cellular ROS was measured by flow cytometry and shown in both curve and bar charts ( n = 3 , biological replicates , mean ± SEM , t test ) Data information : * for P < 0 . 05 ; * * for P < 0 . 01 . Control referred to the corresponding plasmid vector and si - NC was the abbreviation of commercial normal control of siRNA .

O O O B-SMALL_MOLECULE 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 O O O O O O O O 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. Cellular ROS was measured by flow cytometry and shown in both curve and bar charts (n = 3, biological replicates, mean±SEM, t test) Data information: * for P < 0.05; ** for P < 0.01. Control referred to the corresponding plasmid vector and si-NC was the abbreviation of commercial normal control of siRNA.


E . MMP detection . Scale bar , 10 µm . F . IF detection of H2AFX for DNA damage by L - OHP . Scale bar , 10 µm .

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

E. MMP detection. Scale bar, 10 µm. F. IF detection of H2AFX for DNA damage by L-OHP. Scale bar, 10 µm.


E . Cellular ROS was measured by flow cytometry and shown in both curve ( left ) and bar ( right ) charts ( n = 3 , biological replicates , mean ± SEM , Dunnett - t test ) Data information : * for P < 0 . 05 ; * * for P < 0 . 01 ; NS for no significance . Control referred to the corresponding plasmid vector and si - NC was the abbreviation of commercial normal control of siRNA .

O O O B-SMALL_MOLECULE 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 O O O O O O O O O O O O O O O O O O O O O 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. Cellular ROS was measured by flow cytometry and shown in both curve (left) and bar (right) charts (n = 3, biological replicates, mean±SEM, Dunnett-t test) Data information: * for P < 0.05; ** for P < 0.01; NS for no significance. Control referred to the corresponding plasmid vector and si-NC was the abbreviation of commercial normal control of siRNA.


F . MMP detection with down - or up - regulation of lnc - AP / pep - AP in two groups of CRC cells . Scale bar , 10 µm .

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

F. MMP detection with down- or up-regulation of lnc-AP/pep-AP in two groups of CRC cells. Scale bar, 10 µm.


C . Flow cytometry of the cell apoptosis ratio under L - OHP with down - or up - regulation of lnc - AP / pep - AP in two groups of CRC cells , which was quantified in bar charts on the right ( n = 3 , biological replicates , mean ± SEM , Dunnett - t test ) . Data information : * for P < 0 . 05 ; * * for P < 0 . 01 ; NS for no significance . Control referred to the corresponding plasmid vector and si - NC was the abbreviation of commercial normal control of siRNA .

O O B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-DISEASE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Flow cytometry of the cell apoptosis ratio under L-OHP with down- or up-regulation of lnc-AP/pep-AP in two groups of CRC cells, which was quantified in bar charts on the right (n = 3, biological replicates, mean±SEM, Dunnett-t test). Data information: * for P < 0.05; ** for P < 0.01; NS for no significance. Control referred to the corresponding plasmid vector and si-NC was the abbreviation of commercial normal control of siRNA.


E . CCK - 8 assay of the inhibition ratio by L - OHP with inhibitors of apoptosis Z - VAD - FMK or ferroptosis Fer - 1 in HCT116 / L - OHP and SW480 / L - OHP cells ( n = 3 , biological replicates , mean ± SEM ) . Data information : * Control referred to the corresponding plasmid

E. CCK-8 assay of the inhibition ratio by L-OHP with inhibitors of apoptosis Z-VAD-FMK or ferroptosis Fer-1 in HCT116/L-OHP and SW480/L-OHP cells (n = 3, biological replicates, mean±SEM). Data information: * Control referred to the corresponding plasmid vector


B . Alterations of tumor volume in different groups with or without pep - AP OE ( n = 6 , biological replicates , mean ± SEM ) . Data information : * for P < 0 . 05 ; * * for P < 0 . 01 ; NS for no significance . Control referred to the corresponding plasmid vector .

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 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. Alterations of tumor volume in different groups with or without pep-AP OE (n = 6, biological replicates, mean±SEM). Data information: * for P < 0.05; ** for P < 0.01; NS for no significance. Control referred to the corresponding plasmid vector.


F WB analysis of pep - AP , TALDO1 , PARP , cleaved - PARP and cleaved - caspase - 3 within the tumors in C ( a ) .

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F WB analysis of pep-AP, TALDO1, PARP, cleaved-PARP and cleaved-caspase-3 within the tumors in C(a).


G . Relative NADPH / NADP + ratio within the tumors in C ( a ) ( n = 6 , mean ± SEM , Dunnett - t test ) . H . GSH contents within the tumors in C ( a ) ( n = 6 , mean ± SEM , Dunnett - t test ) . Data information : * for P < 0 . 05 ; * * for P < 0 . 01 ; NS for no significance . Control referred to the corresponding plasmid vector .

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 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 O O O O O O O O O

G. Relative NADPH/NADP+ ratio within the tumors in C(a) (n = 6, mean±SEM, Dunnett-t test). H. GSH contents within the tumors in C(a) (n = 6, mean±SEM, Dunnett-t test). Data information: * for P < 0.05; ** for P < 0.01; NS for no significance. Control referred to the corresponding plasmid vector.


I . IF detection of pep - AP , TALDO1 and H2AFX , TUNEL of DNA fragmentation and IHC of Ki67 within the tumors in C ( a ) .

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

I. IF detection of pep-AP, TALDO1 and H2AFX, TUNEL of DNA fragmentation and IHC of Ki67 within the tumors in C(a).


( a , b ) HeLa cells were treated with 10 μg ml − 1 N - Shh peptide for 24 h . ( a ) Endogenous LC3 was detected by immunoblot in total cell lysates , quantified by densitometric analysis and normalized to actin . Relative levels were expressed in percentage with control cells set to 100 and shown in graph .

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

(a,b) HeLa cells were treated with 10 μg ml−1 N-Shh peptide for 24 h. (a) Endogenous LC3 was detected by immunoblot in total cell lysates, quantified by densitometric analysis and normalized to actin. Relative levels were expressed in percentage with control cells set to 100 and shown in graph.


( a , b ) HeLa cells were treated with 10 μg ml − 1 N - Shh peptide for 24 h . ( b ) mRNA was obtained and levels of Ptch1 or Gli1 and actin mRNA were amplified by standard PCR and visualized in agarose gels . Graph shows mRNA levels quantified by densitometric analysis and normalized to actin .

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

(a,b) HeLa cells were treated with 10 μg ml−1 N-Shh peptide for 24 h.(b) mRNA was obtained and levels of Ptch1 or Gli1 and actin mRNA were amplified by standard PCR and visualized in agarose gels. Graph shows mRNA levels quantified by densitometric analysis and normalized to actin.


( c ) 8xGli - luciferase construct was transfected in HeLa cells for 24 h , followed by treatment with 10 μM purmorphamine , or DMSO as a control , for another 24 h . Graph represents the mean value of the firefly luciferase activity relative to Renilla transfection control .

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

(c) 8xGli-luciferase construct was transfected in HeLa cells for 24 h, followed by treatment with 10 μM purmorphamine, or DMSO as a control, for another 24 h. Graph represents the mean value of the firefly luciferase activity relative to Renilla transfection control.


( d ) Endogenous LC3 - II was detected in cell lysates from cells treated with 10 μM purmorphamine for 24 h , either in the absence or presence of trehalose ( 100 mM ) . Where indicated , bafA1 ( 400 nM ) was added for the last 4 h . Quantification by densitometric analysis relative to actin is shown in the graph .

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

(d) Endogenous LC3-II was detected in cell lysates from cells treated with 10 μM purmorphamine for 24 h, either in the absence or presence of trehalose (100 mM). Where indicated, bafA1 (400 nM) was added for the last 4 h. Quantification by densitometric analysis relative to actin is shown in the graph.


( e ) Cells were treated with 10 μM purmorphamine for 24 h under starvation conditions , either in the presence of bafA1 or DMSO as a control , and LC3 - II and actin levels were detected .

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(e) Cells were treated with 10 μM purmorphamine for 24 h under starvation conditions, either in the presence of bafA1 or DMSO as a control, and LC3-II and actin levels were detected.


( f ) HA - HttQ74 construct was transfected into HeLa cells followed by treatment with 10 μM purmorphamine for 24 h . The percentage of transfected cells with aggregates detected by HA immunofluorescence is shown in the graph . P - values were calculated by odds ratio .

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(f) HA-HttQ74 construct was transfected into HeLa cells followed by treatment with 10 μM purmorphamine for 24 h. The percentage of transfected cells with aggregates detected by HA immunofluorescence is shown in the graph. P-values were calculated by odds ratio.


( g ) U20S cells stably expressing HaloTag - p62 were labelled with HaloTag ligand for 15 min and washed out followed by treatment with purmorphamine . After 48 h , cells were lysed , run on a SDS - polyacrylamide gel electrophoresis and fluorescent HaloTag was visualized using a Typhoon 8600 variable mode imager . Fluorescence was normalized to total protein levels , detected on the same gels by Kryton Fluorescence protein stain . A representative gel and its quantitated normalized levels are shown . In all panels , graphs show mean values and error bars represent s . d . from a triplicate experiment representative of at least three independent experiments . Statistical analyses were performed by two - tail Student ' s t - test unless indicated : * * * P0 . 001 ; * * P0 . 01 ; * P0 . 05 . SDS - PAGE , SDS - polyacrylamide gel electrophoresis .

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(g) U20S cells stably expressing HaloTag-p62 were labelled with HaloTag ligand for 15 min and washed out followed by treatment with purmorphamine. After 48 h, cells were lysed, run on a SDS-polyacrylamide gel electrophoresis and fluorescent HaloTag was visualized using a Typhoon 8600 variable mode imager. Fluorescence was normalized to total protein levels, detected on the same gels by Kryton Fluorescence protein stain. A representative gel and its quantitated normalized levels are shown. In all panels, graphs show mean values and error bars represent s.d. from a triplicate experiment representative of at least three independent experiments. Statistical analyses were performed by two-tail Student's t-test unless indicated: ***P0.001; **P0.01; *P0.05. SDS-PAGE, SDS-polyacrylamide gel electrophoresis.


( a ) HeLa cells were transiently transfected with pcDNA ( vector ) , Ptch1 or Ptch2 constructs for 48 h . Where indicated , cells were treated with bafA1 for the last 4 h . LC3 - II levels were detected by western blotting and levels were quantified by densitometric analysis relative to tubulin and shown in graph .

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(a) HeLa cells were transiently transfected with pcDNA (vector), Ptch1 or Ptch2 constructs for 48 h. Where indicated, cells were treated with bafA1 for the last 4 h. LC3-II levels were detected by western blotting and levels were quantified by densitometric analysis relative to tubulin and shown in graph.


( b ) HeLa were transfected with Ptch1 or Ptch2 together with HA - HttQ74 and the percentage of transfected cells with aggregates was assessed by HA immunofluorescence . P - values were calculated by odds ratio .

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(b) HeLa were transfected with Ptch1 or Ptch2 together with HA-HttQ74 and the percentage of transfected cells with aggregates was assessed by HA immunofluorescence. P-values were calculated by odds ratio.


( c ) Atg5 + / + or Atg5 − / − MEFs were transfected with GFP - HttQ74 for 48 h , cells were fixed and the percentage of transfected cells with aggregates was scored by direct fluorescence .

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(c) Atg5+/+ or Atg5−/− MEFs were transfected with GFP-HttQ74 for 48 h, cells were fixed and the percentage of transfected cells with aggregates was scored by direct fluorescence.


( d , e ) Endogenous LC3 - II levels were detected in HeLa cells transfected with control , Ptch1 or Ptch2 siRNA and either left untreated or treated with 100 mM trehalose in the last 24 h ( d ) Quantification and statistical analysis is shown in graphs .

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(d,e) Endogenous LC3-II levels were detected in HeLa cells transfected with control, Ptch1 or Ptch2 siRNA and either left untreated or treated with 100 mM trehalose in the last 24 h (d) Quantification and statistical analysis is shown in graphs.


( d , e ) Endogenous LC3 - II levels were detected in HeLa cells transfected with control , Ptch1 or Ptch2 siRNA and either left untreated or treated with400 nM bafA1 for 4 h ( e ) . Quantification and statistical analysis is shown in

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(d,e) Endogenous LC3-II levels were detected in HeLa cells transfected with control, Ptch1 or Ptch2 siRNA and either left untreated or treated with400 nM bafA1 for 4 h (e). Quantification and statistical analysis is shown in graphs


( f ) siRNA - targeting Ptch2 was transfected into HeLa cells stably expressing mRFP - GFP - LC3 and representative confocal microscopy images are shown . Scale bars represent 26 μm . Percentage of GFP - positive vesicles ( autophagosomes ) , RFP - positive vesicles ( total number of vesicles ) or percentage of autolysosomes ( calculated by subtracting numbers of GFP from RFP vesicles ) , quantified using a Cellomics array scan , was calculated relative to control siRNA - transfected cells . Although an increase in autophagosome biogenesis generally increases both the number of GFP + vesicles and the number of GFP - / RFP + vesicles as a result of an enhanced autophagy flux , defects in autophagosome degradation , such as in bafA1 - treated cells , increase GFP + vesicles but not GFP − / RFP + structures , as GFP remains intact . Graph shows the mean value obtained from four independent experiments in triplicate and with control conditions set to 100 . In all panels , unless indicated , graphs show mean values and error bars represent s . d . from a triplicate experiment representative of at least three independent experiments . Statistical analyses were performed by two - tail Student ' s t - test : * * P0 . 01 ; * P0 . 05 ; NS , not significant .

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(f) siRNA-targeting Ptch2 was transfected into HeLa cells stably expressing mRFP-GFP-LC3 and representative confocal microscopy images are shown. Scale bars represent 26 μm. Percentage of GFP-positive vesicles (autophagosomes), RFP-positive vesicles (total number of vesicles) or percentage of autolysosomes (calculated by subtracting numbers of GFP from RFP vesicles), quantified using a Cellomics array scan, was calculated relative to control siRNA-transfected cells. Although an increase in autophagosome biogenesis generally increases both the number of GFP+ vesicles and the number of GFP-/RFP+ vesicles as a result of an enhanced autophagy flux, defects in autophagosome degradation, such as in bafA1-treated cells, increase GFP+ vesicles but not GFP−/RFP+ structures, as GFP remains intact. Graph shows the mean value obtained from four independent experiments in triplicate and with control conditions set to 100. In all panels, unless indicated, graphs show mean values and error bars represent s.d. from a triplicate experiment representative of at least three independent experiments. Statistical analyses were performed by two-tail Student's t-test: **P0.01; *P0.05; NS, not significant.


( a ) Smo siRNA - treated cells were transfected with HA - HttQ74 for the last 24 h and the percentage of transfected cells with aggregates scored in HA - positive cells by immunofluorescence . Graphs show mean values and error bars represent s . d . of a representative experiment performed in triplicate . P - values were calculated by odds ratio .

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(a) Smo siRNA-treated cells were transfected with HA-HttQ74 for the last 24 h and the percentage of transfected cells with aggregates scored in HA-positive cells by immunofluorescence. Graphs show mean values and error bars represent s.d. of a representative experiment performed in triplicate. P-values were calculated by odds ratio.


( b ) siRNA - targeting Smo was transfected into HeLa cells stably expressing mRFP - GFP - LC3 . Representative confocal microscopy images are shown . Bars represent 26 μm . The number of vesicles was analyzed in a Cellomics array scan and percentage was calculated relative to control siRNA - transfected cells . Statistical tests were performed on data from three independent experiments in triplicate . * P0 . 05 ; * * * P0 . 001 .

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(b) siRNA-targeting Smo was transfected into HeLa cells stably expressing mRFP-GFP-LC3. Representative confocal microscopy images are shown. Bars represent 26 μm. The number of vesicles was analyzed in a Cellomics array scan and percentage was calculated relative to control siRNA-transfected cells. Statistical tests were performed on data from three independent experiments in triplicate. *P0.05; ***P0.001.


( a ) Wild - type Gli + / + or Gli1 − / − , Gli2 − / − and Gli3 − / − MEFs were treated with 100 mM trehalose alone or in combination with 10 μM of Pmph for 24 h , or left untreated . Total cell lysates were subjected to immunoblot with anti - LC3 and anti - actin antibodies .

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(a) Wild-type Gli+/+ or Gli1−/−, Gli2−/− and Gli3−/− MEFs were treated with 100 mM trehalose alone or in combination with 10 μM of Pmph for 24 h, or left untreated. Total cell lysates were subjected to immunoblot with anti-LC3 and anti-actin antibodies.


( b ) Gli2 − / − MEFs were transfected for 48 h with either empty vector or Gli2 DNA construct and treated as in a . with 100 mM trehalose alone or in combination with 10 μM of Pmph for 24 h , or left untreated . Total cell lysates were subjected to immunoblot with anti - LC3 and anti - actin antibodies .

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(b) Gli2−/− MEFs were transfected for 48 h with either empty vector or Gli2 DNA construct and treated as in a. with 100 mM trehalose alone or in combination with 10 μM of Pmph for 24 h, or left untreated. Total cell lysates were subjected to immunoblot with anti-LC3 and anti-actin antibodies.


( c ) LC3 - II levels were detected by western blotting of total lysates from wild - type and Gli2 − / − mice embryos ( E12 . 5 ) . For quantification , LC3 - II levels from a total of five wild - type and five Gli2 − / − embryos were normalized to actin , wild - type was set to 100 . Graphs show mean values and error bars represent s . d . Statistical analysis were performed by two - tail Student ' s t - test * * P0 . 01 . Pmph , purmorphamine .

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(c) LC3-II levels were detected by western blotting of total lysates from wild-type and Gli2−/− mice embryos (E12.5). For quantification, LC3-II levels from a total of five wild-type and five Gli2−/− embryos were normalized to actin, wild-type was set to 100. Graphs show mean values and error bars represent s.d. Statistical analysis were performed by two-tail Student's t-test **P0.01. Pmph, purmorphamine.


( a , b ) Control ( a ) or hh [ ts2 ] ( b ) larvae were incubated at permissive temperature ( 18 ° C ) to the third instar stage , then cultured at non - permissive temperature ( 29 ° C ) in well - fed conditions for 24 h . Dissected fat body tissue was incubated in Lysotracker Red and DAPI and imaged unfixed .

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(a,b) Control (a) or hh[ts2] (b) larvae were incubated at permissive temperature (18 °C) to the third instar stage, then cultured at non-permissive temperature (29 °C) in well-fed conditions for 24 h. Dissected fat body tissue was incubated in Lysotracker Red and DAPI and imaged unfixed.


( c ) Lysotracker Red - labelled fat body containing single - cell flipout clones ( GFP positive ) overexpressing patched . Fat body was dissected from well - fed larvae , incubated in Lysotracker Red / DAPI and imaged unfixed .

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(c) Lysotracker Red-labelled fat body containing single-cell flipout clones (GFP positive) overexpressing patched. Fat body was dissected from well-fed larvae, incubated in Lysotracker Red/DAPI and imaged unfixed.


( d ) Confocal image of a GFP - positive patched - expressing clone , with mCherry - Atg8a expressed in all cells . Fat body was dissected from well - fed larvae and imaged after formaldehyde fixation .

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(d) Confocal image of a GFP-positive patched-expressing clone, with mCherry-Atg8a expressed in all cells. Fat body was dissected from well-fed larvae and imaged after formaldehyde fixation.


( e ) GFP - marked flipout clones of CiCell - expressing cells . Fat body was dissected from well - fed larvae , incubated in Lysotracker Red / DAPI and imaged unfixed .

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(e) GFP-marked flipout clones of CiCell-expressing cells. Fat body was dissected from well-fed larvae, incubated in Lysotracker Red/DAPI and imaged unfixed.


( f ) Confocal image of fixed mCherry - Atg8a expressing fat body containing a GFP - marked clone of CiCell - expressing cells .

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(f) Confocal image of fixed mCherry-Atg8a expressing fat body containing a GFP-marked clone of CiCell-expressing cells.


( g ) Cells homozygous for the null costal2 [ 2 ] allele ( marked by lack of GFP ) were generated by flp / FRT - mediated recombination in mCherry - Atg8a expressing fat body . Larvae were starved 4 h before dissection and fixation .

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(g) Cells homozygous for the null costal2[2] allele (marked by lack of GFP) were generated by flp/FRT-mediated recombination in mCherry-Atg8a expressing fat body. Larvae were starved 4 h before dissection and fixation.


( a ) Graph representing fold change in mRNA levels in cells treated with Shh against untreated cells . Each circle represents an autophagy gene ; those with largest fold - changes are indicated in red . The central line represents no changes in expression ; above the central line , genes whose expression is increased : below , those with reduced levels . Grey lines indicate 2 - fold increase or decrease . ( b ) Table showing the 10 genes with higher fold changes in expression and the values obtained .

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(a) Graph representing fold change in mRNA levels in cells treated with Shh against untreated cells. Each circle represents an autophagy gene; those with largest fold-changes are indicated in red. The central line represents no changes in expression; above the central line, genes whose expression is increased: below, those with reduced levels. Grey lines indicate 2-fold increase or decrease. (b) Table showing the 10 genes with higher fold changes in expression and the values obtained.


( c ) Levels of PERK protein were assessed by western blotting in HeLa cells treated with 10 μM purmorphamine for 24 h . Actin was used as a protein loading control .

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(c) Levels of PERK protein were assessed by western blotting in HeLa cells treated with 10 μM purmorphamine for 24 h. Actin was used as a protein loading control.


( d ) HeLa cells were treated with purmorphamine for 24 h and either left in rich media or in HBSS for the last 4 h . Levels of S51 phoshorylation and total levels of eIF2α were detected by western blotting using LI - COR infra - red imager .

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(d) HeLa cells were treated with purmorphamine for 24 h and either left in rich media or in HBSS for the last 4 h. Levels of S51 phoshorylation and total levels of eIF2α were detected by western blotting using LI-COR infra-red imager.


( e , f ) Wild - type and PERK − / − ( e ) or eIF2α S51A / S51A MEFs ( f ) were treated with DMSO or 10 μM purmorphamine for 24 h and , where indicated , cells were treated with bafA1 for the last 4 h . tubulin - II and tubulin or tubulin , as a loading control , were detected and a representative blot is shown . HBSS , Hank ' s balanced salt solution .

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(e,f) Wild-type and PERK−/− (e) or eIF2α S51A/S51A MEFs (f) were treated with DMSO or 10 μM purmorphamine for 24 h and, where indicated, cells were treated with bafA1 for the last 4 h. tubulin-II and tubulin or tubulin, as a loading control, were detected and a representative blot is shown. HBSS, Hank's balanced salt solution.


A ) Differential interference contrast ( DIC ) image of cortical neurons in culture . The arrowhead points to a neuron expressing Ub - R - GFP . B ) GFP fluorescence before and after exposure to lactacystin ( 10µM ) .

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A) Differential interference contrast (DIC) image of cortical neurons in culture. The arrowhead points to a neuron expressing Ub-R-GFP. B) GFP fluorescence before and after exposure to lactacystin (10µM).


C ) A neuron coexpressing Ub - R - GFP and CFP before ( left ) and 10 hours after exposure to lactacystin ( right ) . Top row - GFP fluorescence ; bottom row - CFP fluorescence . Bars : 50µm ( A , B ) , 20 µm ( C ) . D ) Changes in GFP and CFP fluorescence following exposure to lactacystin at t = 1 hour ( vertical dashed line ) . Changes normalized to initial fluorescence in same cells . 9 neurons from 5 experiments ; averages and SEM . E ) Same as in D , except that here neurons were exposed only to carrier solution . 11 neurons from 5 experiments ; averages and SEM ( barely observable ) . F ) Comparison of GFP fluorescence accumulation rates . Linear fits shown as black lines ; fit parameters shown next to fits . Same data as in D , and E .

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C) A neuron coexpressing Ub-R-GFP and CFP before (left) and 10 hours after exposure to lactacystin (right). Top row - GFP fluorescence; bottom row - CFP fluorescence. Bars: 50µm (A,B), 20 µm (C). D) Changes in GFP and CFP fluorescence following exposure to lactacystin at t=1 hour (vertical dashed line). Changes normalized to initial fluorescence in same cells. 9 neurons from 5 experiments; averages and SEM. E) Same as in D, except that here neurons were exposed only to carrier solution. 11 neurons from 5 experiments; averages and SEM (barely observable). F) Comparison of GFP fluorescence accumulation rates. Linear fits shown as black lines; fit parameters shown next to fits. Same data as in D, and E.


A ) Hippocampal neurons in primary culture immunolabeled against three synaptic proteins ( the postsynaptic scaffold molecule Dlg4 / PSD - 95 , the presynaptic active zone molecule RIM and the presynaptic vesicle protein SV2A ) following 10 hour incubations in carrier solution ( control , upper panels ) or Lactacystin ( 10 μM ; bottom panels ) . Scale bar ,

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A) Hippocampal neurons in primary culture immunolabeled against three synaptic proteins (the postsynaptic scaffold molecule Dlg4/PSD-95, the presynaptic active zone molecule RIM and the presynaptic vesicle protein SV2A) following 10 hour incubations in carrier solution (control, upper panels) or Lactacystin (10 μM; bottom panels). Scale bar, 10μm


B ) Average synaptic immunofluorescence following 10 hour incubations in Lactacystin or carrier solution . Numbers within bars represent the number of fields of view analyzed for each condition ( ~ 170 synapses / field of view , on average ) . Error bars , SEM ; P values - two tailed unpaired t - test . Note that a statistically significant accumulation was observed only for RIM , whereas immunolabeling of all other proteins was either unchanged or reduced .

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B) Average synaptic immunofluorescence following 10 hour incubations in Lactacystin or carrier solution. Numbers within bars represent the number of fields of view analyzed for each condition (~170 synapses/field of view, on average). Error bars, SEM; P values - two tailed unpaired t-test. Note that a statistically significant accumulation was observed only for RIM, whereas immunolabeling of all other proteins was either unchanged or reduced.