G - J Double immunolabeling of PDS5B ( green ) and kinetochores ( ACA , red ) , and staining of the chromatin ( DAPI ) in a metaphase II spermatocyte .

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

G-J Double immunolabeling of PDS5B (green) and kinetochores (ACA, red), and staining of the chromatin (DAPI) in a metaphase II spermatocyte.


Double immunolabeling of PDS5A ( green , A - E ) and SYCP3 ( red ) in Pds5AB cKO spread spermatocytes at the indicated stages .

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

Double immunolabeling of PDS5A (green, A-E) and SYCP3 (red) in Pds5AB cKO spread spermatocytes at the indicated stages.


Double immunolabeling of PDS5B ( green , F - J ) and SYCP3 ( red ) in Pds5AB cKO spread spermatocytes at the indicated stages .

Double immunolabeling of PDS5B (green, F-J) and SYCP3 (red) in Pds5AB cKO spread spermatocytes at the indicated stages.


K - M Preparations from the same mice were immunolabeled with the testis - specific histone variant H1t ( green ) and SYCP3 ( red ) .

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

K-M Preparations from the same mice were immunolabeled with the testis-specific histone variant H1t (green) and SYCP3 (red).


N Scatter dot - plot graph of total SC length measured in the autosomal pairs of 33 early pachytene cells from three different individuals of each genotype . Bars indicate mean and standard error . Statistical significance was assessed using an unpaired two - tailed T - test function ( * * * * , p < 0 . 0001 ) .

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

N Scatter dot-plot graph of total SC length measured in the autosomal pairs of 33 early pachytene cells from three different individuals of each genotype. Bars indicate mean and standard error. Statistical significance was assessed using an unpaired two-tailed T-test function (****, p < 0.0001).


A - H Double immunolabeling of SYCP1 ( green ) and SYCP3 ( red ) in wild - type ( A - D ) and Pds5AB cKO ( E - H ) spread spermatocytes at the indicated stages . I - L Double immunolabeling of HORMAD2 ( pseudocolored in white ) and SYCP3 ( red ) . Data information : Sex bivalents ( XY ) are indicated . Scale bar , 10 μm .

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

A-H Double immunolabeling of SYCP1 (green) and SYCP3 (red) in wild-type (A-D) and Pds5AB cKO (E-H) spread spermatocytes at the indicated stages. I-L Double immunolabeling of HORMAD2 (pseudocolored in white) and SYCP3 (red). Data information: Sex bivalents (XY) are indicated. Scale bar, 10 μm.


Double immunolabeling of SYCP3 ( red ) and the cohesin subunits SMC3 ( green , in Pds5AB cKO spread spermatocytes at the indicated stages . Sex bivalents ( XY ) are indicated . Scale bar , 10 μm .

O B-EXP_ASSAY O B-GENEPROD O O O O O B-SUBCELLULAR O B-GENEPROD O O O O B-GENEPROD O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O

Double immunolabeling of SYCP3 (red) and the cohesin subunits SMC3 (green, in Pds5AB cKO spread spermatocytes at the indicated stages. Sex bivalents (XY) are indicated. Scale bar, 10 μm.


Double immunolabeling of SYCP3 ( red ) and the cohesin subunits REC8 ( green in Pds5AB cKO spread spermatocytes at the indicated stages . Sex bivalents ( XY ) are indicated . Scale bar , 10 μm .

O B-EXP_ASSAY O B-GENEPROD O O O O O B-SUBCELLULAR O B-GENEPROD O O O B-GENEPROD O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O

Double immunolabeling of SYCP3 (red) and the cohesin subunits REC8 (green in Pds5AB cKO spread spermatocytes at the indicated stages. Sex bivalents (XY) are indicated. Scale bar, 10 μm.


Double immunolabeling of SYCP3 ( red ) and the cohesin subunits SMC1β ( green , in Pds5AB cKO spread spermatocytes at the indicated stages . Sex bivalents ( XY ) are indicated . Scale bar , 10 μm .

Double immunolabeling of SYCP3 (red) and the cohesin subunits SMC1β (green, in Pds5AB cKO spread spermatocytes at the indicated stages. Sex bivalents (XY) are indicated. Scale bar, 10 μm.


Double immunolabeling of SYCP3 ( red ) and the cohesin cofactors Sororin ( green , in Pds5AB cKO spread spermatocytes at the indicated stages . Sex bivalents ( XY ) are indicated . Scale bar , 10 μm .

Double immunolabeling of SYCP3 (red) and the cohesin cofactors Sororin (green, in Pds5AB cKO spread spermatocytes at the indicated stages. Sex bivalents (XY) are indicated. Scale bar, 10 μm.


Double immunolabeling of SYCP3 ( red ) and the cohesin cofactors WAPL ( green , in Pds5AB cKO spread spermatocytes at the indicated stages . Sex bivalents ( XY ) are indicated . Scale bar , 10 μm .

Double immunolabeling of SYCP3 (red) and the cohesin cofactors WAPL (green, in Pds5AB cKO spread spermatocytes at the indicated stages. Sex bivalents (XY) are indicated. Scale bar, 10 μm.


Double immunolabeling of SYCP3 ( red ) and either γH2AX ( pseudocolored in blue in Pds5AB cKO spread spermatocytes at the indicated stages .

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

Double immunolabeling of SYCP3 (red) and either γH2AX (pseudocolored in blue in Pds5AB cKO spread spermatocytes at the indicated stages.


Double immunolabeling of SYCP3 ( red ) and RAD51 ( green in Pds5AB cKO spread spermatocytes at the indicated stages . Data information : Sex bivalents ( XY ) are indicated . Scale bar , 10 μm .

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

Double immunolabeling of SYCP3 (red) and RAD51 (green in Pds5AB cKO spread spermatocytes at the indicated stages. Data information: Sex bivalents (XY) are indicated. Scale bar, 10 μm.


I Scatter dot - plot graph of the quantification of RAD51 foci in in 15 zygotene nuclei randomly selected from three different individuals of each genotype . Bars indicate mean and standard error and statistical significance was assessed with an unpaired two - tailed T - test function ( * * * * , p < 0 . 0001 ) .

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

I Scatter dot-plot graph of the quantification of RAD51 foci in in 15 zygotene nuclei randomly selected from three different individuals of each genotype. Bars indicate mean and standard error and statistical significance was assessed with an unpaired two-tailed T-test function (****, p < 0.0001).


J - L Double immunolabeling of MLH1 ( pseudocolored in white ) and SYCP3 ( red ) in Pds5AB cKO spread spermatocytes . Data information : Sex bivalents ( XY ) are indicated . Scale bar , 10 μm .

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

J-L Double immunolabeling of MLH1 (pseudocolored in white) and SYCP3 (red) in Pds5AB cKO spread spermatocytes. Data information: Sex bivalents (XY) are indicated. Scale bar, 10 μm.


Double immunolabeling of SYCP3 ( red ) and TRF1 ( green , in Pds5AB cKO spread spermatocytes at the indicated stages . Data information : Sex bivalents ( XY ) are indicated . Scale bar , 10 μm .

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

Double immunolabeling of SYCP3 (red) and TRF1 (green, in Pds5AB cKO spread spermatocytes at the indicated stages. Data information: Sex bivalents (XY) are indicated. Scale bar, 10 μm.


Double immunolabeling of SYCP3 ( red ) and RAP1 ( green , in Pds5AB cKO spread spermatocytes at the indicated stages . Data information : Sex bivalents ( XY ) are indicated . Scale bar , 10 μm .

Double immunolabeling of SYCP3 (red) and RAP1 (green, in Pds5AB cKO spread spermatocytes at the indicated stages. Data information: Sex bivalents (XY) are indicated. Scale bar, 10 μm.


I Selected zygotene - like and pachytene - like bivalents presenting altered TRF1 distribution as ( from left to right ) ( i ) telomere stretches , ( ii ) multiple telomere , ( iii ) distant telomeres and ( iv ) telomere - less . Arrowheads in ( I ) indicate the position of altered telomeres . J Quantification of telomeres with a regular or altered disposition of TRF1 in Pds5AB cKO spermatocytes at the indicated stages ( n = 1646 telomeres in zygotene - like spermatocytes and 738 in pachytene - like ones ) . Data information : Sex bivalents ( XY ) are indicated . Scale bar , 10 μm .

O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O O O O O O O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-CELL_TYPE O O O O O O O O B-SUBCELLULAR O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O

I Selected zygotene-like and pachytene-like bivalents presenting altered TRF1 distribution as (from left to right) (i) telomere stretches, (ii) multiple telomere, (iii) distant telomeres and (iv) telomere-less. Arrowheads in (I) indicate the position of altered telomeres. J Quantification of telomeres with a regular or altered disposition of TRF1 in Pds5AB cKO spermatocytes at the indicated stages (n=1646 telomeres in zygotene-like spermatocytes and 738 in pachytene-like ones). Data information: Sex bivalents (XY) are indicated. Scale bar, 10 μm.


K , L As in ( I , J ) , respectively , but for RAP1 staining ( n = 1376 telomeres in zygotene - like spermatocytes and 328 in pachytene - like ones ) . Arrowheads in ( K ) indicate the position of altered telomeres . Data information : Sex bivalents ( XY ) are indicated . Scale bar , 10 μm .

O O O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O O O O B-SUBCELLULAR O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O

K, L As in (I, J), respectively, but for RAP1 staining (n=1376 telomeres in zygotene-like spermatocytes and 328 in pachytene-like ones). Arrowheads in (K) indicate the position of altered telomeres. Data information: Sex bivalents (XY) are indicated. Scale bar, 10 μm.


M - P Double immunolabeling of SUN1 ( green ) and SYCP3 ( red ) in Pds5AB cKO spread spermatocytes at the indicated stages . 0 , 1 and 2 in ( N and O ) indicate the number of chromosome ends labelled by SUN1 in a given bivalent . Data information : Sex bivalents ( XY ) are indicated . Scale bar , 10 μm .

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

M-P Double immunolabeling of SUN1 (green) and SYCP3 (red) in Pds5AB cKO spread spermatocytes at the indicated stages. 0, 1 and 2 in (N and O) indicate the number of chromosome ends labelled by SUN1 in a given bivalent. Data information: Sex bivalents (XY) are indicated. Scale bar, 10 μm.


Q As in ( I ) , but for SUN1 staining . Arrowheads in ( Q ) indicate the position of altered telomeres . Data information : Sex bivalents ( XY ) are indicated . Scale bar , 10 μm .

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

Q As in (I), but for SUN1 staining. Arrowheads in (Q) indicate the position of altered telomeres. Data information: Sex bivalents (XY) are indicated. Scale bar, 10 μm.


R Selected zygotene - like and early pachytene - like bivalents from Pds5AB cKO spermatocytes immunolabeled with SYCP3 ( red ) and SUN1 ( green ) , and their respective percentage of appearance . S Quantification of telomeres with a regular or altered disposition of SUN1 ( n = 715 telomeres in zygotene - like spermatocytes and 410 in pachytene - like ones ) . Data information : Sex bivalents ( XY ) are indicated . Scale bar , 10 μm .

O O O O O O O O O O O O B-GENEPROD O B-CELL_TYPE B-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O O O O O B-EXP_ASSAY O O O O O O B-SUBCELLULAR O O O O O B-EXP_ASSAY O B-GENEPROD O O O O B-SUBCELLULAR O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O

R Selected zygotene-like and early pachytene-like bivalents from Pds5AB cKO spermatocytes immunolabeled with SYCP3 (red) and SUN1 (green), and their respective percentage of appearance. S Quantification of telomeres with a regular or altered disposition of SUN1 (n=715 telomeres in zygotene-like spermatocytes and 410 in pachytene-like ones). Data information: Sex bivalents (XY) are indicated. Scale bar, 10 μm.


A - C Immunolabeling of SYCP3 ( red ) and telomere FISH ( PNA - Tel , green ) in Pds5AB cKO spread spermatocytes at the indicated stages . Data information : Sex bivalents ( XY ) are indicated . Scale bar , 10 μm .

O O O B-EXP_ASSAY O B-GENEPROD O O O O B-SUBCELLULAR B-EXP_ASSAY O O O B-SUBCELLULAR O O O O B-GENEPROD O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O

A-C Immunolabeling of SYCP3 (red) and telomere FISH (PNA-Tel, green) in Pds5AB cKO spread spermatocytes at the indicated stages. Data information: Sex bivalents (XY) are indicated. Scale bar, 10 μm.


D 300 % magnification of selected zygotene - like and pachytene - like bivalents presenting regular ( left ) or altered ( i - iv ) telomere FISH signals .

O O 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 B-EXP_ASSAY O O

D 300% magnification of selected zygotene-like and pachytene-like bivalents presenting regular (left) or altered (i-iv) telomere FISH signals.


Double immunolabeling of SYCP3 ( red ) and RAP1 ( blue ) and telomere FISH ( PNA - Tel , green ) in Pds5AB cKO spread

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

Double immunolabeling of SYCP3 (red) and RAP1 (blue) and telomere FISH (PNA-Tel, green) in Pds5AB cKO spread spermatocytes


Double immunolabeling of SYCP3 ( red ) and RAP1 ( blue ) and telomere FISH ( PNA - Tel , green ) in Pds5AB cKO spread spermatocytes at the indicated stages . Asterisks indicate the position of the enlarged chromosomes / bivalents shown in a 300 % magnification displaying telomere stretches , multiple telomere , distant telomeres and telomere - less . White arrowheads indicate telomeres with an altered organization of telomeric DNA into multiple telomere signals with RAP1 signals and telomere stretches Data information : Sex bivalents ( XY ) are indicated . Scale bar , 10 μm .

O B-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O O B-SUBCELLULAR B-EXP_ASSAY O O O B-SUBCELLULAR O O O O B-GENEPROD O O B-CELL_TYPE O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O B-SUBCELLULAR O O O B-SUBCELLULAR O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O B-EXP_ASSAY O B-SUBCELLULAR O O O B-SUBCELLULAR 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

Double immunolabeling of SYCP3 (red) and RAP1 (blue) and telomere FISH (PNA-Tel, green) in Pds5AB cKO spread spermatocytes at the indicated stages. Asterisks indicate the position of the enlarged chromosomes/bivalents shown in a 300% magnification displaying telomere stretches, multiple telomere, distant telomeres and telomere-less. White arrowheads indicate telomeres with an altered organization of telomeric DNA into multiple telomere signals with RAP1 signals and telomere stretches Data information: Sex bivalents (XY) are indicated. Scale bar, 10 μm.


Double immunolabeling of SYCP3 ( red ) and RAP1 ( blue ) and telomere FISH ( PNA - Tel , green ) in Pds5AB cKO spread spermatocytes at the indicated stages . Asterisks indicate the position of the enlarged chromosomes / bivalents shown in a 300 % magnification displaying telomere stretches , multiple telomere , distant telomeres and telomere - less . White arrowheads indicate telomeres with an altered organization of telomeric DNA into multiple telomere signals without RAP1 signals telomere stretches Pink arrowheads in ( T ) indicate a telomere presenting a regular telomere FISH signal without a RAP1 signal . Data information : Sex bivalents ( XY ) are indicated . Scale bar , 10 μm .

O B-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O O B-SUBCELLULAR B-EXP_ASSAY O O O B-SUBCELLULAR O O O O B-GENEPROD O O B-CELL_TYPE O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O B-SUBCELLULAR O O O B-SUBCELLULAR O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O B-EXP_ASSAY O B-SUBCELLULAR O O O B-SUBCELLULAR O O B-GENEPROD O B-SUBCELLULAR O O O O O O O O O B-SUBCELLULAR O O O B-SUBCELLULAR B-EXP_ASSAY O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O

Double immunolabeling of SYCP3 (red) and RAP1 (blue) and telomere FISH (PNA-Tel, green) in Pds5AB cKO spread spermatocytes at the indicated stages. Asterisks indicate the position of the enlarged chromosomes/bivalents shown in a 300% magnification displaying telomere stretches, multiple telomere, distant telomeres and telomere-less. White arrowheads indicate telomeres with an altered organization of telomeric DNA into multiple telomere signals without RAP1 signals telomere stretches Pink arrowheads in (T) indicate a telomere presenting a regular telomere FISH signal without a RAP1 signal. Data information: Sex bivalents (XY) are indicated. Scale bar, 10 μm.


Double immunolabeling of SYCP3 ( red ) and RAP1 ( blue ) and telomere FISH ( PNA - Tel , green ) in Pds5AB cKO spread spermatocytes at the indicated stages . Asterisks indicate the position of the enlarged chromosomes / bivalents shown in a 300 % magnification displaying telomere stretches , multiple telomere , distant telomeres and telomere - less .

O B-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O O B-SUBCELLULAR B-EXP_ASSAY O O O B-SUBCELLULAR O O O O B-GENEPROD O O B-CELL_TYPE O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O B-SUBCELLULAR O O O B-SUBCELLULAR O O B-SUBCELLULAR O B-SUBCELLULAR O O O

Double immunolabeling of SYCP3 (red) and RAP1 (blue) and telomere FISH (PNA-Tel, green) in Pds5AB cKO spread spermatocytes at the indicated stages. Asterisks indicate the position of the enlarged chromosomes/bivalents shown in a 300% magnification displaying telomere stretches, multiple telomere, distant telomeres and telomere-less.


Double immunolabeling of TRF1 ( green ) and SYCP3 ( red ) in squashed spermatocytes from wild - type or Pds5AB cKO mice M - P ) . The first three columns correspond to z - projections of 15 focal planes throughout the Top , Equator and Bottom regions of the nucleus . The projection of 65 focal planes across the same spermatocyte nucleus is shown at the fourth column ( Z - Projection ) . White arrowheads in ( F indicate the presence of TRF1 signals non - attached to the NE . Scale bar , 10 μm .

O B-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O O O B-CELL_TYPE O O O O O B-GENEPROD O 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 B-SUBCELLULAR O O O O O O O O O O B-CELL_TYPE B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O B-SUBCELLULAR O O O O O O O

Double immunolabeling of TRF1 (green) and SYCP3 (red) in squashed spermatocytes from wild-type or Pds5AB cKO mice M-P). The first three columns correspond to z-projections of 15 focal planes throughout the Top, Equator and Bottom regions of the nucleus. The projection of 65 focal planes across the same spermatocyte nucleus is shown at the fourth column (Z-Projection). White arrowheads in (F indicate the presence of TRF1 signals non-attached to the NE. Scale bar, 10 μm.


Double immunolabeling of TRF1 ( green ) and SYCP3 ( red ) in squashed spermatocytes from wild - type or Pds5AB cKO mice The first three columns correspond to z - projections of 15 focal planes throughout the Top , Equator and Bottom regions of the nucleus . The projection of 65 focal planes across the same spermatocyte nucleus is shown at the fourth column ( Z - Projection ) . White arrowheads in N ) indicate the presence of TRF1 signals non - attached to the NE . Scale bar , 10 μm .

O B-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O O O B-CELL_TYPE O O O O O B-GENEPROD O 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 B-SUBCELLULAR O O O O O O O O O O B-CELL_TYPE B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O B-SUBCELLULAR O O O O O O O

Double immunolabeling of TRF1 (green) and SYCP3 (red) in squashed spermatocytes from wild-type or Pds5AB cKO mice The first three columns correspond to z-projections of 15 focal planes throughout the Top, Equator and Bottom regions of the nucleus. The projection of 65 focal planes across the same spermatocyte nucleus is shown at the fourth column (Z-Projection). White arrowheads in N) indicate the presence of TRF1 signals non-attached to the NE. Scale bar, 10 μm.


Q - T Selected bivalents from Pds5AB cKO spermatocytes displaying structural telomere abnormalities as telomere stretches ( Q ) , multiple telomere ( R ) , distant telomere ( S ) and telomere - less ( T ) .

O O O O O O B-GENEPROD O B-CELL_TYPE O O B-SUBCELLULAR B-EXP_ASSAY O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O O O

Q-T Selected bivalents from Pds5AB cKO spermatocytes displaying structural telomere abnormalities as telomere stretches (Q), multiple telomere (R), distant telomere (S) and telomere-less (T).


HCT116 and U87 cells were treated with 20 µM LXR623 for 24h . Transcriptome and gene set enrichment analysis was performed . Shown are enrichment plots . NES : normalized enrichment score .

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

HCT116 and U87 cells were treated with 20 µM LXR623 for 24h. Transcriptome and gene set enrichment analysis was performed. Shown are enrichment plots. NES: normalized enrichment score.


HCT116 cells were treated with 20 µM LXR623 for 24h . Transcriptome and gene set enrichment analysis was performed . Shown are enrichment plots . NES : normalized enrichment score .

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

HCT116 cells were treated with 20 µM LXR623 for 24h. Transcriptome and gene set enrichment analysis was performed. Shown are enrichment plots. NES: normalized enrichment score.


U87 GBM cells were treated with 20 µM LXR623 or 50 µM Simvastatin ( Sim ) for 48h . Thereafter , lysates were collected and analyzed for total cholesterol levels . Shown are means and SD ( n = 3 ) .

B-CELL_LINE B-DISEASE O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE 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

U87 GBM cells were treated with 20 µM LXR623 or 50 µM Simvastatin (Sim) for 48h. Thereafter, lysates were collected and analyzed for total cholesterol levels. Shown are means and SD (n = 3).


Shown is a volcano plot of patients from the TCGA ( database ) with high vs . low levels of ABCA1 . A close - up of the most significantly down - regulated genes is presented ( mRNAs related to the electron transport chain and mitochondrial metabolism ) . The x - axis highlights the fold changes ( log2 scale ) ( down - regulated genes , negative values ) , whereas the y - axis indicates the level of statistical significance ( shown in - log10 format ) .

O O O O O O B-ORGANISM O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O B-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

Shown is a volcano plot of patients from the TCGA (database) with high vs. low levels of ABCA1. A close-up of the most significantly down-regulated genes is presented (mRNAs related to the electron transport chain and mitochondrial metabolism). The x-axis highlights the fold changes (log2 scale) (down-regulated genes, negative values), whereas the y-axis indicates the level of statistical significance (shown in -log10 format).


GBM12 GBM cells ( short term patient - derived xenograft ) , U87 GBM cells , HCT116 colonic carcinoma cells and A375 melanoma cells were treated with the indicated concentrations of ABT263 , LXR623 or the combination for 72h . Thereafter , cellular viability was analyzed and statistical analysis was performed .

O B-DISEASE O O O O B-ORGANISM O O B-EXP_ASSAY O O B-CELL_LINE B-DISEASE O O B-CELL_LINE B-DISEASE I-DISEASE O O B-CELL_LINE B-DISEASE O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O

GBM12 GBM cells (short term patient-derived xenograft), U87 GBM cells, HCT116 colonic carcinoma cells and A375 melanoma cells were treated with the indicated concentrations of ABT263, LXR623 or the combination for 72h. Thereafter, cellular viability was analyzed and statistical analysis was performed.


U87 , T98G and LN229 cells were treated with 1 µM ABT263 , 20 µM LXR623 or the combination of both for 72h . Mewo melanoma and HCT116 colon carcinoma cells were treated with 1 µM ABT263 , 10 µM LXR623 or the combination of both for 72h ( MeWo ) or for 48h ( HCT116 ) . Thereafter , cells were stained with annexin V / propidium iodide and analyzed by multi - parametric flow cytometry .

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

U87, T98G and LN229 cells were treated with 1 µM ABT263, 20 µM LXR623 or the combination of both for 72h. Mewo melanoma and HCT116 colon carcinoma cells were treated with 1 µM ABT263, 10 µM LXR623 or the combination of both for 72h (MeWo) or for 48h (HCT116). Thereafter, cells were stained with annexin V/propidium iodide and analyzed by multi-parametric flow cytometry.


, Stem - like GBM cells , NCH644 , NCH421k and NCH690 were treated with 1 µM ABT263 , 20 µM LXR623 or the combination of both for 48h . Thereafter , cells were stained with annexin V / propidium iodide and analyzed by multi - parametric flow cytometry .

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

, Stem-like GBM cells, NCH644, NCH421k and NCH690 were treated with 1 µM ABT263, 20 µM LXR623 or the combination of both for 48h. Thereafter, cells were stained with annexin V/propidium iodide and analyzed by multi-parametric flow cytometry.


U87 ( 72h treatment ) and HCT116 ( 48h treatment ) were treated with 1 µM ABT263 , 20 µM ( U87 ) or 10 µM ( HCT116 ) LXR623 or the combination of both , stained with TMRE and analyzed by flow cytometric analysis for dissipation of mitochondrial membrane potential .

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

U87 (72h treatment) and HCT116 (48h treatment) were treated with 1 µM ABT263, 20 µM (U87) or 10 µM (HCT116) LXR623 or the combination of both, stained with TMRE and analyzed by flow cytometric analysis for dissipation of mitochondrial membrane potential.


HCT116 colonic carcinoma cells were treated with ABT263 , LXR623 or the combination of both . Whole protein lysates were collected and subjected to capillary electrophoresis for the expression / cleavage of PARP , total caspase - 9 ( CP9 ) and Vinculin . CF : cleaved fragment , FL : full length .

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

HCT116 colonic carcinoma cells were treated with ABT263, LXR623 or the combination of both. Whole protein lysates were collected and subjected to capillary electrophoresis for the expression/cleavage of PARP, total caspase-9 (CP9) and Vinculin. CF: cleaved fragment, FL: full length.


HCT116 colonic carcinoma or NCH644 glioblastoma stem cells were treated with selective BH3 - mimetics , WEHI - 539 ( Bcl - xL inhibitor ) , ABT199 ( Bcl - 2 inhibitor ) or A1210477 ( Mcl - 1 inhibitor ) in the presence or absence of LXR623 for 48h . Thereafter , cells were labeled with annexin V / propidium iodide and analyzed by multi - parametric flow cytometry .

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

HCT116 colonic carcinoma or NCH644 glioblastoma stem cells were treated with selective BH3-mimetics, WEHI-539 (Bcl-xL inhibitor), ABT199 (Bcl-2 inhibitor) or A1210477 (Mcl-1 inhibitor) in the presence or absence of LXR623 for 48h. Thereafter, cells were labeled with annexin V/propidium iodide and analyzed by multi-parametric flow cytometry.


, LN229 , T98G , U87 , U87 - EGFRvIII and HCT116 cells were treated with increasing concentrations of LXR623 for 72h . Thereafter , protein lysates were collected and subjected to standard western blot ( LN229 , T98G and U87 ) or capillary electrophoresis ( U87 - EGFR - vIII and HCT116 ) for the expression of Usp9X , Mcl - 1 , Bcl - 2 , Bcl - xL , Noxa , BIM and / or Actin and Vinculin . In U87 - EGFRvIII and HCT116 , Vinculin was used in lieu of Actin .

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

, LN229, T98G, U87, U87-EGFRvIII and HCT116 cells were treated with increasing concentrations of LXR623 for 72h. Thereafter, protein lysates were collected and subjected to standard western blot (LN229, T98G and U87) or capillary electrophoresis (U87-EGFR-vIII and HCT116) for the expression of Usp9X, Mcl-1, Bcl-2, Bcl-xL, Noxa, BIM and/or Actin and Vinculin. In U87-EGFRvIII and HCT116, Vinculin was used in lieu of Actin.


LN229 , T98G , U87 , U87 - EGFRvIII and HCT116 cells were treated with LXR623 as described in A . Thereafter , protein lysates were collected and subjected to capillary electrophoresis for the expression of ABCA1 and Vinculin .

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LN229, T98G, U87, U87-EGFRvIII and HCT116 cells were treated with LXR623 as described in A. Thereafter, protein lysates were collected and subjected to capillary electrophoresis for the expression of ABCA1 and Vinculin.


LN229 were transfected with siNT , siNoxa 1 , siNoxa 2 or BAK - siRNA for 72h . Thereafter , cells were treated with the combination treatment of 1 µM ABT263 and 20 µM LXR623 for another 24h . After conclusion of the treatment , cells were harvested , fixed , stained with propidium iodide and analyzed by flow cytometry for DNA - fragmentation .

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

LN229 were transfected with siNT, siNoxa 1, siNoxa 2 or BAK-siRNA for 72h. Thereafter, cells were treated with the combination treatment of 1 µM ABT263 and 20 µM LXR623 for another 24h. After conclusion of the treatment, cells were harvested, fixed, stained with propidium iodide and analyzed by flow cytometry for DNA - fragmentation.


LN229 were transfected as described in B . Thereafter , whole cell protein lysates were collected and analyzed by capillary electrophoresis for the expression of Noxa , BAK and Vinculin .

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LN229 were transfected as described in B. Thereafter, whole cell protein lysates were collected and analyzed by capillary electrophoresis for the expression of Noxa, BAK and Vinculin.


LN229 GBM cells were treated with 10 µM LXR623 ( LXR ) , 1 µM ABT263 or the combination for 48h . Thereafter , protein lysates were prepared and immunoprecipitated with an antibody against BAK . Standard western blotting was performed ( immunoprecipitation and the corresponding inputs ) with antibody against BAK and Mcl - 1 . The arrows highlight the specific protein bands , while stars indicate the immunoglobulin light - chains .

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

LN229 GBM cells were treated with 10 µM LXR623 (LXR), 1 µM ABT263 or the combination for 48h. Thereafter, protein lysates were prepared and immunoprecipitated with an antibody against BAK. Standard western blotting was performed (immunoprecipitation and the corresponding inputs) with antibody against BAK and Mcl-1. The arrows highlight the specific protein bands, while stars indicate the immunoglobulin light-chains.


U87 cells were treated with 20 µM LXR623 for 7h and 24h . Thereafter , cells were stained with annexin V / propidium iodide and analyzed by multi - parametric flow cytometry . The lower left quadrant indicates the percentage of viable cells .

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

U87 cells were treated with 20 µM LXR623 for 7h and 24h. Thereafter, cells were stained with annexin V/propidium iodide and analyzed by multi-parametric flow cytometry. The lower left quadrant indicates the percentage of viable cells.


U87 cells were treated with LXR623 as indicated for 7h and analyzed for total ATP levels by a luminescence assay . To account for dead cells , assays were normalized to DNA - content ( cell number ) .

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

U87 cells were treated with LXR623 as indicated for 7h and analyzed for total ATP levels by a luminescence assay. To account for dead cells, assays were normalized to DNA-content (cell number).


U87 cells were treated with LXR623 as indicated for 24h . Cell lysates were prepared and subjected to LC / MS for the determination of ATP levels .

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

U87 cells were treated with LXR623 as indicated for 24h. Cell lysates were prepared and subjected to LC/MS for the determination of ATP levels.


U87 cells were treated with increasing concentrations of LXR623 for 7h and 24h . Thereafter , whole cell protein lysates were collected and analyzed by capillary electrophoresis for the expression of phosphorylated AMPK ( threonine 172 ) and total AMPK .

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

U87 cells were treated with increasing concentrations of LXR623 for 7h and 24h. Thereafter, whole cell protein lysates were collected and analyzed by capillary electrophoresis for the expression of phosphorylated AMPK (threonine 172) and total AMPK.


U87 cells were treated with increasing concentrations of LXR623 in the presence or absence of exogenous ATP for 7h . Thereafter , whole cell protein lysates were collected and analyzed by capillary electrophoresis for the expression of ATF4 .

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

U87 cells were treated with increasing concentrations of LXR623 in the presence or absence of exogenous ATP for 7h. Thereafter, whole cell protein lysates were collected and analyzed by capillary electrophoresis for the expression of ATF4.


U87 cells were treated with increasing concentrations of LXR623 for 24h in the presence or absence of cholesterol . Thereafter , whole cell protein lysates were collected and analyzed by capillary electrophoresis for the expression of phosphorylated AMPK ( threonine 172 ) and total AMPK .

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

U87 cells were treated with increasing concentrations of LXR623 for 24h in the presence or absence of cholesterol. Thereafter, whole cell protein lysates were collected and analyzed by capillary electrophoresis for the expression of phosphorylated AMPK (threonine 172) and total AMPK.


Cells were treated as in F and whole cell protein lysates were collected and analyzed by capillary electrophoresis for the expression of ATF4 and Vinculin ( G ) and by the standard western blot for the expression of Noxa and Actin .

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

Cells were treated as in F and whole cell protein lysates were collected and analyzed by capillary electrophoresis for the expression of ATF4 and Vinculin (G) and by the standard western blot for the expression of Noxa and Actin.


U87 cells were treated with increasing concentrations of LXR623 . Thereafter , whole cell protein lysates were prepared and analyzed for classical ER - stress markers , GRP78 , ATF4 and Noxa by capillary electrophoresis .

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U87 cells were treated with increasing concentrations of LXR623. Thereafter, whole cell protein lysates were prepared and analyzed for classical ER-stress markers, GRP78, ATF4 and Noxa by capillary electrophoresis.


U87 cells were transfected with non - targeting siRNA ( n . t . - siRNA ) , ATF3 - siRNA , ATF4 siRNA or the combination of both ATF4 and ATF3 siRNAs . After 48h , cells were treated with LXR623 , whole cell protein lysates were harvested and analyzed by capillary electrophoresis for the expression of ATF3 , ATF4 , Noxa and Vinculin .

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

U87 cells were transfected with non-targeting siRNA (n.t.-siRNA), ATF3-siRNA, ATF4 siRNA or the combination of both ATF4 and ATF3 siRNAs. After 48h, cells were treated with LXR623, whole cell protein lysates were harvested and analyzed by capillary electrophoresis for the expression of ATF3, ATF4, Noxa and Vinculin.


U87 cells were treated as in A . Shown are the levels of TCA - cycle metabolites .

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

U87 cells were treated as in A. Shown are the levels of TCA-cycle metabolites.


U87 cells were incubated in DMEM ( devoid of phenol red , glucose , pyruvate and glutamine ) supplemented with 25 mM U - 13C - glucose , 4 mM glutamine and 1 . 5 % dialyzed FBS in the presence or absence of 20 µM LXR623 for 24 hours . Cells were then harvested for LC / MS analysis . The fractions of each different isotopologue of each metabolite were calculated ( percentage of the entire pool ) . Shown are the isotopologues of the TCA cycle intermediates labeled by glucose carbons and non - labeled isotopologues ( m + 0 ) .

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

U87 cells were incubated in DMEM (devoid of phenol red, glucose, pyruvate and glutamine) supplemented with 25 mM U-13C-glucose, 4 mM glutamine and 1.5% dialyzed FBS in the presence or absence of 20 µM LXR623 for 24 hours. Cells were then harvested for LC/MS analysis. The fractions of each different isotopologue of each metabolite were calculated (percentage of the entire pool). Shown are the isotopologues of the TCA cycle intermediates labeled by glucose carbons and non-labeled isotopologues (m+0).


U87 cells were incubated in DMEM ( devoid of phenol red , glucose , pyruvate and glutamine ) supplemented with 25 mM U - 13C - glucose , 4 mM glutamine and 1 . 5 % dialyzed FBS in the presence or absence of 20 µM LXR623 for 24 hours . Cells were then harvested for LC / MS analysis . The fractions of each different isotopologue of each metabolite were calculated ( percentage of the entire pool ) . Shown are the isotopologues of non - essential aminoacids ( glutamatic acid and aspartic acid ) .

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

U87 cells were incubated in DMEM (devoid of phenol red, glucose, pyruvate and glutamine) supplemented with 25 mM U-13C-glucose, 4 mM glutamine and 1.5% dialyzed FBS in the presence or absence of 20 µM LXR623 for 24 hours. Cells were then harvested for LC/MS analysis. The fractions of each different isotopologue of each metabolite were calculated (percentage of the entire pool). Shown are the isotopologues of non-essential aminoacids (glutamatic acid and aspartic acid).


U87 cells were incubated in DMEM ( devoid of phenol red , glucose , pyruvate and glutamine ) supplemented with 25 mM U - 13C - glucose , 4 mM glutamine and 1 . 5 % dialyzed FBS in the presence or absence of 20 µM LXR623 for 24 hours . Cells were then harvested for LC / MS analysis . The fractions of each different isotopologue of each metabolite were calculated ( percentage of the entire pool ) . Shown are the isotopologues of non - essential aminoacids ( glutamatic

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

U87 cells were incubated in DMEM (devoid of phenol red, glucose, pyruvate and glutamine) supplemented with 25 mM U-13C-glucose, 4 mM glutamine and 1.5% dialyzed FBS in the presence or absence of 20 µM LXR623 for 24 hours. Cells were then harvested for LC/MS analysis. The fractions of each different isotopologue of each metabolite were calculated (percentage of the entire pool). Shown are the isotopologues of non-essential aminoacids (glutamatic acid


U87 cells were incubated in DMEM ( devoid of phenol red , glucose , pyruvate and glutamine ) supplemented with 25 mM U - 13C - glucose , 4 mM glutamine and 1 . 5 % dialyzed FBS in the presence or absence of 20 µM LXR623 for 24 hours . Cells were then harvested for LC / MS analysis . The fractions of each different isotopologue of each metabolite were calculated ( percentage of the entire pool ) . Shown are the isotopologues of glutathione and nucleotides .

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

U87 cells were incubated in DMEM (devoid of phenol red, glucose, pyruvate and glutamine) supplemented with 25 mM U-13C-glucose, 4 mM glutamine and 1.5% dialyzed FBS in the presence or absence of 20 µM LXR623 for 24 hours. Cells were then harvested for LC/MS analysis. The fractions of each different isotopologue of each metabolite were calculated (percentage of the entire pool). Shown are the isotopologues of glutathione and nucleotides.


U87 cells were incubated in DMEM ( devoid of phenol red , glucose , pyruvate and glutamine ) supplemented with 25 mM U - 13C - glucose , 4 mM glutamine and 1 . 5 % dialyzed FBS in the presence or absence of 20 µM LXR623 for 24 hours . Cells were then harvested for LC / MS analysis . The fractions of each different isotopologue of each metabolite were calculated ( percentage of the entire pool ) .

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

U87 cells were incubated in DMEM (devoid of phenol red, glucose, pyruvate and glutamine) supplemented with 25 mM U-13C-glucose, 4 mM glutamine and 1.5% dialyzed FBS in the presence or absence of 20 µM LXR623 for 24 hours. Cells were then harvested for LC/MS analysis. The fractions of each different isotopologue of each metabolite were calculated (percentage of the entire pool).


U87 cells were treated with DMSO or LXR623 20 µM for 24h . Thereafter , cells were processed for polar metabolite analysis by LC / MS . Shown are the levels of NAD and NADH2 .

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

U87 cells were treated with DMSO or LXR623 20 µM for 24h. Thereafter, cells were processed for polar metabolite analysis by LC/MS. Shown are the levels of NAD and NADH2.


U87 cells were treated with LXR623 for 1h , 3h or 5h and subjected to extracellular flux analysis on the Seahorse XFp instrument in the context of a mitochondrial stress assay ( A - C ) . From this assay , coupled respiration was calculated ( D - F ) .

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

U87 cells were treated with LXR623 for 1h, 3h or 5h and subjected to extracellular flux analysis on the Seahorse XFp instrument in the context of a mitochondrial stress assay (A-C). From this assay, coupled respiration was calculated (D-F).


U87 cells were treated with LXR623 , cholesterol or the combination of both for 24h and subjected to extracellular flux analysis on the Seahorse XFp instrument in the context of a mitochondrial stress assay ( G ) . From this assay , mitochondrial OCR and coupled respiration was calculated ( H - I ) .

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

U87 cells were treated with LXR623, cholesterol or the combination of both for 24h and subjected to extracellular flux analysis on the Seahorse XFp instrument in the context of a mitochondrial stress assay (G). From this assay, mitochondrial OCR and coupled respiration was calculated (H-I).


U87 cells were treated with DMSO or LXR623 20 µM with the indicated time point . Thereafter , whole cell protein lysates were collected and subjected to standard western blot for the expression of the respiratory complexes ( OXPHOS ) , Noxa , and ATF4 . A star indicates an unspecific band .

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

U87 cells were treated with DMSO or LXR623 20 µM with the indicated time point. Thereafter, whole cell protein lysates were collected and subjected to standard western blot for the expression of the respiratory complexes (OXPHOS), Noxa, and ATF4. A star indicates an unspecific band.


U87 cells were treated with DMSO or LXR623 20 µM in the presence or absence of cycloheximide for 7h . Thereafter , whole cell protein lysates were collected and the protein levels of the components of complex V were analyzed by standard western blot .

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

U87 cells were treated with DMSO or LXR623 20 µM in the presence or absence of cycloheximide for 7h. Thereafter, whole cell protein lysates were collected and the protein levels of the components of complex V were analyzed by standard western blot.


U87 cells were treated with increasing concentration of LXR623 in the presence or absence of cholesterol . Thereafter , whole cell protein lysates were collected and analyzed the protein levels of the respiratory complexes ( OXPHOS ) by standard western blot .

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

U87 cells were treated with increasing concentration of LXR623 in the presence or absence of cholesterol. Thereafter, whole cell protein lysates were collected and analyzed the protein levels of the respiratory complexes (OXPHOS) by standard western blot.


U87 cells transduced with non - targeting or LXRβ shRNA were treated with vehicle or 20 µM LXR623 for 24h . Thereafter , extracellular flux analysis was performed in the context of a mitochondrial stress assay . From this assay , basal respiration and coupled respiration was calculated ( M - N ) .

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

U87 cells transduced with non-targeting or LXRβ shRNA were treated with vehicle or 20 µM LXR623 for 24h. Thereafter, extracellular flux analysis was performed in the context of a mitochondrial stress assay. From this assay, basal respiration and coupled respiration was calculated (M-N).


U87 cells transduced with non - targeting or LXRβ shRNA were treated with vehicle or 20 µM LXR623 for 24h . RNA and protein were collected and analyzed for the expression of LXRβ mRNA ( O ) or LXRβ protein by capillary electrophoresis ( P ) . Shown are means and SD ( n = 5 ) . Statistical significance was determined by two - sided student ' s t . test .

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

U87 cells transduced with non-targeting or LXRβ shRNA were treated with vehicle or 20 µM LXR623 for 24h. RNA and protein were collected and analyzed for the expression of LXRβ mRNA (O) or LXRβ protein by capillary electrophoresis (P). Shown are means and SD (n = 5). Statistical significance was determined by two-sided student's t.test.


1x106 HCT116 colon carcinoma cells were implanted subcutaneously . Animals were treated intraperitoneally with vehicle , LXR623 ( 100 - 200 mg / kg ) , ABT263 ( 100 mg / kg ) or both agents ( 3 days per week for 1 . 5 weeks . Tumor growth curves show the development of tumor size for each treatment group . Scatter plots display the quantitative representation of the tumor size among the different treatments toward the end of the experiment . Shown are means and SD ( n ≥ 5 ) . * p = 0 . 022 , * * p = 0 . 0063 , * * * * p < 0 . 0001 . Statistical significance was determined by one - way ANOVA . B , E , GBM43 PDXs were implanted subcutaneously . Animals were treated intraperitoneally with vehicle , LXR623 ( 100 mg / kg ) , ABT263 ( 75 mg / kg ) or both agents ( 3 days per week for 1 . 5 weeks ) . Tumor growth curves show the development of tumor size for each treatment group . Scatter plots display the quantitative representation of the tumor size among the different treatments toward the end of the experiment . Shown are means and SD ( n ≥ 5 ) . * p = 0 . 022 , * * p = 0 . 0031 . Statistical significance was determined by one - way ANOVA . C , F , A375 BRAF V600E mutated melanomas were implanted subcutaneously . Animals were treated intraperitoneally with vehicle , LXR623 ( 100 mg / kg ) , ABT263 ( 75 mg / kg ) or both agents ( 3 days per week for 1 . 5 weeks ) . Tumor growth curves show the development of tumor size for each treatment group . Scatter plots display the quantitative representation of the tumor size among the different treatments toward the end of the experiment . Shown are means and SD ( n ≥ 5 ) . * p = 0 . 0109 ( GW3965 vs Combination ) , * p = 0 . 0166 ( ABT263 vs Combination ) , * * p = 0 . 0047 . Statistical significance was determined by one - way ANOVA .

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

1x106 HCT116 colon carcinoma cells were implanted subcutaneously. Animals were treated intraperitoneally with vehicle, LXR623 (100-200 mg/kg), ABT263 (100 mg/kg) or both agents (3 days per week for 1.5 weeks. Tumor growth curves show the development of tumor size for each treatment group. Scatter plots display the quantitative representation of the tumor size among the different treatments toward the end of the experiment. Shown are means and SD (n ≥ 5). *p=0.022, **p=0.0063, ****p<0.0001. Statistical significance was determined by one-way ANOVA. B, E, GBM43 PDXs were implanted subcutaneously. Animals were treated intraperitoneally with vehicle, LXR623 (100 mg/kg), ABT263 (75 mg/kg) or both agents (3 days per week for 1.5 weeks). Tumor growth curves show the development of tumor size for each treatment group. Scatter plots display the quantitative representation of the tumor size among the different treatments toward the end of the experiment. Shown are means and SD (n ≥ 5). *p=0.022, **p=0.0031. Statistical significance was determined by one-way ANOVA. C, F, A375 BRAF V600E mutated melanomas were implanted subcutaneously. Animals were treated intraperitoneally with vehicle, LXR623 (100 mg/kg), ABT263 (75 mg/kg) or both agents (3 days per week for 1.5 weeks). Tumor growth curves show the development of tumor size for each treatment group. Scatter plots display the quantitative representation of the tumor size among the different treatments toward the end of the experiment. Shown are means and SD (n ≥ 5). *p=0.0109 (GW3965 vs Combination), *p=0.0166 (ABT263 vs Combination), **p=0.0047. Statistical significance was determined by one-way ANOVA.


Related to the HCT116 colonic xenograft model , representative histopathological images ( hematoxylin and eosin stain ) are shown from each treatment group . Scale bar : 50 µm . Sections from the same treatment groups as in G were stained with TUNEL and SDHB , repectively . Displayed are representative images of the staining . Scale bar 50 µM .

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

Related to the HCT116 colonic xenograft model, representative histopathological images (hematoxylin and eosin stain) are shown from each treatment group. Scale bar: 50 µm. Sections from the same treatment groups as in G were stained with TUNEL and SDHB, repectively. Displayed are representative images of the staining. Scale bar 50 µM.


Sections from the experiment in 7A were quantified for apoptotic bodies


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

, Sections from the experiment in 7A were quantified for , number of TUNEL positive cells


Sections from the experiment in 7A were quantified for number of mitosis per multiple high - power fields .

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

Sections from the experiment in 7A were quantified for number of mitosis per multiple high-power fields.


Total carbonyl - bearing proteins detected by shotgun redox proteomic measurement in three biological replicates each of E . coli and D . radiodurans with and without irradiation . The left axis is the number of sequence - unique proteins detected as carbonylated . The right axis is the number of sites in total detected as carbonylated ( red ) or not oxidized ( black ) in peptides bearing at least one carbonyl . Stripes indicate carbonylated proteins and carbonylatable sites detected only in irradiated samples .

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

Total carbonyl-bearing proteins detected by shotgun redox proteomic measurement in three biological replicates each of E. coli and D. radiodurans with and without irradiation. The left axis is the number of sequence-unique proteins detected as carbonylated. The right axis is the number of sites in total detected as carbonylated (red) or not oxidized (black) in peptides bearing at least one carbonyl. Stripes indicate carbonylated proteins and carbonylatable sites detected only in irradiated samples.


Volcano plots for relative protein abundance changes measured by mass spectrometry in E . coli ( left ) and D . radiodurans ( right ) after irradiation using the same biological replicates as in Fig 2A . Black - circled points are those proteins with significant changes ( paired , 2 - sided t - test p - value < 0 . 05 ) of > 2 - fold or < 0 . 5 - fold . Red points are proteins with at least one carbonylated peptide detected . Fold change and p - value cutoffs considered for significance are indicated by dashed lines .

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

Volcano plots for relative protein abundance changes measured by mass spectrometry in E. coli (left) and D. radiodurans (right) after irradiation using the same biological replicates as in Fig 2A. Black-circled points are those proteins with significant changes (paired, 2-sided t-test p-value < 0.05) of >2-fold or <0.5-fold. Red points are proteins with at least one carbonylated peptide detected. Fold change and p-value cutoffs considered for significance are indicated by dashed lines.


Prevalence of individual RKPT residues and prevalence of carbonylated form in experimentally measured peptides combining all 3 biological replicates of both conditions for each organism . Ratios are given above each pair of bars . All proportions are significantly different between each RKPT and their respective carbonylation state by two - tailed z - test of two proportions ( p - values < 0 . 01 ; see Materials and Methods ) , meaning carbonylated proportions are not determined simply by relative prevalence of RKPT . See also Appendix Fig S1 .

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 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

Prevalence of individual RKPT residues and prevalence of carbonylated form in experimentally measured peptides combining all 3 biological replicates of both conditions for each organism. Ratios are given above each pair of bars. All proportions are significantly different between each RKPT and their respective carbonylation state by two-tailed z-test of two proportions (p-values < 0.01; see Materials and Methods), meaning carbonylated proportions are not determined simply by relative prevalence of RKPT. See also Appendix Fig S1.


Prevalence of all canonical amino acids before irradiation of E . coli and D . radiodurans , combining all 3 biological replicates for each condition . Ratios are given above each pair of bars . All proportions are significantly different between species by two - tailed z - test of two proportions ( p - values < 0 . 01 ) . See also Fig EV1 and EV2 .

O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O B-ORGANISM I-ORGANISM I-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 O O O O O O O O O O O O O O O

Prevalence of all canonical amino acids before irradiation of E. coli and D. radiodurans, combining all 3 biological replicates for each condition. Ratios are given above each pair of bars. All proportions are significantly different between species by two-tailed z-test of two proportions (p-values < 0.01). See also Fig EV1 and EV2.


A , B . RT - qPCR analysis of Zfp207 and Oct4 in mouse ESCs during ( A ) retinoic acid ( RA ) - induced and ( B ) embryoid body ( EB ) - mediated differentiation . Nestin was used as a neuronal differentiation marker to monitor RA - mediated differentiation . mRNA levels are relative to the expression at day 0 .

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

A, B. RT-qPCR analysis of Zfp207 and Oct4 in mouse ESCs during (A) retinoic acid (RA)-induced and (B) embryoid body (EB)-mediated differentiation. Nestin was used as a neuronal differentiation marker to monitor RA-mediated differentiation. mRNA levels are relative to the expression at day 0.


J , K . ( J ) AP staining of shScr and Zfp207 - depleted ( sh1 and sh2 ) mouse ESCs . Scale bars , 20 µM . ( K ) Percentage of fully differentiated ( FD ) , partially differentiated ( PD ) and undifferentiated ( UN ) ESC colonies in shScr , sh1 and sh2 .

O O O O O O O B-GENEPROD B-EXP_ASSAY O O O B-GENEPROD O O O O O O O B-ORGANISM B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O

J, K. (J) AP staining of shScr and Zfp207-depleted (sh1 and sh2) mouse ESCs. Scale bars, 20 µM. (K) Percentage of fully differentiated (FD), partially differentiated (PD) and undifferentiated (UN) ESC colonies in shScr, sh1 and sh2.


M , N . ( M ) Western blot of ZFP207 , OCT4 , NANOG , and SOX2 in shScr , sh1 and sh2 ESCs and ( N ) RT - qPCR analysis of Oct4 , Nanog , and Sox2 in shScr , sh1 and sh2 ESCs ; data is relative to shScr .

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

M, N. (M) Western blot of ZFP207, OCT4, NANOG, and SOX2 in shScr, sh1 and sh2 ESCs and (N) RT-qPCR analysis of Oct4, Nanog, and Sox2 in shScr, sh1 and sh2 ESCs; data is relative to shScr.


( O ) Western blot of OCT4 during a 9 - h cycloheximide ( CHX ) time course treatment in shScr , sh1 and sh2 ESCs .

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

(O) Western blot of OCT4 during a 9-h cycloheximide (CHX) time course treatment in shScr, sh1 and sh2 ESCs.


( Q ) Western blot of OCT4 during 4 and 8 h treatment with the proteasome inhibitor MG132 .

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

(Q) Western blot of OCT4 during 4 and 8 h treatment with the proteasome inhibitor MG132.


D . RT - qPCR of Zfp207 , the pluripotency genes ( Oct4 , Nanog and Sox2 ) , the mesodermal markers ( Msx1 and Brachyury , T ) , the endodermal markers ( Foxa2 , Sox17 ) , and the neural - associated genes ( Pax6 , Sox11 and Nestin ) in shScr , sh1 and sh2 ESCs along the time - course of EB - mediated differentiation . mRNA levels are relative to the expression of shScr at day 0 .

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

D. RT-qPCR of Zfp207, the pluripotency genes (Oct4, Nanog and Sox2), the mesodermal markers (Msx1 and Brachyury, T), the endodermal markers (Foxa2, Sox17), and the neural-associated genes (Pax6, Sox11 and Nestin) in shScr, sh1 and sh2 ESCs along the time-course of EB-mediated differentiation. mRNA levels are relative to the expression of shScr at day 0.


B . Immunostaining of NESTIN ( green ) and TUJ1 ( red ) of neural progenitors generated from shScr on day 8 and 10 of the neuroectodermal differentiation . Nuclei were counterstained with DAPI . Scale bar , 20 μM .

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

B. Immunostaining of NESTIN (green) and TUJ1 (red) of neural progenitors generated from shScr on day 8 and 10 of the neuroectodermal differentiation. Nuclei were counterstained with DAPI. Scale bar, 20 μM.


( E ) quantification of the signal in shScr , sh1 and sh2 at day 5 of neuroectodermal differentiation .

O O O O O O O O O O O O O O O O O B-TISSUE O O

(E) quantification of the signal in shScr, sh1 and sh2 at day 5 of neuroectodermal differentiation.


G . RT - qPCR of Zfp207 and neural - associated markers in shScr , sh1 and sh2 along the course of neural differentiation . mRNA levels are relative to shScr at day 0 .

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

G. RT-qPCR of Zfp207 and neural-associated markers in shScr, sh1 and sh2 along the course of neural differentiation. mRNA levels are relative to shScr at day 0.


( A ) Volcano plots of common differentially expressed genes in shScr and Zfp207 - depleted ( sh1 and sh2 ) mouse ESCs . Up - regulated ( Up ) and down - regulated ( Down ) genes are indicated in red and blue , respectively ( p < 0 . 05 ; > 1 . 5 - fold ) . Zfp207 is depicted in black . Grey dots indicate non - significant ( NS ) and < 1 . 5 - fold differential expressed genes .

O O O O O O O O O O O O O B-GENEPROD O O O O O O O B-ORGANISM B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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

(A) Volcano plots of common differentially expressed genes in shScr and Zfp207-depleted (sh1 and sh2) mouse ESCs. Up-regulated (Up) and down-regulated (Down) genes are indicated in red and blue, respectively (p < 0.05; > 1.5-fold). Zfp207 is depicted in black. Grey dots indicate non-significant (NS) and < 1.5-fold differential expressed genes.


A . Heatmap of column z - scores of log2 transformed values of genes involved in alternative splicing ( AS ) in shScr , sh1 and sh2 mouse ESCs .

O O O 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-ORGANISM B-CELL_TYPE O

A. Heatmap of column z-scores of log2 transformed values of genes involved in alternative splicing (AS) in shScr, sh1 and sh2 mouse ESCs.


F . RT - PCR of Zfp207 - regulated AS events . For Mta1 , AS1 ( alternative splicing 1 ) and AS2 ( alternative splicing 2 ) . # 1 and # 2 indicate distinct biological replicates . * Denotes an isoform that was not taken in consideration for the quantification . The structure of each isoform is indicated ( not to scale ) . Alternative exons are blue . The percent spliced in ( PSI ) was quantified for each condition .

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

F. RT-PCR of Zfp207-regulated AS events. For Mta1, AS1 (alternative splicing 1) and AS2 (alternative splicing 2). #1 and #2 indicate distinct biological replicates. *Denotes an isoform that was not taken in consideration for the quantification. The structure of each isoform is indicated (not to scale). Alternative exons are blue. The percent spliced in (PSI) was quantified for each condition.


( E ) FISH of poly ( A ) + RNA distribution in shScr , sh1 and sh2 stained with 5 ´ Cys3 - oligo dT ( 50 ) . Nuclei were counterstained with DAPI . Scale bar , 20 µM .

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

(E) FISH of poly(A)+ RNA distribution in shScr, sh1 and sh2 stained with 5´Cys3-oligo dT(50). Nuclei were counterstained with DAPI. Scale bar, 20 µM.


Quantification of ( F ) nuclear , ( G ) cytoplasmic , and ( H ) of nuclear / cytoplasmic poly ( A ) + FISH signals . ( I ) Relative nuclear and cytosolic total RNA levels in sh1 and sh2 compared to shScr . Data are presented as mean ± SEM of n ≥ 3 independent biological experiments . * P < 0 . 05 , * * * P < 0 . 001 , * * * * P < 0 . 0001 ( shScr vs . sh1 or sh2 ) ; unpaired Student ' s t - test .

O O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O B-SUBCELLULAR B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-EXP_ASSAY 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 (F) nuclear, (G) cytoplasmic, and (H) of nuclear/cytoplasmic poly(A)+ FISH signals. (I) Relative nuclear and cytosolic total RNA levels in sh1 and sh2 compared to shScr. Data are presented as mean ± SEM of n ≥ 3 independent biological experiments. *P < 0.05, ***P < 0.001, ****P < 0.0001 (shScr vs. sh1 or sh2); unpaired Student's t-test.


( a ) Flow cytometry of LPS - stimulated B cells stained with LysoTracker Red .

(a) Flow cytometry of LPS-stimulated B cells stained with LysoTracker Red.


( b ) Autophagic flux of GFP - LC3 splenic B cells stimulated with LPS and then left untreated ( UT ) or treated for 1 h with bafimolycin A1 ( BafA1 ) , assessed by live - cell fluorescence microscopy ( left ) and automated quantification of spots ( right ) . Scale bar ( left ) , 5 μm .

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

(b) Autophagic flux of GFP-LC3 splenic B cells stimulated with LPS and then left untreated (UT) or treated for 1 h with bafimolycin A1 (BafA1), assessed by live-cell fluorescence microscopy (left) and automated quantification of spots (right). Scale bar (left), 5 μm.


( c ) Immunoblot analysis of the processing of endogenous unconjugated LC3 - I to lipid - conjugated LC3 - II in LPS - stimulated B cells with ( + NH4Cl ) or without treatment with NH4Cl ( left ) . Actin serves as a loading control throughout . Right , quantification of LC3 - II band intensity in the absence of NH4Cl , presented relative to that of actin .

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

(c) Immunoblot analysis of the processing of endogenous unconjugated LC3-I to lipid-conjugated LC3-II in LPS-stimulated B cells with (+NH4Cl) or without treatment with NH4Cl (left). Actin serves as a loading control throughout. Right, quantification of LC3-II band intensity in the absence of NH4Cl, presented relative to that of actin.


( d ) Quantitative RT - PCR analysis of transcripts encoding autophagy - related molecules in ASCs 3 d after stimulation with LPS , presented relative to that in unstimulated B cells .

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

(d) Quantitative RT-PCR analysis of transcripts encoding autophagy-related molecules in ASCs 3 d after stimulation with LPS, presented relative to that in unstimulated B cells.


( e ) Immunoblot analysis of p62 in LPS - stimulated B cells ( bottom ) . Above , quantification of band intensity ( as in c ) .

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

(e) Immunoblot analysis of p62 in LPS-stimulated B cells (bottom). Above, quantification of band intensity (as in c).


( f ) Live - cell fluorescence microscopy of CD138 + PCs purified from the spleen of GFP - LC3 mice 5 d after the injection of NP - Ficoll ( PCs ) and of splenic B cells ( B cells ) and bone marrow CD138 + PCs ( BM PCs ) purified from unstimulated GFP - LC3 mice ( right ) and automated quantification of spots ( left ) . Scale bar ( right ) , 5 μm . * P 0 . 05 , * * P 0 . 01 and * * * P 0 . 001 ( Student ' s t - test ) . Data are representative of three or more independent experiments ( a , c - e ) or one experiment or more with > 40 cells ( b , f ; average and s . e . m . ) .

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

(f) Live-cell fluorescence microscopy of CD138+ PCs purified from the spleen of GFP-LC3 mice 5 d after the injection of NP-Ficoll (PCs) and of splenic B cells (B cells) and bone marrow CD138+ PCs (BM PCs) purified from unstimulated GFP-LC3 mice (right) and automated quantification of spots (left). Scale bar (right), 5 μm. *P 0.05, **P 0.01 and ***P 0.001 (Student's t-test). Data are representative of three or more independent experiments (a,c-e) or one experiment or more with >40 cells (b,f; average and s.e.m.).


( a ) Proliferation of B cells purified from Atg5f / fCD19 - Cre and Atg5f / f control mice and activated for 3 d with LPS , assessed by flow cytometry analysis of dilution of the membrane dye CFSE . ( b ) Size of B cells purified as in a and activated for various times ( horizontal axis ) with LPS , assessed by the intensity of forward scatter ( FSC ) by flow cytometry . ( c ) CD138 expression of B cells purified and activated as in a . APC , allophycocyanin ; PE , phycoerythrin .

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

(a) Proliferation of B cells purified from Atg5f/fCD19-Cre and Atg5f/f control mice and activated for 3 d with LPS, assessed by flow cytometry analysis of dilution of the membrane dye CFSE. (b) Size of B cells purified as in a and activated for various times (horizontal axis) with LPS, assessed by the intensity of forward scatter (FSC) by flow cytometry. (c) CD138 expression of B cells purified and activated as in a. APC, allophycocyanin; PE, phycoerythrin.


( d ) Immunoblot analysis of ER proteins in lysates of Atg5f / f and Atg5f / fCD19 - Cre B cells 3 d after stimulation with LPS ( left ) , and quantification of band intensity ( right ; as in Fig . 1c ) . IgM - H , IgM heavy chain . NS , not significant ; * P 0 . 05 and * * P 0 . 01 ( Student ' s t - test ) .

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

(d) Immunoblot analysis of ER proteins in lysates of Atg5f/f and Atg5f/fCD19-Cre B cells 3 d after stimulation with LPS (left), and quantification of band intensity (right; as in Fig. 1c). IgM-H, IgM heavy chain. NS, not significant; *P 0.05 and **P 0.01 (Student's t-test).


( a ) Electron microscopy of Atg5f / f and Atg5f / fCD19 - Cre B cells 3 d after stimulation with LPS ( left and middle ) , and quantification of the ER by stereology ( right ) . Scale bars ( left and middle ) , 1 μm .

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

(a) Electron microscopy of Atg5f/f and Atg5f/fCD19-Cre B cells 3 d after stimulation with LPS (left and middle), and quantification of the ER by stereology (right). Scale bars (left and middle), 1 μm.


( b ) G6Pase electron - microscopy cytochemistry of differentiating PCs 3 d after stimulation with LPS ( left ) , and quantification of G6Pase + ( ER ) area , relative to total cell area ( right ) . Scale bars ( left ) , 1 μm .

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

(b) G6Pase electron-microscopy cytochemistry of differentiating PCs 3 d after stimulation with LPS (left), and quantification of G6Pase+ (ER) area, relative to total cell area (right). Scale bars (left), 1 μm.


( c ) Quantitative RT - PCR analysis of transcripts encoding spliced XBP - 1 ( sXBP - 1 ) , total XBP - 1 ( tXBP - 1 ) and BiP in differentiating PCs at 0 d and 3 d after stimulation with LPS , presented relative to that of unstimulated Atg5f / f B cells at day 3 .

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

(c) Quantitative RT-PCR analysis of transcripts encoding spliced XBP-1 (sXBP-1), total XBP-1 (tXBP-1) and BiP in differentiating PCs at 0 d and 3 d after stimulation with LPS, presented relative to that of unstimulated Atg5f/f B cells at day 3.


( d ) Quantitative RT - PCR analysis of CHOP mRNA in Atg5f / f and Atg5f / fCD19 - Cre B cells stimulated for 3 d with LPS ( left and middle ) , and in Atg5f / f B cells stimulated for 3 d with LPS and treated for the final 24 h with tunicamycin ( Atg5f / f 24 h tun ; 0 . 5 μg / ml ; positive control ) , presented relative to that in Atg5f / f B cells stimulated for 3 d with LPS .

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

(d) Quantitative RT-PCR analysis of CHOP mRNA in Atg5f/f and Atg5f/fCD19-Cre B cells stimulated for 3 d with LPS (left and middle), and in Atg5f/f B cells stimulated for 3 d with LPS and treated for the final 24 h with tunicamycin (Atg5f/f 24 h tun; 0.5 μg/ml; positive control), presented relative to that in Atg5f/f B cells stimulated for 3 d with LPS.


( e ) Immunoblot analysis ( left ) of ER proteins in Atg5f / f B cells left untreated ( UT ) or treated with lysosomal protease inhibitors ( 10 μM leupeptin and E - 64d ) for the final 8 h of the third day of LPS - induced differentiation ( Leu + E - 64d ( 8 h ) ) . Right , quantification of band intensity ( presented as in Fig . 1c ) . * P 0 . 05 , * * P 0 . 01 and * * * P 0 . 001 ( Student ' s t - test ) .

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

(e) Immunoblot analysis (left) of ER proteins in Atg5f/f B cells left untreated (UT) or treated with lysosomal protease inhibitors (10 μM leupeptin and E-64d) for the final 8 h of the third day of LPS-induced differentiation (Leu + E-64d (8 h)). Right, quantification of band intensity (presented as in Fig. 1c). *P 0.05, **P 0.01 and ***P 0.001 (Student's t-test).


( a ) Enzyme - linked immunosorbent assay ( ELISA ) of IgM in supernatants of Atg5f / fCD19 - Cre and Atg5f / f B cells stimulated for 3 d with LPS and plated for 4 h ( as trypan blue - negative cells at a density of 1 × 106 cells per ml in dedicated media ) , presented relative to results obtained for Atg5f / f cells .

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

(a) Enzyme-linked immunosorbent assay (ELISA) of IgM in supernatants of Atg5f/fCD19-Cre and Atg5f/f B cells stimulated for 3 d with LPS and plated for 4 h (as trypan blue-negative cells at a density of 1 × 106 cells per ml in dedicated media), presented relative to results obtained for Atg5f/f cells.


( b , c ) Immunoglobulin assembly ( b ) and total intracellular ( IC ) and secreted ( Sec ) IgM heavy chain ( IgM - H , c ) in Atg5f / f and Atg5f / fCD19 - CreB cells stimulated for 3 d with LPS , then pulse - labeled and chased ( time , above lanes ) , followed by immunoprecipitation of the μ - chain from lysates and SDS - PAGE ( left ) in nonreducing conditions ( b ) or reducing conditions ( c ) . Right , quantification of intracellular IgM polymers by densitometry ( b ) and of secretion efficiency , estimated as the amount of radioactive secreted μ - chain at the end of the chase relative to intracellular μ - chain at the end of the pulse ( c ) . AU , arbitrary units .

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

(b,c) Immunoglobulin assembly (b) and total intracellular (IC) and secreted (Sec) IgM heavy chain (IgM-H, c) in Atg5f/f and Atg5f/fCD19-CreB cells stimulated for 3 d with LPS, then pulse-labeled and chased (time, above lanes), followed by immunoprecipitation of the μ-chain from lysates and SDS-PAGE (left) in nonreducing conditions (b) or reducing conditions (c). Right, quantification of intracellular IgM polymers by densitometry (b) and of secretion efficiency, estimated as the amount of radioactive secreted μ-chain at the end of the chase relative to intracellular μ-chain at the end of the pulse (c). AU, arbitrary units.


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 B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD B-CELL_TYPE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O 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 O O O O O O O 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 ) Quantitative RT - PCR analysis of mRNA encoding Blimp - 1 and the secreted μ - chain in Atg5f / f and Atg5f / fCD19 - Cre B cells stimulated for 3 d with LPS , presented relative to that of Atg5f / f cells .

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

(d) Quantitative RT-PCR analysis of mRNA encoding Blimp-1 and the secreted μ-chain in Atg5f/f and Atg5f/fCD19-Cre B cells stimulated for 3 d with LPS, presented relative to that of Atg5f/f cells.


( e ) Quantitative RT - PCR analysis of transcripts encoding spliced XBP - 1 , BiP , Blimp - 1 and secreted μ - chain in Atg5f / f B cells stimulated for 3 d with LPS and given no further treatment ( − tun ) or treated for the final 24 h with tunicamycin ( tun 24 h ; 0 . 5 μg / ml ) , presented relative to results obtained with LPS - stimulated cells given no further treatment . * P 0 . 05 , * * P 0 . 01 and * * * P 0 . 001 ( Student ' s t - test ) .

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

(e) Quantitative RT-PCR analysis of transcripts encoding spliced XBP-1, BiP, Blimp-1 and secreted μ-chain in Atg5f/f B cells stimulated for 3 d with LPS and given no further treatment (−tun) or treated for the final 24 h with tunicamycin (tun 24 h; 0.5 μg/ml), presented relative to results obtained with LPS-stimulated cells given no further treatment. *P 0.05, **P 0.01 and ***P 0.001 (Student's t-test).


( a ) Intracellular ATP in fresh lysates of Atg5f / f and Atg5f / fCD19 - Cre splenic B cells activated for 3 d with LPS ( 3 ) or left untreated ( 0 ) .

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

(a) Intracellular ATP in fresh lysates of Atg5f/f and Atg5f/fCD19-Cre splenic B cells activated for 3 d with LPS (3) or left untreated (0).


( b ) Death of Atg5f / f and Atg5f / fCD19 - Cre splenic B cells activated for various times ( horizontal axis ) with LPS , quantified by flow cytometry by propidium iodide ( PI ) staining .

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

(b) Death of Atg5f/f and Atg5f/fCD19-Cre splenic B cells activated for various times (horizontal axis) with LPS, quantified by flow cytometry by propidium iodide (PI) staining.


( c ) Quantitative PCR analysis of genomic DNA in differentiating B cells from Atg5f / f and Atg5f / fCD19 - Cre mice , assessing nonrecombinant ( Undeleted ; left ) and recombinant ( Deleted ; right ) loxP - flanked Atg5 alleles ; results are presented relative to those of unstimulated Atg5f / fCD19 - Cre B cells .

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

(c) Quantitative PCR analysis of genomic DNA in differentiating B cells from Atg5f/f and Atg5f/fCD19-Cre mice, assessing nonrecombinant (Undeleted; left) and recombinant (Deleted; right) loxP-flanked Atg5 alleles; results are presented relative to those of unstimulated Atg5f/fCD19-Cre B cells.


( d ) Quantitative RT - PCR analysis of Atg5 mRNA in differentiating Atg5f / f and Atg5f / fCD19 - Cre B cells at 0 and 3 d after stimulation with LPS , presented relative to that in untreated Atg5f / fCD19 - Cre B cells . * P 0 . 05 , * * P 0 . 01 and * * * P 0 . 001 ( Student ' s t - test ) .

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

(d) Quantitative RT-PCR analysis of Atg5 mRNA in differentiating Atg5f/f and Atg5f/fCD19-Cre B cells at 0 and 3 d after stimulation with LPS, presented relative to that in untreated Atg5f/fCD19-Cre B cells. *P 0.05, **P 0.01 and ***P 0.001 (Student's t-test).


( a ) ELISA of anti - pneumovax IgM titers in diluted serum 1 day before and 4 , 10 and 14 d after intraperitoneal immunization of Atg5f / f and Atg5f / fCD19 - Cre mice with 1 μg pneumovax .

O O O B-EXP_ASSAY O O O B-SMALL_MOLECULE B-SUBCELLULAR 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 O O B-GENEPROD B-ORGANISM O O O B-SMALL_MOLECULE O

(a) ELISA of anti-pneumovax IgM titers in diluted serum 1 day before and 4, 10 and 14 d after intraperitoneal immunization of Atg5f/f and Atg5f/fCD19-Cre mice with 1 μg pneumovax.


( b ) ELISA of anti - pneumovax IgG3 titers in serum 14 d after injection as in a . Results are presented as absorbance ( A ) . * P 0 . 05 , * * P 0 . 01 and * * * P 0 . 001 ( Student ' s t - test ) .

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

(b) ELISA of anti-pneumovax IgG3 titers in serum 14 d after injection as in a. Results are presented as absorbance (A). *P 0.05, **P 0.01 and ***P 0.001 (Student's t-test).


( a ) Frequency of CD138hiB220lo cells ( numbers above outlined areas , bottom ) among the IRF4hi population ( numbers above outlined areas , top ) , assessed by flow cytometry ( left ) . 488 , Alexa Fluor 488 . Right , quantification of bone marrow PCs ( each symbol represents an individual mouse ; small horizontal lines indicate mean ( and s . e . m . ) .

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

(a) Frequency of CD138hiB220lo cells (numbers above outlined areas, bottom) among the IRF4hi population (numbers above outlined areas, top), assessed by flow cytometry (left). 488, Alexa Fluor 488. Right, quantification of bone marrow PCs (each symbol represents an individual mouse; small horizontal lines indicate mean (and s.e.m.).


( b ) Quantitative RT - PCR analysis of mRNA encoding IRF4 and Blimp - 1 in CD138hiB220lo bone marrow PCs and B220hiCD138lo spleen B lymphocytes ( control ) sorted from Atg5f / f and Atg5f / fCD19 - Cre mice , to confirm PC phenotype ; results are presented relative to that of histone H3 mRNA ( loading control ) . ( c ) Quantitative PCR analysis of genomic DNA assessing undeleted and deleted loxP - flanked Atg5 alleles in sorted B cells and PCs ( as in b ; presented as in Fig . 5c ) .

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

(b) Quantitative RT-PCR analysis of mRNA encoding IRF4 and Blimp-1 in CD138hiB220lo bone marrow PCs and B220hiCD138lo spleen B lymphocytes (control) sorted from Atg5f/f and Atg5f/fCD19-Cre mice, to confirm PC phenotype; results are presented relative to that of histone H3 mRNA (loading control). (c) Quantitative PCR analysis of genomic DNA assessing undeleted and deleted loxP-flanked Atg5 alleles in sorted B cells and PCs (as in b; presented as in Fig. 5c).


( d ) Immunofluorescence of endogenous LC3 ( left , top row ) and immunoglobulin content ( left , bottom row ) in bone marrow PCs sorted from Atg5f / f and Atg5f / fCD19 - Cre mice , and quantitative RT - PCR analysis of Atg5 mRNA in bone marrow PCs and spleen B cell populations ( right ) sorted from Atg5f / f and Atg5f / f CD19 - Cre mice . Nuclei ( left ) are stained blue with Hoechst . Scale bar ( left ) , 5 μm . * P 0 . 05 , * * P 0 . 01 and * * * P 0 . 001 ( Student ' s t - test ) .

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

(d) Immunofluorescence of endogenous LC3 (left, top row) and immunoglobulin content (left, bottom row) in bone marrow PCs sorted from Atg5f/f and Atg5f/fCD19-Cre mice, and quantitative RT-PCR analysis of Atg5 mRNA in bone marrow PCs and spleen B cell populations (right) sorted from Atg5f/f and Atg5f/f CD19-Cre mice. Nuclei (left) are stained blue with Hoechst. Scale bar (left), 5 μm. *P 0.05, **P 0.01 and ***P 0.001 (Student's t-test).


A - F ( A - C ) Exercise performance on a treadmill apparatus using an intense exercise ( incremental speed paradigm ) that relies solely on anaerobic metabolism . ( D - F ) Exercise performance on a treadmill apparatus using a moderate exercise ( constant sub - maximal speed ) that relies on the recruitment of aerobic metabolism and that is representative of endurance exercise . ( A , D ) Kaplan - Meier curves representing values corresponding to the percentage of mice still running at a given time point . ( B , E ) Median values of the time that mice ran are represented in the min to max graphical representation ; 15 . 5 min for wild - type ( WT ) and 14 . 33 min for SOD1G86R in ( B ) ; 59 . 33 min for WT and 65 . 43 min for SOD1G86R in ( E ) . ( C , F ) Mean distance run ± SEM by WT and SOD1G86R mice in ( C ) and by WT and SOD1G86R mice in ( F ) .

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 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 B-ORGANISM O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O 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 B-GENEPROD O O O O O O O O O O O O O O O O O O B-GENEPROD B-ORGANISM O O O O O O O O B-GENEPROD B-ORGANISM O O O O O

A-F (A-C) Exercise performance on a treadmill apparatus using an intense exercise (incremental speed paradigm) that relies solely on anaerobic metabolism. (D-F) Exercise performance on a treadmill apparatus using a moderate exercise (constant sub-maximal speed) that relies on the recruitment of aerobic metabolism and that is representative of endurance exercise. (A, D) Kaplan-Meier curves representing values corresponding to the percentage of mice still running at a given time point. (B, E) Median values of the time that mice ran are represented in the min to max graphical representation; 15.5 min for wild-type (WT) and 14.33 min for SOD1G86R in (B); 59.33 min for WT and 65.43 min for SOD1G86R in (E). (C, F) Mean distance run ± SEM by WT and SOD1G86R mice in (C) and by WT and SOD1G86R mice in (F).