1 × 106 CD14 + myeloid cells from healthy donors were polarized by 1 ng / ml GM - CSF in the presence of BSA - control ( C ) , 0 . 2 mM oleate ( O ) or 0 . 2 mM stearate ( S ) over a 6 - day period . The expression of CD206 , CD204 and CD38 was analyzed via flow cytometry .

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

1×106 CD14+ myeloid cells from healthy donors were polarized by 1 ng/ml GM-CSF in the presence of BSA-control (C), 0.2 mM oleate (O) or 0.2 mM stearate (S) over a 6-day period. The expression of CD206, CD204 and CD38 was analyzed via flow cytometry.


The level of lipid droplets was determined via BODIPY staining .

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

The level of lipid droplets was determined via BODIPY staining.


The oxygen consumption rate ( OCR ) of differentiated myeloid cells was monitored as described in Materials and Methods .

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The oxygen consumption rate (OCR) of differentiated myeloid cells was monitored as described in Materials and Methods.


( D ) The purified CFSE - labeled autologous CD4 + T cells were co - cultured with polarized myeloid cells with indicated treatment for T cell proliferation assay .

(D) The purified CFSE-labeled autologous CD4+ T cells were co-cultured with polarized myeloid cells with indicated treatment for T cell proliferation assay.


Tumor tissue ( tumor ) as well as corresponding non - tumorous adjacent tissue ( control ) were collected from colorectal cancer patients and prepared for histopathology . The expression of CD68 ( red ) , CD206 ( green ) and ADRP ( blue ) indicate the lipid droplets in tumor infiltrating myeloid cells . Nuclei ( white ) were stained using DAPI . The absolute number of positive cells was quantified in 5 high - power fields ( hpf ) ( scale bar = 20 µm ) .

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

Tumor tissue (tumor) as well as corresponding non-tumorous adjacent tissue (control) were collected from colorectal cancer patients and prepared for histopathology. The expression of CD68 (red), CD206 (green) and ADRP (blue) indicate the lipid droplets in tumor infiltrating myeloid cells. Nuclei (white) were stained using DAPI. The absolute number of positive cells was quantified in 5 high-power fields (hpf) (scale bar = 20 µm).


A . Immunofluorescence microscopy of control , parental and patient fibroblasts labeled for LAMP - 1 . Patient fibroblasts ( VPS41S285P / R662 * and VPS41c . 1423 - 2A > G / R662 * ) show more LAMP - 1 puncta , which are distributed throughout the cell . ( A ' ) Quantification shows a significant increase in the number of LAMP - 1 positive compartments for both patients . > 15 Cells per condition were quantified ( n = 3 ) . Scale bars , 10µm .

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

A. Immunofluorescence microscopy of control, parental and patient fibroblasts labeled for LAMP-1. Patient fibroblasts (VPS41S285P/R662* and VPS41c.1423-2A>G/R662*) show more LAMP-1 puncta, which are distributed throughout the cell. (A') Quantification shows a significant increase in the number of LAMP-1 positive compartments for both patients. >15 Cells per condition were quantified (n=3). Scale bars, 10µm.


D . HeLaVPS41KO cells transfected with VPS41WT - APEX2 - V5 , VPS41S285P - APEX2 - V5 or VPS41R662 * - APEX2 - V5 constructs labeled for LAMP - 1 and V5 immunofluorescence microscopy . All VPS41 variants co - localize with LAMP - 1 , indicating that VPS41S285P and VPS41R662 * are recruited to late endosomes / lysosomes . Scale bars 10µm ; zoom of squared area , 1µm .

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

D. HeLaVPS41KO cells transfected with VPS41WT-APEX2-V5, VPS41S285P-APEX2-V5 or VPS41R662*-APEX2-V5 constructs labeled for LAMP-1 and V5 immunofluorescence microscopy. All VPS41 variants co-localize with LAMP-1, indicating that VPS41S285P and VPS41R662* are recruited to late endosomes/lysosomes. Scale bars 10µm; zoom of squared area, 1µm.


H . VPS41WT / WT , VPS41WT / R662 * and VPS41S285P / R662 * primary fibroblasts incubated with Dextran - 568 for 0 . 5 , 2 , 5 , 8 and 24 hours . Colocalization of Dextran - 568 and SiR - Lysosome Cathepsin D ( SiR - Lyso ) reveals a delay in lysosomal delivery in maternal ( VPS41WT / R662 * ) and patient ( VPS41S285P / R662 * ) cells at 2 hours of Dextran uptake . After 5 hours , maternal cells show similar to control colocalization levels . Patient cells show only after 24 hours colocalization levels similar to control , indicating a delay rather than a block in late endosome - lysosome fusion . > 10 Cells per cell line were quantified .

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

H. VPS41WT/WT, VPS41WT/R662* and VPS41S285P/R662* primary fibroblasts incubated with Dextran-568 for 0.5, 2, 5, 8 and 24 hours. Colocalization of Dextran-568 and SiR-Lysosome Cathepsin D (SiR-Lyso) reveals a delay in lysosomal delivery in maternal (VPS41WT/R662*) and patient (VPS41S285P/R662*) cells at 2 hours of Dextran uptake. After 5 hours, maternal cells show similar to control colocalization levels. Patient cells show only after 24 hours colocalization levels similar to control, indicating a delay rather than a block in late endosome - lysosome fusion. >10 Cells per cell line were quantified.


A . Western blot of LC3 expression levels in control and patient fibroblasts . In steady state ( 0 hours ) conditions , patient fibroblasts have a higher ratio of lipidated LC3 ( LC3II ) : LC3I than control cells . Induction of autophagy by nutrient starvation ( 2 hours incubation with minimal EBSS medium ) results in a raise in LC3II : LC3I ratio in both control and patient cells , but in VPS41S285P / R662 * fibroblasts this increase is only modest ( quantified in A ' ) ( n = 2 ) .

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

A. Western blot of LC3 expression levels in control and patient fibroblasts. In steady state (0 hours) conditions, patient fibroblasts have a higher ratio of lipidated LC3 (LC3II):LC3I than control cells. Induction of autophagy by nutrient starvation (2 hours incubation with minimal EBSS medium) results in a raise in LC3II:LC3I ratio in both control and patient cells, but in VPS41S285P/R662* fibroblasts this increase is only modest (quantified in A') (n=2).


E . Immunofluorescence of HeLaWT and HeLaVPS41KO cells transfected with LC3GFP / RFP tandem construct . The increased percentage of GFP / RFP - positive compartments in HeLaVPS41KO cells indicate a block in autophagic flux ( quantified in E ' ) . > 12 Cells per cell line were quantified . Scale bars 10µm ; zoom , 1µm .

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

E. Immunofluorescence of HeLaWT and HeLaVPS41KO cells transfected with LC3GFP/RFP tandem construct. The increased percentage of GFP/RFP-positive compartments in HeLaVPS41KO cells indicate a block in autophagic flux (quantified in E'). >12 Cells per cell line were quantified. Scale bars 10µm; zoom, 1µm.


A . Immunofluorescence of control , parental and patient fibroblasts labeled for LAMP - 1 and mTOR . In steady state conditions ( 0 hours ) VPS41S285P / R662 * fibroblasts show less colocalization between mTOR and LAMP - 1 . VPS41WT / WT , VPS41WT / S285P and VPS41WT / R662 * show an appropriate mTOR response upon nutrient deprivation ( 2 hours ) or restimulation ( 2 hours starvation followed by 15 minutes restimulation ) ( Appendix Fig S5A ) . > 10 Cells per cell line were quantified ( A ' ) . Quantifications are performed relative to colocalization under steady state conditions per cell line ( n = 3 ) . Scale bars , 10µm ; zoom , 1µm .

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

A. Immunofluorescence of control, parental and patient fibroblasts labeled for LAMP-1 and mTOR. In steady state conditions (0 hours) VPS41S285P/R662* fibroblasts show less colocalization between mTOR and LAMP-1. VPS41WT/WT, VPS41WT/S285P and VPS41WT/R662* show an appropriate mTOR response upon nutrient deprivation (2 hours) or restimulation (2 hours starvation followed by 15 minutes restimulation) (Appendix Fig S5A). >10 Cells per cell line were quantified (A'). Quantifications are performed relative to colocalization under steady state conditions per cell line (n=3). Scale bars, 10µm; zoom, 1µm.


D . Rescue experiments of HeLaVPS41KO cells . Expression of VPS41WT - APEX2 - V5 , VPS41S285P - APEX2 - V5 or VPS41R662 * - APEX2 - V5 . Reintroduction of VPS41WT rescues TFE3 localization , whereas expression of mutant VPS41 has no effect ( quantified in D ' ) . > 83 Cells per condition were quantified ( n = 3 ) . Scale bars , 10µm .

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

D. Rescue experiments of HeLaVPS41KO cells. Expression of VPS41WT-APEX2-V5, VPS41S285P-APEX2-V5 or VPS41R662*-APEX2-V5. Reintroduction of VPS41WT rescues TFE3 localization, whereas expression of mutant VPS41 has no effect (quantified in D'). >83 Cells per condition were quantified (n=3). Scale bars, 10µm.


B . Western blot on cellular or secreted secretogranin II ( SgII ) of PC12 cells VPS41KO , or VPS41KO transduced with HA - VPS41WT or HA - VPS41S285P lentivirus . Cells were washed and incubated for 30 minutes in Tyrode ' s solution containing 2 . 5 mM K + ( basal ) or 90 mM K + ( stimulated ) . No difference in secreted sgII levels was observed between VPS41WT and VPS41S285P .

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

B. Western blot on cellular or secreted secretogranin II (SgII) of PC12 cells VPS41KO, or VPS41KO transduced with HA-VPS41WT or HA-VPS41S285P lentivirus. Cells were washed and incubated for 30 minutes in Tyrode's solution containing 2.5 mM K+ (basal) or 90 mM K+ (stimulated). No difference in secreted sgII levels was observed between VPS41WT and VPS41S285P.


C . Graph representing the percentage of animals with 6 normal dopaminergic neurons in the anterior region of Pdat - 1 : : GFP ; Pdat - 1 : : α - syn ( strain UA44 ) animals with heterozygous expression of hVPS41 variants . Heterozygous expression of hVPS41WT with either variant ( strains UA389 or UA390 ) significantly rescues neurons from α - synuclein - induced degeneration at day 7 whereas compound heterozygous expression ( strain UA391 ) fails to rescue neurodegeneration . At day 10 , none of the heterozygous backgrounds significantly rescues α - synuclein - induced neurodegeneration . n = 30 Adult worms for each of 3 independent experiments for the α - synuclein ( α - syn ) strain ( total of 90 worms ) and n = 90 for each independent transgenic strain ( 30 worms / trial x 3 independent transgenic lines = 270 worms ) for each of 3 independent experiments . D . Representative images of DA neurons from C . elegans expressing Pdat - 1 : : GFP ; Pdat - 1 : : α - syn , with or without hVPS41 variants , as described in ( C ) . Intact DA neurons are indicated with arrowheads and missing neurons are indicated with arrows . Scale bar 20µm .

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

C. Graph representing the percentage of animals with 6 normal dopaminergic neurons in the anterior region of Pdat-1::GFP; Pdat-1:: α-syn (strain UA44) animals with heterozygous expression of hVPS41 variants. Heterozygous expression of hVPS41WT with either variant (strains UA389 or UA390) significantly rescues neurons from α-synuclein-induced degeneration at day 7 whereas compound heterozygous expression (strain UA391) fails to rescue neurodegeneration. At day 10, none of the heterozygous backgrounds significantly rescues α-synuclein-induced neurodegeneration. n=30 Adult worms for each of 3 independent experiments for the α-synuclein (α-syn) strain (total of 90 worms) and n=90 for each independent transgenic strain (30 worms/trial x 3 independent transgenic lines = 270 worms) for each of 3 independent experiments. D. Representative images of DA neurons from C. elegans expressing Pdat-1::GFP; Pdat-1:: α-syn, with or without hVPS41 variants, as described in (C). Intact DA neurons are indicated with arrowheads and missing neurons are indicated with arrows. Scale bar 20µm.


A Gene targeting strategy . Left : max genomic locus , targeting construct and max targeted locus . In vitro Flipase ( Flip ) deletion of the selectable markers lacZ and neo results in a max targeted locus with exons 4 and 5 ( coding for bHLHLZip domain ) flanked by two loxP sites ( fl , flox allele ) . Arrows represent PCR primers used for genotyping MaxF9 and MaxF10 . Right : genomic PCR analysis of tail DNA from F1 mice . PCR products of max wt ( 430 bp ) and max flox ( 526 bp ) alleles . M , 1kb ladder .

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

A Gene targeting strategy. Left: max genomic locus, targeting construct and max targeted locus. In vitro Flipase (Flip) deletion of the selectable markers lacZ and neo results in a max targeted locus with exons 4 and 5 (coding for bHLHLZip domain) flanked by two loxP sites (fl, flox allele). Arrows represent PCR primers used for genotyping MaxF9 and MaxF10. Right: genomic PCR analysis of tail DNA from F1 mice. PCR products of max wt (430 bp) and max flox (526 bp) alleles. M, 1kb ladder.


Flow cytometry analyses of B cell populations ( GFP + ) in Bone Marrow ( BM ) and spleen . Single - cell suspensions were prepared from BM or spleen of MaxKO - mb1 , MycKO - mb1 , DKO - mb1 or heterozygous HET - mb1 control mice , stained with antibodies against the indicated markers , and analyzed by flow cytometry . For BM analysis , cells were isolated from femora and tibiae . Pro - B cells ( Ly - 6c - CD49b - B220 + IgM - CD43 + ) , pre - B cells ( Ly - 6c - CD49b - B220 + IgM - CD43 - ) , immature B cells ( Ly - 6c - CD49b - B220loIgM + CD43 - ) , and mature B cells ( Ly - 6c - CD49b - B220hiIgM + CD43 - ) To identify KO cells ( GFP + ) all the plots show gated GFP + B lymphocytes .

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

Flow cytometry analyses of B cell populations (GFP+) in Bone Marrow (BM) and spleen. Single-cell suspensions were prepared from BM or spleen of MaxKO-mb1, MycKO-mb1, DKO-mb1 or heterozygous HET-mb1 control mice, stained with antibodies against the indicated markers, and analyzed by flow cytometry. For BM analysis, cells were isolated from femora and tibiae. Pro-B cells (Ly-6c-CD49b-B220+IgM-CD43+), pre-B cells (Ly-6c-CD49b-B220+IgM-CD43-), immature B cells (Ly-6c-CD49b-B220loIgM+CD43-), and mature B cells (Ly-6c-CD49b-B220hiIgM+CD43-) To identify KO cells (GFP+) all the plots show gated GFP+ B lymphocytes.


Flow cytometry analyses of B cell populations ( GFP + ) in Bone Marrow ( BM ) Single - cell suspensions were prepared from BM of MaxKO - mb1 , MycKO - mb1 , DKO - mb1 or heterozygous HET - mb1 control mice , stained with antibodies against the indicated markers , and analyzed by flow cytometry . For BM analysis , cells were isolated from femora and tibiae . Pro - B cells ( Ly - 6c - CD49b - B220 + IgM - CD43 + ) , pre - B cells ( Ly - 6c - CD49b - B220 + IgM - CD43 - )

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

Flow cytometry analyses of B cell populations (GFP+) in Bone Marrow (BM) Single-cell suspensions were prepared from BM of MaxKO-mb1, MycKO-mb1, DKO-mb1 or heterozygous HET-mb1 control mice, stained with antibodies against the indicated markers, and analyzed by flow cytometry. For BM analysis, cells were isolated from femora and tibiae. Pro-B cells (Ly-6c-CD49b-B220+IgM-CD43+), pre-B cells (Ly-6c-CD49b-B220+IgM-CD43-)


Flow cytometry analyses of B cell populations ( GFP + ) in spleen . Single - cell suspensions were prepared from spleen of MaxKO - mb1 , MycKO - mb1 , DKO - mb1 or heterozygous HET - mb1 control mice , stained with antibodies against the indicated markers , and analyzed by flow cytometry . spleen follicular B cells ( B220 + CD23 + CD21int ) , marginal zone B cells ( B220 + CD23 - CD21hi ) and immature ( newly formed / transitional stage 1 ) B cells ( B220 + CD23 - CD21lo ) . To identify KO cells ( GFP + ) all the plots show gated GFP + B lymphocytes .

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

Flow cytometry analyses of B cell populations (GFP+) in spleen. Single-cell suspensions were prepared from spleen of MaxKO-mb1, MycKO-mb1, DKO-mb1 or heterozygous HET-mb1 control mice, stained with antibodies against the indicated markers, and analyzed by flow cytometry. spleen follicular B cells (B220+CD23+CD21int), marginal zone B cells (B220+CD23-CD21hi) and immature (newly formed/transitional stage 1) B cells (B220+CD23-CD21lo). To identify KO cells (GFP+) all the plots show gated GFP+ B lymphocytes.


F , G , H Absolute numbers of B lymphocytes in Bone Marrow ( F , G ) and spleen ( F , H ) from MaxKO - mb1 ( n = 6 ) , MycKO - mb - 1 ( n = 3 ) , DKO - mb1 ( n = 5 ) and control HET - mb1 ( n = 4 ) mice . Columns represent mean values and error bars standard deviations . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . The different B cell populations were identified by flow cytometry Experiment representative of at least three independent experiments .

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

F, G, H Absolute numbers of B lymphocytes in Bone Marrow (F, G) and spleen (F, H) from MaxKO-mb1 (n=6), MycKO-mb-1 (n=3), DKO-mb1 (n=5) and control HET-mb1 (n=4) mice. Columns represent mean values and error bars standard deviations. *p<0.05, **p<0.01, ***p<0.001. The different B cell populations were identified by flow cytometry Experiment representative of at least three independent experiments.


I Cell cycle analysis by flow cytometry of sorted GFP + pre - and pro - B ( B220 + IgM - ) , immature ( B220loIgM + ) and mature ( B220hiIgM + ) bone marrow B cells from MaxKO - mb1 , MycKO - mb1 , DKO - mb1 and HET - mb1 control mice . Mice ( n = 5 ) were injected ( i . p . ) with 200 µg of EdU and analyzed 4 h later . Single - cell suspensions were prepared , stained and sorted . Sorted cells were subsequently stained with propidium iodide for DNA content . Experiment representative of at least three independent experiments . Dotted line is shown for EdU MFI comparison .

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

I Cell cycle analysis by flow cytometry of sorted GFP+ pre- and pro-B (B220+IgM-), immature (B220loIgM+) and mature (B220hiIgM+) bone marrow B cells from MaxKO-mb1, MycKO-mb1, DKO-mb1 and HET-mb1 control mice. Mice (n=5) were injected (i.p.) with 200 µg of EdU and analyzed 4 h later. Single-cell suspensions were prepared, stained and sorted. Sorted cells were subsequently stained with propidium iodide for DNA content. Experiment representative of at least three independent experiments. Dotted line is shown for EdU MFI comparison.


J Percentage of annexin - V + GFP + cells in the BM of MaxKO - mb1 ( n = 5 ) , MycKO - mb1 ( n = 3 ) , DKO - mb1 ( n = 3 ) and control ( n = 4 ) mice . Pro - B , pre - B , immature and mature B lymphocyte subpopulations were identified by flow cytometry as indicated in E . * p < 0 . 05 , * * * p < 0 . 001 . Experiment representative of at least three independent experiments .

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

J Percentage of annexin-V+GFP+ cells in the BM of MaxKO-mb1(n=5), MycKO-mb1(n=3), DKO-mb1 (n=3) and control (n=4) mice. Pro-B, pre-B, immature and mature B lymphocyte subpopulations were identified by flow cytometry as indicated in E. *p<0.05, ***p<0.001. Experiment representative of at least three independent experiments.


Flow cytometry analysis and absolute numbers of B220loCD138 + ( plasmablasts ) ( A , B ) from HET - cd19 ( n = 6 ) , MaxKO - cd19 ( n = 8 ) , MycKO - cd19 ( n = 4 ) , and DKO - cd19 ( n = 5 ) mice . Spleen B220 + GFP + B lymphocytes were sorted and activated in vitro with LPS plus IL - 4 for 72 h . Cells were stained with monoclonal antibodies against the indicated markers and analyzed by flow cytometry . * p < 0 . 05 , * * p < 0 . 01 . Columns represent mean values and error bars standard deviations . Experiment representative of at least three independent experiments .

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

Flow cytometry analysis and absolute numbers of B220loCD138+ (plasmablasts) (A, B) from HET-cd19 (n=6), MaxKO-cd19 (n=8), MycKO-cd19 (n=4), and DKO-cd19 (n=5) mice. Spleen B220+GFP+ B lymphocytes were sorted and activated in vitro with LPS plus IL-4 for 72 h. Cells were stained with monoclonal antibodies against the indicated markers and analyzed by flow cytometry. *p<0.05, **p<0.01. Columns represent mean values and error bars standard deviations. Experiment representative of at least three independent experiments.


Flow cytometry analysis and absolute numbers of B220 + IgG1 + switched B cells ( C , D ) from HET - cd19 ( n = 6 ) , MaxKO - cd19 ( n = 8 ) , MycKO - cd19 ( n = 4 ) , and DKO - cd19 ( n = 5 ) mice . Spleen B220 + GFP + B lymphocytes were sorted and activated in vitro with LPS plus IL - 4 for 72 h . Cells were stained with monoclonal antibodies against the indicated markers and analyzed by flow cytometry . * p < 0 . 05 , * * p < 0 . 01 . Columns represent mean values and error bars standard deviations . Experiment representative of at least three independent experiments .

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

Flow cytometry analysis and absolute numbers of B220+IgG1+ switched B cells (C, D) from HET-cd19 (n=6), MaxKO-cd19 (n=8), MycKO-cd19 (n=4), and DKO-cd19 (n=5) mice. Spleen B220+GFP+ B lymphocytes were sorted and activated in vitro with LPS plus IL-4 for 72 h. Cells were stained with monoclonal antibodies against the indicated markers and analyzed by flow cytometry. *p<0.05, **p<0.01. Columns represent mean values and error bars standard deviations. Experiment representative of at least three independent experiments.


E Spleen B220 + GFP + B lymphocytes were stained with 7AAD and annexin - V to monitor apoptosis . 7 - AAD - annexin - v - ( alive ) , 7 - AAD - annexin - v + ( early apoptotic ) , 7 - AAD + annexin - v + ( late apoptotic ) , 7 - AAD + annexin - v - ( necrosis ) . F Relative numbers of live , early apoptotic , late apoptotic and necrotic cells from E .

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

E Spleen B220+GFP+ B lymphocytes were stained with 7AAD and annexin-V to monitor apoptosis. 7-AAD- annexin-v- (alive), 7-AAD- annexin-v+ (early apoptotic), 7-AAD+ annexin-v+ (late apoptotic), 7-AAD+ annexin-v- (necrosis). F Relative numbers of live, early apoptotic, late apoptotic and necrotic cells from E.


G CD69 surface expression in purified B220 + GFP + mature spleen cells . Sorted B cells were activated in vitro with LPS plus IL - 4 for 72 h , stained and analyzed by flow cytometry . The solid grey peak represents the expression levels of CD69 marker in non - activated mature B lymphocytes at day 0 . H Relative numbers of CD69 + B lymphocytes in the cultures from G ( n = 4 ) . Experiment representative of at least three independent experiments .

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

G CD69 surface expression in purified B220+GFP+ mature spleen cells. Sorted B cells were activated in vitro with LPS plus IL-4 for 72 h, stained and analyzed by flow cytometry. The solid grey peak represents the expression levels of CD69 marker in non-activated mature B lymphocytes at day 0. H Relative numbers of CD69+ B lymphocytes in the cultures from G (n=4). Experiment representative of at least three independent experiments.


I Analysis of germinal center ( GC ) formation in the spleen of MaxKO - cd19 , Myc - KO - cd19 , DKO - cd19 and heterozygous control mice immunized with TNP - KLH . Representative images of frozen spleen sections stained with IgM ( grey / blue ) , PNA ( GC marker ; red ) , and GFP ( Max - , c - Myc - or c - Myc / Max - deficient B cells ; green ) . Scale bar , 80µm .

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

I Analysis of germinal center (GC) formation in the spleen of MaxKO-cd19, Myc-KO-cd19, DKO-cd19 and heterozygous control mice immunized with TNP-KLH. Representative images of frozen spleen sections stained with IgM (grey/blue), PNA (GC marker; red), and GFP (Max-, c-Myc- or c-Myc/Max-deficient B cells; green). Scale bar, 80µm.


J Upper , Number of GCs per mouse in spleen sections of immunized MaxKO - cd19 ( n = 3 mice , n = 186 follicles analyzed ) , MycKO - cd19 ( n = 3 mice , n = 178 follicles analyzed ) , DKO - cd19 ( n = 3 mice , n = 191 follicles analyzed ) and heterozygous control mice ( n = 3 mice , n = 203 follicles analyzed ) . Lower , Frequencies of GC ( PNA + ) GFP + ( deleted cells ) or GFP - ( non - deleted cells ) in immunized MaxKO - cd19 ( n = 13 GCs ) , MycKO - cd19 ( n = 21 GCs ) , DKO - cd19 ( n = 27 GCs ) and heterozygous control ( n = 62 GCs ) mice . Analysis was performed at 13 days post - immunization . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

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

J Upper, Number of GCs per mouse in spleen sections of immunized MaxKO-cd19 (n=3 mice, n=186 follicles analyzed), MycKO-cd19 (n=3 mice, n=178 follicles analyzed), DKO-cd19 (n=3 mice, n=191 follicles analyzed) and heterozygous control mice (n=3 mice, n=203 follicles analyzed). Lower, Frequencies of GC (PNA+) GFP+ (deleted cells) or GFP- (non-deleted cells) in immunized MaxKO-cd19 (n=13 GCs), MycKO-cd19 (n=21 GCs), DKO-cd19 (n=27 GCs) and heterozygous control (n=62 GCs) mice. Analysis was performed at 13 days post-immunization. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.


A Number of cell divisions in activated mature B lymphocytes . Sorted B220 + GFP + spleen cells from HET - cd19 ( n = 5 ) , MaxKO - cd19 ( n = 5 ) , MycKO - cd19 ( n = 3 ) , and DKO - cd19 ( n = 3 ) mice were stained with a cell tracer , activated in vitro with LPS plus IL - 4 for 72 h and analyzed by flow cytometry . Experiment representative of at least three independent experiments B Cell counts from the cultures C Photographs of B cell cultures Scale bar , 500 µm . 4 × magnification . D Forward scatter ( FSC ) histograms of activated B lymphocytes An arbitrary dashed line is shown for comparison .

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

A Number of cell divisions in activated mature B lymphocytes. Sorted B220+GFP+ spleen cells from HET-cd19 (n=5), MaxKO-cd19 (n=5), MycKO-cd19 (n=3), and DKO-cd19 (n=3) mice were stained with a cell tracer, activated in vitro with LPS plus IL-4 for 72 h and analyzed by flow cytometry. Experiment representative of at least three independent experiments B Cell counts from the cultures C Photographs of B cell cultures Scale bar, 500 µm. 4× magnification. D Forward scatter (FSC) histograms of activated B lymphocytes An arbitrary dashed line is shown for comparison.


Cell cycle analysis of sorted B220 + GFP + B lymphocytes activated in vitro . Cells were activated and EdU was added 4 h before harvesting . Cells were stained with propidium iodide to measure DNA content . Experiment representative of at least three independent experiments . An arbitrary dashed line shows the difference of EdU MFI . S phase mean fluorescence intensity ( MFI ) : 7871 HET , 6512 MaxKO , 5860 MycKO , 6420

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

Cell cycle analysis of sorted B220+GFP+ B lymphocytes activated in vitro. Cells were activated and EdU was added 4 h before harvesting. Cells were stained with propidium iodide to measure DNA content. Experiment representative of at least three independent experiments. An arbitrary dashed line shows the difference of EdU MFI. S phase mean fluorescence intensity (MFI): 7871 HET, 6512 MaxKO, 5860 MycKO, 6420 DKO


Representation of labeling with nucleotide analogues CldU ( red ) and IdU ( green ) and photographs of DNA fibers from sorted B220 + GFP + B lymphocytes activated for 48 h . Scale bar , 10 µm .

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

Representation of labeling with nucleotide analogues CldU (red) and IdU (green) and photographs of DNA fibers from sorted B220+GFP+ B lymphocytes activated for 48 h. Scale bar, 10 µm.


H Fork rate of DNA fibres from primary B lymphocytes sorted and activated Data is pooled from three replicate experiments . Horizontal bars represent median values . 150 - 250 forks were measured per condition . * * * , p < 0 . 001 ; * * , p < 0 .

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

H Fork rate of DNA fibres from primary B lymphocytes sorted and activated Data is pooled from three replicate experiments. Horizontal bars represent median values. 150-250 forks were measured per condition. ***, p<0.001; **, p<0.01


I Western blotting of DNA replication proteins in activated B lymphocytes Sorted B220 + GFP + spleen cells were activated in vitro with LPS plus IL - 4 for 72 h . MEK2 , SMC1 are shown as loading controls . The blots shown are representative of three independent experiments .

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

I Western blotting of DNA replication proteins in activated B lymphocytes Sorted B220+GFP+ spleen cells were activated in vitro with LPS plus IL-4 for 72 h. MEK2, SMC1 are shown as loading controls. The blots shown are representative of three independent experiments.


A Bar plot showing the number of DEGs ( adj . p - value < 0 . 01 , FC + 1 . 5 in red , 1 . 5 < FC > - 1 . 5 in white , FC < - 1 . 5 in blue ) . Sorted B220 + GFP + spleen B cells from MaxKO - cd19 , MycKO - cd19 , DKO - cd19 and control HET - cd19 mice were activated with LPS plus IL - 4 for 72 h and analyzed . Two samples for each condition were used .

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

A Bar plot showing the number of DEGs (adj. p-value <0.01, FC +1.5 in red, 1.5 < FC >-1.5 in white, FC< -1.5 in blue). Sorted B220+GFP+ spleen B cells from MaxKO-cd19, MycKO-cd19, DKO-cd19 and control HET-cd19 mice were activated with LPS plus IL-4 for 72 h and analyzed. Two samples for each condition were used.


C Hierarchical clustering heat map of DEGs in any comparison ( two samples per condition ) . Dark blue in left panel indicates significance for that contrast column . Colour code : red represents upregulation , white , unchanged , and blue downregulation of expression . Gender : colour code for genetic condition . Pink are female and blue colour male mice .

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C Hierarchical clustering heat map of DEGs in any comparison (two samples per condition). Dark blue in left panel indicates significance for that contrast column. Colour code: red represents upregulation, white, unchanged, and blue downregulation of expression. Gender: colour code for genetic condition. Pink are female and blue colour male mice.


D Venn diagrams of up - ( left ) and down - regulated genes ( right ) for all three conditions ( + / - 1 . 5 - FC and p < 0 . 01 ) . Heatmaps of genes only deregulated in MaxKO - cd19 , MycKO - cd19 , or DKO - cd19 mice are shown .

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

D Venn diagrams of up- (left) and down-regulated genes (right) for all three conditions (+/-1.5-FC and p<0.01). Heatmaps of genes only deregulated in MaxKO-cd19, MycKO-cd19, or DKO-cd19 mice are shown.


B Heatmaps of genes involved in the relevant pathways Colour code : red represents upregulation , white , unchanged , and blue downregulation of expression .

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 Heatmaps of genes involved in the relevant pathways Colour code: red represents upregulation, white, unchanged, and blue downregulation of expression.


C qPCR analysis of up - and down - regulated genes Pooled RNA was prepared from sorted B220 + GFP + spleen B cells of MaxKO - cd19 ( n = 4 ) , MycKO - cd19 ( n = 4 ) , or DKO - cd19 ( n = 4 ) and control HET - cd19 ( n = 4 ) mice activated Genes were randomly selected and qPCR was performed using specific primers for cd72 , med13l , card11 , ssh2 , cdc7 , mrpl12 , rpl39 , and cysc . max and c - myc were included as controls and actin was used for normalization .

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

C qPCR analysis of up- and down-regulated genes Pooled RNA was prepared from sorted B220+GFP+ spleen B cells of MaxKO-cd19 (n=4), MycKO-cd19 (n=4), or DKO-cd19 (n=4) and control HET-cd19 (n=4) mice activated Genes were randomly selected and qPCR was performed using specific primers for cd72, med13l, card11, ssh2, cdc7, mrpl12, rpl39, and cysc. max and c-myc were included as controls and actin was used for normalization.


Bacterial growth assessed by color change due to medium acidification for both the undefined ATCC 1161 and semi - defined CSY media with or without sucrose , 4 % and 1 % respectively . ( + ) , inoculated wells ; ( - ) , non - inoculated control . The picture was taken after a 24 - hour incubation period .

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Bacterial growth assessed by color change due to medium acidification for both the undefined ATCC 1161 and semi-defined CSY media with or without sucrose, 4% and 1% respectively. (+), inoculated wells; (-), non-inoculated control. The picture was taken after a 24-hour incubation period.


Impact of horse serum ( HS ) and yeast extract ( YE ) dilution on M . florum doubling time . The green and red dotted lines indicate HS and YE concentrations found in ATCC 1161 medium ( 20 % HS , 1 . 35 % YE ) and CSY medium ( 0 . 313 % HS , 0 . 02 % YE ) , respectively . Doubling times were measured in a CMRL - 1066 base medium using colorimetric assays . Dots and error bars indicate the mean and standard deviation calculated from three biological replicates .

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

Impact of horse serum (HS) and yeast extract (YE) dilution on M. florum doubling time. The green and red dotted lines indicate HS and YE concentrations found in ATCC 1161 medium (20% HS, 1.35% YE) and CSY medium (0.313% HS, 0.02% YE), respectively. Doubling times were measured in a CMRL-1066 base medium using colorimetric assays. Dots and error bars indicate the mean and standard deviation calculated from three biological replicates.


The maximal biomass concentration observed for M . florum cultures growing in CSY medium with varying initial concentrations of sucrose . Biomass was measured using colony forming units ( CFU / ml ; left axis ) and converted to grams of dry weight ( gDW / L ; right axis ) . A rectangular hyperbola fit is shown ( yellow ) , and the dotted line represents the maximal biomass value predicted by the fit ( 1e8 x 5 . 95 CFU / ml ; 0 . 013 gDW / L ) .

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

The maximal biomass concentration observed for M. florum cultures growing in CSY medium with varying initial concentrations of sucrose. Biomass was measured using colony forming units (CFU/ml; left axis) and converted to grams of dry weight (gDW/L; right axis). A rectangular hyperbola fit is shown (yellow), and the dotted line represents the maximal biomass value predicted by the fit (1e8 x 5.95 CFU/ml; 0.013 gDW/L).


Sucrose specific uptake rate ( e ) and combined lactate / acetate specific secretion rates ( f ) at varying initial sucrose concentrations in CSY medium . Boxplots represent the median and interquartile range of uptake or secretion rate values calculated at different time intervals during the exponential growth phase of M . florum cultures . Whiskers indicate minimal and maximal values . Dotted lines indicate the selected uptake ( - 5 . 26 mmol ∙ gDW - 1 ∙ hr - 1 ) and secretion rate ( 8 . 69 mmol ∙ gDW - 1 ∙ hr - 1 ) values used for modeling . Sucrose quantifications were performed in technical duplicate , whereas lactate and acetate quantifications were performed in single replicates . See Appendix Figure S5 for further details .

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

Sucrose specific uptake rate (e) and combined lactate/acetate specific secretion rates (f) at varying initial sucrose concentrations in CSY medium. Boxplots represent the median and interquartile range of uptake or secretion rate values calculated at different time intervals during the exponential growth phase of M. florum cultures. Whiskers indicate minimal and maximal values. Dotted lines indicate the selected uptake (-5.26 mmol∙gDW-1∙hr-1) and secretion rate (8.69 mmol∙gDW-1∙hr-1) values used for modeling. Sucrose quantifications were performed in technical duplicate, whereas lactate and acetate quantifications were performed in single replicates. See Appendix Figure S5 for further details.


Week 10 COs stained for the ventricular radial glia ( vRGs ) marker CRYAB , together with WWOX and the pan - radial glia marker SOX2 . The images on the right are enlargements of the boxed area . The dashed line defines the ventricular space . Scale = 50µm ( left ) , 25µm ( right ) ( WT : n = 8 from 3 batches , KO : n = 8 from 3 batches ) .

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

Week 10 COs stained for the ventricular radial glia (vRGs) marker CRYAB, together with WWOX and the pan-radial glia marker SOX2. The images on the right are enlargements of the boxed area. The dashed line defines the ventricular space. Scale= 50µm (left), 25µm (right) (WT: n=8 from 3 batches, KO: n=8 from 3 batches).


Immunofluorescent ( IF ) staining for the glutamatergic neurons marker VGLUT1 and GABAergic neurons marker GAD67 ( GAD1 ) in week 10 COs ( WT : n = 8 from 3 batches , KO : n = 8 from 3 batches , W - AAV : n = 4 from 1 batch ) . Scale = 100µm . Quantification of the images represented in figure 1E . VGLUT1 and GAD67 were quantified as the surface area covered by the staining and were normalized to the number of nuclei in each image . The boxplot represents the 1st and 3rd quartile , with its whiskers showing the minimum and maximum points and a central band representing the median . As the samples were not normally distributed , statistical significance was determined using Kruskal - Wallis test with Dunn ' s multiple comparisons test . Outliers were removed using the ROUT test ( Q = 1 % ) . Data information : n . s ( non - significant ) , * p ≤ 0 . 05 , * * * * p ≤ 0 . 0001 .

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

Immunofluorescent (IF) staining for the glutamatergic neurons marker VGLUT1 and GABAergic neurons marker GAD67 (GAD1) in week 10 COs (WT: n=8 from 3 batches, KO: n=8 from 3 batches, W-AAV: n=4 from 1 batch). Scale = 100µm.   Quantification of the images represented in figure 1E. VGLUT1 and GAD67 were quantified as the surface area covered by the staining and were normalized to the number of nuclei in each image. The boxplot represents the 1st and 3rd quartile, with its whiskers showing the minimum and maximum points and a central band representing the median. As the samples were not normally distributed, statistical significance was determined using Kruskal-Wallis test with Dunn's multiple comparisons test. Outliers were removed using the ROUT test (Q=1%). Data information: n.s (non-significant), *p ≤ 0.05, ****p ≤ 0.0001.


Sample recordings from 7 - week - old hESC - derived Cerebral Organoids ( COs ) . Sample traces show visible differences in local field potential , with WWOX - KO COs ( Red ) showing increased activity compared to WT ( Blue ) in baseline condition ( Left ) and in the presence of 100µM 4AP ( Right ) .

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

Sample recordings from 7-week-old hESC-derived Cerebral Organoids (COs). Sample traces show visible differences in local field potential, with WWOX-KO COs (Red) showing increased activity compared to WT (Blue) in baseline condition (Left) and in the presence of 100µM 4AP (Right).


Sample recordings from 7 - week - old hESC - derived Cerebral Organoids ( COs ) . WWOX ' s coding sequence was re - introduced into week 6 WWOX - KO COs using lentiviral transduction ( lenti - WWOX ) ( F ) Immunofluorescent staining showing WWOX expression in different populations in WWOX - KO organoids following infection with lentivirus . NT = non - treated . Scale = 50µm .

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

Sample recordings from 7-week-old hESC-derived Cerebral Organoids (COs). WWOX's coding sequence was re-introduced into week 6 WWOX-KO COs using lentiviral transduction (lenti-WWOX) (F) Immunofluorescent staining showing WWOX expression in different populations in WWOX-KO organoids following infection with lentivirus. NT= non-treated. Scale = 50µm .


Week 15 and week 24 COs stained for the astrocytic and radial glia marker GFAP , and the astrocyte - specific marker S100β ( WT W15 : n = 9 from 3 batches , KO W15 : n = 16 from 3 batches , W - AAV W15 : n = 4 organoids from 1 batch . WT W24 : n = 10 from 4 batches , KO W24 : n = 9 from 3 batches , W - AAV W24 : n = 4 from 1 batch ) . Scale =

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

Week 15 and week 24 COs stained for the astrocytic and radial glia marker GFAP, and the astrocyte-specific marker S100β (WT W15: n=9 from 3 batches, KO W15: n=16 from 3 batches, W-AAV W15: n=4 organoids from 1 batch. WT W24: n=10 from 4 batches, KO W24: n=9 from 3 batches, W-AAV W24: n=4 from 1 batch). Scale = 100µm


IF staining of week 6 COs for astrocytic markers in the surrounding of VZs ( WT : n = 6 from 2 batches , KO : n = 6 from 2 batches ) . Scale = 100µm ( left ) , 50µm ( right ) . Staining for the DNA damage markers γH2AX and 53BP1 in the nuclei of cells in the VZ of week 6 COs at physiological conditions , together with the pan - radial glia marker SOX2 ( WT : n = 8 from 3 individual batches , KO : n = 12 from 3 individual batches , W - AAV : n = 4 from 1 batch ) . Scale = 50µm ( left ) , 25µm ( right ) . Quantification of γH2AX ( top ) and 53BP1 foci ( bottom ) in the nuclei of cells composing the innermost layer of the VZ , normalized to the total number of nuclei in this layer . The boxplot represents the 1st and 3rd quartile , with its whiskers showing the minimum and maximum points and a central band representing the median . Statistical significance was determined using one - way ANOVA with Tukey ' s multiple comparisons test ( WT : n = 8 organoids from 3 batches , KO : n = 12 organoids from 3 batches , W - AAV : n = 4 from 1 batch ) . Data information : n . s ( non - significant ) , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * * p ≤ 0 . 0001 .

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

IF staining of week 6 COs for astrocytic markers in the surrounding of VZs (WT: n=6 from 2 batches, KO: n=6 from 2 batches). Scale = 100µm (left) , 50µm (right).   Staining for the DNA damage markers γH2AX and 53BP1 in the nuclei of cells in the VZ of week 6 COs at physiological conditions, together with the pan-radial glia marker SOX2 (WT: n=8 from 3 individual batches, KO: n=12 from 3 individual batches, W-AAV: n=4 from 1 batch). Scale = 50µm (left), 25µm (right).   Quantification of γH2AX (top) and 53BP1 foci (bottom) in the nuclei of cells composing the innermost layer of the VZ, normalized to the total number of nuclei in this layer. The boxplot represents the 1st and 3rd quartile, with its whiskers showing the minimum and maximum points and a central band representing the median. Statistical significance was determined using one-way ANOVA with Tukey's multiple comparisons test (WT: n=8 organoids from 3 batches, KO: n=12 organoids from 3 batches, W-AAV: n=4 from 1 batch).  Data information: n.s (non-significant), *p ≤ 0.05, **p ≤ 0.01, ****p ≤ 0.0001.


RNA sequencing ( RNA - seq ) of week 15 COs and transcriptome analysis ( WT : n = 2 , KO : n = 4 ) . qPCR analysis for selected Wnt target genes validating the results of the RNA - seq . Y - axis indicates relative expression fold change . Data are represented as mean ± SEM . Statistical significance was determined using one - way ANOVA with Tukey ' s multiple comparisons test ( WT : n = 4 from 1 batch , KO : n = 4 from 1 batch , W - AAV : n = 4 from 1 batch ) . Data information : * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001 .

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

RNA sequencing (RNA-seq) of week 15 COs and transcriptome analysis (WT: n=2, KO: n=4). qPCR analysis for selected Wnt target genes validating the results of the RNA- seq. Y-axis indicates relative expression fold change. Data are represented as mean ± SEM. Statistical significance was determined using one-way ANOVA with Tukey's multiple comparisons test (WT: n=4 from 1 batch, KO: n=4 from 1 batch, W-AAV: n=4 from 1 batch). Data information: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.


Week 16 COs were sub - fractionated into a cytoplasmic ( C ) and nuclear ( N ) fractions . The experiment was run twice with a total of 2 WT organoids and 4 KOs organoids ( 2 for each KO line ) . KAP - 1 marks the nucleus and HSP90 marks the cytoplasm . The numbers at the bottom are a quantification of the nuclear fraction of β - catenin band intensities normalized to the cytosolic fraction [ β - C ( N / C ) ] .

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

Week 16 COs were sub-fractionated into a cytoplasmic (C) and nuclear (N) fractions. The experiment was run twice with a total of 2 WT organoids and 4 KOs organoids (2 for each KO line). KAP-1 marks the nucleus and HSP90 marks the cytoplasm. The numbers at the bottom are a quantification of the nuclear fraction of β-catenin band intensities normalized to the cytosolic fraction [β-C (N/C)].


IF staining in week 15 COs validating the decreased levels of the deep - layer cortical markers CTIP2 ( BCL11B ) and TBR1 , and superficial - layer marker SATB2 ( WT : n = 3 , KO : n = 4 , W - AAV : n = 4 ) . Scale = 50µm . Quantification of the cortical markers seen in G , normalized to the total number of nuclei . Y - axis indicates the fold change compared to the average of the WT COs . The boxplot represents the 1st and 3rd quartile , with its whiskers showing the minimum and maximum points and a central band representing the median . Statistical significance was determined using one - way ANOVA with Tukey ' s multiple comparisons test ( WT : n = 9 from 3 batches , KO : n = 16 from 3 batches , W - AAV : n = 4 organoids from 1 batch ) . Data information : * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001 .

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

IF staining in week 15 COs validating the decreased levels of the deep-layer cortical markers CTIP2 (BCL11B) and TBR1, and superficial-layer marker SATB2 (WT: n=3, KO: n=4, W-AAV: n=4). Scale = 50µm.   Quantification of the cortical markers seen in G, normalized to the total number of nuclei. Y-axis indicates the fold change compared to the average of the WT COs. The boxplot represents the 1st and 3rd quartile, with its whiskers showing the minimum and maximum points and a central band representing the median. Statistical significance was determined using one-way ANOVA with Tukey's multiple comparisons test (WT: n=9 from 3 batches, KO: n=16 from 3 batches, W-AAV: n=4 organoids from 1 batch).   Data information: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.


Peripheral blood mononuclear cells ( PBMCs ) were isolated from a WOREE patient and from his healthy parents and were reprogrammed into iPSCs , and subsequently were differentiated into COs . Week 10 WSM COs stained for the progenitor marker SOX2 , neuronal marker β3 - Tubulin and WWOX ( WSM F1 : n = 2 from 1 batch , WSM M2 : n = 2 from 1 batch , WSM S2 : n = 2 from 1 batch , WSM S5 : n = 2 from 1 batch , WSM S5 W - AAV3 : n = 2 from 1 batch , WSM S5 W - AAV6 : n = 2 from 1 batch ) . Scale = 50µm .

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

Peripheral blood mononuclear cells (PBMCs) were isolated from a WOREE patient and from his healthy parents and were reprogrammed into iPSCs, and subsequently were differentiated into COs.   Week 10 WSM COs stained for the progenitor marker SOX2, neuronal marker β3-Tubulin and WWOX (WSM F1: n=2 from 1 batch, WSM M2: n=2 from 1 batch, WSM S2: n=2 from 1 batch, WSM S5: n=2 from 1 batch, WSM S5 W-AAV3: n=2 from 1 batch, WSM S5 W-AAV6: n=2 from 1 batch). Scale = 50µm.


Peripheral blood mononuclear cells ( PBMCs ) were isolated from a WOREE patient and from his healthy parents and were reprogrammed into iPSCs , and subsequently were differentiated into COs . Average firing rate over 24 neurons from WSM P COs ( 4 organoids ) , 41 neurons from WSM S COs ( 3 organoids ) and 40 neurons from WSM S W - AAV organoids ( 3 organoids ) . Statistical significance was determined using one - way ANOVA with Tukey ' s multiple comparisons test . Bars represents the mean ± SEM . Data information : ns ( non - significant ) , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001 .

B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O O O O O B-DISEASE B-ORGANISM O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O B-CELL_TYPE 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Peripheral blood mononuclear cells (PBMCs) were isolated from a WOREE patient and from his healthy parents and were reprogrammed into iPSCs, and subsequently were differentiated into COs. Average firing rate over 24 neurons from WSM P COs (4 organoids), 41 neurons from WSM S COs (3 organoids) and 40 neurons from WSM S W-AAV organoids (3 organoids). Statistical significance was determined using one-way ANOVA with Tukey's multiple comparisons test. Bars represents the mean ± SEM. Data information: ns (non-significant), *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.


Peripheral blood mononuclear cells ( PBMCs ) were isolated from a WOREE patient and from his healthy parents and were reprogrammed into iPSCs , and subsequently were differentiated into COs . GAD67 and VGLUT1 immunostaining in week 10 WSM COs ( WSM F1 : n = 2 from 1 batch , WSM M2 : n = 2 from 1 batch , WSM S2 : n = 2 from 1 batch , WSM S5 : n = 2 from 1 batch , WSM S5 W - AAV3 : n = 2 from 1 batch , WSM S5 W - AAV6 : n = 2 from 1 batch ) . Scale = 50µm . Quantification of the data shown in fig 5D . Statistical significance was determined using one - way ANOVA with Tukey ' s multiple comparisons test ( WSM F1 : n = 2 from 1 batch , WSM M2 : n = 2 from 1 batch , WSM S2 : n = 2 from 1 batch , WSM S5 : n = 2 from 1 batch , WSM S5 W - AAV3 : n = 2 from 1 batch , WSM S5 W - AAV6 : n = 2 from 1 batch ) . Data information : ns ( non - significant ) , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001 .

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

Peripheral blood mononuclear cells (PBMCs) were isolated from a WOREE patient and from his healthy parents and were reprogrammed into iPSCs, and subsequently were differentiated into COs. GAD67 and VGLUT1 immunostaining in week 10 WSM COs (WSM F1: n=2 from 1 batch, WSM M2: n=2 from 1 batch, WSM S2: n=2 from 1 batch, WSM S5: n=2 from 1 batch, WSM S5 W-AAV3: n=2 from 1 batch, WSM S5 W-AAV6: n=2 from 1 batch). Scale = 50µm.   Quantification of the data shown in fig 5D. Statistical significance was determined using one-way ANOVA with Tukey's multiple comparisons test (WSM F1: n=2 from 1 batch, WSM M2: n=2 from 1 batch, WSM S2: n=2 from 1 batch, WSM S5: n=2 from 1 batch, WSM S5 W-AAV3: n=2 from 1 batch, WSM S5 W-AAV6: n=2 from 1 batch).  Data information: ns (non-significant), *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.


Week 15 WSM COs stained for astrocytic markers GFAP and S100β . ( WSM F1 : n = 2 from 1 batch , WSM M2 : n = 3 from 1 batch , WSM S2 : n = 2 from 1 batch , WSM S5 : n = 2 from 1 batch , WSM S5 W - AAV3 : n = 2 from 1 batch , WSM S5 W - AAV6 : n = 2 from 1 batch ) . Scale = 50µm . Quantification of DNA damage foci , marked by γH2AX ( left ) and 53BP1 ( right ) , in the nuclei of the cells in the VZs of week 6 WSM COs . The boxplot represents the 1st and 3rd quartile , with its whiskers showing the minimum and maximum points and a central band representing the median . Statistical significance was determined using one - way ANOVA with Tukey ' s multiple comparisons test ( WSM F1 : n = 4 from 1 batch , WSM M2 : n = 4 from 1 batch , WSM S2 : n = 4 from 1 batch , WSM S5 : n = 4 from 1 batch , WSM S5 W - AAV3 : n = 4 from 1 batch , WSM S5 W - AAV6 : n = 4 from 1 batch ) . Data information : * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001 .

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

Week 15 WSM COs stained for astrocytic markers GFAP and S100β. (WSM F1: n=2 from 1 batch, WSM M2: n=3 from 1 batch, WSM S2: n=2 from 1 batch, WSM S5: n=2 from 1 batch, WSM S5 W-AAV3: n=2 from 1 batch, WSM S5 W-AAV6: n=2 from 1 batch). Scale = 50µm.   Quantification of DNA damage foci, marked by γH2AX (left) and 53BP1 (right), in the nuclei of the cells in the VZs of week 6 WSM COs. The boxplot represents the 1st and 3rd quartile, with its whiskers showing the minimum and maximum points and a central band representing the median. Statistical significance was determined using one-way ANOVA with Tukey's multiple comparisons test (WSM F1: n=4 from 1 batch, WSM M2: n=4 from 1 batch, WSM S2: n=4 from 1 batch, WSM S5: n=4 from 1 batch, WSM S5 W-AAV3: n=4 from 1 batch, WSM S5 W-AAV6: n=4 from 1 batch).   Data information: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.


qPCR analysis for selected Wnt target genes in week 10 WSM COs . Y - axis indicates relative expression fold change . Data are represented as mean ± SEM . Statistical significance was determined using one - way ANOVA with Tukey ' s multiple comparisons test ( WSM P : n = 6 from 1 batch , WSM S : n = 4 from 1 batch , WSM S W - AAV : n = 4 from 1 batch ) . Data information : * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001 .

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

qPCR analysis for selected Wnt target genes in week 10 WSM COs. Y-axis indicates relative expression fold change. Data are represented as mean ± SEM. Statistical significance was determined using one-way ANOVA with Tukey's multiple comparisons test (WSM P: n=6 from 1 batch, WSM S: n=4 from 1 batch, WSM S W-AAV: n=4 from 1 batch). Data information: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.


Week 15 WSM COs stained for the cortical layers ' markers CTIP2 and SATB2 . ( WSM F1 : n = 2 from 1 batch , WSM M2 : n = 3 from 1 batch , WSM S2 : n = 2 from 1 batch , WSM S5 : n = 2 from 1 batch , WSM S5 W - AAV3 : n = 2 from 1 batch , WSM S5 W - AAV6 : n = 2 from 1 batch ) . Scale = 50µm . Quantification of the staining presented in E . The boxplot represents the 1st and 3rd quartile , with its whiskers showing the minimum and maximum points and a central band representing the median . Statistical significance was determined using one - way ANOVA with Tukey ' s multiple comparisons test ( WSM F1 : n = 2 from 1 batch , WSM M2 : n = 3 from 1 batch , WSM S2 : n = 2 from 1 batch , WSM S5 : n = 2 from 1 batch , WSM S5 W - AAV3 : n = 2 from 1 batch , WSM S5 W - AAV6 : n = 2 from 1 batch ) . Data information : * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001 . E

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

Week 15 WSM COs stained for the cortical layers' markers CTIP2 and SATB2. (WSM F1: n=2 from 1 batch, WSM M2: n=3 from 1 batch, WSM S2: n=2 from 1 batch, WSM S5: n=2 from 1 batch, WSM S5 W-AAV3: n=2 from 1 batch, WSM S5 W-AAV6: n=2 from 1 batch). Scale = 50µm.   Quantification of the staining presented in E. The boxplot represents the 1st and 3rd quartile, with its whiskers showing the minimum and maximum points and a central band representing the median. Statistical significance was determined using one-way ANOVA with Tukey's multiple comparisons test (WSM F1: n=2 from 1 batch, WSM M2: n=3 from 1 batch, WSM S2: n=2 from 1 batch, WSM S5: n=2 from 1 batch, WSM S5 W-AAV3: n=2 from 1 batch, WSM S5 W-AAV6: n=2 from 1 batch).  Data information: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. E


C Calculated BRET ratios for the indicated protein pairs . The positive control protein PA - mCit - NL and the interacting fusion proteins NL - BCL2L1 and PA - mCit - BAD show high BRET ratios .

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

C Calculated BRET ratios for the indicated protein pairs. The positive control protein PA-mCit-NL and the interacting fusion proteins NL-BCL2L1 and PA-mCit-BAD show high BRET ratios.


D Calculated LuC ratios for the indicated protein pairs . The positive control proteins PA - NL and PA - mCit - NL and the interacting proteins NL - BCL2L1 and PA - mCit - BAD show high LuC ratios .

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

D Calculated LuC ratios for the indicated protein pairs. The positive control proteins PA-NL and PA-mCit-NL and the interacting proteins NL-BCL2L1 and PA-mCit-BAD show high LuC ratios.


Reproducibility of LuTHy PPI mapping experiments with positive and negative reference sets . The scatter plots show the mean cBRET ( A ) ratios from two independent experiments ( Exp 1 and Exp 2 ) , the calculated two - tailed Pearson correlation coefficient is indicated ; * * * p < 0 . 001 .

O O B-EXP_ASSAY I-EXP_ASSAY 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

Reproducibility of LuTHy PPI mapping experiments with positive and negative reference sets. The scatter plots show the mean cBRET (A) ratios from two independent experiments (Exp 1 and Exp 2), the calculated two-tailed Pearson correlation coefficient is indicated; ***p<0.001.


Reproducibility of LuTHy PPI mapping experiments with positive and negative reference sets . The scatter plots show the mean cLuC ( B ) ratios from two independent experiments ( Exp 1 and Exp 2 ) , the calculated two - tailed Pearson correlation coefficient is indicated ; * * * p < 0 . 001 .

Reproducibility of LuTHy PPI mapping experiments with positive and negative reference sets. The scatter plots show the mean cLuC (B) ratios from two independent experiments (Exp 1 and Exp 2), the calculated two-tailed Pearson correlation coefficient is indicated; ***p<0.001.


C Receiver operating characteristic ( ROC ) analysis of cBRET ( AUC = 0 . 80 ± 0 . 04 ) and cLuC ( AUC = 0 . 72 ± 0 . 04 ) data determines the thresholds to define true positive binary interactions .

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

C Receiver operating characteristic (ROC) analysis of cBRET (AUC=0.80±0.04) and cLuC (AUC=0.72±0.04) data determines the thresholds to define true positive binary interactions.


D Performance of BRET and LuC assays in benchmarking studies with hPRS and hRRS .

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D Performance of BRET and LuC assays in benchmarking studies with hPRS and hRRS.


C Recovery rates of PPIs from affinity - based sub - groups with BRET and LuC assays . Significance was calculated by two - sided Fisher ' s exact test ; * p < 0 . 05 . D Success of PPI detection from AIRS sub - groups considering single and double LuTHy readouts .

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

C Recovery rates of PPIs from affinity-based sub-groups with BRET and LuC assays. Significance was calculated by two-sided Fisher's exact test; *p<0.05. D Success of PPI detection from AIRS sub-groups considering single and double LuTHy readouts.


E Scatter plot depicting the relationships between published binding affinities ( KD ) and in - cell BRET50 measurements for 25 interactions in AIRS ; BRET50 values are the mean from two independent experiments . Significance was calculated by two - tailed Spearman correlation ; * * * p < 0 . 001 .

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

E Scatter plot depicting the relationships between published binding affinities (KD) and in-cell BRET50 measurements for 25 interactions in AIRS; BRET50 values are the mean from two independent experiments. Significance was calculated by two-tailed Spearman correlation; ***p<0.001.


A Live - cell bioluminescence imaging ( BLI ) of HEK293 cells co - producing the indicated fusion proteins . mCitrine was excited at 500 nm , emitted fluorescence was detected at 535 nm . After substrate addition , short ( 460 ) and long ( 535 ) band - pass ( BP ) filters in a dual - view adapter were used to detect the emitted luminescence simultaneously at the respective wavelengths . BRET images were calculated by dividing the 535 BP by the 460 BP images using ImageJ . Scale bar = 20 μm .

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

A Live-cell bioluminescence imaging (BLI) of HEK293 cells co-producing the indicated fusion proteins. mCitrine was excited at 500 nm, emitted fluorescence was detected at 535 nm. After substrate addition, short (460) and long (535) band-pass (BP) filters in a dual-view adapter were used to detect the emitted luminescence simultaneously at the respective wavelengths. BRET images were calculated by dividing the 535 BP by the 460 BP images using ImageJ. Scale bar = 20 μm.


B Quantification of the rapamycin - induced interaction between NL - FKBP12 and PA - mCit - FRB . Plasmids encoding the fusion proteins were co - transfected in HEK293 cells and 24 h later treated with the indicated concentrations of rapamycin . After an additional incubation for 4 h BRET was quantified upon substrate addition . After cell lysis in buffer with indicated rapamycin concentrations , LuC ratios were determined . EC50 values were obtained with non - linear curve fitting .

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

B Quantification of the rapamycin-induced interaction between NL-FKBP12 and PA-mCit-FRB. Plasmids encoding the fusion proteins were co-transfected in HEK293 cells and 24 h later treated with the indicated concentrations of rapamycin. After an additional incubation for 4 h BRET was quantified upon substrate addition. After cell lysis in buffer with indicated rapamycin concentrations, LuC ratios were determined. EC50 values were obtained with non-linear curve fitting.


Plasmids encoding the fusion proteins were co - transfected in HEK293 cells and After an additional incubation for 4 h BRET was quantified upon substrate addition . LuC ratios were determined . EC50 values were obtained with non - linear curve fitting . C Effects of Nutlin - 3 on the interaction between NL - MDM2 and PA - mCit - p53 . for 6 h treatment with Nutlin - 3 .

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

Plasmids encoding the fusion proteins were co-transfected in HEK293 cells and After an additional incubation for 4 h BRET was quantified upon substrate addition. LuC ratios were determined. EC50 values were obtained with non-linear curve fitting. C Effects of Nutlin-3 on the interaction between NL-MDM2 and PA-mCit-p53. for 6 h treatment with Nutlin-3.


D Time resolved HSF1 oligomerization by Hsp90 inhibition . HEK293 cells co - producing NL - HSF1 and PA - mCit - HSF1 for 24 h were transferred to 384 - well plates containing Ganetespib or Geldanamycin to reach a final concentration of 1 µM . Luminescence was measured at the indicated time points and the calculated BRET ratios normalized to the respective untreated control . Data are the mean of two biological replicates . All values are mean ± sem . Significance was calculated by two - way ANOVA followed by Dunnett ' s multiple comparison ' s post - hoc test .

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

D Time resolved HSF1 oligomerization by Hsp90 inhibition. HEK293 cells co-producing NL-HSF1 and PA-mCit-HSF1 for 24 h were transferred to 384-well plates containing Ganetespib or Geldanamycin to reach a final concentration of 1 µM. Luminescence was measured at the indicated time points and the calculated BRET ratios normalized to the respective untreated control. Data are the mean of two biological replicates. All values are mean ± sem. Significance was calculated by two-way ANOVA followed by Dunnett's multiple comparison's post-hoc test.


E Schematic depiction of a heat shock experiment . HEK293 cells co - producing NL - HSF1 / PA - mCit - HSF1 for 48h were subjected to a heat shock at 42 ° C for 6 h , after which the cells were recovered at 37 ° C for 24h . Luminescence was measured at the indicated time points : immediately before the heat shock , during the heat shock at 90 , 180 and 360 min and after a recovery phase of 24 h . Calculated cBRET ratios were normalized to a control plate that was not heat treated . All values are mean ± sem from three independent experiments . Significance was calculated by one - way ANOVA followed by Dunnett ' s multiple comparisons post - hoc test ; * p < 0 . 05 .

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

E Schematic depiction of a heat shock experiment. HEK293 cells co-producing NL-HSF1/PA-mCit-HSF1 for 48h were subjected to a heat shock at 42°C for 6 h, after which the cells were recovered at 37°C for 24h. Luminescence was measured at the indicated time points: immediately before the heat shock, during the heat shock at 90, 180 and 360 min and after a recovery phase of 24 h. Calculated cBRET ratios were normalized to a control plate that was not heat treated. All values are mean ± sem from three independent experiments. Significance was calculated by one-way ANOVA followed by Dunnett's multiple comparisons post-hoc test; *p<0.05.


B , C , D VCP and the UBX domain - containing proteins were co - expressed as N - and C - terminal NL and PA - mCit fusion proteins in eight different orientations for which cBRET ( B ) and cLuC ( C ) ratios were generated . cBRET and cLuC values over the threshold of ≥ 0 . 01 and ≥ 0 . 03 , respectively , are colored in cyan . D The cBRET and cLuC ratios were normalized to the highest of the obtained eight values to easily identify the orientation among the eight tested interactions with the highest cBRET and cLuC value .

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

B,C,D VCP and the UBX domain-containing proteins were co-expressed as N- and C-terminal NL and PA-mCit fusion proteins in eight different orientations for which cBRET (B) and cLuC (C) ratios were generated. cBRET and cLuC values over the threshold of ≥0.01 and ≥0.03, respectively, are colored in cyan. D The cBRET and cLuC ratios were normalized to the highest of the obtained eight values to easily identify the orientation among the eight tested interactions with the highest cBRET and cLuC value.


Tukey box plots of the mean cBRET ( B ) ratios obtained from PPI experiments with wild - type CSPα - mCit - PA and its mutant variants .

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Tukey box plots of the mean cBRET (B) ratios obtained from PPI experiments with wild-type CSPα-mCit-PA and its mutant variants.


Tukey box plots of the mean cLuC ratios ( C ) obtained from PPI experiments with wild - type CSPα - mCit - PA and its mutant variants .

Tukey box plots of the mean cLuC ratios (C) obtained from PPI experiments with wild-type CSPα-mCit-PA and its mutant variants.


D Number of screened presynaptic proteins manually annotated according to their function and localization in the presynaptic terminal . Number of proteins identified with wild - type ( wt ) CSPα - mCit - PA or its mutant variants ( ΔL116 and L115R ) are indicated .

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

D Number of screened presynaptic proteins manually annotated according to their function and localization in the presynaptic terminal. Number of proteins identified with wild-type (wt) CSPα-mCit-PA or its mutant variants (ΔL116 and L115R) are indicated.


E Influence of mutations in CSPα ( ΔL116 , L115R ) on the interactions with ZDHHC17 in primary hippocampal neurons . BRET and LuC ratios were systematically quantified for interactions with wild - type and mutant CSPα fusion proteins .

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

E Influence of mutations in CSPα (ΔL116, L115R) on the interactions with ZDHHC17 in primary hippocampal neurons. BRET and LuC ratios were systematically quantified for interactions with wild-type and mutant CSPα fusion proteins.


F Immunoblot analysis of SHEP cells transfected with plasmids encoding wild - type CSPα - mCit - PA and mutant variants . Blot was developed with an anti - CSP antibody ; loading control : anti - Histone - H3 antibody . Arrowhead and bracket indicate monomeric CSPα - mCit - PA and SDS - insoluble protein aggregates , respectively .

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F Immunoblot analysis of SHEP cells transfected with plasmids encoding wild-type CSPα-mCit-PA and mutant variants. Blot was developed with an anti-CSP antibody; loading control: anti-Histone-H3 antibody. Arrowhead and bracket indicate monomeric CSPα-mCit-PA and SDS-insoluble protein aggregates, respectively.


G Immunofluorescence image of SHEP cells co - transfected with NL - ZDHHC17 and CSPα - mCit - wt , - ΔL116 or - L115R constructs lacking the PA - tag . NL - ZDHHC17 was stained with an anti NanoLuc antibody . Scale bar : 20 µm .

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

G Immunofluorescence image of SHEP cells co-transfected with NL-ZDHHC17 and CSPα-mCit-wt, -ΔL116 or -L115R constructs lacking the PA-tag. NL-ZDHHC17 was stained with an anti NanoLuc antibody. Scale bar: 20 µm.


A Flow cytometric quantification of lymphocytes , monocytes , B - cells , natural killer ( NK ) cells , dendritic cells ( DCs ) and granulocytes in peripheral venous blood samples from non - diabetic ( ND ) and type - 2 diabetic ( T2D ) patients with COVID - 19 .

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

A Flow cytometric quantification of lymphocytes, monocytes, B-cells, natural killer (NK) cells, dendritic cells (DCs) and granulocytes in peripheral venous blood samples from non-diabetic (ND) and type-2 diabetic (T2D) patients with COVID-19.


B Quantification of monocyte subpopulation phenotypes as classical ( CD14Hi CD16 - ) , intermediate ( CD14Hi CD16 + ) or non - classical ( CD14Lo CD16 + ) .

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

B Quantification of monocyte subpopulation phenotypes as classical (CD14Hi CD16-), intermediate (CD14Hi CD16+) or non-classical (CD14Lo CD16+).


C Principle component analysis of lymphocyte and monocyte population frequencies in ND and T2D COVID - 19 patients and in T2D patients without COVID - 19 .

O O O O O B-CELL_TYPE O B-CELL_TYPE B-EXP_ASSAY I-EXP_ASSAY O O O B-DISEASE I-DISEASE I-DISEASE I-DISEASE B-ORGANISM O O B-DISEASE B-ORGANISM O B-DISEASE I-DISEASE I-DISEASE O

C Principle component analysis of lymphocyte and monocyte population frequencies in ND and T2D COVID-19 patients and in T2D patients without COVID-19.


A Quantification of CD14 expression and size ( FSC ) in monocyte populations from non diabetic ( ND ) and type - 2 diabetic ( T2D ) COVID - 19 patients .

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

A Quantification of CD14 expression and size (FSC) in monocyte populations from non diabetic (ND) and type-2 diabetic (T2D) COVID-19 patients.


B Frequency of conventional ( FSCLo ) and large ( FSCHi ) monocytes in ND and T2D COVID - 19 patients .

O O O O O O O O O O O O B-CELL_TYPE O O O B-DISEASE I-DISEASE I-DISEASE I-DISEASE B-ORGANISM O

B Frequency of conventional (FSCLo) and large (FSCHi) monocytes in ND and T2D COVID-19 patients.


C Expression of CD16 , CD14 and HLA - DR in FSCLo and FSCHi monocytes from COVID - 19 patients .

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

C Expression of CD16, CD14 and HLA-DR in FSCLo and FSCHi monocytes from COVID-19 patients.


D Proportions of classical , intermediate and non - classical monocytes within FSCLo and FSCHi monocytes from COVID - 19 patients .

O B-EXP_ASSAY O O O O O O O O B-CELL_TYPE O O O O B-CELL_TYPE O B-DISEASE I-DISEASE I-DISEASE B-ORGANISM O

D Proportions of classical, intermediate and non-classical monocytes within FSCLo and FSCHi monocytes from COVID-19 patients.


E Correlative analyses of IL8 expression in peripheral blood mononuclear cells ( PBMCs ) to monocyte FSC .

E Correlative analyses of IL8 expression in peripheral blood mononuclear cells (PBMCs) to monocyte FSC.


F IL8 , IL6 and CCL2 mRNA expression in PBMCs from ND and T2D COVID - 19 patients .

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

F IL8, IL6 and CCL2 mRNA expression in PBMCs from ND and T2D COVID-19 patients.


G Monocyte size quantified in ND and T2D COVID - 19 patients treated in general wards ( Gen ) or in intensive care unit ( ICU ) .

O B-CELL_TYPE B-EXP_ASSAY O O O O B-DISEASE I-DISEASE I-DISEASE I-DISEASE B-ORGANISM O O O O O O O O O O O O O O O O

G Monocyte size quantified in ND and T2D COVID-19 patients treated in general wards (Gen) or in intensive care unit (ICU).


A Quantification of IRF5 and IFNB1 mRNA expression in peripheral blood mononuclear cells of non - diabetic ( ND ) and type 2 diabetic ( T2D ) COVID - 19 patients ( A ) .

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

A Quantification of IRF5 and IFNB1 mRNA expression in peripheral blood mononuclear cells of non-diabetic (ND) and type 2 diabetic (T2D) COVID-19 patients (A).


B Histograms of IRF5 expression on different populations analysed by flow cytometry on venous blood samples .

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

B Histograms of IRF5 expression on different populations analysed by flow cytometry on venous blood samples.


C IRF5 median fluorescence intensity ( MFI ) in monocytes of ND and T2D patients with COVID - 19 .

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

C IRF5 median fluorescence intensity (MFI) in monocytes of ND and T2D patients with COVID-19.


D IRF5 expression overlaid onto monocyte phenotypic gating .

D IRF5 expression overlaid onto monocyte phenotypic gating.


E IRF5 expression ( MFI ) in monocyte subtypes from ND and T2D COVID - 19 patients .

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

E IRF5 expression (MFI) in monocyte subtypes from ND and T2D COVID-19 patients.


F HLA - DR and IRF5 expression in monocytes from COVID - 19 patients .

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

F HLA-DR and IRF5 expression in monocytes from COVID-19 patients.


G IRF5 expression in monocytes of ND or T2D COVID - 19 patients admitted to intensive care unit ( ICU ) or treated exclusively in general wards ( Gen ) .

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

G IRF5 expression in monocytes of ND or T2D COVID-19 patients admitted to intensive care unit (ICU) or treated exclusively in general wards (Gen) .


H IRF5 expression in monocytes of ND or T2D COVID - 19 patients admitted ICU or treated exclusively in Gen .

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

H IRF5 expression in monocytes of ND or T2D COVID-19 patients admitted ICU or treated exclusively in Gen.


B Proportions of lymphocytes and monocytes in peripheral venous blood from T2D patients with COVID - 19 either treated in Gen or requiring ICU admission .

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

B Proportions of lymphocytes and monocytes in peripheral venous blood from T2D patients with COVID-19 either treated in Gen or requiring ICU admission.


C Phenotypic analysis of lymphocyte and monocyte subpopulations in peripheral venous blood from T2D COVID - 19 patients treated in Gen or requiring ICU treatment .

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

C Phenotypic analysis of lymphocyte and monocyte subpopulations in peripheral venous blood from T2D COVID-19 patients treated in Gen or requiring ICU treatment.