Immunoblot analysis of proteins from nuclear fractions from naïve THP1 - WT or TFEB - S401A knock - in ( clone I11 ) cells treated with 50 ng / ml PMA or 250 nM Torin - 1 for 1 h . Immunoblots are representative of at least three independent experiments .

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Immunoblot analysis of proteins from nuclear fractions from naïve THP1-WT or TFEB-S401A knock-in (clone I11) cells treated with 50 ng/ml PMA or 250 nM Torin-1 for 1 h. Immunoblots are representative of at least three independent experiments.


Immunoblot analysis of proteins from nuclear and cytosolic fractions from naïve THP1 - WT or TFEB - S401A knock - in ( clone I11 ) cells treated with 50 ng / ml PMA for 6 h .

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Immunoblot analysis of proteins from nuclear and cytosolic fractions from naïve THP1-WT or TFEB-S401A knock-in (clone I11) cells treated with 50 ng/ml PMA for 6 h.


Immunoblot analysis of protein lysates and cell culture medium from PMA - differentiated ( Rested ) THP1 - WT or TFEB - S401A knock - ins ( clones I11 and M17 ) cells incubated with 0 . 1 μg / ml LPS for 4 h prior to the addition of 15 μM Nigericin for 45 min . C - F . Quantification of immunoblot data shown in ( B ) . Data are presented as mean ± SD using one - way ANOVA ( unpaired ) followed by Tukey ' s multiple comparisons test , ( ns ) not significant , ( * ) p < 0 . 05 , ( * * ) p < 0 . 01 , ( * * * ) p < 0 . 001 and ( * * * * ) p < 0 . 0001 from three independent experiments . Data information : n ≥ 3 biological replicates ( each dot represents a biological replicate ) .

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

Immunoblot analysis of protein lysates and cell culture medium from PMA-differentiated (Rested) THP1-WT or TFEB-S401A knock-ins (clones I11 and M17) cells incubated with 0.1 μg/ml LPS for 4 h prior to the addition of 15 μM Nigericin for 45 min. C-F. Quantification of immunoblot data shown in (B). Data are presented as mean ± SD using one-way ANOVA (unpaired) followed by Tukey's multiple comparisons test, (ns) not significant, (*)p<0.05, (**)p<0.01, (***)p< 0.001 and (****)p< 0.0001 from three independent experiments. Data information: n ≥ 3 biological replicates (each dot represents a biological replicate).


Fluorescence confocal microscopy of PMA - differentiated ( Rested ) THP1 - WT or TFEB - S401A knock - ins ( clones I11 and M17 ) cells incubated with 1 μg / ml LPS for 6 h prior to the detection of dead cells with LIVE / DEAD fixable blue dead cell stain . Scale bars , 10 μm . Quantification of the percentage of dead cells shown in ( G ) . Data are presented as mean ± SD using one - way ANOVA ( unpaired ) followed by Tukey ' s multiple comparisons test , ( ns ) not significant and ( * * * * ) p < 0 . 0001 as compared to their corresponding untreated ( Control ) cells , with > 200 cells counted per trial from three independent experiments . Data information : n ≥ 3 biological replicates ( each dot represents a biological replicate ) .

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

Fluorescence confocal microscopy of PMA-differentiated (Rested) THP1-WT or TFEB-S401A knock-ins (clones I11 and M17) cells incubated with 1 μg/ml LPS for 6 h prior to the detection of dead cells with LIVE/DEAD fixable blue dead cell stain. Scale bars, 10 μm. Quantification of the percentage of dead cells shown in (G). Data are presented as mean ± SD using one-way ANOVA (unpaired) followed by Tukey's multiple comparisons test, (ns) not significant and (****)p< 0.0001 as compared to their corresponding untreated (Control) cells, with >200 cells counted per trial from three independent experiments. Data information: n ≥ 3 biological replicates (each dot represents a biological replicate).


A . From left to right , Surface representation of the three - dimensional structures of C3b - MCP ( CCP1 - 4 ) ( blue ) , C3b - SPICE ( CCP1 - 4 ) ( green ) , C3b - FH ( CCP1 - 4 ) ( orange ) , C3b - CR1 ( CCP15 - 17 ) ( dark red ) , C3b - DAF ( CCP2 - 4 ) ( purple ) .

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A. From left to right, Surface representation of the three-dimensional structures of C3b-MCP (CCP1-4) (blue), C3b-SPICE (CCP1-4) (green), C3b-FH (CCP1-4) (orange), C3b-CR1 (CCP15-17) (dark red), C3b-DAF (CCP2-4) (purple).


B . Cartoon representation of the five regulators . The width of the cartoon is proportional to the B - factors observed in the structural data . The B - factor coloring ( from blue to red ) is in relative scale according to the resolution of the crystal structures .

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B. Cartoon representation of the five regulators. The width of the cartoon is proportional to the B-factors observed in the structural data. The B-factor coloring (from blue to red) is in relative scale according to the resolution of the crystal structures.


C . Cartoon representation of the five C3b molecules in the C3b - regulator complexes . Coloring and style as in B .

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C. Cartoon representation of the five C3b molecules in the C3b-regulator complexes. Coloring and style as in B.


D . Highlight of electron density maps ( 2Fo - Fc , blue , contour level 1 . 2σ ; Fo - Fc , green and red , contour level 3σ and - 3σ , resp . ) for the regions of the C3b - regulator complexes showing flexibility . From left to right : the putative site for MCP CCP2 , SPICE CCP4 , CR1 CCP15 , and DAF CCP2 .

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D. Highlight of electron density maps (2Fo-Fc, blue, contour level 1.2σ; Fo-Fc, green and red, contour level 3σ and -3σ, resp.) for the regions of the C3b-regulator complexes showing flexibility. From left to right: the putative site for MCP CCP2, SPICE CCP4, CR1 CCP15, and DAF CCP2.


A . " Open - book " surface representation of C3b and regulator molecules , highlighting their domain organizations using different colors as shown in the neighboring schemes . Domains in complement regulators are labeled using domain numbering from CCPi to CCPiv based on their contact sites on C3b . The corresponding CCP domains in each regulator described in this work are shown in the bottom - left scheme . Domains CCP1 - 2 of MCP are shown in shaded grey because they are not involved in C3b binding . The table reports the extent of the regulator contact surface on C3b and the associated buried surface area .

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

A. "Open-book" surface representation of C3b and regulator molecules, highlighting their domain organizations using different colors as shown in the neighboring schemes. Domains in complement regulators are labeled using domain numbering from CCPi to CCPiv based on their contact sites on C3b. The corresponding CCP domains in each regulator described in this work are shown in the bottom-left scheme. Domains CCP1-2 of MCP are shown in shaded grey because they are not involved in C3b binding. The table reports the extent of the regulator contact surface on C3b and the associated buried surface area.


B . Opened view of the molecular footprint of the regulator - C3b interaction ( as shown in Fig . 2A ) , highlighting the C3b domains on the regulator surface ( top ) and the regulator domains on C3b ( bottom ) .

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B. Opened view of the molecular footprint of the regulator-C3b interaction (as shown in Fig. 2A), highlighting the C3b domains on the regulator surface (top) and the regulator domains on C3b (bottom).


A . Superposition of MCP and FH domains CCP3 - 4 ( shown as blue and orange cartoons , resp . ) on the surface of C3b .

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A. Superposition of MCP and FH domains CCP3-4 (shown as blue and orange cartoons, resp.) on the surface of C3b.


B . Cartoon representation of the superposition of domains CCP3 - 4 of MCP ( blue ) , FH ( orange ) and SPICE ( green ) . Despite the strong structural similarity and the conserved angle between domains CCP3 and CCP4 , MCP and SPICE show a rotation of approximately 100 ° in the position of CCP4 compared to FH .

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B. Cartoon representation of the superposition of domains CCP3-4 of MCP (blue), FH (orange) and SPICE (green). Despite the strong structural similarity and the conserved angle between domains CCP3 and CCP4, MCP and SPICE show a rotation of approximately 100° in the position of CCP4 compared to FH.


C . The reported mutagenesis data on MCP CCP3 and CCP4 ( data from Liszewki et al . ( Liszewski et al , 2000 ) ) are consistent with the crystal structure . Shown are two orientations of the surface of MCP CCP3 - 4 as in the crystal structure of the C3b - MCP complex , highlighting the contact sites with the C3b platform ( purple ) , the MCP mutations identified as critical for C3b binding ( red ) and those not affecting the interaction ( blue ) . The GB24 antibody epitope is shown as yellow contour on the MCP surface .

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C. The reported mutagenesis data on MCP CCP3 and CCP4 (data from Liszewki et al. (Liszewski et al, 2000)) are consistent with the crystal structure. Shown are two orientations of the surface of MCP CCP3-4 as in the crystal structure of the C3b-MCP complex, highlighting the contact sites with the C3b platform (purple), the MCP mutations identified as critical for C3b binding (red) and those not affecting the interaction (blue). The GB24 antibody epitope is shown as yellow contour on the MCP surface.


D . Model of the putative positioning of the flexible CCP1 - 2 domains of MCP . The model has been generated by superposing the structure of CCP1 - 4 of MCP from PDB 3O8E ( Persson et al , 2010 ) onto the structure of the C3b - MCP ( CCP1 - 4 ) complex . CCP1 - 2 domains of MCP , not visible in the C3b - MCP ( CCP1 - 4 ) structure , are shown in grey .

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D. Model of the putative positioning of the flexible CCP1-2 domains of MCP. The model has been generated by superposing the structure of CCP1-4 of MCP from PDB 3O8E (Persson et al, 2010) onto the structure of the C3b-MCP (CCP1-4) complex. CCP1-2 domains of MCP, not visible in the C3b-MCP (CCP1-4) structure, are shown in grey.


E . Highlight of the primary sequence differences between SPICE and VCP . The 11 mutations distinguishing the two viral regulators are shown on the surface of SPICE in purple . The contact surface of C3b on the regulator in the C3b - SPICE ( CCP1 - 4 ) complex is shown in green on the right panel .

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E. Highlight of the primary sequence differences between SPICE and VCP. The 11 mutations distinguishing the two viral regulators are shown on the surface of SPICE in purple. The contact surface of C3b on the regulator in the C3b-SPICE (CCP1-4) complex is shown in green on the right panel.


( A ) Mapping of known disease - related mutations on C3b ( Table 3 ) . The high - resolution C3b three - dimensional structure is used as structural template , shown as white surface . The regulator contact surfaces ( based on C3b - FH ( CCP1 - 4 ) ( Wu et al , 2009 ) and C3d - FH ( CCP19 - 20 ) ( Kajander et al , 2011 ; Morgan et al , 2011 ) ) are shown as black contour . All mutations reported in Table 3 are shown in blue on the C3b surface . Disease - related mutants reported to affect regulator binding are shown in red and labeled in the right magnified panel .

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(A) Mapping of known disease-related mutations on C3b (Table 3). The high-resolution C3b three-dimensional structure is used as structural template, shown as white surface. The regulator contact surfaces (based on C3b-FH (CCP1-4) (Wu et al, 2009) and C3d-FH (CCP19-20) (Kajander et al, 2011; Morgan et al, 2011)) are shown as black contour. All mutations reported in Table 3 are shown in blue on the C3b surface. Disease-related mutants reported to affect regulator binding are shown in red and labeled in the right magnified panel.


( B ) Mapping of disease - related mutations on the FH ( CCP1 - 4 ) surface using data from Table 4 . Colors as in Fig . 4A .

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(B) Mapping of disease-related mutations on the FH (CCP1-4) surface using data from Table 4. Colors as in Fig. 4A.


( C ) Mapping of disease - related mutations on the MCP ( CCP3 - 4 ) surface using data from Table 4 . Colors as in Fig . 4A .

(C) Mapping of disease-related mutations on the MCP (CCP3-4) surface using data from Table 4. Colors as in Fig. 4A.


( A ) N - glycan composition of PD - 1 protein expressed from 293T cells by ESI - MS analysis . N - glycans of representative peaks from ESI - MS were identified by MS / MS analysis . H , mannose ; N , N - acetylglucosamine ; F , fucose . Structural formulas : blue square , N - acetylglucosamine ; red triangle , fucose ; green circle , mannose ; yellow circle , galactose .

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(A) N-glycan composition of PD-1 protein expressed from 293T cells by ESI-MS analysis. N-glycans of representative peaks from ESI-MS were identified by MS/MS analysis. H, mannose; N, N-acetylglucosamine; F, fucose. Structural formulas: blue square, N-acetylglucosamine; red triangle, fucose; green circle, mannose; yellow circle, galactose.


( B , C ) Accelerated thermal stability of PD - 1 proteins obtained from 293T cells ( green line ) , insect cells ( pink line ) or E . coli ( blue line ) is characterized in all - in - one UNcle platform . Protein unfolding is observed as an increase in barycentric mean ( B ) while small particle formation of the samples is observed as an increase in SLS intensity at 266 nm ( C ) .

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(B,C) Accelerated thermal stability of PD-1 proteins obtained from 293T cells (green line), insect cells (pink line) or E. coli (blue line) is characterized in all-in-one UNcle platform. Protein unfolding is observed as an increase in barycentric mean (B) while small particle formation of the samples is observed as an increase in SLS intensity at 266 nm (C).


( D ) SPR analysis of the binding characteristics of camrelizumab with PD - 1 expressed in 293T cells , insect cells or E . coli cell , which enabled different levels of glycosylations . The binding affinity is presented as KD . The data presented here is a representative of two independent experiments with similar results .

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(D) SPR analysis of the binding characteristics of camrelizumab with PD-1 expressed in 293T cells, insect cells or E. coli cell, which enabled different levels of glycosylations. The binding affinity is presented as KD. The data presented here is a representative of two independent experiments with similar results.


( A ) Gel filtration profiles of PD - 1 ( orange ) , camrelizumab - scFv ( blue ) , and the PD - 1 / camrelizumab - scFv complex ( red ) are analyzed by size - exclusion chromatography as indicated . The SDS - PAGE analyses are shown with peak 1 of camrelizumab - scFv , peak 2 of camrelizumab - scFv / PD - 1 complex , and peak 3 of PD - 1 .

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(A) Gel filtration profiles of PD-1 (orange), camrelizumab-scFv (blue), and the PD-1/camrelizumab-scFv complex (red) are analyzed by size-exclusion chromatography as indicated. The SDS-PAGE analyses are shown with peak 1 of camrelizumab-scFv, peak 2 of camrelizumab-scFv/PD-1 complex, and peak 3 of PD-1.


( E ) SPR analysis of the binding of camrelizumab with N58A mutated PD - 1 expressed in 293T cells .

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(E) SPR analysis of the binding of camrelizumab with N58A mutated PD-1 expressed in 293T cells.


( F ) The SPR analysis of the binding of camrelizumab with WT ( upper ) or PNGase F treated PD - 1 proteins ( lower ) expressed in 293T cells .

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(F) The SPR analysis of the binding of camrelizumab with WT (upper) or PNGase F treated PD-1 proteins (lower) expressed in 293T cells.


( G The SPR analysis of the binding of nivolumab with WT ( upper ) or N58A mutated ( lower ) PD - 1 expressed in 293T

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(G The SPR analysis of the binding of nivolumab with WT (upper) or N58A mutated (lower) PD-1 expressed in 293T cells


H ) The SPR analysis of the binding of pembrolizumab with WT ( upper ) or N58A mutated ( lower ) PD - 1 proteins .

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H) The SPR analysis of the binding of pembrolizumab with WT (upper) or N58A mutated (lower) PD-1 proteins.


( A , B ) The blocking of the binding of WT PD - 1 - mFc ( A ) or N58A mutated PD - 1 - mFc ( B ) to PD - L1 transiently expressed on the surface of 293T cells are analyzed with varied concentrations of camrelizumab . The PD - L1 expressing 293T cells stained with PBS alone is used as negative controls , whereas the cells stained with WT or N58A mutated PD - 1 - mFc proteins are used as a positive control . The frequency of PD - 1 - mFc staining positive cells in PD - L1 - GFP positive cells is labeled in the upright corner . The reduced frequency of the PD - L1 - mFc - staining - positive subpopulation compared with positive control indicates the blockage of the PD - 1 / PD - L1 interaction . The density of events at a given position in the plot is color - coded , with red representing the highest number of events at that point in the graph , while yellow , green , and blue represent progressively lower event densities . The results presented here are representative of three independent experiments with similar results .

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

(A,B) The blocking of the binding of WT PD-1-mFc (A) or N58A mutated PD-1-mFc (B) to PD-L1 transiently expressed on the surface of 293T cells are analyzed with varied concentrations of camrelizumab. The PD-L1 expressing 293T cells stained with PBS alone is used as negative controls, whereas the cells stained with WT or N58A mutated PD-1-mFc proteins are used as a positive control. The frequency of PD-1-mFc staining positive cells in PD-L1-GFP positive cells is labeled in the upright corner. The reduced frequency of the PD-L1-mFc-staining-positive subpopulation compared with positive control indicates the blockage of the PD-1/PD-L1 interaction. The density of events at a given position in the plot is color-coded, with red representing the highest number of events at that point in the graph, while yellow, green, and blue represent progressively lower event densities. The results presented here are representative of three independent experiments with similar results.


A . Volcano blot representation of the differently expressed genes in FCRLS - and CD11b - positive Grn - / - microglia in comparison to wt microglia isolated from 5 . 5 - month - old mice ( male , n = 5 ) . 116 genes out of 529 genes analyzed , are significantly changed , and from these 58 genes are up - and 58 genes are down regulated . Eight genes with the highest fold change are highlighted .

A. Volcano blot representation of the differently expressed genes in FCRLS- and CD11b- positive Grn-/- microglia in comparison to wt microglia isolated from 5.5-month-old mice (male, n = 5). 116 genes out of 529 genes analyzed, are significantly changed, and from these 58 genes are up- and 58 genes are down regulated. Eight genes with the highest fold change are highlighted.


B . Heatmap of significantly affected genes ( p < 0 . 05 ) in FCRLS - and CD11b - positive Grn - / - microglia in comparison to Grn + / + microglia isolated from 5 . 5 - month - old mice ( n = 5 per genotype ) . For the significantly affected genes of the Grn - / - microglia , mRNA expression data of the Trem2 - / - microglia in comparison to the corresponding wt microglia were taken from previously published data ( Mazaheri et al , 2017 ) . The RNA counts for each gene and sample were normalized to the mean value of wt followed by a log2 transformation ( n ≥ 5 per genotype ) . * labelled genes were not analyzed or below detection limit in Trem2 - / - microglia mRNA expression data set ( Mazaheri et al , 2017 ) .

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B. Heatmap of significantly affected genes (p < 0.05) in FCRLS- and CD11b-positive Grn-/- microglia in comparison to Grn+/+ microglia isolated from 5.5-month-old mice (n = 5 per genotype). For the significantly affected genes of the Grn-/- microglia, mRNA expression data of the Trem2-/- microglia in comparison to the corresponding wt microglia were taken from previously published data (Mazaheri et al, 2017). The RNA counts for each gene and sample were normalized to the mean value of wt followed by a log2 transformation (n ≥ 5 per genotype). * labelled genes were not analyzed or below detection limit in Trem2-/- microglia mRNA expression data set (Mazaheri et al, 2017).


D . Gene expression levels of FCRLS - and CD11b - positive Grn - / - microglia normalized to wt in comparison to the published normalized data set of Trem2 - / - microglia ( Mazaheri et al , 2017 ) .

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D. Gene expression levels of FCRLS- and CD11b-positive Grn-/- microglia normalized to wt in comparison to the published normalized data set of Trem2-/- microglia (Mazaheri et al, 2017).


A . Immunoblot analysis of ApoE , CLEC7A , TREM2 and CD68 in lysates of acutely isolated microglia from Grn - / - and Grn + / + mice ( 9 months of age , n = 3 per genotype , female ) . IBA1 was used as loading control .

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A. Immunoblot analysis of ApoE, CLEC7A, TREM2 and CD68 in lysates of acutely isolated microglia from Grn-/- and Grn+/+ mice (9 months of age, n=3 per genotype, female). IBA1 was used as loading control.


B . Quantification of protein expression normalized to levels of Grn + / + microglia ( n = 3 ) . Data represent the mean ± SD .

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B. Quantification of protein expression normalized to levels of Grn+/+ microglia (n=3). Data represent the mean ± SD.


C . ELISA mediated quantification of sTREM2 in brain homogenates of Grn - / - and Grn + / + mice Data are shown as mean ± SEM ( n = 3 - 6 ) . D . ELISA mediated quantification of sTREM2 in serum of Grn - / - and Grn + / + mice . Data are shown as mean ± SEM ( n = 4 - 13 ) .

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C. ELISA mediated quantification of sTREM2 in brain homogenates of Grn-/- and Grn+/+ mice Data are shown as mean ± SEM (n=3-6). D. ELISA mediated quantification of sTREM2 in serum of Grn-/- and Grn+/+ mice. Data are shown as mean ± SEM (n=4-13).


E . Microglial expression of P2RY12 in cortical sections of wt , Grn - / - and Trem2 - / - mice . ( 9 months of age , female ) F . Quantification of P2RY12 positive microglia . Data are shown as mean ± SD ( n = 3 per genotype , female , except one male for Trem2 - / - ) .

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E. Microglial expression of P2RY12 in cortical sections of wt, Grn-/-and Trem2-/- mice. (9 months of age, female) F. Quantification of P2RY12 positive microglia. Data are shown as mean ± SD (n=3 per genotype, female, except one male for Trem2-/-).


G . Immunoblot analysis of secreted PGRN and ApoE ( ApoEmed ) in conditioned media and PGRN , ApoE , CLEC7A , P2RY12 and TREM2 in lysates of BV2 wild type ( Grnwt ) and PGRN deficient ( Grnmut ) cells . Soluble amyloid precursor protein ( APPs ) and calnexin were used as a loading control .

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G. Immunoblot analysis of secreted PGRN and ApoE (ApoEmed) in conditioned media and PGRN, ApoE, CLEC7A, P2RY12 and TREM2 in lysates of BV2 wild type (Grnwt) and PGRN deficient (Grnmut) cells. Soluble amyloid precursor protein (APPs) and calnexin were used as a loading control.


H . Quantification of Immunoblots normalized to BV2 Grnwt levels ( n = 3 - 5 ) . Data represent the mean ± SD .

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H. Quantification of Immunoblots normalized to BV2 Grnwt levels (n=3-5). Data represent the mean ± SD.


I . ELISA mediated quantification of sTREM2 in conditioned media of BV2 Grnwt and Grnmut cells . Data represent the mean ±

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

I. ELISA mediated quantification of sTREM2 in conditioned media of BV2 Grnwt and Grnmut cells. Data represent the mean ± SD


J . Quantification of relative mRNA levels of Apoe , Clec7a , P2ry12 and Trem2 in BV2 Grnwt and Grnmut cells . Data represent the mean ± SD .

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J. Quantification of relative mRNA levels of Apoe, Clec7a, P2ry12 and Trem2 in BV2 Grnwt and Grnmut cells. Data represent the mean ± SD.


A . Flow cytometric analysis of phagocytic capacity in BV2wt and BV2mut cells using pHrodo green E . coli as target particles . Phagocytosis was terminated after 30 and 60 minutes ( min ) of incubation . Data are presented as mean percentage of cells positive for pHrodo uptake ± S . D ( n = 3 ) .

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A. Flow cytometric analysis of phagocytic capacity in BV2wt and BV2mut cells using pHrodo green E. coli as target particles. Phagocytosis was terminated after 30 and 60 minutes (min) of incubation. Data are presented as mean percentage of cells positive for pHrodo uptake ± S.D (n=3).


B . Enhanced migration of CD68 positive , PGRN deficient cells in an ex vivo model . Immunofluorescence analysis of cultured Grn + / + and Grn - / - organotypic brain at 7 DIV immunostained using microglial marker CD68 . Nuclei were counterstained using DAPI . Images of boxed regions in left panels are shown at higher magnifications in right panels . Scale bars : 500 µm ( left panels ) , 250 µm ( right panels ) . Quantitative analysis reveals that Grn - / - microglia migrate larger distances than Grn + / + . Data are shown as mean ± SD ( n = 3 independent experiments ) Quantification displays an increased number of migrating Grn - / - cells compared to Grn + / + cells . Data are shown as mean ± SD ( n = 3 independent experiments )

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

B. Enhanced migration of CD68 positive, PGRN deficient cells in an ex vivo model. Immunofluorescence analysis of cultured Grn+/+ and Grn-/- organotypic brain at 7 DIV immunostained using microglial marker CD68. Nuclei were counterstained using DAPI. Images of boxed regions in left panels are shown at higher magnifications in right panels. Scale bars: 500 µm (left panels), 250 µm (right panels). Quantitative analysis reveals that Grn-/- microglia migrate larger distances than Grn+/+. Data are shown as mean ± SD (n=3 independent experiments) Quantification displays an increased number of migrating Grn-/- cells compared to Grn+/+ cells. Data are shown as mean ± SD (n=3 independent experiments)


E . Enhanced microglial clustering around amyloid plaques in the absence of PGRN . Left : IBA1 - stained microglia clustering around X - 34 - positive amyloid plaque cores in 4 - month - old APPPS1 / Grn + / + mice . Right : Age - matched APPPS1 / Grn - / - mice display microglial hyper - clustering around amyloid plaques compared to APPPS1 / Grn + / + . Dotted white boxes indicate the area that is shown at higher magnification . Scale bars indicate 10µm and 3µm in inset . Immunohistochemical quantification of IBA1 positive microglial cell number per

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Enhanced microglial clustering around amyloid plaques in the absence of PGRN. Left: IBA1-stained microglia clustering around X-34-positive amyloid plaque cores in 4-month-old APPPS1/Grn+/+ mice. Right: Age-matched APPPS1/Grn-/- mice display microglial hyper-clustering around amyloid plaques compared to APPPS1/Grn+/+. Dotted white boxes indicate the area that is shown at higher magnification. Scale bars indicate 10µm and 3µm in inset. Immunohistochemical quantification of IBA1 positive microglial cell number per plaqu


A . TSPO - µPET in rodents : Axial slices ( A1 , A2 , A3 ) of averaged % - TSPO - PET differences between Grn - / - or Trem2 - / - mice and wt indicate increased microglial activity in the cerebrum of Grn - / - mice ( hot color scale ) and decreased microglial activity in the cerebrum of Trem2 - / - mice ( cold color scale ) . Scatter plot depicts single TSPO - µPET values deriving from a neocortical volume - of - interest . µPET results are illustrated upon an MRI template ; 6 - 10 female mice per group ( Grn - / - n = 6 at 8 months , Trem2 - / - n = 8 at 11 months ; wt n = 10 at 8 - 11 months ) . Statistics derive from one - way ANOVA with Tukey post - hoc test .

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

A. TSPO-µPET in rodents: Axial slices (A1, A2, A3) of averaged %-TSPO-PET differences between Grn-/-or Trem2-/- mice and wt indicate increased microglial activity in the cerebrum of Grn-/- mice (hot color scale) and decreased microglial activity in the cerebrum of Trem2-/- mice (cold color scale). Scatter plot depicts single TSPO-µPET values deriving from a neocortical volume-of-interest. µPET results are illustrated upon an MRI template; 6-10 female mice per group (Grn-/- n=6 at 8 months, Trem2-/- n=8 at 11 months; wt n=10 at 8-11 months). Statistics derive from one-way ANOVA with Tukey post-hoc test.


B . PGRN serum levels of the patients subjected to TSPO - PET confirmed the GRN mutation and non - GRN mutation carrier . PGRN was measured by ELISA and normalized to serum levels of a healthy control .

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B. PGRN serum levels of the patients subjected to TSPO-PET confirmed the GRN mutation and non-GRN mutation carrier. PGRN was measured by ELISA and normalized to serum levels of a healthy control.


C . TSPO - PET in human FTLD patients : Visual interpretation of axial TSPO - PET SUVR images in upper cranial layers reveals increased microglial activity in frontal and parietal cortices ( white arrows ) of a FTLD patient with a GRN loss of function mutation in comparison to a FTLD patient without a GRN mutation . PET results are illustrated upon an MRI template ; 60 - 80 min p . i . emission recording ; global mean scaling . Both patients were low - affinity binder .

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

C. TSPO-PET in human FTLD patients: Visual interpretation of axial TSPO-PET SUVR images in upper cranial layers reveals increased microglial activity in frontal and parietal cortices (white arrows) of a FTLD patient with a GRN loss of function mutation in comparison to a FTLD patient without a GRN mutation. PET results are illustrated upon an MRI template; 60-80 min p.i. emission recording; global mean scaling. Both patients were low-affinity binder.


FDG - µPET in rodents : Axial slices ( as indicated in Fig . 4A ) of averaged % - FDG - µPET differences between Grn - / - or Trem2 - / - mice and wt indicate decreased glucose metabolism in the cerebrum of Grn - / - and Trem2 - / - ( both cold color scale ) when compared to wt . Scatter plot depicts single FDG - PET values deriving from a neocortical volume - of - interest . µPET results are illustrated upon an MRI template ; 6 - 10 female mice per group ( Grn - / - n = 6 at 8 months , Trem2 - / - n = 6 at 11 months ; wt n = 10 at 8 - 11 months ) .

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

FDG-µPET in rodents: Axial slices (as indicated in Fig. 4A) of averaged %-FDG-µPET differences between Grn-/-or Trem2-/- mice and wt indicate decreased glucose metabolism in the cerebrum of Grn-/- and Trem2-/- (both cold color scale) when compared to wt. Scatter plot depicts single FDG-PET values deriving from a neocortical volume-of-interest. µPET results are illustrated upon an MRI template; 6-10 female mice per group (Grn-/- n=6 at 8 months, Trem2-/- n=6 at 11 months; wt n=10 at 8-11 months).


( A _ B ) In situs on adult brain sections reveal that ( A ) opn4 . 1 and ( B ) opn4xb are coexpressed in many brain domains including the epiphysis cerebri ( indicated with E ) or pineal . A horizontal line through a representation of the brain above the sagittal section indicates the location within the brain . Scale bar : 1 . 0 mm .

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

(A_B) In situs on adult brain sections reveal that (A) opn4.1 and (B) opn4xb are coexpressed in many brain domains including the epiphysis cerebri (indicated with E) or pineal. A horizontal line through a representation of the brain above the sagittal section indicates the location within the brain. Scale bar: 1.0 mm.


( J ) ish with asmt probe on larvae sampled on the 6th dpf at ZT3 shows that asmt mRNA is confined to the pineal and indicates that the asmt gene is not ectopically expressed in opn4 dko larvae . The higher asmt mRNA level measured by qPCR in the opn4 dko therefore points at a defect in the pineal . Note that the eyes were removed after the ish for optimal visibility of expression in the brain . Scale bar : 0 . 5 mm .

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(J) ish with asmt probe on larvae sampled on the 6th dpf at ZT3 shows that asmt mRNA is confined to the pineal and indicates that the asmt gene is not ectopically expressed in opn4 dko larvae. The higher asmt mRNA level measured by qPCR in the opn4 dko therefore points at a defect in the pineal. Note that the eyes were removed after the ish for optimal visibility of expression in the brain. Scale bar: 0.5 mm.


( N ) ish with ddc probe on wild - type and opn4 dko larvae sampled on the 6th dpf represents ddc expression in pineals . Biological replicates indicated with BR . Scale bar : 0 . 05 mm ( O ) Quantification of the adjusted intensity in the pineals from larvae presented in N , implies a significant higher ddc transcript level in the opn4 dko pineal . Data information : boxplot divides the data in quartiles : the box indicates the interquartile range , with the horizontal line in the box denoting the median of the data set , the whiskers extend to the minimum and maximum , and meet the box at the median of the lower ( quartile 1 ) and median of the upper ( quartile 3 ) half of the dataset . Black dots indicate biological replicates O : n = 8 ) , the red dot indicates the mean , red error bars indicate the confidence interval ( 95 % ) , asterisks indicate significance ( 0 . 01 < p ( ✱ ) < 0 . 05 , 0 . 001 < p ( ✱ ✱ ) < 0 . 01 , p ( ✱ ✱ ✱ ) < 0 . 001 , ns = not significant ) .

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

(N) ish with ddc probe on wild-type and opn4 dko larvae sampled on the 6th dpf represents ddc expression in pineals. Biological replicates indicated with BR. Scale bar: 0.05 mm (O) Quantification of the adjusted intensity in the pineals from larvae presented in N, implies a significant higher ddc transcript level in the opn4 dko pineal. Data information: boxplot divides the data in quartiles: the box indicates the interquartile range, with the horizontal line in the box denoting the median of the data set, the whiskers extend to the minimum and maximum, and meet the box at the median of the lower (quartile 1) and median of the upper (quartile 3) half of the dataset. Black dots indicate biological replicates O: n=8), the red dot indicates the mean, red error bars indicate the confidence interval (95%), asterisks indicate significance (0.01<p(✱)<0.05, 0.001<p(✱✱)<0.01, p(✱✱✱)<0.001, ns = not significant).


Neuroblastoma PDX and SK - N - BE ( 2 ) c cells treated with IBL inhibitors at indicated concentrations for 48 hours . pAkt ( at Ser473 ( A ) and Thr308 ( B ) sites ) levels in LU - NB - 3 and SK - N - BE ( 2 ) c cells determined by western blotting . Total Akt levels were used as loading control .

Neuroblastoma PDX and SK-N-BE(2)c cells treated with IBL inhibitors at indicated concentrations for 48 hours. pAkt (at Ser473 (A) and Thr308 (B) sites) levels in LU-NB-3 and SK-N-BE(2)c cells determined by western blotting. Total Akt levels were used as loading control.


Neuroblastoma PDX cells treated with IBL inhibitors at indicated concentrations for 48 hours . C . p - p70S6K and p - p85S6K levels in LU - NB - 3 cells determined by western blotting . Actin , p70S6K , and p85S6K levels were used as loading controls .

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Neuroblastoma PDX cells treated with IBL inhibitors at indicated concentrations for 48 hours. C. p-p70S6K and p-p85S6K levels in LU-NB-3 cells determined by western blotting. Actin, p70S6K, and p85S6K levels were used as loading controls.


D . Brightfield photomicrographs of LU - NB - 3 and SK - N - BE ( 2 ) c cells treated with 0 . 36μM IBL - 202 or 0 . 05 μM IBL - 301 . Scale bars represent 100 μm ( LU - NB - 3 ) or 200 μm ( SK - N - BE ( 2 ) c ) . Arrows indicate neurite outgrowths and asterisks indicate where inserts are magnified . IBL - 301 treated cells were stained for Tuj1 . DAPI was used to visualize nuclei . E . Quantification of neurite outgrowth presented as number of neurites / cell in LU - NB - 3 PDX and SK - N - BE ( 2 ) cells treated with IBL - 301 . For LU - NB - 3 PDX cells , representative areas ( n = 2 ) were used and n = 344 and n = 240 cells / condition for CTRL and IBL - 301 , respectively , were counted . For SK - N - BE ( 2 ) c cells , representative areas ( n = 2 and n = 3 for CTRL and IBL - 301 respectively ) were used and n = 141 and n = 130 cells / condition for CTRL and IBL - 301 , respectively , were counted . Statistical significance was determined by two - sided student ' s t - tes . P = 0 . 003 for LU - NB - 3 and p = 0 . 08 for SK - N - BE ( 2 ) c .

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

D. Brightfield photomicrographs of LU-NB-3 and SK-N-BE(2)c cells treated with 0.36μM IBL-202 or 0.05 μM IBL-301. Scale bars represent 100 μm (LU-NB-3) or 200 μm (SK-N-BE(2)c). Arrows indicate neurite outgrowths and asterisks indicate where inserts are magnified. IBL-301 treated cells were stained for Tuj1. DAPI was used to visualize nuclei. E. Quantification of neurite outgrowth presented as number of neurites/cell in LU-NB-3 PDX and SK-N-BE(2) cells treated with IBL-301. For LU-NB-3 PDX cells, representative areas (n=2) were used and n=344 and n=240 cells/condition for CTRL and IBL-301, respectively, were counted. For SK-N-BE(2)c cells, representative areas (n=2 and n=3 for CTRL and IBL-301 respectively) were used and n=141 and n=130 cells/condition for CTRL and IBL-301, respectively, were counted. Statistical significance was determined by two-sided student's t-tes. P=0.003 for LU-NB-3 and p=0.08 for SK-N-BE(2)c.


Neuroblastoma PDX and SK - N - BE ( 2 ) c cells treated with IBL inhibitors at indicated concentrations for 48 hours . F . Gap43 protein levels in LU - NB - 3 and SK - N - BE ( 2 ) c cells determined by western blotting . SDHA levels were used as loading control .

Neuroblastoma PDX and SK-N-BE(2)c cells treated with IBL inhibitors at indicated concentrations for 48 hours. F. Gap43 protein levels in LU-NB-3 and SK-N-BE(2)c cells determined by western blotting. SDHA levels were used as loading control.


Neuroblastoma PDX cells treated with IBL inhibitors at indicated concentrations for 48 hours . G . N - Myc levels in LU - NB - 2 and LU - NB - 3 cells determined by western blotting . SDHA levels were used as loading control .

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Neuroblastoma PDX cells treated with IBL inhibitors at indicated concentrations for 48 hours. G. N-Myc levels in LU-NB-2 and LU-NB-3 cells determined by western blotting. SDHA levels were used as loading control.


A . Viability IBL - 202 or IBL - 301 treated cells determined by CellTiterGlo .

A. Viability IBL-202 or IBL-301 treated cells determined by CellTiterGlo.


B - C . Cell cycle distribution of LU - NB - 3 cells determined by flow cytometry . Bars show mean values from two independent experiments .

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B-C. Cell cycle distribution of LU-NB-3 cells determined by flow cytometry. Bars show mean values from two independent experiments.


D . Cleaved caspase 3 ( Cl Casp3 ) levels in LU - NB - 3 cells determined by western blotting . SDHA levels were used as loading control .

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D. Cleaved caspase 3 (Cl Casp3) levels in LU-NB-3 cells determined by western blotting. SDHA levels were used as loading control.


E . Flow cytometry analyses of Annexin V and PI stainings following treatment with 360 nM IBL - 202 or 50 nM IBL - 301 . F . Quantification of live and dead cells from the AnnexinV / PI stainings . Dead cells = PI positive , Live cells = PI negative . Dot plots from two independent experiments and error bars represent SEM .

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E. Flow cytometry analyses of Annexin V and PI stainings following treatment with 360 nM IBL-202 or 50 nM IBL-301. F. Quantification of live and dead cells from the AnnexinV/PI stainings. Dead cells=PI positive, Live cells=PI negative. Dot plots from two independent experiments and error bars represent SEM.


Neuroblastoma cells were treated with indicated concentrations of IBL - 302 for 48 hours A . Expression of pAkt ( Ser473 ) in LU - NB - 3 and SK - N - BE ( 2 ) c cells . Total Akt and actin were used as loading controls . B . Expression of pAkt ( Thr308 ) in LU - NB - 3 and SK - N - BE ( 2 ) c cells . Total Akt and actin were used as loading controls .

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

Neuroblastoma cells were treated with indicated concentrations of IBL-302 for 48 hours A. Expression of pAkt(Ser473) in LU-NB-3 and SK-N-BE(2)c cells. Total Akt and actin were used as loading controls. B. Expression of pAkt(Thr308) in LU-NB-3 and SK-N-BE(2)c cells. Total Akt and actin were used as loading controls.


Neuroblastoma cells were treated with indicated concentrations of IBL - 302 for 48 hours C . Brightfield photomicrographs of LU - NB - 3 and SK - N - BE ( 2 ) c cells treated with 50 nM IBL - 302 . Scale bars represent 100 μm ( LU - NB - 3 ) or 200 μm ( SK - N - BE ( 2 ) c ) . D . Quantification of neurite outgrowth presented as number of neurites / cell in LU - NB - 3 PDX and SK - N - BE ( 2 ) c cells treated with IBL - 302 . For LU - NB - 3 PDX cells , representative areas ( n = 4 ) were used and n = 460 and n = 216 cells / condition for CTRL and IBL - 302 , respectively , were counted . For SK - N - BE ( 2 ) c cells , representative areas ( n = 5 and n = 7 for CTRL and IBL - 302 respectively ) were used and n = 124 and n = 260 cells / condition for CTRL and IBL - 301 , respectively , were counted . Statistical significance was determined by two - sided student ' s t - test . p = 0 . 009 for LU - NB - 3 and p = 0 . 0003 for SK - N - BE ( 2 ) c .

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

Neuroblastoma cells were treated with indicated concentrations of IBL-302 for 48 hours C. Brightfield photomicrographs of LU-NB-3 and SK-N-BE(2)c cells treated with 50 nM IBL-302. Scale bars represent 100 μm (LU-NB-3) or 200 μm (SK-N-BE(2)c). D. Quantification of neurite outgrowth presented as number of neurites/cell in LU-NB-3 PDX and SK-N-BE(2)c cells treated with IBL-302. For LU-NB-3 PDX cells, representative areas (n=4) were used and n=460 and n=216 cells/condition for CTRL and IBL-302, respectively, were counted. For SK-N-BE(2)c cells, representative areas (n=5 and n=7 for CTRL and IBL-302 respectively) were used and n=124 and n=260 cells/condition for CTRL and IBL-301, respectively, were counted. Statistical significance was determined by two-sided student's t-test. p=0.009 for LU-NB-3 and p=0.0003 for SK-N-BE(2)c.


Neuroblastoma cells were treated with indicated concentrations of IBL - 302 for 48 hours E . N - Myc protein expression determined by western blotting . SDHA was used as loading control .

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Neuroblastoma cells were treated with indicated concentrations of IBL-302 for 48 hours E. N-Myc protein expression determined by western blotting. SDHA was used as loading control.


Neuroblastoma cells were treated with indicated concentrations of IBL - 302 for 72 hours F . Cell viability determined by CellTiterGlo .

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Neuroblastoma cells were treated with indicated concentrations of IBL-302 for 72 hours F. Cell viability determined by CellTiterGlo.


Neuroblastoma cells were treated with indicated concentrations of IBL - 302 for 48 hours G . Flow cytometry analyses of Annexin V and PI stainings following treatment with 50nM IBL - 302 H . Quantification of live and dead cells . Dead cells = PI positive , Live cells = PI negative . Dot plots from two independent experiments and error bars represent SEM .

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Neuroblastoma cells were treated with indicated concentrations of IBL-302 for 48 hours G. Flow cytometry analyses of Annexin V and PI stainings following treatment with 50nM IBL-302 H. Quantification of live and dead cells. Dead cells=PI positive, Live cells=PI negative. Dot plots from two independent experiments and error bars represent SEM.


I . Neuroblastoma SK - N - BE ( 2 ) c carrying mice ( n = 5 in each group ) were treated with vehicle ( CTRL ) or 40 mg / kg IBL - 302 for up to 35 days and tumor growth was followed over time . Asterisks indicate each occasion a mouse within that particular group was sacrificed .

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I. Neuroblastoma SK-N-BE(2)c carrying mice (n=5 in each group) were treated with vehicle (CTRL) or 40 mg/kg IBL-302 for up to 35 days and tumor growth was followed over time. Asterisks indicate each occasion a mouse within that particular group was sacrificed.


J . Kaplan - Meier survival curves comparing mice treated with vehicle ( CTRL ) or IBL - 302 . Log - rank test was used to determine statistical significance . p = 0 . 0577 .

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J. Kaplan-Meier survival curves comparing mice treated with vehicle (CTRL) or IBL-302. Log-rank test was used to determine statistical significance. p=0.0577.


A - B . LU - NB - 3 PDX , SK - N - BE ( 2 ) c and SK - N - SH cells were treated with indicated concentrations of cisplatin and / or 0 . 36 μM IBL - 202 , 0 . 05 μM IBL - 301 , or 0 . 05 μM IBL - 302 for 48 hours . Cell viability determined by CellTiterGlo . Graphs show mean values and SEM from three independent experiments . Theoretical additive curves were evaluated by calculating the combination index ( CI ) based on the Bliss Independence model .

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A-B. LU-NB-3 PDX, SK-N-BE(2)c and SK-N-SH cells were treated with indicated concentrations of cisplatin and/or 0.36 μM IBL-202, 0.05 μM IBL-301, or 0.05 μM IBL-302 for 48 hours. Cell viability determined by CellTiterGlo. Graphs show mean values and SEM from three independent experiments. Theoretical additive curves were evaluated by calculating the combination index (CI) based on the Bliss Independence model.


C - D . Cell cycle distribution in LU - NB - 3 PDX cells after treatment with 320 nM cisplatin , 50 nM IBL - 302 or the combination of these , determined by flow cytometry . Bars show mean values from two independent experiments .

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C-D. Cell cycle distribution in LU-NB-3 PDX cells after treatment with 320 nM cisplatin, 50 nM IBL-302 or the combination of these, determined by flow cytometry. Bars show mean values from two independent experiments.


E . Tumor size in individual mice from Neuroblastoma PDX carrying mice ( n = 5 in each group ) were treated with vehicle ( CTRL ) , low - dose ( 20mg / kg ) IBL - 302 , low - dose ( 1mg / kg ) cisplatin , or the combination of low - dose IBL - 302 and low - dose cisplatin for up to 70 days . Tumor size was measured over time . Asterisks indicate each occasion a mouse within that particular group was sacrificed .

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E. Tumor size in individual mice from Neuroblastoma PDX carrying mice (n=5 in each group) were treated with vehicle (CTRL), low-dose (20mg/kg) IBL-302, low-dose (1mg/kg) cisplatin, or the combination of low-dose IBL-302 and low-dose cisplatin for up to 70 days. Tumor size was measured over time. Asterisks indicate each occasion a mouse within that particular group was sacrificed.


F . Neuroblastoma PDX carrying mice ( n = 5 in each group ) were treated with vehicle ( CTRL ) , low - dose ( 20mg / kg ) IBL - 302 , low - dose ( 1mg / kg ) cisplatin , or the combination of low - dose IBL - 302 and low - dose cisplatin for up to 70 days . Tumor size was measured over time . Asterisks indicate each occasion a mouse within that particular group was sacrificed .

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F. Neuroblastoma PDX carrying mice (n=5 in each group) were treated with vehicle (CTRL), low-dose (20mg/kg) IBL-302, low-dose (1mg/kg) cisplatin, or the combination of low-dose IBL-302 and low-dose cisplatin for up to 70 days. Tumor size was measured over time. Asterisks indicate each occasion a mouse within that particular group was sacrificed.


G . Kaplan - Meier survival curves comparing mice treated with vehicle ( CTRL ) , IBL - 302 , cisplatin , or the combination . Log - rank test was used to determine statistical significance . p = 0 . 0014 .

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G. Kaplan-Meier survival curves comparing mice treated with vehicle (CTRL), IBL-302, cisplatin, or the combination. Log-rank test was used to determine statistical significance. p=0.0014.


H . Immunohistochemical staining of N - Myc expression in tumors Scale bars represent 100 μm .

H. Immunohistochemical staining of N-Myc expression in tumors Scale bars represent 100 μm.


LU - NB - 3 PDX cells were treated with 50 nM of IBL - 302 for 48 hours and subsequently analyzed by RNAseq and mass spectrometry . A - C . Heat map of significantly differentially expressed genes across treatment groups from RNAseq data ( FDR < 0 . 1 ) ( A ) . Genes defined as differentially expressed that were also present in MS and phospho - MS are visualized in ( B ) and ( C ) , respectively .

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LU-NB-3 PDX cells were treated with 50 nM of IBL-302 for 48 hours and subsequently analyzed by RNAseq and mass spectrometry. A-C. Heat map of significantly differentially expressed genes across treatment groups from RNAseq data (FDR<0.1) (A). Genes defined as differentially expressed that were also present in MS and phospho-MS are visualized in (B) and (C), respectively.


( A ) Waterfall plot showing the percentage of tumor volume change in olaparib - treated tumors compared to the tumor volume on day 1 . + 20 % and - 30 % are marked by doted lines to indicate the range of PR , SD and PD . The box underneath summarizes different characteristics of each model and the clinical context at the moment of PDX implantation . TNBC , Triple Negative Breast Cancer ; ER + BC , Estrogen Receptor positive Breast Cancer ; P , primary ; M , metastasis . Error bars indicate SEM from independent tumors ( n ≥ 3 ) .

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(A) Waterfall plot showing the percentage of tumor volume change in olaparib-treated tumors compared to the tumor volume on day 1. +20% and -30% are marked by doted lines to indicate the range of PR, SD and PD. The box underneath summarizes different characteristics of each model and the clinical context at the moment of PDX implantation. TNBC, Triple Negative Breast Cancer; ER+BC, Estrogen Receptor positive Breast Cancer; P, primary; M, metastasis. Error bars indicate SEM from independent tumors (n ≥ 3).


( B ) Graph showing the percentage of tumor volume change during olaparib treatment in PDXs from cohort - 1 . Olaparib - sensitive models are represented with discontinuous lines . Acquisition of PARPi - resistance in PDX302 , STG201 and PDX093 after prolonged exposure to olaparib is shown .

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(B) Graph showing the percentage of tumor volume change during olaparib treatment in PDXs from cohort-1. Olaparib-sensitive models are represented with discontinuous lines. Acquisition of PARPi-resistance in PDX302, STG201 and PDX093 after prolonged exposure to olaparib is shown.


( A ) Levels of BRCA1 promoter hypermethylation , levels BRCA1 mRNA and the presence of BRCA1 nuclear foci by immunofluorescence are shown T127 and T162 were used as positive controls for hypermethylated BRCA1 promoter . Error bars indicate SEM from independent tumors ( n ≥ 2 ) . Dashed line indicates mean of BRCA1 mRNA levels in normal breast . PARPi - response is shown in the summary underneath : white box : PD ; black box : PR / CR . Alterations in HRR - related genes in PDX are also indicated .

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(A) Levels of BRCA1 promoter hypermethylation, levels BRCA1 mRNA and the presence of BRCA1 nuclear foci by immunofluorescence are shown T127 and T162 were used as positive controls for hypermethylated BRCA1 promoter. Error bars indicate SEM from independent tumors (n ≥ 2). Dashed line indicates mean of BRCA1 mRNA levels in normal breast. PARPi-response is shown in the summary underneath: white box: PD; black box: PR/CR. Alterations in HRR-related genes in PDX are also indicated.


( B ) Western blot of PALB2 detected in U2OS cells and PDXs . Three biological replicates of PDX093 are shown ; PDX302 is used as PALB2 wild type PDX control .

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(B) Western blot of PALB2 detected in U2OS cells and PDXs. Three biological replicates of PDX093 are shown; PDX302 is used as PALB2 wild type PDX control.


( C ) YFP - PALB2 recruitment to laser - induced DSBs is impaired in HeLa cells expressing PALB2 p . M296Nfs ( n = 4 , unpaired t - test at 16min ) .

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(C) YFP-PALB2 recruitment to laser-induced DSBs is impaired in HeLa cells expressing PALB2 p.M296Nfs (n=4, unpaired t-test at 16min).


Gene - targeting efficiency using Cas9 / mClover - LMNA1 homologous recombination assay of ( D ) siRNA PALB2 cells ( n = 4 , one - way ANOVA ) or ( E ) cells with no PALB2 depletion complemented with wild type and p . M296Nfs siRNA - resistant constructs ( n = 7 , one - way ANOVA ) . Western blots of PALB2 wild type and PALB2 p . M296Nfs for each condition are shown . Error bars indicate SEM from independent experiments .

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Gene-targeting efficiency using Cas9/mClover-LMNA1 homologous recombination assay of (D) siRNA PALB2 cells (n=4, one-way ANOVA) or (E) cells with no PALB2 depletion complemented with wild type and p.M296Nfs siRNA-resistant constructs (n=7, one-way ANOVA). Western blots of PALB2 wild type and PALB2 p.M296Nfs for each condition are shown. Error bars indicate SEM from independent experiments.


( A ) Percentage of geminin - positive , RAD51 nuclear foci - containing cells detected by immunofluorescence in FFPE samples from PDX tumors treated with vehicle or PARPi Error bars indicate SEM from independent tumors ( n ≥ 2 ) . PARPi - response is shown in the summary underneath : white box : PD ; black box : PR / CR . Immunofluorescence staining of RAD51 foci in PARPi - and vehicle - treated PDX tumors is shown . Alterations in HRR - related genes are also summarized : hBRCA1 : BRCA1 promoter hypermethylation and lack of BRCA1 expression and BRCA1 nuclear foci formation .

(A) Percentage of geminin-positive, RAD51 nuclear foci-containing cells detected by immunofluorescence in FFPE samples from PDX tumors treated with vehicle or PARPi Error bars indicate SEM from independent tumors (n ≥ 2). PARPi-response is shown in the summary underneath: white box: PD; black box: PR/CR. Immunofluorescence staining of RAD51 foci in PARPi- and vehicle-treated PDX tumors is shown. Alterations in HRR-related genes are also summarized: hBRCA1: BRCA1 promoter hypermethylation and lack of BRCA1 expression and BRCA1 nuclear foci formation.


( B ) Restoration of RAD51 foci formation in PARPi acquired - resistant PDXs . Immunofluorescence staining of BRCA1 and RAD51 foci in PARPi - treated tumors from STG201 , PDX302 and the corresponding PARPi acquired - resistant models ( STG201OR and PDX302OR ) . Scale bars : 10µm .

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(B) Restoration of RAD51 foci formation in PARPi acquired-resistant PDXs. Immunofluorescence staining of BRCA1 and RAD51 foci in PARPi-treated tumors from STG201, PDX302 and the corresponding PARPi acquired-resistant models (STG201OR and PDX302OR). Scale bars: 10µm.


( C ) Quantification of geminin - positive cells that exhibit γH2AX nuclear foci following treatment with vehicle and olaparib . Paired t test in PARPi - sensitive ( CR / PR ) vs . PARPi - resistant ( PD ) PDXs .

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(C) Quantification of geminin-positive cells that exhibit γH2AX nuclear foci following treatment with vehicle and olaparib. Paired t test in PARPi-sensitive (CR/PR) vs. PARPi-resistant (PD) PDXs.


( A ) Percentage of geminin - positive , RAD51 nuclear foci - containing cells in FFPE samples from untreated PDX tumors . Color bars indicate the presence of pathogenic variants in the indicated genes . Error bars indicate SEM from independent tumors ( n ≥ 2 ) . PARPi - response is shown in the summary underneath : black box : PR / CR ; white box : PD . Box colors indicate the PARP inhibitor treatment . Boxes with two colors indicate the same response to both treatments . Olaparib 100mg / kg & 50mg / kg indicates that both doses were tested and resulted in the same response categorization .

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(A) Percentage of geminin-positive, RAD51 nuclear foci-containing cells in FFPE samples from untreated PDX tumors. Color bars indicate the presence of pathogenic variants in the indicated genes. Error bars indicate SEM from independent tumors (n ≥ 2). PARPi-response is shown in the summary underneath: black box: PR/CR; white box: PD. Box colors indicate the PARP inhibitor treatment. Boxes with two colors indicate the same response to both treatments. Olaparib 100mg/kg & 50mg/kg indicates that both doses were tested and resulted in the same response categorization.


( B ) ROC curves of the RAD51 score and HRD score , for PARPi response prediction capacity in the PDX cohort - 2 . Bootstrap statistical test .

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(B) ROC curves of the RAD51 score and HRD score, for PARPi response prediction capacity in the PDX cohort-2. Bootstrap statistical test.


( C ) Immunofluorescence staining of 53BP1 and BRCA1 nuclear foci [ with an antibody towards the N - terminus ( B1 - NT ) or C - terminus ( B1 - CT ) of BRCA1 ] in three BRCA1 - mutant , PARPi - resistant models from PDX cohort - 2 . The location of the mutation within the gene is indicated . Scale bars : 10µm .

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(C) Immunofluorescence staining of 53BP1 and BRCA1 nuclear foci [with an antibody towards the N-terminus (B1-NT) or C-terminus (B1-CT) of BRCA1] in three BRCA1-mutant, PARPi-resistant models from PDX cohort-2. The location of the mutation within the gene is indicated. Scale bars: 10µm.


( B ) Percentage of geminin - positive , RAD51 or γH2AX nuclear foci - containing cells in FFPE tumor samples from patients with HBOC syndrome . The box underneath summarizes the patient ' s young onset ( < 35y : < 35 years ) , her family history ( FH , purple box for PALB2 - related tumors ) and the presence of BRCA1 nuclear foci in the analyzed tumor samples .

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(B) Percentage of geminin-positive, RAD51 or γH2AX nuclear foci-containing cells in FFPE tumor samples from patients with HBOC syndrome. The box underneath summarizes the patient's young onset (<35y: <35 years), her family history (FH, purple box for PALB2-related tumors) and the presence of BRCA1 nuclear foci in the analyzed tumor samples.


( C ) Immunofluorescence staining of γH2AX , BRCA1 and RAD51 foci in three representative FFPE tumors from patients : one RAD51 - and BRCA1 - positive tumor ( Pt03 ) , one RAD51 - and BRCA1 - negative tumor ( Pt11 ) and one PALB2 - related tumor , with BRCA1 but not RAD51 nuclear foci ( Pt20 . 1 ) . Scale bars : 10µm .

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(C) Immunofluorescence staining of γH2AX, BRCA1 and RAD51 foci in three representative FFPE tumors from patients: one RAD51- and BRCA1-positive tumor (Pt03), one RAD51- and BRCA1-negative tumor (Pt11) and one PALB2-related tumor, with BRCA1 but not RAD51 nuclear foci (Pt20.1). Scale bars: 10µm.


( A ) The mRNA levels of QKI relative to those of 36B4 in BAT and ingWAT from HFD mice ( n = 8 ) and lean controls ( n = 8 ) .

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(A)The mRNA levels of QKI relative to those of 36B4 in BAT and ingWAT from HFD mice (n = 8) and lean controls (n = 8).


WT fl / fl and QKI KO fl / fl : AP2 - Cre mice fed a NCD or HFD for 20 weeks . The arrow indicates the time when the high fat diet （ HFD ） starts , and body weight was monitored weekly . ( n = 10 , per genotype )

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WT fl/fl and QKI KO fl/fl:AP2-Cre mice fed a NCD or HFD for 20 weeks. The arrow indicates the time when the high fat diet（HFD）starts, and body weight was monitored weekly. (n = 10, per genotype)


Representative pictures of adipocyte - specific QKI - deficient mice under NCD or HFD conditions .

Representative pictures of adipocyte-specific QKI-deficient mice under NCD or HFD conditions.


Gross appearance of tissues from BAT , eWAT and ingWAT fat pads from WT fl / fl and QKI KO fl / fl : AP2 - Cre mice after 20 weeks on the NCD or HFD .

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Gross appearance of tissues from BAT, eWAT and ingWAT fat pads from WT fl/fl and QKI KO fl/fl:AP2-Cre mice after 20 weeks on the NCD or HFD.


H & E staining of BAT , eWAT and ingWAT from WT fl / fl and QKI KO fl / fl : AP2 - Cre mice fed the NCD or HFD for 20 weeks . ( Scale bar represents 50 μm )

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H&E staining of BAT, eWAT and ingWAT from WT fl/fl and QKI KO fl/fl:AP2-Cre mice fed the NCD or HFD for 20 weeks. (Scale bar represents 50 μm)


The gross appearance of BAT , eWAT and ingWAT fat pads after administration of AAV - ShQKI or AAV - NC vectors in local adipose ( ingWAT and BAT ) .

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The gross appearance of BAT, eWAT and ingWAT fat pads after administration of AAV-ShQKI or AAV-NC vectors in local adipose (ingWAT and BAT).


The increase in relative body weights following HFD feeding . ( n = 8 , per group )

The increase in relative body weights following HFD feeding. (n = 8, per group)


H & E staining of the liver , BAT and ingWAT from AAV - NC or AAV - ShQKI mice . ( Scale bar represents 50 μm ) .

H&E staining of the liver, BAT and ingWAT from AAV-NC or AAV-ShQKI mice. (Scale bar represents 50 μm).


Representative CT images of WT fl / fl and QKI KO fl / fl : AP2 - Cre mice on an NCD or HFD , showing the whole - body fat composition .

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Representative CT images of WT fl/fl and QKI KO fl/fl:AP2-Cre mice on an NCD or HFD, showing the whole-body fat composition.


Glucose tolerance tests ( GTT ) and Insulin tolerance tests ( ITT ) were performed on WT fl / fl and QKI KO fl / fl : AP2 - Cre mice fed the NCD or HFD for 20 weeks . Blood glucose concentrations during GTT and ITT were monitored . ( n = 10 , per genotype )

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Glucose tolerance tests (GTT) and Insulin tolerance tests (ITT) were performed on WT fl/fl and QKI KO fl/fl:AP2-Cre mice fed the NCD or HFD for 20 weeks. Blood glucose concentrations during GTT and ITT were monitored. (n= 10, per genotype)


Blood glucose and plasma insulin levels in WT fl / fl and QKI KO fl / fl : AP2 - Cre mice fed the NCD or HFD for 20 weeks . ( n = 8 , per genotype )

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Blood glucose and plasma insulin levels in WT fl/fl and QKI KO fl/fl:AP2-Cre mice fed the NCD or HFD for 20 weeks. (n = 8, per genotype)


Plasma Free Fatty Acid ( FFA ) and Glycerol levels in WT fl / fl and QKI KO fl / fl : AP2 - Cre mice fed the NCD or HFD for 20 weeks . ( n = 8 , per genotype )

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Plasma Free Fatty Acid (FFA) and Glycerol levels in WT fl/fl and QKI KO fl/fl:AP2-Cre mice fed the NCD or HFD for 20 weeks. (n = 8, per genotype)


H & E and Oil Red O staining , and liver triglyceride ( TG ) content in WT fl / fl and QKI KO fl / fl : AP2 - Cre mice fed the NCD or HFD for 20 weeks . ( Scale bar represents 50 μm )

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H&E and Oil Red O staining, and liver triglyceride (TG) content in WT fl/fl and QKI KO fl/fl:AP2-Cre mice fed the NCD or HFD for 20 weeks. (Scale bar represents 50 μm)


O2 consumption rates were measured via indirect calorimetry using CLAMS under chow - fed conditions ( n = 8 , per genotype ) . And average light and dark O2 consumption rates are shown on the right .

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O2 consumption rates were measured via indirect calorimetry using CLAMS under chow-fed conditions (n = 8, per genotype). And average light and dark O2 consumption rates are shown on the right.


Heat production rates in WT fl / fl and QKI KO fl / fl : AP2 - Cre mice ( n = 8 , per genotype ) . And average light and dark heat production rates are shown on the right .

Heat production rates in WT fl/fl and QKI KO fl/fl:AP2-Cre mice (n = 8, per genotype). And average light and dark heat production rates are shown on the right.


Heatmaps of log2 ( fold change QKI KO fl / fl : AP2 - Cre BAT vs . WT fl / fl BAT ) for genes involved in thermogenesis and oxidation . ( 3 samples , per group )

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Heatmaps of log2 (fold change QKI KO fl/fl:AP2-Cre BAT vs. WT fl/fl BAT) for genes involved in thermogenesis and oxidation. (3 samples, per group)


Photograph of WT fl / fl and QKI KO fl / fl : AP2 - Cre classical BAT in 4 % paraformaldehyde solution , representative transmission electron micrographs , and UCP1 staining of BAT from WT fl / fl and QKI KO fl / fl : AP2 - Cre mice .

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Photograph of WT fl/fl and QKI KO fl/fl:AP2-Cre classical BAT in 4% paraformaldehyde solution, representative transmission electron micrographs, and UCP1 staining of BAT from WT fl/fl and QKI KO fl/fl:AP2-Cre mice.


The average diameter of lipid droplets and the number of mitochondria per field from WT fl / fl and QKI KO fl / fl : AP2 - Cre BAT . ( n = 3 biological replicates )

The average diameter of lipid droplets and the number of mitochondria per field from WT fl/fl and QKI KO fl/fl:AP2-Cre BAT. (n = 3 biological replicates)


The relative mRNA levels of genes related to thermogenesis in BAT analyzed by RT - PCR . ( n = 4 biological replicates )

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The relative mRNA levels of genes related to thermogenesis in BAT analyzed by RT-PCR. (n = 4 biological replicates)


Total oxygen consumption rates ( OCR ) per gram in BAT from WT fl / fl and QKI KO fl / fl : AP2 - Cre mice under basal conditions were measured using a Clark electrode . ( n = 3 biological replicates )

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

Total oxygen consumption rates (OCR) per gram in BAT from WT fl/fl and QKI KO fl/fl:AP2-Cre mice under basal conditions were measured using a Clark electrode. (n = 3 biological replicates)


The relative mRNA levels of genes related to thermogenesis in ingWAT analyzed by RT - PCR . ( n = 4 biological replicates )

The relative mRNA levels of genes related to thermogenesis in ingWAT analyzed by RT-PCR. (n = 4 biological replicates)


Total oxygen consumption rates ( OCR ) per gram in ingWAT from WT fl / fl and QKI KO fl / fl : AP2 - Cre mice under basal conditions . ( n = 3 biological replicates )

Total oxygen consumption rates (OCR) per gram in ingWAT from WT fl/fl and QKI KO fl/fl:AP2-Cre mice under basal conditions. (n = 3 biological replicates)


Representative recording of Continuous measurement of oxygen consumption rate ( OCR ) in isolated mitochondria from BAT of WT fl / fl and QKI KO fl / fl : AP2 - Cre mice on NCD ; Oxygen consumption was performed under basal conditions , following the addition of 0 . 25 mg mitochondria ( Mit ) , 5 mM substrate glycerol - 3 - phosphate ( G3P ) , 2 mM GDP , and 450 mM ADP , 2 μg / ml oligomycin , and 1 . 5 μM FCCP . Quantification of experimental data . ( n = 3 biological replicates , per group )

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

Representative recording of Continuous measurement of oxygen consumption rate (OCR) in isolated mitochondria from BAT of WT fl/fl and QKI KO fl/fl:AP2-Cre mice on NCD; Oxygen consumption was performed under basal conditions, following the addition of 0. 25 mg mitochondria (Mit), 5 mM substrate glycerol-3-phosphate (G3P), 2 mM GDP, and 450 mM ADP, 2 μg/ml oligomycin, and 1.5 μM FCCP. Quantification of experimental data. (n=3 biological replicates, per group)


UCP1 - dependent oxygen consumption rates estimated as GDP - inhabitable rates , based on the data shown in ( K ) . ( n = 3 biological replicates )

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

UCP1-dependent oxygen consumption rates estimated as GDP-inhabitable rates, based on the data shown in (K). (n=3 biological replicates)


The protein levels of PGC1α and QKI in siRNA - mediated QKI knockdown differentiated cell , and the quantification graph was shown on the right . ( n = 3 biological replicates )

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

The protein levels of PGC1α and QKI in siRNA-mediated QKI knockdown differentiated cell, and the quantification graph was shown on the right. (n=3 biological replicates)


Immunostaining of PGC1α and UCP1 in NC , Si - QKI 1 , and Si - QKI 2 adipocytes . ( Scale bar represents 20 μm ) .

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

Immunostaining of PGC1α and UCP1 in NC, Si-QKI 1, and Si-QKI 2 adipocytes. (Scale bar represents 20 μm).


The relative mRNA levels of genes related to thermogenesis in adipocytes treated with NC , QKI siRNA or both QKI and PGC1α siRNA . ( n = 3 biological replicates )

The relative mRNA levels of genes related to thermogenesis in adipocytes treated with NC, QKI siRNA or both QKI and PGC1α siRNA. (n=3 biological replicates)


The oxygen consumption rates in adipocytes treated with NC , QKI siRNA or both QKI and PGC1α siRNA . ( n = 3 biological replicates )

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

The oxygen consumption rates in adipocytes treated with NC, QKI siRNA or both QKI and PGC1α siRNA. (n=3 biological replicates)


Representative pictures of oil red O staining in three adipocyte types : ShScramble + ovCon cells , QKI knockdown cells ( ShQKI + ovCon ) and QKI knockdown with QKI - 5 re - expression cells ( ShQKI + ovQKI ) .

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

Representative pictures of oil red O staining in three adipocyte types: ShScramble+ovCon cells, QKI knockdown cells (ShQKI+ovCon) and QKI knockdown with QKI-5 re-expression cells (ShQKI+ovQKI).


The protein levels of PGC1α in these adipocytes , and the quantification graph was shown on the right . ( n = 3 biological replicates )

The protein levels of PGC1α in these adipocytes, and the quantification graph was shown on the right. (n=3 biological replicates)


The relative mRNA levels of genes associated with energy dissipation in ShScramble + ovCon , ShQKI + ovCon and ShQKI + ovQKI differentiated cells . The results represent 3 independent experiments .

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

The relative mRNA levels of genes associated with energy dissipation in ShScramble+ovCon, ShQKI+ovCon and ShQKI+ovQKI differentiated cells. The results represent 3 independent experiments.


Construction of a series of luciferase reporter vectors ( PGC1α full - length 3 ' UTR T , A , B , C fragments and PGC1α 3 ' UTR QRE motif sites mutant B M1 , B M2 , C M1 , C M2 fragments ) . Luciferase activity assays to examine the functional QRE motif sites in the PGC1α 3 ' UTR affected by QKI . ( n = 4 biological replicates )

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

Construction of a series of luciferase reporter vectors (PGC1α full-length 3'UTR T, A, B, C fragments and PGC1α 3'UTR QRE motif sites mutant B M1, B M2, C M1, C M2 fragments). Luciferase activity assays to examine the functional QRE motif sites in the PGC1α 3'UTR affected by QKI. (n=4 biological replicates)


Construction of a series of luciferase reporter vectors ( UCP1 full - length 3 ' UTR T fragment and UCP1 3 ' UTR QRE motif sites mutant T M1 , T M2 , T M3 fragments ) . Luciferase activity assays to examine the functional QRE motif sites in the UCP1 3 ' UTR affected by QKI . ( n = 4 biological replicates )

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

Construction of a series of luciferase reporter vectors (UCP1 full-length 3'UTR T fragment and UCP1 3'UTR QRE motif sites mutant T M1, T M2, T M3 fragments). Luciferase activity assays to examine the functional QRE motif sites in the UCP1 3'UTR affected by QKI. (n=4 biological replicates)


Based on RIP - sequencing data , the distribution of QKI - binding peaks within UCP1 and PGC1α transcript regions .

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

Based on RIP-sequencing data, the distribution of QKI-binding peaks within UCP1 and PGC1α transcript regions.


RNA immunoprecipitation followed by real - time PCR . Specific primer for PGC1α 3 ' UTR region 1 , region 2 and UCP1 3 ' UTR to detect these QRE motif sites region bound to endogenous QKI in brown fat tissue , and data showing the PGC1α and UCP1 transcripts abundance in QKI IP compared with IgG IP . ( n = 4 biological replicates )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 immunoprecipitation followed by real-time PCR. Specific primer for PGC1α 3'UTR region 1, region 2 and UCP1 3'UTR to detect these QRE motif sites region bound to endogenous QKI in brown fat tissue, and data showing the PGC1α and UCP1 transcripts abundance in QKI IP compared with IgG IP. (n=4 biological replicates)


293T cells were transfected with PGC1α wild type or QRE sites mutant 3 ' UTR fragments . RNA immunoprecipitation ( RIP ) to identify these QRE motif sites region bound to endogenous QKI by using anti - QKI antibodies . The results showed that endogenous QKI interacted with full - length wild type QRE motif regions within 3 ' UTRs of

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

293T cells were transfected with PGC1α wild type or QRE sites mutant 3'UTR fragments. RNA immunoprecipitation (RIP) to identify these QRE motif sites region bound to endogenous QKI by using anti-QKI antibodies. The results showed that endogenous QKI interacted with full-length wild type QRE motif regions within 3'UTRs of PGC1α


293T cells were transfected with UCP1 wild type or QRE sites mutant 3 ' UTR fragments . RNA immunoprecipitation ( RIP ) to identify these QRE motif sites region bound to endogenous QKI by using anti - QKI antibodies . The results showed that endogenous QKI interacted with full - length wild type QRE motif regions within 3 ' UTRs of UCP1 .

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

293T cells were transfected with UCP1 wild type or QRE sites mutant 3'UTR fragments. RNA immunoprecipitation (RIP) to identify these QRE motif sites region bound to endogenous QKI by using anti-QKI antibodies. The results showed that endogenous QKI interacted with full-length wild type QRE motif regions within 3'UTRs of UCP1.


The mRNA stability in adipocytes was measured by incubating cells with actinomycin D ( 5 μg / ml ) . Actinomycin D was added to stop transcription , and RNAs were harvested at the indicated times ( x - axis ) after transcription inhibition . Real - time PCR was used to determine remaining UCP1 , PGC1α and Actin mRNA levels compared with the starting time . Increasing PGC1α mRNA half - life by depletion of QKI ( ShScramble T1 / 2 = 4 . 73 h , ShQKI T1 / 2 = 8 . 04 h ) . ( n = 4 biological replicates )

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

The mRNA stability in adipocytes was measured by incubating cells with actinomycin D (5 μg/ml). Actinomycin D was added to stop transcription, and RNAs were harvested at the indicated times (x-axis) after transcription inhibition. Real-time PCR was used to determine remaining UCP1, PGC1α and Actin mRNA levels compared with the starting time. Increasing PGC1α mRNA half-life by depletion of QKI (ShScramble T1/2 = 4.73 h, ShQKI T1/2 = 8.04 h). (n=4 biological replicates)


The nuclear and cytoplasmic fractionation was valid by immunoblotting using cytoplasmic and nuclear protein markers ( GAPDH and Lamin B1 ) to show the purity of fractions .

The nuclear and cytoplasmic fractionation was valid by immunoblotting using cytoplasmic and nuclear protein markers (GAPDH and Lamin B1) to show the purity of fractions.


Real - time PCR analysis of mRNA transcript levels of the QKI targets UCP1 and PGC1α in the nuclear and cytoplasmic fractions of differentiated MEF cells transfected with ShScramble or ShQKI . Values represent the cytoplasmic - to - nuclear ratio of each transcript . Actin served as a negative control . ( n = 4 biological replicates )

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

Real-time PCR analysis of mRNA transcript levels of the QKI targets UCP1 and PGC1α in the nuclear and cytoplasmic fractions of differentiated MEF cells transfected with ShScramble or ShQKI. Values represent the cytoplasmic-to-nuclear ratio of each transcript. Actin served as a negative control. (n=4 biological replicates)


RNAs from isolated nuclear and cytoplasmic fractionation were harvested after RNA immunoprecipitation ( RIP ) of RNA - protein complexes using either anti - QKI antibody or control IgG . And RIP - RT - PCR showing the PGC1α and UCP1 transcripts majorly interacted with endogenous QKI in the nucleus . ( n = 3 biological replicates )

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

RNAs from isolated nuclear and cytoplasmic fractionation were harvested after RNA immunoprecipitation (RIP) of RNA-protein complexes using either anti-QKI antibody or control IgG. And RIP-RT-PCR showing the PGC1α and UCP1 transcripts majorly interacted with endogenous QKI in the nucleus. (n=3 biological replicates)


Control ( ShScramble ) and QKI knockdown ( ShQKI ) adipocyte lysates were subjected to polysome profiling , and a representative polysome profile is shown . Total RNA was extracted from each fraction , and the distribution of UCP1 and PGC1α mRNA was measured via real - time PCR ( K and L ) . The results are shown as the percentage of total mRNA ( % mRNA ) in polysome fractions and represent 3 independent experiments .

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

Control (ShScramble) and QKI knockdown (ShQKI) adipocyte lysates were subjected to polysome profiling, and a representative polysome profile is shown. Total RNA was extracted from each fraction, and the distribution of UCP1 and PGC1α mRNA was measured via real-time PCR (K and L). The results are shown as the percentage of total mRNA (% mRNA) in polysome fractions and represent 3 independent experiments.


The rectal temperature of WT fl / fl and QKI KO fl / fl : AP2 - Cre mice was monitored upon acute cold exposure ( 4 ° C ) . ( n = 10 mice , per group )

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

The rectal temperature of WT fl/fl and QKI KO fl/fl:AP2-Cre mice was monitored upon acute cold exposure (4 °C). (n = 10 mice, per group)


H & E staining and UCP1 immunostaining of sections of ingWAT from WT fl / fl and QKI KO fl / fl : AP2 - Cre mice at RT ( 22 ° C ) or following 72 h of cold exposure ( 4 ° C ) . ( Scale bar : 50 μm ) .

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

H&E staining and UCP1 immunostaining of sections of ingWAT from WT fl/fl and QKI KO fl/fl:AP2-Cre mice at RT (22 °C) or following 72 h of cold exposure (4 °C). (Scale bar: 50 μm).


The protein levels of UCP1 in ingWAT from WT fl / fl and QKI KO fl / fl : AP2 - Cre mice at RT or following 72 h of cold exposure ( 4 ° C ) , and quantified protein levels are shown in ( D ) . ( n = 3 biological replicates , per group )

The protein levels of UCP1 in ingWAT from WT fl/fl and QKI KO fl/fl:AP2-Cre mice at RT or following 72 h of cold exposure (4 °C), and quantified protein levels are shown in (D). (n=3 biological replicates, per group)


Red Nile staining in BAT injected with Control or QKI knockdown AAV at RT ( 22 ° C ) or following 6 h and 72 h of cold exposure ( 4 ° C ) .

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

Red Nile staining in BAT injected with Control or QKI knockdown AAV at RT (22 °C) or following 6 h and 72 h of cold exposure (4 °C).


TG content in BAT injected with Control or QKI knockdown AAV at RT ( 22 ° C ) or following 6 h and 72 h of cold exposure ( 4 ° C ) .

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

TG content in BAT injected with Control or QKI knockdown AAV at RT (22 °C) or following 6 h and 72 h of cold exposure (4 °C).


The UCP1 protein level in BAT at RT or following 6 h and 72 h of cold exposure ( 4 ° C ) and the quantified protein levels . ( n = 4 biological replicates , per group )

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

The UCP1 protein level in BAT at RT or following 6 h and 72 h of cold exposure (4 °C) and the quantified protein levels. (n = 4 biological replicates, per group)


The mRNA levels of genes related to energy consumption ( PGC1α , UCP1 and Dio2 ) . ( n = 4 biological replicates , per group )

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

The mRNA levels of genes related to energy consumption (PGC1α, UCP1 and Dio2). (n=4 biological replicates, per group)


The protein levels of QKI in BAT and ingWAT during cold stimulation ( 4 ° C ) and the quantified protein levels . ( n = 3 biological replicates )

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

The protein levels of QKI in BAT and ingWAT during cold stimulation (4 °C) and the quantified protein levels. (n=3 biological replicates)


The relative luminescence of a QKI promoter reporter under cAMP ( Forskolin and IBMX ) stimulus . ( n = 3 biological replicates )

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

The relative luminescence of a QKI promoter reporter under cAMP (Forskolin and IBMX) stimulus. (n=3 biological replicates)


The effect of transcriptional factors on the QKI promoter , assessed by luciferase activity assays . ( n = 3 biological replicates )

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

The effect of transcriptional factors on the QKI promoter, assessed by luciferase activity assays. (n=3 biological replicates)


The relative mRNA levels of genes ( UCP1 , PGC1α , Dio2 ) in differentiated MEFs transfected with ShScramble or ShQKI lentivirus , following cAMP ( Forskolin and IBMX ) stimulation . ( n = 3 biological replicates )

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

The relative mRNA levels of genes (UCP1, PGC1α, Dio2) in differentiated MEFs transfected with ShScramble or ShQKI lentivirus, following cAMP (Forskolin and IBMX) stimulation. (n=3 biological replicates)


( A ) Western blot of Beclin ‐ 1 and FACS profiles following siRNA treatment in HeLa cells and cells stably expressing a HA ‐ Beclin ‐ 1 construct resistant to the siRNA targeting endogenous Beclin ‐ 1 .

(A) Western blot of Beclin‐1 and FACS profiles following siRNA treatment in HeLa cells and cells stably expressing a HA‐Beclin‐1 construct resistant to the siRNA targeting endogenous Beclin‐1.


( B ) FACS profiles of cells treated with indicated siRNA following nocodazole blockage and release .

(B) FACS profiles of cells treated with indicated siRNA following nocodazole blockage and release.


( C , D ) Quantification of cells in mitosis ( C ) and quantification of each step of mitosis ( D ) by H3Ser10P staining for Control and Beclin ‐ 1 siRNA ‐ treated cells . Error bars represent standard deviations ( s . d . ) ( n = number of counted cells ) calculated on three independent experiments . Two and three asterisks indicate significant results ( P = 0 . 0028 and P = 0 . 0006 , respectively ) .

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

(C,D) Quantification of cells in mitosis (C) and quantification of each step of mitosis (D) by H3Ser10P staining for Control and Beclin‐1 siRNA‐treated cells. Error bars represent standard deviations (s.d.) (n=number of counted cells) calculated on three independent experiments. Two and three asterisks indicate significant results (P=0.0028 and P=0.0006, respectively).


( E ) Knockdown of Beclin ‐ 1 increases the number of mitotic cells with misaligned chromosomes . siRNA ‐ treated HeLa cells were fixed in PFA , immunolabelled for α ‐ tubulin ( green ) and stained with DAPI ( blue ) . White arrows indicate misaligned chromosomes . Quantification of cells with misaligned chromosomes is shown at the right ( n = number of counted cells ) . Three asterisks indicate significant results ( P = 0 . 0001 ) . Scale bar , 10 μm . DAPI , 4 ′ , 6 ‐ diamidino ‐ 2 ‐ phenylindole ; HA , haemagglutinin ; PFA , paraformaldehyde ; siRNA , small interfering RNA .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Knockdown of Beclin‐1 increases the number of mitotic cells with misaligned chromosomes. siRNA‐treated HeLa cells were fixed in PFA, immunolabelled for α‐tubulin (green) and stained with DAPI (blue). White arrows indicate misaligned chromosomes. Quantification of cells with misaligned chromosomes is shown at the right (n=number of counted cells). Three asterisks indicate significant results (P=0.0001). Scale bar, 10 μm. DAPI, 4′,6‐diamidino‐2‐phenylindole; HA, haemagglutinin; PFA, paraformaldehyde; siRNA, small interfering RNA.