J , K . Immunofluorescence images ( J ) and quantification ( K ) of Cyclin B1 and MAD1 kinetochore localisation in doxycycline - inducible MAD1α and β knockouts treated with or without dox for 10 days and then arrested in nocodazole . Cells were selected that had full MAD1 knockout in the doxycycline treatment ( this constituted approximately 30 % of cells ) .

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J,K. Immunofluorescence images (J) and quantification (K) of Cyclin B1 and MAD1 kinetochore localisation in doxycycline-inducible MAD1α and β knockouts treated with or without dox for 10 days and then arrested in nocodazole. Cells were selected that had full MAD1 knockout in the doxycycline treatment (this constituted approximately 30% of cells).


L . Relative kinetochore volumes occupied by Cyclin B1 and MAD1 ( relative to CenpC ) in nocodazole - arrested MAD1α and MAD1β cells ( calculated from experiments shown in ( H , I ) .

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L. Relative kinetochore volumes occupied by Cyclin B1 and MAD1 (relative to CenpC) in nocodazole-arrested MAD1α and MAD1β cells (calculated from experiments shown in (H,I).


A . Elution profiles and SDS - PAGE for SEC runs on the indicated column of the Cyclin B1 : CDK1 complex ( blue profile ) , MBP - MAD1 : MAD2 ( red ) , and their combination ( green ) . Note that the same Cyclin B1 : CDK1 elution profile and SDS - PAGE is also displayed as reference in panels D and F to improve clarity . For the same reason , the MBP - MAD1 : MAD2 elution profile and SDS - PAGE is also displayed in panels 2A - C . B . Elution profiles and SDS - PAGE for SEC runs on the indicated column of CDK1 ( blue ) , MBP - MAD1 : MAD2 ( red ) , and their combination ( green ) . C . Elution profiles and SDS - PAGE for SEC runs on the indicated column of Cyclin B1 ( blue ) , MBP - MAD1 : MAD2 ( red ) , and their combination ( green ) . D . Elution profiles and SDS - PAGE for SEC runs of the Cyclin B1 : CDK1 complex ( blue ) , MBP - MAD1 ∆ 93 : MAD2 ( green ) , and their combination ( red ) .

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

A. Elution profiles and SDS-PAGE for SEC runs on the indicated column of the Cyclin B1:CDK1 complex (blue profile), MBP-MAD1:MAD2 (red), and their combination (green). Note that the same Cyclin B1:CDK1 elution profile and SDS-PAGE is also displayed as reference in panels D and F to improve clarity. For the same reason, the MBP-MAD1:MAD2 elution profile and SDS-PAGE is also displayed in panels 2A-C. B. Elution profiles and SDS-PAGE for SEC runs on the indicated column of CDK1 (blue), MBP-MAD1:MAD2 (red), and their combination (green). C. Elution profiles and SDS-PAGE for SEC runs on the indicated column of Cyclin B1 (blue), MBP-MAD1:MAD2 (red), and their combination (green). D. Elution profiles and SDS-PAGE for SEC runs of the Cyclin B1:CDK1 complex (blue), MBP-MAD1∆93:MAD2 (green), and their combination (red).


F . Elution profiles and SDS - PAGE for SEC runs of Cyclin B1 : CDK1 ( blue ) , MBP - MAD13EK : MAD2 ( E52K , E53K , and E56K mutations ; red ) , and their combination ( green ) . In A - D , individual potential binding partners were combined at a concentration of 5 µM .

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F. Elution profiles and SDS-PAGE for SEC runs of Cyclin B1:CDK1 (blue), MBP-MAD13EK:MAD2 (E52K, E53K, and E56K mutations; red), and their combination (green). In A-D, individual potential binding partners were combined at a concentration of 5 µM.


A . Western blot analysis of indicated vsv - MAD1 - WT or 3EK HeLa clones treated with or without doxycycline for 10 days .

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A. Western blot analysis of indicated vsv-MAD1-WT or 3EK HeLa clones treated with or without doxycycline for 10 days.


B . Immunofluorescence images showing MAD1 and ZW10 kinetochore levels in nocodazole - arrested MAD1 - WT - C13 AND 3EK - C14 just after nuclear envelope breakdown ( early prometaphase ) or later in mitosis when the chromatin is condensed ( late prometaphase ) . Note , that early and late prometaphase was defined based on the level of chromatin condensation .

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B. Immunofluorescence images showing MAD1 and ZW10 kinetochore levels in nocodazole-arrested MAD1-WT-C13 AND 3EK-C14 just after nuclear envelope breakdown (early prometaphase) or later in mitosis when the chromatin is condensed (late prometaphase). Note, that early and late prometaphase was defined based on the level of chromatin condensation.


C , D . Relative kinetochore volumes occupied by MAD1 ( C ) and ZW10 ( D ) ( relative to CenpC ) in MAD1 - WT and - 3EK cells in early and late prometaphase .

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C,D. Relative kinetochore volumes occupied by MAD1 (C) and ZW10 (D) (relative to CenpC) in MAD1-WT and -3EK cells in early and late prometaphase.


E . Quantification of MAD1 kinetochore intensity from indicated MAD1 - WT and 3EK clones treated as in ( B ) .

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E. Quantification of MAD1 kinetochore intensity from indicated MAD1-WT and 3EK clones treated as in (B).


A - C . Immunofluorescence images ( A ) and quantifications ( B , C ) of relative MAD1 and MAD2 kinetochore levels in indicated cells lines arrested in nocodazole and treated with / without AZ - 3146 for 30 min to inhibit MPS1 . In all kinetochore intensity graphs , each dot represents a cell , horizontal lines indicate the median and vertical bars show the 95 % confidence interval . Note , when these vertical bars do not overlap the difference is considered statistically significant at a level of at least p < 0 . 05 ( see methods ) . All kinetochore intensity graphs display data that is relative to WT C11 controls in nocodazole , which are normalised to 1 . The mean level of the normalised controls is indicated by the dotted lines . 30 cells from 3 experiments . Scale bars = 5µM . D . Duration of mitotic arrest in indicated cell lines arrested in nocodazole and then treated with 1 . 25 µM AZ - 3146 . Graph shows cumulative mean ( ± SEM ) of 3 experiments , 50 cells per condition per experiment .

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A-C. Immunofluorescence images (A) and quantifications (B,C) of relative MAD1 and MAD2 kinetochore levels in indicated cells lines arrested in nocodazole and treated with/without AZ-3146 for 30 min to inhibit MPS1. In all kinetochore intensity graphs, each dot represents a cell, horizontal lines indicate the median and vertical bars show the 95% confidence interval. Note, when these vertical bars do not overlap the difference is considered statistically significant at a level of at least p<0.05 (see methods). All kinetochore intensity graphs display data that is relative to WT C11 controls in nocodazole, which are normalised to 1. The mean level of the normalised controls is indicated by the dotted lines. 30 cells from 3 experiments. Scale bars = 5µM. D. Duration of mitotic arrest in indicated cell lines arrested in nocodazole and then treated with 1.25 µM AZ-3146. Graph shows cumulative mean (±SEM) of 3 experiments, 50 cells per condition per experiment.


A . Immunofluorescence images of MAD1 and MAD1 - pT716 kinetochore levels in nocodazole - arrested RPE1 cells treated with or without AZ - 3146 . Scale bars = 5µM .

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A. Immunofluorescence images of MAD1 and MAD1-pT716 kinetochore levels in nocodazole-arrested RPE1 cells treated with or without AZ-3146. Scale bars = 5µM.


B . Electron micrographs of rotary - shadowed MAD193 - 718 : MAD2 ( top row ) , MBP - MAD1 : MAD2 ( middle row ) , and MBP - MAD1 : MAD2 in complex with GST - CDK1 : Cyclin B1 ( bottom row ) . Scale bar = 50 nm .

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B. Electron micrographs of rotary-shadowed MAD193-718:MAD2 (top row), MBP-MAD1:MAD2 (middle row), and MBP-MAD1:MAD2 in complex with GST-CDK1:Cyclin B1 (bottom row). Scale bar = 50 nm.


C . Quantifications ( top ) and corresponding immunofluorescence images ( underneath ) of relative kinetochore MAD1 and MAD1 - pT716 levels in nocodazole - arrested of MAD1 - WT - C14 and MAD1 - 3EK - C12 cells treated with different doses of AZ - 3146 for 30 min . MG132 was included at the time of AZ - 3146 addition to prevent mitotic exit . Each dot represents a cell , horizontal lines indicate the median and error bars show the 95 % confidence interval . Note , when these vertical bars do not overlap the difference is considered statistically significant at a level of at least p < 0 . 05 ( see methods ) . 30 cells from 3 experiments . Both kinetochore intensity graphs display data that is relative to the WT - C14 untreated controls , which are normalised to 1 . The mean level of the normalised controls is indicated by the dotted lines .

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

C. Quantifications (top) and corresponding immunofluorescence images (underneath) of relative kinetochore MAD1 and MAD1-pT716 levels in nocodazole-arrested of MAD1-WT-C14 and MAD1-3EK-C12 cells treated with different doses of AZ-3146 for 30 min. MG132 was included at the time of AZ-3146 addition to prevent mitotic exit. Each dot represents a cell, horizontal lines indicate the median and error bars show the 95% confidence interval. Note, when these vertical bars do not overlap the difference is considered statistically significant at a level of at least p<0.05 (see methods). 30 cells from 3 experiments. Both kinetochore intensity graphs display data that is relative to the WT-C14 untreated controls, which are normalised to 1. The mean level of the normalised controls is indicated by the dotted lines.


D . Duration of mitotic arrest in MAD1 - WT C14 or MAD1 - 3EK C12 cells arrested in nocodazole and then treated with indicated concentrations of AZ - 3146 . Note , the 1 . 25 µM AZ - 3146 data is displayed in Figure 4D , but also included here to allow comparison with other drug doses . Graph shows cumulative mean ( ± SEM ) of 3 experiments , 50 cells per condition per experiment .

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D. Duration of mitotic arrest in MAD1-WT C14 or MAD1-3EK C12 cells arrested in nocodazole and then treated with indicated concentrations of AZ-3146. Note, the 1.25 µM AZ-3146 data is displayed in Figure 4D, but also included here to allow comparison with other drug doses. Graph shows cumulative mean (±SEM) of 3 experiments, 50 cells per condition per experiment.


The heatmap shows the dynamic expression of genes in the SF3A / 3B complex ( E ) and SR family ( F ) at each indicated differentiation stage . Both heatmaps were row normalized . The heatmaps were scaled with Z - Score using the log2 ( FPKM ) expression of indicated components . n = 3 technical replicates .

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

The heatmap shows the dynamic expression of genes in the SF3A/3B complex (E) and SR family (F) at each indicated differentiation stage. Both heatmaps were row normalized. The heatmaps were scaled with Z-Score using the log2(FPKM) expression of indicated components. n=3 technical replicates.


The mRNA expression of splicing regulator SRSF2 and splicing factor PCBP2 during hematopoietic differentiation were measured by RT - qPCR . The ACTB gene was used as a control . Results given are mean ± standard deviation ( SD ) . P - values were determined by an unpaired two - tailed Student ' s t - test . n ≥ 3 biological replicates .

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The mRNA expression of splicing regulator SRSF2 and splicing factor PCBP2 during hematopoietic differentiation were measured by RT-qPCR. The ACTB gene was used as a control. Results given are mean ± standard deviation (SD). P-values were determined by an unpaired two-tailed Student's t-test. n ≥ 3 biological replicates.


The experimental schematic ( upper panel ) . APLNR + cells on day 2 were FACS - purified and treated with various dosages of PLB from day 2 . 5 to 5 during inducing hematopoietic differentiation . On day 5 , the generation of CD31 + CD34 + EPCs and CD31 + CD34 + CD73 - HEPs were assessed . The representative FACS plots showed the frequency of APLNR + cells , CD31 + CD34 + EPCs and CD31 + CD34 + CD73 - HEPs without or with PLB treatment . The bar graphs show the percentage of CD31 + CD34 + EPCs and CD31 + CD34 + CD73 - HEPs without or with PLB treatment .

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

The experimental schematic (upper panel). APLNR+ cells on day 2 were FACS-purified and treated with various dosages of PLB from day 2.5 to 5 during inducing hematopoietic differentiation. On day 5, the generation of CD31+CD34+ EPCs and CD31+CD34+CD73- HEPs were assessed. The representative FACS plots showed the frequency of APLNR+ cells, CD31+CD34+ EPCs and CD31+CD34+CD73- HEPs without or with PLB treatment. The bar graphs show the percentage of CD31+CD34+ EPCs and CD31+CD34+CD73- HEPs without or with PLB treatment.


The bar graph represents the percentage of CD31 + CD34 + cells from ( H ) . P - values were calculated by one - way ANOVA followed by Tukey ' s test .

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The bar graph represents the percentage of CD31+CD34+ cells from (H). P-values were calculated by one-way ANOVA followed by Tukey's test.


The bar graph representing the percentage of CD31 + CD34 + cells from ( K ) . P - values were determined by an unpaired two - tailed Student ' s t - test .

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The bar graph representing the percentage of CD31+CD34+ cells from (K). P-values were determined by an unpaired two-tailed Student's t-test.


The representative FACS plots show the frequency of CD31 + CD34 + EPCs in the GFP + population at day 5 with 1 . 25 nM PLB supplementation alone or in combination with NUMB _ S ectopic expression from day 2 . 5 . The bar graph denotes the percentage of CD31 + CD34 + in GFP + cells as described in ( F ) . P - values were determined by an unpaired two - tailed Student ' s t - test .

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

The representative FACS plots show the frequency of CD31+CD34+ EPCs in the GFP+ population at day 5 with 1.25 nM PLB supplementation alone or in combination with NUMB_S ectopic expression from day 2.5. The bar graph denotes the percentage of CD31+CD34+ in GFP+ cells as described in (F). P-values were determined by an unpaired two-tailed Student's t-test.


On day 5 of hematopoietic differentiation , the inclusion or exclusion of NUMB exon 9 were detected by RT - PCR with 1 . 25 nM PLB supplementation or SRSF2 exogenous expression alone or their combinational treatment from day 2 . 5 . The bar graph showing the signaling intensity of each specific band from the RT - PCR electropherogram .

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On day 5 of hematopoietic differentiation, the inclusion or exclusion of NUMB exon 9 were detected by RT-PCR with 1.25 nM PLB supplementation or SRSF2 exogenous expression alone or their combinational treatment from day 2.5. The bar graph showing the signaling intensity of each specific band from the RT-PCR electropherogram.


The top panel depicts the location of specific primers targeting NUMB _ S and NUMB _ L isoform . The bottom bar graph showing the expression of NUMB _ S and NUMB _ L in cells on day 5 of differentiation with 1 . 25 nM PLB supplementation or SRSF2 exogenous expression alone or their combinational treatment from day 2 . 5 with RT - qPCR .

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The top panel depicts the location of specific primers targeting NUMB_S and NUMB_L isoform. The bottom bar graph showing the expression of NUMB_S and NUMB_L in cells on day 5 of differentiation with 1.25 nM PLB supplementation or SRSF2 exogenous expression alone or their combinational treatment from day 2.5 with RT-qPCR.


The heatmap plotting the expression of NUMB _ S and the key components in NOTCH signaling in day 2 - APLNR + cells , day 5 - DMSO treated and day 5 - PLB treated cells ( 2 . 5 nM ) , respectively . The NOTCH components were divided into groups of upregulated , downregulated , and unchanged by comparison between DMSO and PLB treated cells . The heatmap was scaled with Z - Score using the log2 ( FPKM ) expression of indicated genes . n = 2 technical

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

The heatmap plotting the expression of NUMB_S and the key components in NOTCH signaling in day 2-APLNR+ cells, day 5-DMSO treated and day 5-PLB treated cells (2.5 nM), respectively. The NOTCH components were divided into groups of upregulated, downregulated, and unchanged by comparison between DMSO and PLB treated cells. The heatmap was scaled with Z-Score using the log2(FPKM) expression of indicated genes. n=2 technical replicates


The representative FACS plots indicating the generation of CD31 + CD34 + EPCs on day 5 of differentiation with or without HES1 overexpression . DOX was treated from day 2 . 5 to 5 to induce HES1 overexpression . Statistical analysis of the percentage of CD31 + CD34 + EPCs of ( H ) .

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

The representative FACS plots indicating the generation of CD31+CD34+ EPCs on day 5 of differentiation with or without HES1 overexpression. DOX was treated from day 2.5 to 5 to induce HES1 overexpression. Statistical analysis of the percentage of CD31+CD34+ EPCs of (H).


B In vivo transduction profile of luciferase reporter vectors displaying variants of the enriched library capsid sequence motifs or unmodified wild type AAV2 control capsid . Four weeks after i . v . injection of 5x1010 genomic particles / mouse containing a CMV - luciferase reporter gene , luminescence was measured in the brain and control organs . Data are shown as bars ( mean ) with plotted individual data points ( n = 3 animals / group , age 8 - 12 weeks ) .

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B In vivo transduction profile of luciferase reporter vectors displaying variants of the enriched library capsid sequence motifs or unmodified wild type AAV2 control capsid. Four weeks after i.v. injection of 5x1010 genomic particles/mouse containing a CMV-luciferase reporter gene, luminescence was measured in the brain and control organs. Data are shown as bars (mean) with plotted individual data points (n = 3 animals/group, age 8-12 weeks).


A Luciferase reporter gene vectors displaying the brain - targeted peptide NRGTEWD ( " BR1 " ) , unmodified wild type AAV2 capsid , or the previously reported brain - targeting peptide DSPAHPS ( PPS ) . Vectors were intravenously injected into mice ( 5x1010 genomic particles / mouse ) . Panels show representative examples of n = 5 animals , age 8 - 12 weeks per group . Animals were imaged in dorsal ( left panel ) , ventral ( second from left panel ) , and lateral ( second from right and right panel ) position , 14 days after vector injection . B Close - up imaging of AAV - BR1 - treated mice . Mice were imaged in dorsal and lateral position in a different color scheme allowing detailed visualization of the transduced brain in living animals , even as late as at day 264 after vector injection . C Virtual sections : sagittal , coronal , and transaxial ( left panels ) and three - dimensional reconstruction ( right panel ) of the luminescence images of a mouse injected with BR1 vector ( as in A ) . Images were obtained by measuring different wavelengths of the emitted light ( Living Image software ) , confirming the brain as exclusive source of luminescence .

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A Luciferase reporter gene vectors displaying the brain-targeted peptide NRGTEWD ("BR1"), unmodified wild type AAV2 capsid, or the previously reported brain-targeting peptide DSPAHPS (PPS). Vectors were intravenously injected into mice (5x1010 genomic particles/mouse). Panels show representative examples of n = 5 animals, age 8-12 weeks per group. Animals were imaged in dorsal (left panel), ventral (second from left panel), and lateral (second from right and right panel) position, 14 days after vector injection.B Close-up imaging of AAV-BR1-treated mice. Mice were imaged in dorsal and lateral position in a different color scheme allowing detailed visualization of the transduced brain in living animals, even as late as at day 264 after vector injection.C Virtual sections : sagittal, coronal, and transaxial (left panels) and three-dimensional reconstruction (right panel) of the luminescence images of a mouse injected with BR1 vector (as in A). Images were obtained by measuring different wavelengths of the emitted light (Living Image software), confirming the brain as exclusive source of luminescence.


AAV - BR1 vector harboring the luciferase gene under control of the CAG promoter was administered intravenously ( 5x1010 genomic particles / mouse , age 8 weeks ) . Long - term transgene expression was analyzed by in vivo bioluminescence imaging at 16 time points during a 665 - day period ( n = 2 animals ) . Original images of analyzed animals ( above ) and quantification of luminescence in the brain as the region of interest = ROI ( bottom ) .

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AAV-BR1 vector harboring the luciferase gene under control of the CAG promoter was administered intravenously (5x1010 genomic particles/mouse, age 8 weeks). Long-term transgene expression was analyzed by in vivo bioluminescence imaging at 16 time points during a 665-day period (n = 2 animals). Original images of analyzed animals (above) and quantification of luminescence in the brain as the region of interest = ROI (bottom).


Luciferase activity and vector copy numbers were determined in tissue lysates 14 days after vector administration ( 5x1010 genomic particles / mouse , age 8 - 12 weeks ) . A Vector - mediated luminescence . Transgene expression was measured in AAV - BR1 harboring the luciferase gene under control of the CAG promoter and control vectors ( AAV - PPS and wild type rAAV2 ) in brain and off - target control organs ( left panel ) . Comparison of luminescence in the brain mediated by AAV - BR1 and control vectors ( right panel ) , * * * * p < 0 . 0001 ( BR1 : brain vs . all ) , * * * p = 0 . 0002 ( PPS : heart vs . brain / muscle ) , * * * p = 0 . 0001 ( PPS : heart vs . lung / liver / spleen / kidney ) , * p = 0 . 0101 ( WT : heart vs . lung ) , * p = 0 . 0120 ( WT : heart vs . spleen ) , * p = 0 . 0148 ( WT : heart vs . kidney ) , * p = 0 . 0113 ( WT : heart vs . muscle ) , * * p = 0 . 0011 ( Brain : BR1 vs . PPS ) , * * * p = 0 . 0009 ( Brain : BR1 vs . WT ) .

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Luciferase activity and vector copy numbers were determined in tissue lysates 14 days after vector administration (5x1010 genomic particles/mouse, age 8-12 weeks).A Vector-mediated luminescence. Transgene expression was measured in AAV-BR1 harboring the luciferase gene under control of the CAG promoter and control vectors (AAV-PPS and wild type rAAV2) in brain and off-target control organs (left panel). Comparison of luminescence in the brain mediated by AAV-BR1 and control vectors (right panel), ****p < 0.0001 (BR1: brain vs. all), ***p = 0.0002 (PPS: heart vs. brain/ muscle), ***p = 0.0001 (PPS: heart vs. lung/ liver/ spleen/ kidney), *p = 0.0101 (WT: heart vs. lung), *p = 0.0120 (WT: heart vs. spleen), *p = 0.0148 (WT: heart vs. kidney), *p = 0.0113 (WT: heart vs. muscle), **p = 0.0011 (Brain: BR1 vs. PPS), ***p = 0.0009 (Brain: BR1 vs. WT).


Luciferase activity and vector copy numbers were determined in tissue lysates 14 days after vector administration ( 5x1010 genomic particles / mouse , age 8 - 12 weeks ) . B Biodistribution of AAV - BR1 and control vectors ( AAV - PPS and wild type rAAV2 ) in brain and off - target control organs , excluding spleen ( left panel ) . Genome copy numbers of AAV - BR1 and control vectors ( AAV - PPS and wild type rAAV2 ) in the brain ( right panel ) , * * * * p < 0 . 0001 ( BR1 : brain vs . liver / kidney / muscle ) , * * * p = 0 . 0006 ( BR1 : brain vs . heart ) , * * * p = 0 . 0003 ( BR1 : brain vs . lung ) , * * p = 0 . 0025 ( PPS : kidney vs . muscle ) , * p = 0 . 0378 ( PPS : kidney vs . lung ) , * p = 0 . 0114 ( PPS : kidney vs . liver ) , * * * * * p < 0 . 0001 ( WT : liver vs . all ) , * * p = 0 . 0028 ( Brain : BR1 vs . PPS / WT ) .

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Luciferase activity and vector copy numbers were determined in tissue lysates 14 days after vector administration (5x1010 genomic particles/mouse, age 8-12 weeks).B Biodistribution of AAV-BR1 and control vectors (AAV-PPS and wild type rAAV2) in brain and off-target control organs, excluding spleen (left panel). Genome copy numbers of AAV-BR1 and control vectors (AAV-PPS and wild type rAAV2) in the brain (right panel), ****p < 0.0001 (BR1: brain vs. liver/ kidney/ muscle), ***p = 0.0006 (BR1: brain vs. heart), ***p = 0.0003 (BR1: brain vs. lung), **p = 0.0025 (PPS: kidney vs. muscle), *p = 0.0378 (PPS: kidney vs. lung), *p = 0.0114 (PPS: kidney vs. liver), *****p < 0.0001 (WT: liver vs. all), **p = 0.0028 (Brain: BR1 vs. PPS/ WT).


C57BL / 6 mice ( age 8 weeks ) were injected with AAV - BR1 harboring an eGFP reporter gene under control of the CAG promoter . Images show representative examples of n = 6 mice . A Representative images from cerebellum , olfactory bulb , striatum , cerebral cortex , and the spinal cord , 14 days after vector injection . BR1 - eGFP - transduced cells ( green ) were positive for the endothelial marker CD31 ( red ) . Scale bars represent 250 µm .

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C57BL/6 mice (age 8 weeks) were injected with AAV-BR1 harboring an eGFP reporter gene under control of the CAG promoter. Images show representative examples of n = 6 mice.A Representative images from cerebellum, olfactory bulb, striatum, cerebral cortex, and the spinal cord, 14 days after vector injection. BR1-eGFP-transduced cells (green) were positive for the endothelial marker CD31 (red). Scale bars represent 250 µm.


C57BL / 6mice ( age 8 weeks ) were injected with AAV - BR1 harboring an eGFP reporter gene under control of the CAG promoter . Images show representative examples of n = 6 mice . B Higher magnification confocal microscopy images . The endothelial marker CD31 ( red ) colocalizes with vector - mediated eGFP expression ( green ) . Scale bars represent 50 µm ( upper panel ) or 10 µm ( lower panel ) .

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C57BL/6mice (age 8 weeks) were injected with AAV-BR1 harboring an eGFP reporter gene under control of the CAG promoter. Images show representative examples of n = 6 mice.B Higher magnification confocal microscopy images. The endothelial marker CD31 (red) colocalizes with vector-mediated eGFP expression (green). Scale bars represent 50 µm (upper panel) or 10 µm (lower panel).


C57BL / 6 mice ( age 8 weeks ) were injected with AAV - BR1 harboring an eGFP reporter gene under control of the CAG promoter . Images show representative examples of n = 6 mice . C CD13 staining of pericytes . The vector - mediated eGFP expression pattern ( green ) does not colocalize with CD13 ( red ) . Scale bars represent 250 µm ( upper panel ) or 10 µm ( lower panel ) .

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C57BL/6 mice (age 8 weeks) were injected with AAV-BR1 harboring an eGFP reporter gene under control of the CAG promoter. Images show representative examples of n = 6 mice.C CD13 staining of pericytes. The vector-mediated eGFP expression pattern (green) does not colocalize with CD13 (red). Scale bars represent 250 µm (upper panel) or 10 µm (lower panel).


C57BL / 6 mice ( age 8 weeks ) were injected with AAV - BR1 harboring an eGFP reporter gene under control of the CAG promoter . Images show representative examples of n = 6 mice . D Aquaporin 4 staining of astrocyte end - feet . The vector - mediated eGFP expression ( green ) does not colocalize with aquaporin 4 ( red ) . Scale bars represent 100 µm ( upper panel ) or 10 µm ( lower panel ) .

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C57BL/6 mice (age 8 weeks) were injected with AAV-BR1 harboring an eGFP reporter gene under control of the CAG promoter. Images show representative examples of n = 6 mice.D Aquaporin 4 staining of astrocyte end-feet. The vector-mediated eGFP expression (green) does not colocalize with aquaporin 4 (red). Scale bars represent 100 µm (upper panel) or 10 µm (lower panel).


C57BL / 6 mice ( age 8 weeks ) were injected with AAV - BR1 harboring an eGFP reporter gene under control of the CAG promoter . Images show representative examples of n = 6 mice . E Expression of eGFP in primary brain endothelial cells prepared from C57BL / 6 mice , 14 days after injection with AAV - BR1 - eGFP . Scale bars represent 500 µm ( upper panel ) or 100 µm ( lower panel ) .

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C57BL/6 mice (age 8 weeks) were injected with AAV-BR1 harboring an eGFP reporter gene under control of the CAG promoter. Images show representative examples of n = 6 mice.E Expression of eGFP in primary brain endothelial cells prepared from C57BL/6 mice, 14 days after injection with AAV-BR1-eGFP. Scale bars represent 500 µm (upper panel) or 100 µm (lower panel).


Two weeks after vector injection ( 1 . 8 x1011 genomic particles / animal ) , into 16 - weeks old mice , Cre - mediated gene recombination driven by the CAG promoter was observed mainly in brain endothelial cells ( lower panel , red ) . No recombination was observed in control animals without BR1 - iCre - virus injection ( upper panel ) . CD31 ( green ) was used as a marker for brain endothelial cells . Panels show representative examples of n = 3 animals . Scale bars represent 250 µm .

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Two weeks after vector injection (1.8 x1011 genomic particles/ animal), into 16-weeks old mice, Cre-mediated gene recombination driven by the CAG promoter was observed mainly in brain endothelial cells (lower panel, red). No recombination was observed in control animals without BR1-iCre-virus injection (upper panel). CD31 (green) was used as a marker for brain endothelial cells. Panels show representative examples of n = 3 animals. Scale bars represent 250 µm.


BR1 - mediated expression of NEMO or eGFP was driven by the CAG promoter . A Immunostaining of cerebral microvessels . Treatment with AAV - BR1 - NEMO normalized string vessels ( white arrows , highlighted in white square inset ) in Nemo - / + mice as compared to Nemo - / + mice treated with the AAV - BR1 - eGFP control vector at postnatal day 8 ( P8 ) . The upper left panel shows the staining in untreated wild type control mice . String vessels were identified as capillaries that have lost CD31 - positive ( green ) endothelial cells but stain for the basement membrane protein collagen IV ( red ) . Scale bars represent 200 µm . The lower right panel summarizes quantitative analysis of string vessels in Nemo - / + and control mice ( NemoFl or wild type ) at P0 ( n = 3 animals / group ) and at P8 ( n = 6 animals / group , * p = 0 . 0125 ) . String vessels in the cerebral cortex were quantified as percentage of total vessel lengths .

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BR1-mediated expression of NEMO or eGFP was driven by the CAG promoter.A Immunostaining of cerebral microvessels. Treatment with AAV-BR1-NEMO normalized string vessels (white arrows, highlighted in white square inset) in Nemo-/+ mice as compared to Nemo-/+ mice treated with the AAV-BR1-eGFP control vector at postnatal day 8 (P8). The upper left panel shows the staining in untreated wild type control mice. String vessels were identified as capillaries that have lost CD31-positive (green) endothelial cells but stain for the basement membrane protein collagen IV (red). Scale bars represent 200 µm. The lower right panel summarizes quantitative analysis of string vessels in Nemo-/+ and control mice (NemoFl or wild type) at P0 (n=3 animals/ group) and at P8 (n=6 animals/ group, *p = 0.0125). String vessels in the cerebral cortex were quantified as percentage of total vessel lengths.


BR1 - mediated expression of NEMO or eGFP was driven by the CAG promoter . B Quantification of active caspase 3 - positive endothelial cells at P8 ( n = 5 animals / group , * p = 0 . 0201 ) .

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BR1-mediated expression of NEMO or eGFP was driven by the CAG promoter.B Quantification of active caspase 3-positive endothelial cells at P8 (n= 5 animals/ group, *p = 0.0201).


BR1 - mediated expression of NEMO or eGFP was driven by the CAG promoter . C Albumin in brain tissue as indicator for BBB leakage . In Nemo - / + mice treated with AAV - BR1 - NEMO , less albumin was found in brain tissue , indicating less BBB leakage . Representative Western blot with albumin from P8 Nemo - / + mice treated with AAV - BR1 - NEMO or AAV - BR1 - eGFP control vector , respectively , as well as untreated wild type ( WT ) control mice . Right panel : quantitative analysis of the Western blots ( n = 4 animals / group , * p = 0 . 0283 )

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BR1-mediated expression of NEMO or eGFP was driven by the CAG promoter.C Albumin in brain tissue as indicator for BBB leakage. In Nemo-/+ mice treated with AAV-BR1-NEMO, less albumin was found in brain tissue, indicating less BBB leakage. Representative Western blot with albumin from P8 Nemo-/+ mice treated with AAV-BR1-NEMO or AAV-BR1-eGFP control vector, respectively, as well as untreated wild type (WT) control mice. Right panel: quantitative analysis of the Western blots (n = 4 animals/ group, *p = 0.0283)


BR1 - mediated expression of NEMO or eGFP was driven by the CAG promoter . D Body weight of vector - treated mice . WT control or Nemo - / + mice at P8 treated with AAV - BR1 - NEMO or AAV - BR1 - eGFP ( n = 18 animals / group , * p = 0 . 0038 ) .

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BR1-mediated expression of NEMO or eGFP was driven by the CAG promoter.D Body weight of vector-treated mice. WT control or Nemo-/+ mice at P8 treated with AAV-BR1-NEMO or AAV-BR1-eGFP (n= 18 animals/ group, *p = 0.0038).


BR1 - mediated expression of NEMO or eGFP was driven by the CAG promoter . All animals were at the age of 8 - 12 weeks . A Representative immunostainings of cerebral microvessels . String vessels ( white arrows , highlighted in white square inset ) were significantly reduced in NemobeKO mice treated with AAV - BR1 - NEMO compared to NemobeKO mice treated with AAV - BR1 - eGFP control vector . NemoFl mice served as control animals . Scale bars represent 200 µm . B Quantification of string vessel lengths in the cerebral cortex as percentage of total vessel lengths . NemobeKO and NemoFl mice were treated with AAV - BR1 - NEMO or AAV - BR1 - eGFP control vector ( n = 9 NemoFl animals + BR1 - eGFP , 10 NemobeKO animals + BR1 - eGFP , 9 NemoFl animals + BR1 - NEMO , and 13 NemobeKO animals + BR1 - NEMO ) , * * * * p < 0 . 0001 . C Total vessel length measured as total CD31 - positive vessels . Vessels were restored in NemobeKO mice treated with AAV - BR1 - NEMO compared to the AAV - BR1 - eGFP injected mice . NemoFl mice served as a control ( n = 9 NemoFl animals + BR1 - eGFP , 10 NemobeKO animals + BR1 - eGFP , 9 NemoFl animals + BR1 - NEMO , and 13 NemobeKO animals + BR1 - NEMO ) , * * * p = 0 . 0007 , * * p = 0 . 0038 ( NemoKO : eGFP vs . NemoFl : NEMO ) , * * p = 0 . 0014 ( NemoKO : NEMO vs . NemoKO : eGFP ) .

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BR1-mediated expression of NEMO or eGFP was driven by the CAG promoter. All animals were at the age of 8-12 weeks.A Representative immunostainings of cerebral microvessels. String vessels (white arrows, highlighted in white square inset) were significantly reduced in NemobeKO mice treated with AAV-BR1-NEMO compared to NemobeKO mice treated with AAV-BR1-eGFP control vector. NemoFl mice served as control animals. Scale bars represent 200 µm.B Quantification of string vessel lengths in the cerebral cortex as percentage of total vessel lengths. NemobeKO and NemoFl mice were treated with AAV-BR1-NEMO or AAV-BR1-eGFP control vector (n=9 NemoFl animals + BR1-eGFP, 10 NemobeKO animals + BR1-eGFP, 9 NemoFl animals + BR1-NEMO, and 13 NemobeKO animals + BR1-NEMO), ****p < 0.0001.C Total vessel length measured as total CD31-positive vessels. Vessels were restored in NemobeKO mice treated with AAV-BR1-NEMO compared to the AAV-BR1-eGFP injected mice. NemoFl mice served as a control (n= 9 NemoFl animals +BR1-eGFP, 10 NemobeKO animals + BR1-eGFP, 9 NemoFl animals + BR1-NEMO, and 13 NemobeKO animals + BR1-NEMO), ***p = 0.0007, **p = 0.0038 (NemoKO:eGFP vs. NemoFl:NEMO ), **p = 0.0014 (NemoKO:NEMO vs. NemoKO:eGFP).


BR1 - mediated expression of NEMO or eGFP was driven by the CAG promoter . All animals were at the age of 8 - 12 weeks . D IgG and albuminWestern blots of brain lysates . Less leakage in the BBB was detected in NemobeKO mice treated with AAV - BR1 - NEMO than in NemobeKO mice injected with AAV - BR1 - eGFP ( the right panel indicates the quantified gel intensity under the various treatment conditions ( n = 5 animals per group ) . NemoFl mice served as controls , * * * * p < 0 . 0001 .

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BR1-mediated expression of NEMO or eGFP was driven by the CAG promoter. All animals were at the age of 8-12 weeks.D IgG and albuminWestern blots of brain lysates. Less leakage in the BBB was detected in NemobeKO mice treated with AAV-BR1-NEMO than in NemobeKO mice injected with AAV-BR1-eGFP (the right panel indicates the quantified gel intensity under the various treatment conditions (n= 5 animals per group). NemoFl mice served as controls, ****p < 0.0001.


BR1 - mediated expression of NEMO or eGFP was driven by the CAG promoter . All animals were at the age of 8 - 12 weeks . E Quantitative immunoglobulin staining of coronal brain sections of NemoFl and NemobeKO mice . Ig extravasation was significantly reduced in AAV - BR1 - NEMO - treated mice ( n = 9 NemoFl animals + BR1 - eGFP , 10 NemobeKO animals + BR1 - eGFP , 9 NemoFl animals + BR1 - NEMO , and 13 NemobeKO animals + BR1 - NEMO ) , * * * * p < 0 . 0001 .

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BR1-mediated expression of NEMO or eGFP was driven by the CAG promoter. All animals were at the age of 8-12 weeks.E Quantitative immunoglobulin staining of coronal brain sections of NemoFl and NemobeKO mice. Ig extravasation was significantly reduced in AAV-BR1-NEMO-treated mice (n= 9 NemoFl animals + BR1-eGFP, 10 NemobeKO animals + BR1-eGFP, 9 NemoFl animals + BR1-NEMO, and 13 NemobeKO animals + BR1-NEMO), ****p < 0.0001.


BR1 - mediated expression of NEMO or eGFP was driven by the CAG promoter . All animals were at the age of 8 - 12 weeks . F Effect of AAV - BR1 vector on BBB permeability . No vector ( left ) or empty AAV - BR1 vector ( right ) was injected i . v . to wild type mice and BBB permeability was assessed by extravasation of the fluorescent tracer sodium fluorescein ( n = 7 animals per group ) . No significant difference was detected ( n . s . ) .

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BR1-mediated expression of NEMO or eGFP was driven by the CAG promoter. All animals were at the age of 8-12 weeks.F Effect of AAV-BR1 vector on BBB permeability. No vector (left) or empty AAV-BR1 vector (right) was injected i.v. to wild type mice and BBB permeability was assessed by extravasation of the fluorescent tracer sodium fluorescein (n=7 animals per group). No significant difference was detected (n.s.).


A LC3 punctum formation in WT and GCN5 KO HeLa cells ( Scale bars , 10 µm ) .

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A LC3 punctum formation in WT and GCN5 KO HeLa cells (Scale bars, 10 µm).


Immunoblot showing LC3 - II formation in WT or GCN5 KO HeLa cells in the presence or absence of the lysosome inhibitor Baf .

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Immunoblot showing LC3-II formation in WT or GCN5 KO HeLa cells in the presence or absence of the lysosome inhibitor Baf.


Immunoblot showing LC3 - II formation MB - 3 - treated HeLa cells in the presence or absence of the lysosome inhibitor Baf .

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Immunoblot showing LC3-II formation MB-3-treated HeLa cells in the presence or absence of the lysosome inhibitor Baf.


D Formation of LC3 puncta in GCN5 KO HeLa cells overexpressing Myc - tagged GCN5 or GCN5 - E575Q ( Scale bars , 10 µm ) .

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D Formation of LC3 puncta in GCN5 KO HeLa cells overexpressing Myc-tagged GCN5 or GCN5-E575Q (Scale bars, 10 µm).


E Quantification of LC3 puncta in cells The cells were treated with or without Baf ( Graph represents data from three independent experiments with ≥ 30 cells per condition ; mean ± SEM ; * * * p < 0 . 001 , Student ' s t - test ) .

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E Quantification of LC3 puncta in cells The cells were treated with or without Baf (Graph represents data from three independent experiments with ≥ 30 cells per condition; mean ± SEM; ***p < 0.001, Student's t-test).


Formation of LC3 puncta in HeLa cells overexpressing GFP - GCN5 with or without Baf

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Formation of LC3 puncta in HeLa cells overexpressing GFP-GCN5 with or without Baf treatment


Formation of LC3 puncta in HeLa cells overexpressing GFP - GCN5 with or without Baf treatment ( Graph represents data from three independent experiments with ≥ 30 cells per condition ; mean ± SEM ; * p < 0 . 05 , * * p < 0 . 01 , Student ' s t - test ; Scale bar , 10 µm ) .

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Formation of LC3 puncta in HeLa cells overexpressing GFP-GCN5 with or without Baf treatment (Graph represents data from three independent experiments with ≥ 30 cells per condition; mean ± SEM; *p < 0.05, **p < 0.01, Student's t-test; Scale bar, 10 µm).


H LC3 - II formation in GFP - GCN5 - overexpressing HeLa cells .

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H LC3-II formation in GFP-GCN5-overexpressing HeLa cells.


I GFP - p62 levels in HEK293 cells stably expressing GFP - p62 . The cells were cultured with GCN5 siRNA with or without CQ .

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I GFP-p62 levels in HEK293 cells stably expressing GFP-p62. The cells were cultured with GCN5 siRNA with or without CQ.


J PDLIM1 and IFT20 protein levels in GCN5 KO HEK293 cells with or without transfection of GFP - GCN5 and addition of CQ .

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J PDLIM1 and IFT20 protein levels in GCN5 KO HEK293 cells with or without transfection of GFP-GCN5 and addition of CQ.


K Representative images of mCherry - Atg8a ( red ) and DAPI ( blue ) in Drosophila larval fat body in which dGcn5 is overexpressed ( OE ) or silenced ( KD ) using the pan - fat body driver ( cg - GAL4 ) . Drosophila ( cg - GAL4 / + ) was used as the control ( Graph represents data from three independent experiments with ≥ 30 cells per condition ; mean ± SEM ; * p < 0 . 05 , * * * p < 0 . 001 , Student ' s t - test ; Scale bars , 10 µm ) .

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K Representative images of mCherry-Atg8a (red) and DAPI (blue) in Drosophila larval fat body in which dGcn5 is overexpressed (OE) or silenced (KD) using the pan-fat body driver (cg-GAL4). Drosophila (cg-GAL4/+) was used as the control (Graph represents data from three independent experiments with ≥ 30 cells per condition; mean ± SEM; *p < 0.05, ***p < 0.001, Student's t-test; Scale bars, 10 µm).


A LAMP1 puncta ( green ) and DAPI ( blue ) in WT and GCN5 KO HEK293 cells ( Scale bars , 10 µm ) .

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A LAMP1 puncta (green) and DAPI (blue) in WT and GCN5 KO HEK293 cells (Scale bars, 10 µm).


B Fluorescence - activated cell sorting analysis of WT and GCN5 KO HEK293 cells stained with LysoTracker . Fluorescence intensity of 10 , 000 cells per sample was measured .

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B Fluorescence-activated cell sorting analysis of WT and GCN5 KO HEK293 cells stained with LysoTracker. Fluorescence intensity of 10,000 cells per sample was measured.


C Immunoblot showing lysosomal protein levels in three independent clones of GCN5 KO HEK293 cells . CTSD HC , cathepsin D heavy chain .

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C Immunoblot showing lysosomal protein levels in three independent clones of GCN5 KO HEK293 cells. CTSD HC, cathepsin D heavy chain.


D LAMP1 puncta in GCN5 KO HEK293 cells overexpressing Myc - tagged GCN5 or GCN5 - E575Q ( Scale bars , 10 µm ) .

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D LAMP1 puncta in GCN5 KO HEK293 cells overexpressing Myc-tagged GCN5 or GCN5-E575Q (Scale bars, 10 µm).


E Quantification of LAMP1 puncta in ( A ) and ( D ) ( Graph represents data from three independent experiments with ≥ 30 cells per condition ; mean ± SEM ; * * * p < 0 . 001 , Student ' s t - test ) .

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E Quantification of LAMP1 puncta in (A) and (D) (Graph represents data from three independent experiments with ≥ 30 cells per condition; mean ± SEM; ***p < 0.001, Student's t-test).


F Hexosaminidase activity in GCN5 KO HEK293 cells ( mean ± SEM ; n = 3 independent experiments ; * * * p < 0 . 001 , Student ' s t - test ) .

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F Hexosaminidase activity in GCN5 KO HEK293 cells (mean ± SEM; n = 3 independent experiments; ***p < 0.001, Student's t-test).


G Degradation of EGFR in WT and GCN5 KO HEK293 cells in the presence or absence of CQ .

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G Degradation of EGFR in WT and GCN5 KO HEK293 cells in the presence or absence of CQ.


H Representative images of LysoTracker staining ( red ) and DAPI ( blue ) in Drosophila larval fat body in which dGcn5 is overexpressed ( OE ) or silenced ( KD ) . Drosophila ( cg - GAL4 / + ) was used as the control ( Graph represents data from three independent experiments with ≥ 30 cells per condition ; mean ± SEM ; * p < 0 . 05 , * * p < 0 . 01 , Student ' s t - test ; Scale bars , 10 µm ) .

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H Representative images of LysoTracker staining (red) and DAPI (blue) in Drosophila larval fat body in which dGcn5 is overexpressed (OE) or silenced (KD). Drosophila (cg-GAL4/+) was used as the control (Graph represents data from three independent experiments with ≥ 30 cells per condition; mean ± SEM; *p < 0.05, **p < 0.01, Student's t-test; Scale bars, 10 µm).


A Quantification of LC3 puncta in WT , GCN5 KO and GCN5 / TFEB DKO HeLa cells in the presence or absence of Baf ( Graph represents data from three independent experiments with ≥ 30 cells per condition ; mean ± SEM ; * * * p < 0 . 001 , Student ' s t - test ) .

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A Quantification of LC3 puncta in WT, GCN5 KO and GCN5/TFEB DKO HeLa cells in the presence or absence of Baf (Graph represents data from three independent experiments with ≥ 30 cells per condition; mean ± SEM; ***p < 0.001, Student's t-test).


B Immunoblot showing LC3 - II formation in WT , GCN5 KO and GCN5 / TFEB DKO HeLa cells in the presence or absence of Baf .

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B Immunoblot showing LC3-II formation in WT, GCN5 KO and GCN5/TFEB DKO HeLa cells in the presence or absence of Baf.


C Quantification of LAMP1 puncta in WT , GCN5 KO and GCN5 / TFEB DKO HEK293 cells ( Graph represents data from three independent experiments with ≥ 30 cells per condition ; mean ± SEM ; * * * p < 0 . 001 , Student ' s t - test ) .

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C Quantification of LAMP1 puncta in WT, GCN5 KO and GCN5/TFEB DKO HEK293 cells (Graph represents data from three independent experiments with ≥ 30 cells per condition; mean ± SEM; ***p < 0.001, Student's t-test).


D Immunoblot showing LAMP1 levels in WT , GCN5 KO and GCN5 / TFEB DKO HEK293 cells .

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D Immunoblot showing LAMP1 levels in WT, GCN5 KO and GCN5/TFEB DKO HEK293 cells.


E Acetylation of TFEB - Flag in stable TFEB - Flag - expressing HEK293 cells with overexpression of individual histone acetyltransferases as indicated . TFEB - Flag was immunoprecipitated with anti - Flag beads and analyzed by immunoblot using anti - acetyl - lysine ( Ace - lys ) .

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E Acetylation of TFEB-Flag in stable TFEB-Flag-expressing HEK293 cells with overexpression of individual histone acetyltransferases as indicated. TFEB-Flag was immunoprecipitated with anti-Flag beads and analyzed by immunoblot using anti-acetyl-lysine (Ace-lys).


F TFEB - Flag acetylation in the stable TFEB - Flag - expressing HEK293 cells transfected with Myc - tagged GCN5 or GCN5 - E575Q after incubation with GCN5 siRNA for 48 h .

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F TFEB-Flag acetylation in the stable TFEB-Flag-expressing HEK293 cells transfected with Myc-tagged GCN5 or GCN5-E575Q after incubation with GCN5 siRNA for 48 h.


G Acetylation of endogenous TFEB in MEF cells treated with the GCN5 inhibitor MB - 3 or the p300 / CBP inhibitor C646 . H3 , histone H3 . Ace - H3 ( K9 ) and Ace - H3 ( K27 ) were used to show the activity of GCN5 and p300 , respectively .

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G Acetylation of endogenous TFEB in MEF cells treated with the GCN5 inhibitor MB-3 or the p300/CBP inhibitor C646. H3, histone H3. Ace-H3 (K9) and Ace-H3 (K27) were used to show the activity of GCN5 and p300, respectively.


H Co - precipitation of GCN5 with TFEB . TFEB was immunoprecipitated from MEF cells using anti - TFEB , and the precipitates were analyzed using anti - GCN5 .

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H Co-precipitation of GCN5 with TFEB. TFEB was immunoprecipitated from MEF cells using anti-TFEB, and the precipitates were analyzed using anti-GCN5.


I In vitro acetylation assays of TFEB . Purified recombinant TFEB was incubated with Myc - GCN5 or Myc - GCN5 - E575Q immunoprecipitated from HEK293 cells .

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I In vitro acetylation assays of TFEB. Purified recombinant TFEB was incubated with Myc-GCN5 or Myc-GCN5-E575Q immunoprecipitated from HEK293 cells.


J Acetylation of Flag - tagged TFEB or TFEB mutants expressed in HEK293 cells . 3KR : Lys 116 , Lys 274 , and Lys 279 were replaced by Arg .

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J Acetylation of Flag-tagged TFEB or TFEB mutants expressed in HEK293 cells. 3KR: Lys 116, Lys 274, and Lys 279 were replaced by Arg.


A RT - qPCR analysis of the expression of TFEB target genes in WT , GCN5 KO and GCN5 / TFEB DKO HEK293 cells . Data information : In this Figure , data are presented as mean ± SEM ; n = 3 independent experiments ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , Student ' s t - test .

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A RT-qPCR analysis of the expression of TFEB target genes in WT, GCN5 KO and GCN5/TFEB DKO HEK293 cells. Data information: In this Figure, data are presented as mean ± SEM; n = 3 independent experiments; *p < 0.05, **p < 0.01, ***p < 0.001, Student's t-test.


B RT - qPCR analysis of dMitf target gene expression in Drosophila larval fat body with silencing of dGcn5 . Data information : In this Figure , data are presented as mean ± SEM ; n = 3 independent experiments ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , Student ' s t - test .

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B RT-qPCR analysis of dMitf target gene expression in Drosophila larval fat body with silencing of dGcn5. Data information: In this Figure, data are presented as mean ± SEM; n = 3 independent experiments; *p < 0.05, **p < 0.01, ***p < 0.001, Student's t-test.


C Expression of TFEB target genes in HEK293 cells transfected with or without TFEB - WT or TFEB - 3KR . Data information : In this Figure , data are presented as mean ± SEM ; n = 3 independent experiments ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , Student ' s t - test .

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C Expression of TFEB target genes in HEK293 cells transfected with or without TFEB-WT or TFEB-3KR. Data information: In this Figure, data are presented as mean ± SEM; n = 3 independent experiments; *p < 0.05, **p < 0.01, ***p < 0.001, Student's t-test.


D , E Luciferase activity measured in WT , GCN5 KO and GCN5 / TFEB DKO HEK293 cells ( D ) , and in HEK293 cells with TFEB - WT or TFEB - 3KR transfection ( E ) . The cells were transfected or co - transfected with a TFEB - luciferase reporter . 3KR : Lys 116 , Lys 274 , and Lys 279 were replaced by Arg . Data information : In this Figure , data are presented as mean ± SEM ; n = 3 independent experiments ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , Student ' s t - test .

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D, E Luciferase activity measured in WT, GCN5 KO and GCN5/TFEB DKO HEK293 cells (D), and in HEK293 cells with TFEB-WT or TFEB-3KR transfection (E). The cells were transfected or co-transfected with a TFEB-luciferase reporter. 3KR: Lys 116, Lys 274, and Lys 279 were replaced by Arg. Data information: In this Figure, data are presented as mean ± SEM; n = 3 independent experiments; *p < 0.05, **p < 0.01, ***p < 0.001, Student's t-test.


A Subcellular localization of TFEB in WT or GCN5 KO HEK293 cells treated with or without Torin1 ( Scale bars , 10 µm ) .

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A Subcellular localization of TFEB in WT or GCN5 KO HEK293 cells treated with or without Torin1 (Scale bars, 10 µm).


B Subcellular localization of Flag - tagged TFEB or TFEB mutants in HEK293 cells treated with or without Torin1 . Cells were stained with anti - Flag antibody ( Scale bars , 10 µm ) .

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B Subcellular localization of Flag-tagged TFEB or TFEB mutants in HEK293 cells treated with or without Torin1. Cells were stained with anti-Flag antibody (Scale bars, 10 µm).


C ChIP - qPCR analysis of TFEB binding to the promoter of its target genes CLCN7 , GLA and CTSD . Normal mouse IgG and primers against the upstream region lacking CLEAR sites ( up ) were used as negative controls ( mean ± SEM ; n = 3 independent experiments ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , Student ' s t - test ) .

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C ChIP-qPCR analysis of TFEB binding to the promoter of its target genes CLCN7, GLA and CTSD. Normal mouse IgG and primers against the upstream region lacking CLEAR sites (up) were used as negative controls (mean ± SEM; n = 3 independent experiments; *p < 0.05, **p < 0.01, ***p < 0.001, Student's t-test).


D Electrophoresis mobility shift assay of TFEB binding to the promoter of GLA . A DNA fragment from the GLA promoter containing the TFEB binding site was incubated with purified recombinant TFEB or each of the TFEB mutants , and was subjected to electrophoresis . Asterisk indicates the DNA fragment from the GAPDH promoter which was used as a TFEB non - binding DNA control .

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D Electrophoresis mobility shift assay of TFEB binding to the promoter of GLA. A DNA fragment from the GLA promoter containing the TFEB binding site was incubated with purified recombinant TFEB or each of the TFEB mutants, and was subjected to electrophoresis. Asterisk indicates the DNA fragment from the GAPDH promoter which was used as a TFEB non-binding DNA control.


F Co - precipitation of Flag - tagged TFEB and TFEB mutants with Myc - TFEB in HEK293T cells . Immunoprecipitation was carried out with anti - Myc beads , and the precipitates were analyzed using anti - Flag .

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F Co-precipitation of Flag-tagged TFEB and TFEB mutants with Myc-TFEB in HEK293T cells. Immunoprecipitation was carried out with anti-Myc beads, and the precipitates were analyzed using anti-Flag.


Co - precipitation of Flag - tagged TFEB and TFEB mutants with Myc - tagged MITF ( G ) in HEK293T cells .

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Co-precipitation of Flag-tagged TFEB and TFEB mutants with Myc-tagged MITF (G) in HEK293T cells.


Co - precipitation of Flag - tagged TFEB and TFEB mutants with Myc - tagged TFE3 ( H ) in HEK293T cells .

Co-precipitation of Flag-tagged TFEB and TFEB mutants with Myc-tagged TFE3 (H) in HEK293T cells.


I TFEB homodimer formation detected by glutaraldehyde ( GA ) crosslinking . Purified recombinant GST - tagged TFEB or TFEB mutants was incubated with or without glutaraldehyde , then the products were analyzed by immunoblotting using anti - TFEB .

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I TFEB homodimer formation detected by glutaraldehyde (GA) crosslinking. Purified recombinant GST-tagged TFEB or TFEB mutants was incubated with or without glutaraldehyde, then the products were analyzed by immunoblotting using anti-TFEB.


A Acetylation of TFEB - Flag , Myc - GCN5 and histone H3 in HEK293 cells . The cells were either starved of glucose or amino acids , or treated with Torin1 or MB - 3 .

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A Acetylation of TFEB-Flag, Myc-GCN5 and histone H3 in HEK293 cells. The cells were either starved of glucose or amino acids, or treated with Torin1 or MB-3.


B Acetylation of endogenous TFEB in HeLa cells .

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B Acetylation of endogenous TFEB in HeLa cells.


C GCN5 activity measured by in vitro acetylation assay using Myc - GCN5 immunoprecipitated from HEK293T cells and purified histone H3 as the substrate . ( mean ± SEM ; n = 3 independent experiments ; * * * p < 0 . 001 , Student ' s t - test ) .

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C GCN5 activity measured by in vitro acetylation assay using Myc-GCN5 immunoprecipitated from HEK293T cells and purified histone H3 as the substrate. (mean ± SEM; n = 3 independent experiments; ***p < 0.001, Student's t-test).


D Co - precipitation of TFEB - Flag with Myc - TFEB in HEK293

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D Co-precipitation of TFEB-Flag with Myc-TFEB in HEK293 cells


E LC3 - II formation in HeLa cells transfected with Flag - tagged TFEB or TFEB mutants after TFEB RNAi . The cells were treated with or without amino acid starvation .

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E LC3-II formation in HeLa cells transfected with Flag-tagged TFEB or TFEB mutants after TFEB RNAi. The cells were treated with or without amino acid starvation.


F GFP - p62 levels in HEK293 cells stably expressing GFP - p62 . The cells were treated with or without addition of CQ .

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F GFP-p62 levels in HEK293 cells stably expressing GFP-p62. The cells were treated with or without addition of CQ.


Representative images of mCherry - Atg8a ( red ) ( G ) in Drosophila larval fat body in which dMitf or dMitf - 2KQ ( K445Q / K450Q ) was overexpressed . Drosophila ( cg - GAL4 / + ) was used as the control and DAPI staining ( blue ) was used to indicate the cell nucleus ( Scale bars , 10 µm ) .

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Representative images of mCherry-Atg8a (red) (G) in Drosophila larval fat body in which dMitf or dMitf-2KQ (K445Q/K450Q) was overexpressed. Drosophila (cg-GAL4/+) was used as the control and DAPI staining (blue) was used to indicate the cell nucleus (Scale bars, 10 µm).


Representative images of LysoTracker ( red ) staining ( H ) in Drosophila larval fat body in which dMitf or dMitf - 2KQ ( K445Q / K450Q ) was overexpressed . Drosophila ( cg - GAL4 / + ) was used as the control and DAPI staining ( blue ) was used to indicate the cell nucleus ( Scale bars , 10 µm ) .

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Representative images of LysoTracker (red) staining (H) in Drosophila larval fat body in which dMitf or dMitf-2KQ (K445Q/K450Q) was overexpressed. Drosophila (cg-GAL4/+) was used as the control and DAPI staining (blue) was used to indicate the cell nucleus (Scale bars, 10 µm).


Quantification of mCherry - ATG8a puncta ( I ) ( Graph represents data from three independent experiments with ≥ 30 cells per condition ; mean ± SEM ; * * * p < 0 . 001 , Student ' s t - test ) .

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Quantification of mCherry-ATG8a puncta (I) (Graph represents data from three independent experiments with ≥ 30 cells per condition; mean ± SEM; ***p < 0.001, Student's t-test).


Quantification of LysoTracker puncta ( J ) ( Graph represents data from three independent experiments with ≥ 30 cells per condition ; mean ± SEM ; * * * p < 0 . 001 , Student ' s t - test ) .

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Quantification of LysoTracker puncta (J) (Graph represents data from three independent experiments with ≥ 30 cells per condition; mean ± SEM; ***p < 0.001, Student's t-test).


The level of insoluble Tau protein in the head of adult Drosophila with or without CQ feeding . The indicated genes were transgenically expressed in the eyes of the Drosophila using the GMR - Gal4 driver . Drosophila ( GMR - Gal4 / + ) was used as the control .

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The level of insoluble Tau protein in the head of adult Drosophila with or without CQ feeding. The indicated genes were transgenically expressed in the eyes of the Drosophila using the GMR-Gal4 driver. Drosophila (GMR-Gal4/+) was used as the control.


C Representative light micrographs of adult Drosophila eyes expressing the indicated transgenes under the control of the GMR - Gal4 driver . The Drosophila were fed with or without CQ . Drosophila ( GMR - Gal4 / + ) was used as the control .

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C Representative light micrographs of adult Drosophila eyes expressing the indicated transgenes under the control of the GMR-Gal4 driver. The Drosophila were fed with or without CQ. Drosophila (GMR-Gal4/+) was used as the control.


D Distribution of the different eye phenotypes following genetic manipulations The Drosophila were fed with or without CQ ( 30 flies per condition were randomly selected for analyses ; * p < 0 . 05 , * * * p < 0 . 001 , ꭓ2 test ) .

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D Distribution of the different eye phenotypes following genetic manipulations The Drosophila were fed with or without CQ (30 flies per condition were randomly selected for analyses; *p < 0.05, ***p < 0.001, ꭓ2 test).


( A ) EAE severity in 6 - to - 8 - week - old female ( left panel ) and male ( right panel ) wild - type ( WT ) and ERα - / - mice ( n = 7 - 9 mice per group ) . ( B ) EAE sensitivity to IFNβ in 6 - to - 8 - week - old female ( top panel ) and male ( bottom panel ) WT and ERα - / - mice . Vehicle ( Veh ) or IFNβ ( 47 μg / kg ) were i . p . injected . every other day from 0 to 10 dpi ( n = 8 - 12 mice per group ) .

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(A) EAE severity in 6-to-8-week-old female (left panel) and male (right panel) wild-type (WT) and ERα-/- mice (n=7-9 mice per group). (B) EAE sensitivity to IFNβ in 6-to-8-week-old female (top panel) and male (bottom panel) WT and ERα-/- mice. Vehicle (Veh) or IFNβ (47 μg/kg) were i.p. injected. every other day from 0 to 10 dpi (n= 8-12 mice per group).


( D ) Sensitivity of EAE to IFNβ in Cyp19 - / - ovary - transferred female WT mice and WT ovary - transferred Cyp19 - / - mice ( n = 5 - 6 mice ) .

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(D) Sensitivity of EAE to IFNβ in Cyp19-/- ovary-transferred female WT mice and WT ovary-transferred Cyp19-/- mice (n=5-6 mice).


( E ) Estradiol levels in LNs ( A : axillary , B : branchial , C : cervical , and I : inguinal ) in sham - operated and OVX mice ( n = 5 mice per group ) .

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(E) Estradiol levels in LNs (A: axillary, B: branchial, C: cervical, and I: inguinal) in sham-operated and OVX mice (n=5 mice per group).


( A ) Brightfield micrograph of ventral horns of Golgi - Cox stained lumber ( L ) 5 spinal cord section of male WT and ERα - / - EAE mice at 33 dpi with quantitation of area stained in gray matter . The white dotted line indicates the division between white and gray matter , V . H . represents the ventral horn ( n = 5 mice per group ) .

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(A) Brightfield micrograph of ventral horns of Golgi-Cox stained lumber (L)5 spinal cord section of male WT and ERα-/- EAE mice at 33 dpi with quantitation of area stained in gray matter. The white dotted line indicates the division between white and gray matter, V.H. represents the ventral horn (n=5 mice per group).


( C ) Representative confocal reflection maximum intensity projection micrograph of Golgi - Cox stained dendrites in the lumbar ventral horn of male WT and ERα - / - mice at 33 dpi with quantitative analysis of dendrite spine density ( n = 4 - 5 mice per group , 20 dendrites analyzed per animal ) .

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(C) Representative confocal reflection maximum intensity projection micrograph of Golgi-Cox stained dendrites in the lumbar ventral horn of male WT and ERα-/- mice at 33 dpi with quantitative analysis of dendrite spine density (n=4-5 mice per group, 20 dendrites analyzed per animal).


( A ) Upregulated and downregulated genes in RNAseq analysis were evaluated by qPCR . Splenic CD4 + T cells from male WT EAE and ERα - / - EAE mice at 9 dpi were used ( n = 5 - 8 mice ) .

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(A) Upregulated and downregulated genes in RNAseq analysis were evaluated by qPCR. Splenic CD4+ T cells from male WT EAE and ERα-/- EAE mice at 9 dpi were used (n=5-8 mice).


( D ) Neurite outgrowth of the neurons in Golgi - stained spinal sections of Tcra - / - recipient mice that received passive intrathecal injections of CD4 + splenic cells from male WT EAE and ERα - / - EAE mice at 14 dpi quantified using the Sholl technique . Sholl rings are separated by 10 μm ( n = 4 mice per group , 20 neurons analyzed per animal ) with an end radius of 100 μm .

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(D) Neurite outgrowth of the neurons in Golgi-stained spinal sections of Tcra-/- recipient mice that received passive intrathecal injections of CD4+ splenic cells from male WT EAE and ERα-/- EAE mice at 14 dpi quantified using the Sholl technique. Sholl rings are separated by 10 μm (n=4 mice per group, 20 neurons analyzed per animal) with an end radius of 100 μm.


( C ) Neurite outgrowth of the neurons in Golgi - stained spinal sections of Tcra - / - recipient mice that received passive intrathecal injections of CD4 + splenic cells from male WT EAE and cERα - / - EAE mice at 14 dpi quantified using the Sholl analysis method . Sholl rings are separated by 10 μm ( n = 4 mice per group , 20 neurons analyzed per animal ) with an end radius of 100 μm .

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(C) Neurite outgrowth of the neurons in Golgi-stained spinal sections of Tcra-/- recipient mice that received passive intrathecal injections of CD4+ splenic cells from male WT EAE and cERα-/- EAE mice at 14 dpi quantified using the Sholl analysis method. Sholl rings are separated by 10 μm (n=4 mice per group, 20 neurons analyzed per animal) with an end radius of 100 μm.


( A ) Number of membrane - bound lymphotoxin ( mLT ) + DCs ( derived from spleen and LNs ) in male cERα - / - EAE mice at 9 dpi ( n = 5 mice per group ) . DC population were gated as Ly6G - and CD11c + ( EV Fig . 1 A ) .

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

(A) Number of membrane-bound lymphotoxin (mLT)+ DCs (derived from spleen and LNs) in male cERα-/- EAE mice at 9 dpi (n=5 mice per group). DC population were gated as Ly6G- and CD11c+ (EV Fig.1 A).


( B ) Tuj1 - stained differentiated N2a cells co - cultured with nothing , non - treated CD4 + T cell , and rLT - treated CD4 + T cell ( 10 ng / ml ) for 24 h . Panels depict representative fields for each condition . Quantification of percentage differentiated neuron and average neurite length . The results are derived from at least six biological samples .

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

(B) Tuj1-stained differentiated N2a cells co-cultured with nothing, non-treated CD4+ T cell, and rLT-treated CD4+ T cell (10 ng/ml) for 24 h. Panels depict representative fields for each condition. Quantification of percentage differentiated neuron and average neurite length. The results are derived from at least six biological samples.


( A ) Lta mRNA expression in BMDCs developed from bone marrows of WT and ERα - / - mice 3 h after treatment with LPS ( 100 ng / ml ) with vehicle ( Veh ) or PPT ( 1 µM ) pre - treatment for 18 h ( n = 4 samples per group ) . ( B ) Il1b and Ifnb mRNA expression 3 h after treatment with LPS ( 100 ng / ml ) with vehicle ( Veh ) or PPT ( 1 µM ) pre - treatment for 18 h ( n = 4 - 5 samples per group ) .

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

(A) Lta mRNA expression in BMDCs developed from bone marrows of WT and ERα-/- mice 3 h after treatment with LPS (100 ng/ml) with vehicle (Veh) or PPT (1 µM) pre-treatment for 18 h (n=4 samples per group). (B) Il1b and Ifnb mRNA expression 3 h after treatment with LPS (100 ng/ml) with vehicle (Veh) or PPT (1 µM) pre-treatment for 18 h (n=4-5 samples per group).


( C ) Representative Western blot of TRAF3 and HSC70 in WT BMDCs and ERα - / - BMDCs . TRAF3 expression in BMDCs following vehicle ( Veh ) or PPT ( 1 µM ) pre - treatment for 18 h was evaluated by normalization of HSC70 ( n = 5 - 7 per group ) .

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

(C) Representative Western blot of TRAF3 and HSC70 in WT BMDCs and ERα-/- BMDCs. TRAF3 expression in BMDCs following vehicle (Veh) or PPT (1 µM) pre-treatment for 18 h was evaluated by normalization of HSC70 (n=5-7 per group).


( F ) Nuclear localization of IRF3 in BMDCs treated with vehicle or with LPS for 2 h prior upon a vehicle or PPT ( 1 µM ) pre - treatment for 18 h . The results are presented as a ratio of the nuclear IRF3 signal to the cytosol IRF3 signal ( n = 5 - 9 biological samples )

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

(F) Nuclear localization of IRF3 in BMDCs treated with vehicle or with LPS for 2 h prior upon a vehicle or PPT (1 µM) pre-treatment for 18 h. The results are presented as a ratio of the nuclear IRF3 signal to the cytosol IRF3 signal (n=5-9 biological samples)


( C ) Fluorescence polarization assays demonstrating that a peptide containing the BH3 ‐ like domain of Beclin ‐ 1 interacts with Bcl ‐ XL . Recombinant Bcl ‐ XL ΔTM was incubated with carboxyfluorescein ‐ labeled Bak BH3 peptide , in the absence or presence of wild ‐ type ( WT ) Beclin ‐ 1 BH3 or either of the two mutant peptides described in B .

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

(C) Fluorescence polarization assays demonstrating that a peptide containing the BH3‐like domain of Beclin‐1 interacts with Bcl‐XL. Recombinant Bcl‐XL ΔTM was incubated with carboxyfluorescein‐labeled Bak BH3 peptide, in the absence or presence of wild‐type (WT) Beclin‐1 BH3 or either of the two mutant peptides described in B.


( D ) Affinity determination of the interaction between recombinant Bcl ‐ XLΔTM and fluorescent Beclin ‐ 1 BH3 , by fluorescence polarization . A comparison between WT Bcl ‐ XL ( squares ) and G138A Bcl ‐ XL ( triangles ) is shown ( means ± s . d . , n = 3 separate experiments ) .

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

(D) Affinity determination of the interaction between recombinant Bcl‐XLΔTM and fluorescent Beclin‐1 BH3, by fluorescence polarization. A comparison between WT Bcl‐XL(squares) and G138A Bcl‐XL (triangles) is shown (means±s.d., n=3 separate experiments).


( E ) Co ‐ immunoprecipitation of Bcl ‐ XL and WT or mutant Beclin ‐ 1 . HeLa cells were transfected with the indicated constructs , followed by immunoprecipitation of Flag ‐ tagged Bcl ‐ XL and immunochemical detection of Beclin ‐ 1 .

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

(E) Co‐immunoprecipitation of Bcl‐XL and WT or mutant Beclin‐1. HeLa cells were transfected with the indicated constructs, followed by immunoprecipitation of Flag‐tagged Bcl‐XL and immunochemical detection of Beclin‐1.


( F ) Co ‐ immunoprecipitation of Beclin ‐ 1 with ‐ WT and mutant ( G138A ) Bcl ‐ XL . Results are representative of at least three experiments yielding similar results .

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

(F) Co‐immunoprecipitation of Beclin‐1 with‐WT and mutant (G138A) Bcl‐XL. Results are representative of at least three experiments yielding similar results.


( A ) Competition between Beclin ‐ 1 BH3 and ABT737 for Bcl ‐ XL binding . A fluorescent 25 ‐ mer peptide containing the BH3 ‐ like domain of Beclin ‐ 1 ( Figure 1D ) was docked to recombinant Bcl ‐ XL ΔTM protein in the absence or presence of ABT737 . The IC50 of ABT737 , as measured in the presence of 15 nM of peptide and 100 nM of Bcl ‐ XL ΔTM , was 1 . 7 μM .

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

(A) Competition between Beclin‐1 BH3 and ABT737 for Bcl‐XL binding. A fluorescent 25‐mer peptide containing the BH3‐like domain of Beclin‐1 ( Figure 1D) was docked to recombinant Bcl‐XL ΔTM protein in the absence or presence of ABT737. The IC50 of ABT737, as measured in the presence of 15 nM of peptide and 100 nM of Bcl‐XL ΔTM, was 1.7 μM.


( B , C ) Abolition of the interaction between Bcl ‐ XL and Beclin ‐ 1 by ABT737 in intact cells . Co ‐ immunoprecipitation assays were performed on HeLa cells transfected 48 h earlier with the indicated constructs as in Figure 1E . Sixteen hours before the immunoprecipitation , cells were exposed to ABT737 . Similar results were obtained for co ‐ transfected Bcl ‐ XL and Beclin ‐ 1 ( B ) and for endogenous Beclin ‐ 1 interacting with Flag ‐ tagged Bcl ‐ XL ( C ) .

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

(B, C) Abolition of the interaction between Bcl‐XL and Beclin‐1 by ABT737 in intact cells. Co‐immunoprecipitation assays were performed on HeLa cells transfected 48 h earlier with the indicated constructs as in Figure 1E. Sixteen hours before the immunoprecipitation, cells were exposed to ABT737. Similar results were obtained for co‐transfected Bcl‐XL and Beclin‐1 (B) and for endogenous Beclin‐1 interacting with Flag‐tagged Bcl‐XL (C).


( D , E ) Differential effect of ABT737 on the interaction between Bcl ‐ 2 ( D ) or Mcl ‐ 1 ( E ) and Beclin ‐ 1 . This experiment was designed as ( B ) . All experiments have been performed at least three times , with similar results .

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

(D, E) Differential effect of ABT737 on the interaction between Bcl‐2 (D) or Mcl‐1 (E) and Beclin‐1. This experiment was designed as (B). All experiments have been performed at least three times, with similar results.