Figure 5C57BL / 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 ) . 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 ) . 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 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5C57BL/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).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).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).


Figure 6Two 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6Two 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.


Figure 7BR1 - 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 ) . 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 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7BR1-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).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).


Figure 8BR1 - 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 . 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 . ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8BR1-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.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.).


Figure 1A 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 . 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 ) . 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 treatmentFormation 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 . 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 . 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 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O B-PANEL_START O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A 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.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).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 treatmentFormation 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.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.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).


Figure 2A 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 . 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 ) . 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 ) . 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 ) .

O B-PANEL_START O 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-PANEL_START O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A 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.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).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).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).


Figure 3A 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 . 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 . 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 . 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 . 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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Figure 3A 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.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.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.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.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.


Figure 4A 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 . 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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Figure 4A 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.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.


Figure 5A 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 ) . 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 . 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 . 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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Figure 5A 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).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.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.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.


Figure 6A 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 . 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 cellsE 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 . 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 ) . 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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Figure 6A 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.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 cellsE 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.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).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).


Figure 7The 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 . 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 . 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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Figure 7The 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.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.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).


Figure 1 ( 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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Figure 1(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).


Figure 2 ( 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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Figure 2(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).


Figure 3 ( 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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Figure 3(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).


Figure 4 ( 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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Figure 4(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.


Figure 5 ( 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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Figure 5(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.


Figure 6 ( 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 .

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Figure 6(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.


Figure 7 ( 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 ) . ( 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 )

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Figure 7(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).(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)


Figure 1 ( 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 ) . ( 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 .

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Figure 1(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).(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.


Figure 2 ( 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 ) . ( 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 .

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Figure 2(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).(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.


Figure 3 ( A , B ) Detection of autophagic vacuoles by LC3 ‐ GFP and their modulation by ABT737 and by Beclin ‐ 1 ‐ specific siRNAs . HeLa cells were transfected with control or Beclin ‐ 1 ‐ specific siRNAs , 24 h later re ‐ transfected with LC3 ‐ GFP , cultured in complete medium ( CM ) for 24 h , and finally kept 12 h either in CM or in nutrient ‐ free ( NF ) conditions , in the presence or absence of 1 μM ABT737 . Representative microphotographs of cells cultured in NF medium are shown in ( A ) and the percentage ( means ± s . d . , n = 3 separate experiments ) of LC3 ‐ GFP ‐ transfected cells bearing LC3 ‐ GFP aggregates in the cytoplasm ( LC3 ‐ GFPvac ) are quantified in ( B ) . The insert in ( B ) demonstrates the efficiency of the Beclin ‐ 1 ‐ specific siRNAs , as quantified by immunoblot . ( C , D ) Detection of cytoplasmicvacuoles using chloromethylfluorescein diacetate ( CMFDA ) . Cells were transfected with control or Beclin ‐ 1 ‐ specific siRNAs , cultured for 48 h in CM , washed , cultured in CM ( D ) or NF ( C , D ) for 12 h , stained with CMFDA , and either photographed ( C ) or subjected to the quantification of the cells that bear at least one discernible cytoplasmicvacuole ( arrow head ) ( means ± s . d . , n = 3 separate experiments ) . ( E ) Ultrastructure of autophagic vacuoles induced by ABT737 . Transmission electron microphotographs are shown . ( F ) Detection of dead and dying cells in the cultures . Cells treated as in ( A ) were stained with the ΔΨm ‐ sensitive dye DiOC6 ( 3 ) and the vital dye propidium iodide ( PI ) . The black portions of the columns refer to the DiOC6 ( 3 ) low PI + population ( dead ) and the remaining part of the column corresponds to the DiOC6 ( 3 ) low PI − ( dying ) population . Results are means ± s . d . of three independent experiments .

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Figure 3(A, B) Detection of autophagic vacuoles by LC3‐GFP and their modulation by ABT737 and by Beclin‐1‐specific siRNAs. HeLa cells were transfected with control or Beclin‐1‐specific siRNAs, 24 h later re‐transfected with LC3‐GFP, cultured in complete medium (CM) for 24 h, and finally kept 12 h either in CM or in nutrient‐free (NF) conditions, in the presence or absence of 1 μM ABT737. Representative microphotographs of cells cultured in NF medium are shown in (A) and the percentage (means±s.d., n=3 separate experiments) of LC3‐GFP‐transfected cells bearing LC3‐GFP aggregates in the cytoplasm (LC3‐GFPvac) are quantified in (B). The insert in (B) demonstrates the efficiency of the Beclin‐1‐specific siRNAs, as quantified by immunoblot.(C, D) Detection of cytoplasmicvacuoles using chloromethylfluorescein diacetate (CMFDA). Cells were transfected with control or Beclin‐1‐specific siRNAs, cultured for 48 h in CM, washed, cultured in CM (D) or NF (C, D) for 12 h, stained with CMFDA, and either photographed (C) or subjected to the quantification of the cells that bear at least one discernible cytoplasmicvacuole (arrow head) (means±s.d., n=3 separate experiments).(E) Ultrastructure of autophagic vacuoles induced by ABT737. Transmission electron microphotographs are shown.(F) Detection of dead and dying cells in the cultures. Cells treated as in (A) were stained with the ΔΨm‐sensitive dye DiOC6(3) and the vital dye propidium iodide (PI). The black portions of the columns refer to the DiOC6(3)low PI+ population (dead) and the remaining part of the column corresponds to the DiOC6(3)low PI− (dying) population. Results are means±s.d. of three independent experiments.


Figure 4Quantification of autophagic vacuoles induced by ABT737 . HeLa cells transfected with the indicated siRNAs ( specific for Emerin or for Beclin ‐ 1 at 0 h ) were re ‐ transfected with LC3 ‐ GFP finally cultured in nutrient ‐ free ( NF ) conditions ( 60 - 72 h ) , in the presence or absence 1 μM ABT737 and then subjected to electron microscopy detection of immature ( AV1 ) or mature ( AV2 ) autophagic vacuoles . Representative pictures are shown in ( A ) . The number of AV1 and AV2 was determined for a minimum of 50 cells ( means ± s . e . m . ) ( B ) .

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Figure 4Quantification of autophagic vacuoles induced by ABT737. HeLa cells transfected with the indicated siRNAs (specific for Emerin or for Beclin‐1 at 0 h) were re‐transfected with LC3‐GFP finally cultured in nutrient‐free (NF) conditions (60-72 h), in the presence or absence 1 μM ABT737 and then subjected to electron microscopy detection of immature (AV1) or mature (AV2) autophagic vacuoles. Representative pictures are shown in (A).The number of AV1 and AV2 was determined for a minimum of 50 cells (means±s.e.m.) (B).


Figure 5 ( A - D ) Modulation of Beclin ‐ 1 ‐ induced autophagy by BH3 mutants and ABT737 . Cells were transfected with GFP ‐ LC3 together with an empty control vector or Beclin ‐ 1 ( WT , L116A , F123A ) and cultured for 48 h , followed by overnight culture in CM ( B ) or NF ( A , B ) in the absence or presence of 1 μM ABT737 . Representative microphotographs are shown in ( A ) and the LC3 ‐ GFP ‐ positive vacuoles per cell are quantified in ( B ) ( means ± s . d . ; n = 3 separate experiments ) . Alternatively , cells were not transfected and stained with CMFDA to detect vacuoles , as shown in ( C ) and ( D ) , yielding similar results as for the LC3 ‐ GFP method . ( E ) BH3 ‐ dependent modulation of Beclin ‐ 1 ‐ induced autophagy by anti ‐ apoptotic Bcl ‐ 2 proteins . Cells were transfected simultaneously with Beclin ‐ 1 ( or control vector ) and equivalent amounts of plasmids coding for WT Bcl ‐ XL , Bcl ‐ XL G138A , Bcl ‐ 2 or Mcl ‐ 1 ( or control vector ) , as well as GFP ‐ LC3 . The culture in CM or NF in the absence or presence of ABT737 was performed during the last 12 h of the 60 h ‐ long experiment . The percentage of cells exhibiting the accumulation of LC3 ‐ GFP in vacuoles ( LC3 ‐ GFPvac ) is quantified as means ± s . d . ( n = 3 separate experiments ) .

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Figure 5(A-D) Modulation of Beclin‐1‐induced autophagy by BH3 mutants and ABT737. Cells were transfected with GFP‐LC3 together with an empty control vector or Beclin‐1 (WT, L116A, F123A) and cultured for 48 h, followed by overnight culture in CM (B) or NF (A, B) in the absence or presence of 1 μM ABT737. Representative microphotographs are shown in (A) and the LC3‐GFP‐positive vacuoles per cell are quantified in (B) (means±s.d.; n=3 separate experiments). Alternatively, cells were not transfected and stained with CMFDA to detect vacuoles, as shown in (C) and (D), yielding similar results as for the LC3‐GFP method.(E) BH3‐dependent modulation of Beclin‐1‐induced autophagy by anti‐apoptotic Bcl‐2 proteins. Cells were transfected simultaneously with Beclin‐1 (or control vector) and equivalent amounts of plasmids coding for WT Bcl‐XL, Bcl‐XL G138A, Bcl‐2 or Mcl‐1 (or control vector), as well as GFP‐LC3. The culture in CM or NF in the absence or presence of ABT737 was performed during the last 12 h of the 60 h‐long experiment. The percentage of cells exhibiting the accumulation of LC3‐GFP in vacuoles (LC3‐GFPvac) is quantified as means±s.d. (n=3 separate experiments).


Figure 6 ( A ) Immunoprecipitation of endogenous Bcl ‐ 2 and Beclin ‐ 1 . Untransfected HeLa or MV4 . 11 cells were treated by culture in nutrient ‐ free EBSS or with ABT737 ( 1 μM ) . ( B ) Immunoprecipitation of wild ‐ type and ER ‐ targeted Bcl ‐ 2 with Beclin ‐ 1 . Cells stably transfected with wild type , ER ‐ and mitochondrion ‐ targeted Bcl ‐ 2 were subjected to starvation or treated with ABT737 , followed by immunoprecipitation of Bcl ‐ 2 and immunodetection of Beclin ‐ 1 . ( C , D ) Immunoprecipitation of Bcl ‐ 2 and Beclin ‐ 1 in subcellular fractions . Cells treated as in ( B ) were lysed and subjected to subcellular fractionation to enrich either ER vesicles ( microsomes , ( C ) ) or mitochondria ( heavy membrane fraction , ( D ) ) , followed by immunoprecipitation of Bcl ‐ 2 and immunodetection of Beclin ‐ 1 . Calreticulin and Hsp60 were used as internal controls . Results are representative of three independent determinations .

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Figure 6(A) Immunoprecipitation of endogenous Bcl‐2 and Beclin‐1. Untransfected HeLa or MV4.11 cells were treated by culture in nutrient‐free EBSS or with ABT737 (1 μM).(B) Immunoprecipitation of wild‐type and ER‐targeted Bcl‐2 with Beclin‐1. Cells stably transfected with wild type, ER‐ and mitochondrion‐targeted Bcl‐2 were subjected to starvation or treated with ABT737, followed by immunoprecipitation of Bcl‐2 and immunodetection of Beclin‐1.(C, D) Immunoprecipitation of Bcl‐2 and Beclin‐1 in subcellular fractions. Cells treated as in (B) were lysed and subjected to subcellular fractionation to enrich either ER vesicles (microsomes, (C)) or mitochondria (heavy membrane fraction, (D)), followed by immunoprecipitation of Bcl‐2 and immunodetection of Beclin‐1. Calreticulin and Hsp60 were used as internal controls. Results are representative of three independent determinations.


Figure 7 ( A , B ) Interactions between Bcl ‐ XL , Beclin ‐ 1 and Bad in conditions of autophagy induction . Cells were either treated by nutrient depletion ( A ) or addition of 1 μM rapamycin , 1 mM lithium chloride , 100 μM L ‐ 690 , 330 or 50 μM carbamazepine ( B ) , followed by immunprecipitation of Bcl ‐ XL ( as in Figure 2C ) and revealing the immunoblots by antibodies specific for Bcl ‐ XL , Beclin ‐ 1 or Bad . ( C ) Interaction between endogenous Bad and Bcl ‐ 2 . HeLa cells were treated with ABT737 ( 1 μM ) or nutrient ‐ depleted , followed by immunoprecipitation of Bcl ‐ 2 and immunodetection of Bad . ( D , E ) Impact of Bad depletion on autophagy . Cells were transfected with LC3 ‐ GFP together with siRNAs specific for Bad , siRNAs specific for emerin or the Beclin ‐ 1 ‐ associated PI3 kinase Vps34 . Forty ‐ eight hours later , when the siRNAs had down ‐ regulated the proteins of interest ( D ) , cells were subjected by nutrient depletion ( NF ) or addition of rapamycin ( E ) and the frequency of cells exhibiting LC3 ‐ GFP aggregation as a marker of autophagy was assessed after 18 h ( means ± s . d . , * P0 . 05 , n = 3 separate experiments ) . ( F ) Effect of the Bad knock ‐ out on autophagy . Wild Type ( WT ) or Bad − / − MEF were transfected with LC3 ‐ GFP and then subjected to nutrient depletion ( NF ) and / or treatment with ABT737 ( 18h ; means ± s . d . , n = 3 ) . The asterisks denotes a significant ( P0 . 05 ) effect of Bad deficiency . ( G ) Effect of the Bad knockout on the Beclin ‐ 1 / Bcl ‐ 2 interaction . WT or Bad − / − MEF were subjected to the indicated treatments , followed by immunoprecipitation of Bcl ‐ 2 and immunobloting of Beclin ‐ 1 . ( H ) Overexpression of Bad triggers autophagy . Cells were transfected with LC3 ‐ GFP alone ( Control ) , together with vector ‐ only or with a vector encoding Bad , and subjected 48 h later to nutrient depletion ( NF ) . Cells were cultured in the continuous presence or absence of the pan ‐ caspase inhibitor Z ‐ VAD ‐ fmk ( 50 μM ) for 16 h , followed by assessment of autophagy as in ( E ) ( means ± s . d . , n = 3 separate experiments , * P0 . 01 ) . Immunoblots are representative of at least three independent experiments .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7(A, B) Interactions between Bcl‐XL, Beclin‐1 and Bad in conditions of autophagy induction. Cells were either treated by nutrient depletion (A) or addition of 1 μM rapamycin, 1 mM lithium chloride, 100 μM L‐690,330 or 50 μM carbamazepine (B), followed by immunprecipitation of Bcl‐XL (as in Figure 2C) and revealing the immunoblots by antibodies specific for Bcl‐XL, Beclin‐1 or Bad.(C) Interaction between endogenous Bad and Bcl‐2. HeLa cells were treated with ABT737 (1 μM) or nutrient‐depleted, followed by immunoprecipitation of Bcl‐2 and immunodetection of Bad.(D, E) Impact of Bad depletion on autophagy. Cells were transfected with LC3‐GFP together with siRNAs specific for Bad, siRNAs specific for emerin or the Beclin‐1‐associated PI3 kinase Vps34. Forty‐eight hours later, when the siRNAs had down‐regulated the proteins of interest (D), cells were subjected by nutrient depletion (NF) or addition of rapamycin (E) and the frequency of cells exhibiting LC3‐GFP aggregation as a marker of autophagy was assessed after 18 h (means±s.d., *P0.05, n=3 separate experiments).(F) Effect of the Bad knock‐out on autophagy. Wild Type (WT) or Bad−/− MEF were transfected with LC3‐GFP and then subjected to nutrient depletion (NF) and/or treatment with ABT737 (18h; means±s.d., n=3). The asterisks denotes a significant (P0.05) effect of Bad deficiency.(G) Effect of the Bad knockout on the Beclin‐1/Bcl‐2 interaction. WT or Bad−/− MEF were subjected to the indicated treatments, followed by immunoprecipitation of Bcl‐2 and immunobloting of Beclin‐1.(H) Overexpression of Bad triggers autophagy. Cells were transfected with LC3‐GFP alone (Control), together with vector‐only or with a vector encoding Bad, and subjected 48 h later to nutrient depletion (NF). Cells were cultured in the continuous presence or absence of the pan‐caspase inhibitor Z‐VAD‐fmk (50 μM) for 16 h, followed by assessment of autophagy as in (E) (means±s.d., n=3 separate experiments, *P0.01). Immunoblots are representative of at least three independent experiments.


Figure 8 ( A ) Representative images of wt and egl ‐ 1 mutant embryos , bearing the plgg − 1DsRED ( B ) Quantification of LGG ‐ 1 ∷ DsRED fluorescence as determined in ( A ) . Each point represents the measurement for one individual embryo . Horizontal bars denote means and gray areas denote standard errors .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8(A) Representative images of wt and egl‐1 mutant embryos, bearing the plgg−1DsRED(B) Quantification of LGG‐1∷DsRED fluorescence as determined in (A). Each point represents the measurement for one individual embryo. Horizontal bars denote means and gray areas denote standard errors.


Figure 1A 293FT cells were transfected with 3xFlag - Usp27x ( pFCMV7 . 1 vector backbone ) together with a construct driving expression of untagged BimEL or 3xHA - BimEL ( both pMIG vector backbone ) . Cells were lysed , and 3xHA - BimEL was immunoprecipitated with anti - HA - antibodies . IP products were tested by Western blotting for the presence of Bim and FLAG - Usp27x probing with antibodies against Bim or against the FLAG - peptide . See also Appendix Fig . S1B . Western blots show representative of n ≥ 3 independent experiments . B Usp27x binds a mutant of Bim incapable of binding to anti - apoptotic Bcl - 2 - proteins . 293FT cells transfected with constructs encoding 3xFLAG - Usp27x and 3xHA - tagged BimEL ( see A , 2µg each ) or 3xHA - tagged BimEL ∆ ∆ ( a mutant with two mutations in the BH3 - domain , incapable of binding antiapoptotic Bcl - 2proteins [ 50 ] ) were immunoprecipitated from whole cell extracts using anti - HA - resin . Bim and Usp27x were detected with anti - HA - and anti - FLAG - antibodies as indicated ; antiapoptotic proteins : Mcl - 1 , Bcl - XL , Bcl - 2 . The caspase - inhibitor Q - VD - OPh ( QVD ) was added to the cultures described in A and B to inhibit Bim - induced apoptosis . Western blots are representative of n = 3 independent experiments . C Usp27x interacts with endogenous BimEL independently of its catalytic activity293FT cells either carrying 3xFlag - Usp27x ( 293FT - TetR - 3xFlag - usp27x ) or the catalytic inactive mutant 3xFlag - Usp27xC87A under the control of the Tet - repressor were treated for 24h with dox to induce expression of Usp27x or Usp27xC87A . In all conditions PMA ( to induce Bim - ubiquitination , 16 . 2 nM ) , and Q - VD - OPh ( to inhibit apoptosis , 10 µM , see Fig . 3 ) were added at the time of Usp27x - induction . MG132 ( to prevent Bim - degradation , 40 µM ) was added in all conditions 4h prior to cell lysis . 3xFlag tagged Usp27x or Usp27xC87A was immunoprecipitated from whole cell lysates using anti - Flag resin . Interaction with BimEL or β - TrCP was detected by Western blotting using anti - Bim or anti - β - TrCP antibodies . Western blots are representative of n ≥ 3 independent experiments . D Usp27x expression does not inhibit interaction of BimEL to β - TrCP293FT - TetR - 3xFlag - Usp27x cells were transfected with pMIG - 3xHA - BimEL in the presence of both PMA and QVD . At the same time dox ( to induce 3xFlag - Usp27x ) was added as indicated and 20h later cells were treated with MG132 [ 40µM ] for additional 4h . Cells were lysed and 3xHA - BimEL was immunoprecipitated as described above . As a control HA - matrix was used without lysate to rule out any unspecific signal that might come from the immobilized anti HA - antibody ( beads ) . Western blots show representative of n = 2 independent experiments . E Binding of Usp27x to BimEL can be blocked by the MEK - inhibitor UO126293FT - TetR - 3xFlag - Usp27x cells ( see C ) were treated for 24h with dox to induce expression of Usp27x . In all conditions PMA [ 16 . 2 nM ] , and Q - VD - OPh [ 10 µM ] were added at the time of Usp27x - induction . As a control , cells were pretreated with UO126 [ 10µM ] for 30 minutes to block the PMA stimulated ERK pathway before the addition of dox plus PMA plus QVD . 3xFlag tagged Usp27x was immuno - precipitated from whole cell lysates using anti - Flag resin . Interaction with BimEL was detected by Western blotting using anti - Bim antibodies . As a control Flag - matrix ( beads ) was used alongside the IP under same conditions but without addition of protein lysates . Western blots are representative of n = 4 independent experiments ( see also Appendix Fig . S2B ) . F Binding of Usp27xto BimEL does not require β - TrCP293FT - TetR - 3xFlag - Usp27x cells were transfected with control siRNA ( siCo3 ) or siRNA specific for β - TrCP . 48h later cells were stimulated with PMA plus dox plus QVD for additional 24h . After cell lysis , Flag - Usp27x was immuno - precipitated using anti - Flag - matrix , and BimEL and β - TrCP bound to Usp27x were identified by Western blotting . Blots are representative of n = 2 independent experiments . G Usp27x binds specifically to BimEL293FT - TetR - 3xFlag - Usp27x cells or 293FT - 3xFlag - Usp27x cells with a specific deletion of the BimELprotein were treated with dox , PMA and QVD as in C . Cells were lysed , and lysates were subjected to anti - Flagimmunoprecipitation . 3xFlag - Usp27x was detected using anti - FLAG antibodies . Blots are representative of n = 3 independent experiments .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A 293FT cells were transfected with 3xFlag-Usp27x (pFCMV7.1 vector backbone) together with a construct driving expression of untagged BimEL or 3xHA-BimEL(both pMIG vector backbone). Cells were lysed, and 3xHA-BimEL was immunoprecipitated with anti-HA-antibodies. IP products were tested by Western blotting for the presence of Bim and FLAG-Usp27x probing with antibodies against Bim or against the FLAG-peptide. See also Appendix Fig. S1B. Western blots show representative of n≥3 independent experiments.B Usp27x binds a mutant of Bim incapable of binding to anti-apoptotic Bcl-2-proteins. 293FT cells transfected with constructs encoding 3xFLAG-Usp27x and 3xHA-tagged BimEL (see A, 2µg each) or 3xHA-tagged BimEL∆∆(a mutant with two mutations in the BH3-domain, incapable of binding antiapoptotic Bcl-2proteins [50]) were immunoprecipitated from whole cell extracts using anti-HA-resin. Bim and Usp27x were detected with anti-HA- and anti-FLAG-antibodies as indicated; antiapoptotic proteins: Mcl-1, Bcl-XL, Bcl-2. The caspase-inhibitor Q-VD-OPh (QVD) was added to the cultures described in A and B to inhibit Bim-induced apoptosis. Western blots are representative of n=3 independent experiments.C Usp27x interacts with endogenous BimEL independently of its catalytic activity293FT cells either carrying 3xFlag-Usp27x (293FT-TetR-3xFlag-usp27x) or the catalytic inactive mutant 3xFlag-Usp27xC87A under the control of the Tet-repressor were treated for 24h with dox to induce expression of Usp27x or Usp27xC87A. In all conditions PMA (to induce Bim-ubiquitination, 16.2 nM), and Q-VD-OPh (to inhibit apoptosis, 10 µM, see Fig. 3) were added at the time of Usp27x-induction. MG132 (to prevent Bim-degradation, 40 µM) was added in all conditions 4h prior to cell lysis. 3xFlag tagged Usp27x or Usp27xC87A was immunoprecipitated from whole cell lysates using anti-Flag resin. Interaction with BimEL or β-TrCP was detected by Western blotting using anti-Bim or anti-β-TrCP antibodies. Western blots are representative of n≥3 independent experiments.D Usp27x expression does not inhibit interaction of BimEL to β-TrCP293FT-TetR-3xFlag-Usp27x cells were transfected with pMIG-3xHA-BimEL in the presence of both PMA and QVD. At the same time dox (to induce 3xFlag-Usp27x) was added as indicated and 20h later cells were treated with MG132 [40µM] for additional 4h.Cells were lysed and 3xHA-BimEL was immunoprecipitated as described above. As a control HA-matrix was used without lysate to rule out any unspecific signal that might come from the immobilized anti HA-antibody (beads). Western blots show representative of n=2 independent experiments.E Binding of Usp27x to BimEL can be blocked by the MEK-inhibitor UO126293FT-TetR-3xFlag-Usp27x cells (see C) were treated for 24h with dox to induce expression of Usp27x. In all conditions PMA [16.2 nM], and Q-VD-OPh [10 µM] were added at the time of Usp27x-induction. As a control, cells were pretreated with UO126 [10µM] for 30 minutes to block the PMA stimulated ERK pathway before the addition of dox plus PMA plus QVD. 3xFlag tagged Usp27x was immuno-precipitated from whole cell lysates using anti-Flag resin. Interaction with BimEL was detected by Western blotting using anti-Bim antibodies. As a control Flag-matrix (beads) was used alongside the IP under same conditions but without addition of protein lysates. Western blots are representative of n=4 independent experiments (see also Appendix Fig. S2B).F Binding of Usp27xto BimEL does not require β-TrCP293FT-TetR-3xFlag-Usp27x cells were transfected with control siRNA (siCo3) or siRNA specific for β-TrCP. 48h later cells were stimulated with PMA plus dox plus QVD for additional 24h. After cell lysis, Flag-Usp27x was immuno-precipitated using anti-Flag-matrix, and BimEL and β-TrCP bound to Usp27x were identified by Western blotting. Blots are representative of n=2 independent experiments.G Usp27x binds specifically to BimEL293FT-TetR-3xFlag-Usp27x cells or 293FT-3xFlag-Usp27x cells with a specific deletion of the BimELprotein were treated with dox, PMA and QVD as in C. Cells were lysed, and lysates were subjected to anti-Flagimmunoprecipitation. 3xFlag-Usp27x was detected using anti-FLAG antibodies. Blots are representative of n=3 independent experiments.


Figure 2A Usp27x inhibits the PMA - induced degradation of BimEL in 293FT cells . 293FT - TetR - 3xFlagUsp27x or TetR - 3xFlag - Usp27xC87A cells were treated with dox to induce 3xFlag - Usp27x or Usp27xC87A , or with PMA [ 16 . 2 nM ] ( to induce BimEL degradation ) , or with the combination of dox , PMA and QVD [ 10µM ] ( to inhibit apoptosis ) as indicated for 24 h ( left ) or 48 h ( right ) . Cell lysates were analysed by Western blotting . The blots are representative of n = 4 similar experiments . See also Appendix Fig . S5B . * Probably modified form of wild - type 3xFlag - Usp27x ( such as dimer , ubiquitinated Usp27x or a stable complex of Usp27x with another interacting partner ) ; this form is only seen if high expression levels of catalytically active Usp27x are reached ( see Appendix Fig . S5A where Usp27x was transfected into 293FT cells ) or if wt Usp27x is enriched during immunoprecipitation . * * Probably degradation product of 3xFlag - Usp27x . These forms of Usp27x were not seen in every experiment . B 293FT - TetR - 3xFlagUsp27x or TetR - 3xFlag - Usp27xC87A cells treated as in A were analysed using conditions of higher resolution . A band very likely corresponding to phosphorylated BimEL is detectable . Similar results were obtained in n = 2 separate experiments . C Usp27x stabilizes expression of p - BimEL ( Ser69 ) in Bim - degrading conditions without reducing β - TrCP levels293FT - TetR - 3xFlagUsp27x were treated with dox , PMA [ 16 . 2 nM ] , UO126 [ 10µM ] or QVD [ 10µM ] as indicated for 24h , and levels of phosphorylated BimEL at serine69 were analysed by Western blotting using a phospho - Bim ( Ser69 ) specific antibody . Scans are from the same membrane and exposure times . Similar results were obtained in n = 2 separate experiments . D UO126 reduces phosphorylation - associated shift of BimEL293FT - TetR - 3xFlagUsp27x were treated for 24h with the indicated drug combinations and Bim was detected by Western blotting . UO126 was added 30 minutes prior other stimulation . Similar results were obtained in n = 3 separate experiments ( see Appendix Fig . S5D ) . E Usp27x - expression reduces ubiquitination of Bim293FT - TetR - 3xFlagUsp27x cells were transfected with a vector coding for 6His - ubiquitin ( left ) or GFP ( right ) . After 24h cells were treated with PMA and QVD , and 3xFlag - Usp27x was induced by dox at the same time . After additional 20h cells were treated for 4h with MG132 ( 40 µM ) to block proteasomal degradation of ubiquitinated proteins . Cells were lysed under denaturing conditions and His - ubiquitin - labelled proteins or proteins from only GFP expressing cells were purified by Ni2 + - NTAaffinity chromatography . Ubiquitinated Bim ( Bim - Ubn ) was detected by Western blot using anti - Bim antibodies ( left ) . Similar results were obtained in n = 3 separate experiments ( another experiment including a Coomassie stain is shown in Appendix Fig . S3E ) . * Indicates non - His - ubiquitinated BimEL that was bound unspecifically to the Ni2 + - agarose

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A Usp27x inhibits the PMA-induced degradation of BimEL in 293FT cells. 293FT-TetR-3xFlagUsp27x or TetR-3xFlag-Usp27xC87A cells were treated with dox to induce 3xFlag-Usp27x or Usp27xC87A, or with PMA [16.2 nM] (to induce BimEL degradation), or with the combination of dox, PMA and QVD [10µM] (to inhibit apoptosis) as indicated for 24 h (left) or 48 h (right). Cell lysates were analysed by Western blotting. The blots are representative of n=4 similar experiments. See also Appendix Fig. S5B. * Probably modified form of wild-type 3xFlag-Usp27x (such as dimer, ubiquitinated Usp27x or a stable complex of Usp27x with another interacting partner); this form is only seen if high expression levels of catalytically active Usp27x are reached (see Appendix Fig. S5A where Usp27x was transfected into 293FT cells) or if wt Usp27x is enriched during immunoprecipitation.** Probably degradation product of 3xFlag-Usp27x. These forms of Usp27x were not seen in every experiment.B 293FT-TetR-3xFlagUsp27x or TetR-3xFlag-Usp27xC87A cells treated as in A were analysed using conditions of higher resolution. A band very likely corresponding to phosphorylated BimEL is detectable. Similar results were obtained in n=2 separate experiments.C Usp27x stabilizes expression of p-BimEL (Ser69) in Bim-degrading conditions without reducing β-TrCP levels293FT-TetR-3xFlagUsp27x were treated with dox, PMA [16.2 nM], UO126 [10µM] or QVD [10µM] as indicated for 24h, and levels of phosphorylated BimEL at serine69 were analysed by Western blotting using a phospho-Bim (Ser69) specific antibody. Scans are from the same membrane and exposure times. Similar results were obtained in n=2 separate experiments.D UO126 reduces phosphorylation-associated shift of BimEL293FT-TetR-3xFlagUsp27x were treated for 24h with the indicated drug combinations and Bim was detected by Western blotting. UO126 was added 30 minutes prior other stimulation. Similar results were obtained in n=3 separate experiments (see Appendix Fig. S5D).E Usp27x-expression reduces ubiquitination of Bim293FT-TetR-3xFlagUsp27x cells were transfected with a vector coding for 6His-ubiquitin (left) or GFP (right). After 24h cells were treated with PMA and QVD, and 3xFlag-Usp27x was induced by dox at the same time. After additional 20h cells were treated for 4h with MG132 (40 µM) to block proteasomal degradation of ubiquitinated proteins. Cells were lysed under denaturing conditions and His-ubiquitin-labelled proteins or proteins from only GFP expressing cells were purified by Ni2+-NTAaffinity chromatography. Ubiquitinated Bim (Bim-Ubn) was detected by Western blot using anti-Bim antibodies (left). Similar results were obtained in n=3 separate experiments (another experiment including a Coomassie stain is shown in Appendix Fig. S3E). *Indicates non-His-ubiquitinated BimEL that was bound unspecifically to the Ni2+-agarose beads


Figure 3A 293FT - TetR - 3xFlag - Usp27x or 3xFlag - Usp27xC87A , a polyclonal derivative where the Bim - locus had been targeted by CRISPR / Cas9 - and four single clones obtained by serial dilution from the polyclonal line were treated as indicated ( PMA [ 16 . 2 nM ] , QVD [ 10 µM ] ) . Apoptosis was measured after 24 h of treatment by staining for active caspase - 3 , followed by flow cytometric analysis . Data ( means / SEM ) are from n = 14 ( 3xFlag - Usp27x line and 3xFlag - Usp27xBim2KO polyclonal line ) , n = 5 ( 3xFlag - Usp27xC87A , and 3xFlag - Usp27xBim2KO clone # 14 ) , n = 7 ( 3xFlag - Usp27xBim2KO clone # 11 ) or n = 4 ( 3xFlag - Usp27xBim2KO clone # 4 and # 7 ) separate experiments . P - values ( t - test ) for statistically significant differences are shown . B The same cells as in A ( except Usp27xC87A mutant ) were treated as in A and stained for active Bax . Data ( means / SEM ) are from n = 8 ( 3xFlag - Usp27x line and 3xFlag - Usp27xBim2KO polyclonal line ) , n = 5 ( 3xFlag - Usp27xBim2KO clone # 11 and # 14 ) or n = 4 ( 3xFlag - Usp27xBim2KO clone # 4 and # 7 ) separate experiments . P - values ( t - test ) for statistically significant differences are shown . Expression levels of Bim , β - TrCP and Flag - Usp27x of the different cell lines are shown in Appendix Fig . S5E .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A 293FT-TetR-3xFlag-Usp27x or 3xFlag-Usp27xC87A, a polyclonal derivative where the Bim-locus had been targeted by CRISPR/Cas9- and four single clones obtained by serial dilution from the polyclonal line were treated as indicated (PMA [16.2 nM], QVD [10 µM]). Apoptosis was measured after 24 h of treatment by staining for active caspase-3, followed by flow cytometric analysis. Data (means/SEM) are from n=14 (3xFlag-Usp27x line and 3xFlag-Usp27xBim2KO polyclonal line), n=5 (3xFlag-Usp27xC87A, and 3xFlag-Usp27xBim2KO clone #14), n=7 (3xFlag-Usp27xBim2KO clone #11) or n=4 (3xFlag-Usp27xBim2KO clone #4 and #7) separate experiments. P-values (t-test) for statistically significant differences are shown.B The same cells as in A (except Usp27xC87A mutant) were treated as in A and stained for active Bax. Data (means/SEM) are from n=8 (3xFlag-Usp27x line and 3xFlag-Usp27xBim2KO polyclonal line), n=5 (3xFlag-Usp27xBim2KO clone #11 and #14) or n=4 (3xFlag-Usp27xBim2KO clone #4 and #7) separate experiments. P-values (t-test) for statistically significant differences are shown. Expression levels of Bim, β-TrCP and Flag-Usp27x of the different cell lines are shown in Appendix Fig. S5E.