C . Analysis of IDH1 activity in HEK293T cells treated with the indicated concentration of tubacin ( n = 6 - 9 tests from three independent experiments ) . D . Analysis of the α - KG level in HEK293T cells treated with the indicated concentration of tubacin ( n = 4 independent experiments ) . Data information : All values are presented as mean ± SEM . * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 ANOVA

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C. Analysis of IDH1 activity in HEK293T cells treated with the indicated concentration of tubacin (n = 6-9 tests from three independent experiments). D. Analysis of the α-KG level in HEK293T cells treated with the indicated concentration of tubacin (n = 4 independent experiments). Data information: All values are presented as mean ± SEM. **P < 0.01, ***P < 0.001, ****P < 0.0001 ANOVA test


Immunoblotting ( E ) in HEK293T cells transfected with plasmids expressing GFP , GFP - IDH1 , GFP - IDH1 K233R , or GFP - IDH1 K233Q . In panel E , the black arrowhead indicates exogenous IDH1 , while the white arrowhead marks endogenous IDH1 .

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Immunoblotting (E) in HEK293T cells transfected with plasmids expressing GFP, GFP-IDH1, GFP-IDH1 K233R, or GFP-IDH1 K233Q. In panel E, the black arrowhead indicates exogenous IDH1, while the white arrowhead marks endogenous IDH1.


G - H . Immunoblotting ( G ) and IDH1 activity analysis ( H , n = 6 - 9 tests from three independent experiments ) in HEK293T cells transfected with control or HDAC6 siRNAs and plasmids expressing GFP , GFP - IDH1 , GFP - IDH1 K233R , or GFP - IDH1 K233Q . In panel G , the black arrowhead indicates exogenous IDH1 , while the white arrowhead marks endogenous IDH1 . Data information : All values are presented as mean ± SEM . * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 ANOVA

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G-H. Immunoblotting (G) and IDH1 activity analysis (H, n = 6-9 tests from three independent experiments) in HEK293T cells transfected with control or HDAC6 siRNAs and plasmids expressing GFP, GFP-IDH1, GFP-IDH1 K233R, or GFP-IDH1 K233Q. In panel G, the black arrowhead indicates exogenous IDH1, while the white arrowhead marks endogenous IDH1. Data information: All values are presented as mean ± SEM. **P < 0.01, ***P < 0.001, ****P < 0.0001 ANOVA test


Coomassie blue staining ( I ) of the purified proteins .

Coomassie blue staining (I) of the purified proteins.


K . Representative images of the colony formation assay . Scale bar , 200 µm . L . Number of colonies formed by wild - type and Hdac6 knockout mice bone marrow cells treated or untreated with 10 µM GSK321 ( n = 6 mice per group ) . Data information : All values are presented as mean ± SEM . * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 ANOVA

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K. Representative images of the colony formation assay. Scale bar, 200 µm. L. Number of colonies formed by wild-type and Hdac6 knockout mice bone marrow cells treated or untreated with 10 µM GSK321 (n = 6 mice per group). Data information: All values are presented as mean ± SEM. **P < 0.01, ***P < 0.001, ****P < 0.0001 ANOVA test


E . IGV screenshots showing elevated hydroxymethylation of multiple candidate genes associated with negative regulation of the cell cycle .

E. IGV screenshots showing elevated hydroxymethylation of multiple candidate genes associated with negative regulation of the cell cycle.


F . IGV screenshots of TET2 - independent genes showing no significant changes in hydroxymethylation .

F. IGV screenshots of TET2-independent genes showing no significant changes in hydroxymethylation.


Heatmaps based on the GO enrichment analysis showing genes that negatively regulate the cell cycle .

Heatmaps based on the GO enrichment analysis showing genes that negatively regulate the cell cycle.


qPCR showing that knockdown of HDAC6 significantly upregulates the expression of TET2 target genes with hyper - hydroxymethylation . Data information : All values are presented as mean ± SEM . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 , ns , not significant ( ANOVA test ) .

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qPCR showing that knockdown of HDAC6 significantly upregulates the expression of TET2 target genes with hyper-hydroxymethylation. Data information: All values are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, not significant (ANOVA test).


qPCR showing that knockdown of HDAC6 significantly upregulates the expression of TET2 target genes with hyper - hydroxymethylation . qPCR showing that treatment with 10 µM GSK321 inhibits the upregulation of the indicated genes in HDAC6 - knockdown HEK293T cells . Data information : All values are presented as mean ± SEM . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 , ns , not significant ( ANOVA test ) .

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qPCR showing that knockdown of HDAC6 significantly upregulates the expression of TET2 target genes with hyper-hydroxymethylation. qPCR showing that treatment with 10 µM GSK321 inhibits the upregulation of the indicated genes in HDAC6-knockdown HEK293T cells. Data information: All values are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, not significant (ANOVA test).


qPCR showing that treatment with 10 µM GSK321 inhibits the upregulation of the indicated genes in HDAC6 - knockdown HEK293T cells . Data information : All values are presented as mean ± SEM . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 , ns , not significant ( ANOVA test ) .

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qPCR showing that treatment with 10 µM GSK321 inhibits the upregulation of the indicated genes in HDAC6-knockdown HEK293T cells. Data information: All values are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, not significant (ANOVA test).


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 . ) .

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.).


( B ) Immunoblot analysis of whole cell samples of the HA - tagged full - length ( FL ) IFT54 , and its deletion mutants as indicated , each expressed in the ift54 null mutant ; wild type ( WT ) and ift54 mutant cells were used as control . Anti - HA was use to visualize the transgenic proteins , antibodies to FLA8 / KIF3B were used as a loading control .

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(B) Immunoblot analysis of whole cell samples of the HA-tagged full-length (FL) IFT54, and its deletion mutants as indicated, each expressed in the ift54 null mutant; wild type (WT) and ift54 mutant cells were used as control. Anti-HA was use to visualize the transgenic proteins, antibodies to FLA8/KIF3B were used as a loading control.


( C ) Histogram showing ciliary length distribution in populations of ift54 mutant cells transformed with the deletion mutant constructs as indicated . Wild type ( WT ) cells were used as a control .

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(C) Histogram showing ciliary length distribution in populations of ift54 mutant cells transformed with the deletion mutant constructs as indicated. Wild type (WT) cells were used as a control.


( D ) Representative DIC images of wild type ( WT ) , ift54 and ift54 cells transformed with the deletion constructs as indicated . Arrows indicate cilia bulges . Scale bar , 5 μm .

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(D) Representative DIC images of wild type (WT), ift54 and ift54 cells transformed with the deletion constructs as indicated. Arrows indicate cilia bulges. Scale bar, 5 μm.


( A ) Immunostaining of IFT54 mutated proteins in various deletion mutants as indicated ; wild - type ( WT ) cells were used as control . Specimens were stained with anti - HA and anti - α - tubulin antibodies followed by imaging using an epifluorescence microscope . Arrows indicate protein accumulation at ciliary tip ( left ) or proximal end of cilia ( right ) . Scale bars , 5 μm .

(A) Immunostaining of IFT54 mutated proteins in various deletion mutants as indicated; wild-type (WT) cells were used as control. Specimens were stained with anti-HA and anti-α-tubulin antibodies followed by imaging using an epifluorescence microscope. Arrows indicate protein accumulation at ciliary tip (left) or proximal end of cilia (right). Scale bars, 5 μm.


( B , C ) Immunostaining analysis of IFT43 and IFT38 in the deletion mutants as indicated . Cells were fixed and stained with antibodies against IFT43 ( an IFT - A subunit ) ( B ) or against IFT38 ( an IFT - B subunit ) ( C ) followed by imaging using both DIC and epifluorescence microscopy . IFT43 and IFT38 accumulated at the ciliary tip in IFT54Δ261 - 275 mutant but at proximal end of cilia in IFT54Δ342 - 356 mutant . Arrows indicate ciliary bulges . WT , wild - type cells . Scale bars , 5 μm .

(B, C) Immunostaining analysis of IFT43 and IFT38 in the deletion mutants as indicated. Cells were fixed and stained with antibodies against IFT43 (an IFT-A subunit) (B) or against IFT38 (an IFT-B subunit) (C) followed by imaging using both DIC and epifluorescence microscopy. IFT43 and IFT38 accumulated at the ciliary tip in IFT54Δ261-275 mutant but at proximal end of cilia in IFT54Δ342-356 mutant. Arrows indicate ciliary bulges. WT, wild-type cells. Scale bars, 5 μm.


( A ) Immunostaining of cells with antibody against IFT dynein subunit D1bLIC . D1bLIC is absent in the cilia bulges at the ciliary tip in IFT54Δ261 - 275 mutant while enriched at the cilia bulges at proximal end of cilia in IFT54Δ342 - 356 mutant . WT , wild type cells . Arrows indicate ciliary bulges . Scale bar , 5 μm . ( B ) Immunostaining of cells with antibody against kinesin - II subunit FLA10 . FLA10 is enriched in cilia bulges at the ciliary tip in IFT54Δ261 - 275 mutant or at proximal end of cilia in IFT54Δ342 - 356 mutant . WT , wild type cells . The insets show enhanced and / or enlarged images . Arrows indicate ciliary bulges . Scale bar , 5 μm .

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(A) Immunostaining of cells with antibody against IFT dynein subunit D1bLIC. D1bLIC is absent in the cilia bulges at the ciliary tip in IFT54Δ261-275 mutant while enriched at the cilia bulges at proximal end of cilia in IFT54Δ342-356 mutant. WT, wild type cells. Arrows indicate ciliary bulges. Scale bar, 5 μm. (B) Immunostaining of cells with antibody against kinesin-II subunit FLA10. FLA10 is enriched in cilia bulges at the ciliary tip in IFT54Δ261-275 mutant or at proximal end of cilia in IFT54Δ342-356 mutant. WT, wild type cells. The insets show enhanced and/or enlarged images. Arrows indicate ciliary bulges. Scale bar, 5 μm.


( C , D ) Immunoblot analysis of whole cells ( C ) and isolated cilia ( D ) of wild type and the three IFT54 deletion mutants . The membranes were probed with antibodies against IFT complex and motor proteins as indicated . Wild - type levels of the various IFT components were detected in the whole cell samples of the deletion strains ( C ) . IFT121 , IFT38 and FLA8 accumulated in cilia of both IFT54Δ261 - 275 and IFT54Δ342 - 356 mutants . D1bLIC was relatively reduced in IFT54Δ261 - 275 mutant cilia but was substantially increased in IFT54Δ342 - 356 mutant cilia ( D ) . Ratios of protein amounts from one representative experiment are in parentheses .

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(C, D) Immunoblot analysis of whole cells (C) and isolated cilia (D) of wild type and the three IFT54 deletion mutants. The membranes were probed with antibodies against IFT complex and motor proteins as indicated. Wild-type levels of the various IFT components were detected in the whole cell samples of the deletion strains (C). IFT121, IFT38 and FLA8 accumulated in cilia of both IFT54Δ261-275 and IFT54Δ342-356 mutants. D1bLIC was relatively reduced in IFT54Δ261-275 mutant cilia but was substantially increased in IFT54Δ342-356 mutant cilia (D). Ratios of protein amounts from one representative experiment are in parentheses.


( A ) Kymograms showing the trajectories of IFT trains inside cilia as visualized with IFT43 - YFP . An ift43 rescue strain expressing IFT43 - YFP ( as a control ) and the IFT54Δ342 - 356 mutant expressing IFT43 - YFP were analyzed by live imaging via TIRF microscopy .

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(A) Kymograms showing the trajectories of IFT trains inside cilia as visualized with IFT43-YFP. An ift43 rescue strain expressing IFT43-YFP (as a control) and the IFT54Δ342-356 mutant expressing IFT43-YFP were analyzed by live imaging via TIRF microscopy.


( B - C ) Velocities ( B ) and frequencies ( C ) of anterograde and retrograde IFT of IFT43 - YFP in control and the IFT54Δ342 - 356 mutant . n represents the number of cilia assayed from three independent experiments . Values show the mean ± S . D . Unpaired two - tailed Student ' s t test analysis , * * * , p < 0 . 0001 .

(B-C) Velocities (B) and frequencies (C) of anterograde and retrograde IFT of IFT43-YFP in control and the IFT54Δ342-356 mutant. n represents the number of cilia assayed from three independent experiments. Values show the mean ± S.D. Unpaired two-tailed Student's t test analysis, ***, p<0.0001.


( D ) Deletion of residues 342 - 356 of IFT54 increases its interaction with kinesin - II . Cell extracts from wild type ( WT ) , ift54 expressing HA - tagged full - length ( FL ) IFT54 or IFT54Δ342 - 356 mutant were subjected to immunoprecipitation with anti - HA antibody followed by immunoblotting with the indicated antibodies . The normalized ratios of FLA8 and FLA10 in the immunoprecipitates ( IFT54 versus the mutant ) are 1 : 3 . 90 and 1 : 3 . 13 , respectively .

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(D) Deletion of residues 342-356 of IFT54 increases its interaction with kinesin-II. Cell extracts from wild type (WT), ift54 expressing HA-tagged full-length (FL) IFT54 or IFT54Δ342-356 mutant were subjected to immunoprecipitation with anti-HA antibody followed by immunoblotting with the indicated antibodies. The normalized ratios of FLA8 and FLA10 in the immunoprecipitates (IFT54 versus the mutant) are 1:3.90 and 1:3.13, respectively.


( E ) ATP treatment induces dissociation of IFT54 but not IFT54Δ342 - 356 mutant from kinesin - II . Immunoprecipitation experiments were performed as shown in ( D ) in the presence or absence of ATP . The normalized ratios of FLA8 and FLA10 in the immunoprecipitates ( IFT54 without ATP / with ATP / mutant without ATP / with ATP ) are 1 : 0 . 37 : 1 . 70 : 1 . 38 and 1 : 0 . 35 : 1 . 47 : 1 . 45 , respectively .

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(E) ATP treatment induces dissociation of IFT54 but not IFT54Δ342-356 mutant from kinesin-II. Immunoprecipitation experiments were performed as shown in (D) in the presence or absence of ATP. The normalized ratios of FLA8 and FLA10 in the immunoprecipitates (IFT54 without ATP/with ATP/mutant without ATP/with ATP) are 1:0.37:1.70:1.38 and 1:0.35:1.47:1.45, respectively.


( F ) Analysis of the interaction of IFT54 and IFT54Δ342 - 356 mutant with kinesin - II by GST - pulldown assay . Bacterial expressed GST , GST - tagged IFT54 or IFT54Δ342 - 356 mutant was mixed respectively with recombinant kinesin - II purified from insect cells in the presence or absence of ATP followed by GST pulldown and immunoblotting . The normalized ratios of KAP , FLA10 and FLA8 in the pull - down samples ( GST - IFT54 without ATP / with ATP / mutant without ATP / with ATP ) are 1 : 0 . 51 : 1 . 31 : 1 . 44 , 1 : 0 . 55 : 1 . 29 : 1 . 19 and 1 : 0 . 50 : 1 . 31 : 1 . 29 , respectively .

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(F) Analysis of the interaction of IFT54 and IFT54Δ342-356 mutant with kinesin-II by GST-pulldown assay. Bacterial expressed GST, GST-tagged IFT54 or IFT54Δ342-356 mutant was mixed respectively with recombinant kinesin-II purified from insect cells in the presence or absence of ATP followed by GST pulldown and immunoblotting. The normalized ratios of KAP, FLA10 and FLA8 in the pull-down samples (GST-IFT54 without ATP/with ATP/mutant without ATP/with ATP) are 1:0.51:1.31:1.44, 1:0.55:1.29:1.19 and 1:0.50:1.31:1.29, respectively.


( G ) Interaction of IFT54 with kinesin - II is conserved in mammalian cells . Cell extracts from HEK293T cells expressing eGFP , eGFP tagged TRAF3IP1 or TRAF3IP1Δ535 - 549 ( corresponding deletion mutant of IFT54Δ342 - 356 ) were analyzed by immunoprecipitation with GFP antibody followed by immunoblotting with anti - GFP and KIF3A antibodies , respectively . Please note , the deletion mutant had an increased interaction with kinesin - II subunit KIF3A relative to the control . The normalized ratio of KIF3A in the immunoprecipitates ( TRAF3IP1 versus mutant ) is 1 : 1 . 76 .

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(G) Interaction of IFT54 with kinesin-II is conserved in mammalian cells. Cell extracts from HEK293T cells expressing eGFP, eGFP tagged TRAF3IP1 or TRAF3IP1Δ535-549 (corresponding deletion mutant of IFT54Δ342-356) were analyzed by immunoprecipitation with GFP antibody followed by immunoblotting with anti-GFP and KIF3A antibodies, respectively. Please note, the deletion mutant had an increased interaction with kinesin-II subunit KIF3A relative to the control. The normalized ratio of KIF3A in the immunoprecipitates (TRAF3IP1 versus mutant) is 1:1.76.


( A ) Kymograms showing the trajectories of IFT trains inside cilia as visualized with D1bLIC - YFP . Wild - type ( WT ) and IFT54Δ261 - 275 mutant cells expressing D1bLIC - YFP were analyzed by live imaging via TIRF microscopy .

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(A) Kymograms showing the trajectories of IFT trains inside cilia as visualized with D1bLIC-YFP. Wild-type (WT) and IFT54Δ261-275 mutant cells expressing D1bLIC-YFP were analyzed by live imaging via TIRF microscopy.


( B , C ) Velocities ( B ) and frequencies ( C ) of anterograde and retrograde IFT of D1bLIC - YFP . n represents the number of cilia assayed from four independent experiments . Values show the mean ± S . D . Unpaired two - tailed Student ' s t test analysis , n . s . : no significance ; * * * , p < 0 . 0001 .

(B, C) Velocities (B) and frequencies (C) of anterograde and retrograde IFT of D1bLIC-YFP. n represents the number of cilia assayed from four independent experiments. Values show the mean ± S.D. Unpaired two-tailed Student's t test analysis, n.s.: no significance; ***, p<0.0001.


( D ) Kymograms of IFT43 - YFP of WT and mutant cilia .

(D) Kymograms of IFT43-YFP of WT and mutant cilia.


( E , F ) Velocities ( E ) and frequencies ( F ) of anterograde and retrograde IFT of IFT43 - YFP . n represents the number of cilia assayed from three independent experiments . Values show the mean ± S . D . Unpaired two - tailed Student ' s t test analysis , n . s . : no significance ; * * * , p < 0 . 0001 .

(E, F) Velocities (E) and frequencies (F) of anterograde and retrograde IFT of IFT43-YFP. n represents the number of cilia assayed from three independent experiments. Values show the mean ± S.D. Unpaired two-tailed Student's t test analysis, n.s.: no significance; ***, p<0.0001.


( G ) Yeast two hybrid assay for interaction between IFT54 and IFT dynein subunits . Yeast cells that were transformed with each pair of constructs as indicated were grown under selection media lacking leucine , tryptophan , histidine , and adenine ( - 4 ) or lacking leucine and tryptophan ( - 2 ) . DHC1bT , DHC1b tail domain ; Empty AD or BD vectors were used as control .

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(G) Yeast two hybrid assay for interaction between IFT54 and IFT dynein subunits. Yeast cells that were transformed with each pair of constructs as indicated were grown under selection media lacking leucine, tryptophan, histidine, and adenine (-4) or lacking leucine and tryptophan (-2). DHC1bT, DHC1b tail domain; Empty AD or BD vectors were used as control.


( H ) Interaction of IFT54 and IFT54Δ261 - 275 with D1bLIC by GST - pulldown assay . Bacterial expressed GST , GST - IFT54 or GST - IFT54Δ261 - 275 were mixed respectively with His - D1bLIC followed by GST pulldown and immunoblotting with anti - GST and anti - His antibodies . The normalized ratio of His - D1bLIC in the pull - down samples ( GST - IFT54 versus mutant ) is 1 : 0 . 32 .

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(H) Interaction of IFT54 and IFT54Δ261-275 with D1bLIC by GST-pulldown assay. Bacterial expressed GST, GST-IFT54 or GST-IFT54Δ261-275 were mixed respectively with His-D1bLIC followed by GST pulldown and immunoblotting with anti-GST and anti-His antibodies. The normalized ratio of His-D1bLIC in the pull-down samples (GST-IFT54 versus mutant) is 1:0.32.


( I ) Co - immunoprecipitation of IFT54 and IFT54Δ261 - 275 with D1bLIC . Cell extracts from wild - type ( WT ) , ift54 expressing HA - tagged full - length ( FL ) IFT54 or IFT54Δ261 - 275 mutant were subjected to immunoprecipitation with anti - HA antibody followed by immunoblotting with the indicated antibodies . Please note , IFT54Δ261 - 275 mutant exhibited weaker interaction with D1bLIC relative to the control . The normalized ratio of D1bLIC in the immunoprecipitates ( IFT54 - HA versus mutant ) is 1 : 0 . 37 .

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(I) Co-immunoprecipitation of IFT54 and IFT54Δ261-275 with D1bLIC. Cell extracts from wild-type (WT), ift54 expressing HA-tagged full-length (FL) IFT54 or IFT54Δ261-275 mutant were subjected to immunoprecipitation with anti-HA antibody followed by immunoblotting with the indicated antibodies. Please note, IFT54Δ261-275 mutant exhibited weaker interaction with D1bLIC relative to the control. The normalized ratio of D1bLIC in the immunoprecipitates (IFT54-HA versus mutant) is 1:0.37.


( J ) Interaction of IFT54 with D1bLIC is conserved in mammalian cells . Cell extracts from HEK293T cells expressing eGFP , eGFP tagged TRAF3IP1 or TRAF3IP1Δ460 - 474 ( corresponding deletion mutant of IFT54Δ261 - 275 ) were analyzed by immunoprecipitation with GFP antibody followed by immunoblotting with anti - GFP and DYNC2LI1 , human homologue of D1bLIC , antibodies , respectively . Please note , the deletion mutant weakened the interaction with IFT dynein subunit DYNC2LI1 relative to the control . The normalized ratio of DYNC2LI1 in the immunoprecipitates ( TRAF3IP1 versus mutant ) is 1 : 0 . 49 .

(J) Interaction of IFT54 with D1bLIC is conserved in mammalian cells. Cell extracts from HEK293T cells expressing eGFP, eGFP tagged TRAF3IP1 or TRAF3IP1Δ460-474 (corresponding deletion mutant of IFT54Δ261-275) were analyzed by immunoprecipitation with GFP antibody followed by immunoblotting with anti-GFP and DYNC2LI1, human homologue of D1bLIC, antibodies, respectively. Please note, the deletion mutant weakened the interaction with IFT dynein subunit DYNC2LI1 relative to the control. The normalized ratio of DYNC2LI1 in the immunoprecipitates (TRAF3IP1 versus mutant) is 1:0.49.


A . Representative fluorescence micrographs of HeLa cells treated with 250 μM lysosomotropic drug LLOMe or equal volume of DMSO ( Ctrl ) for 1 h before fixation and immunostained with Hoechst ( blue ) , anti - CHMP4B ( green ) , anti - GAL3 ( red ) and anti - LAMP1 ( white ) are presented . Cells treated with LLOMe show increased recruitment of ESCRT - III protein to lysosomes when compared to Ctrl cells . Number of foci per cell were quantified and are indicated as mean ± SD . Data from > 86 cells per condition from three independent experiments . CHMP4B , GAL3 , LAMP1 foci per cell ( Ctrl versus LLOMe treatment ) : p = 0 . 0398 , p = 0 . 0033 , p = 0 . 2400 ( Student " s t ‐ test ) , respect Data information : Scale bars : 10 μm and 1 μm ( inset )

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

A. Representative fluorescence micrographs of HeLa cells treated with 250 μM lysosomotropic drug LLOMe or equal volume of DMSO (Ctrl) for 1 h before fixation and immunostained with Hoechst (blue), anti-CHMP4B (green), anti-GAL3 (red) and anti-LAMP1 (white) are presented. Cells treated with LLOMe show increased recruitment of ESCRT-III protein to lysosomes when compared to Ctrl cells. Number of foci per cell were quantified and are indicated as mean ± SD. Data from >86 cells per condition from three independent experiments. CHMP4B, GAL3, LAMP1 foci per cell (Ctrl versus LLOMe treatment): p=0.0398, p=0.0033, p=0.2400 (Student"s t‐test), respect Data information: Scale bars: 10 μm and 1 μm (inset)


B . HeLa cells stably expressing CHMP4B - eGFP and mCherry - Galectin3 treated as in ( A ) and stained with CD63 antibody are shown . Number of foci per cell were quantified and are indicated as mean ± SD . Data is quantified from > 87 cells per condition from three independent experiments . CHMP4B , GAL3 , CD63 foci per cell ( Ctrl versus LLOMe treatment ) : p = 0 . 0111 , p = 0 . 0050 , p = 0 . 3793 ( Student " s t ‐ test ) , respect Data information : Scale bars : 10 μm and 1 μm ( inset )

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

B. HeLa cells stably expressing CHMP4B-eGFP and mCherry-Galectin3 treated as in (A) and stained with CD63 antibody are shown. Number of foci per cell were quantified and are indicated as mean ± SD. Data is quantified from >87 cells per condition from three independent experiments. CHMP4B, GAL3, CD63 foci per cell (Ctrl versus LLOMe treatment): p=0.0111, p=0.0050, p=0.3793 (Student"s t‐test), respect Data information: Scale bars: 10 μm and 1 μm (inset)


To screen for ESCRT proteins that are involved in the endolysosomal repair process , cells were incubated with 250 μM LLOMe for 30 min and processed for immunofluorescence . TSG101 , a component of the ESCRT - I complex , and EAP30 , an ESCRT - II protein , are clearly recruited to the sites of damage compared to the DMSO control ( Ctrl ) . As shown , the ESCRT - III complex together with ALIX is recruited to damaged endolysosomes . In contrast , HRS , a component of the ESCRT - 0 complex , and HD - PTP show no recruitment to damaged endomembranes . Number of foci per cell was quantified from > 85 cells per condition from three independent experiments and are presented as mean ± SD . Statistical significance for number of foci per cell in Ctrl versus LLOMe treatment was determined using Student ' s t ‐ test , the p - values for which are : HRS p = 0 . 9257 , GAL3 p = 0 . 0050 ; TSG101 p = 0 . 0243 , GAL3 p = 0 . 0489 ; EAP30 p = 0 . 0006 , GAL3 p = 0 . 0307 ; CHMP2A p = 0 . 0284 , GAL3 p = 0 . 0260 ; CHMP4B p = 0 . 0028 , GAL3 p = 0 . 0414 ; VPS4A p = 0 . 0309 , GAL3 p = 0 . 0461 ; ALIX p = 0 . 0249 , GAL3 p = 0 . 0471 ; HD - PTP p = 0 . 4898 , GAL3 p = 0 . 0105 . Data information : Scale bars : 5 μm and 1 μm ( inset ) .

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

To screen for ESCRT proteins that are involved in the endolysosomal repair process, cells were incubated with 250 μM LLOMe for 30 min and processed for immunofluorescence. TSG101, a component of the ESCRT-I complex, and EAP30, an ESCRT-II protein, are clearly recruited to the sites of damage compared to the DMSO control (Ctrl). As shown, the ESCRT-III complex together with ALIX is recruited to damaged endolysosomes. In contrast, HRS, a component of the ESCRT-0 complex, and HD-PTP show no recruitment to damaged endomembranes. Number of foci per cell was quantified from >85 cells per condition from three independent experiments and are presented as mean ± SD. Statistical significance for number of foci per cell in Ctrl versus LLOMe treatment was determined using Student's t‐test, the p-values for which are: HRS p=0.9257, GAL3 p=0.0050; TSG101 p=0.0243, GAL3 p=0.0489 ; EAP30 p=0.0006, GAL3 p=0.0307; CHMP2A p=0.0284, GAL3 p=0.0260; CHMP4B p=0.0028, GAL3 p=0.0414; VPS4A p=0.0309, GAL3 p=0.0461; ALIX p=0.0249, GAL3 p=0.0471; HD-PTP p=0.4898, GAL3 p=0.0105. Data information: Scale bars: 5 μm and 1 μm (inset).


HeLa cells stably expressing CHMP4B - eGFP were transfected with siRNAs against : ( A ) HR 48 h post - transfection cells were used for live - cell imaging experiments . LLOMe was added using perfusion system . A . Depletion of HRS does not alter the dynamics of CHMP4B - eGFP recruitment as compared to siCtrl during treatment with LLOMe Data information : The quantification graphs represent average CHMP4B foci per cell quantified from three independent live - cell imaging experiments per condition . Error bars correspond to 95 % confidence intervals . Data from > 56 cells per condition were analyzed in each experiment . Right panels show knockdown efficiency of siRNA oligos as detected by Western blot ( * , nonspecific immunoreactivity )

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

HeLa cells stably expressing CHMP4B-eGFP were transfected with siRNAs against: (A) HR 48 h post-transfection cells were used for live-cell imaging experiments. LLOMe was added using perfusion system. A. Depletion of HRS does not alter the dynamics of CHMP4B-eGFP recruitment as compared to siCtrl during treatment with LLOMe Data information: The quantification graphs represent average CHMP4B foci per cell quantified from three independent live-cell imaging experiments per condition. Error bars correspond to 95% confidence intervals. Data from >56 cells per condition were analyzed in each experiment. Right panels show knockdown efficiency of siRNA oligos as detected by Western blot (*, nonspecific immunoreactivity)


HeLa cells stably expressing CHMP4B - eGFP were transfected with siRNAs agains ( B ) TSG10 48 h post - transfection cells were used for live - cell imaging experiments . LLOMe was added using perfusion system B . Downregulation of TSG101 , with two independent siRNAs causes a delay in CHMP4B - eGFP recruitment as compared to siCtrl , indicating an important role of the ESCRT - I complex in recruiting the downstream components Data information : The quantification graphs represent average CHMP4B foci per cell quantified from three independent live - cell imaging experiments per condition . Error bars correspond to 95 % confidence intervals . Data from > 56 cells per condition were analyzed in each experiment . Right panels show knockdown efficiency of siRNA oligos as detected by Western blot ( * , nonspecific immunoreactivity )

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

HeLa cells stably expressing CHMP4B-eGFP were transfected with siRNAs agains (B) TSG10 48 h post-transfection cells were used for live-cell imaging experiments. LLOMe was added using perfusion system B. Downregulation of TSG101, with two independent siRNAs causes a delay in CHMP4B-eGFP recruitment as compared to siCtrl, indicating an important role of the ESCRT-I complex in recruiting the downstream components Data information: The quantification graphs represent average CHMP4B foci per cell quantified from three independent live-cell imaging experiments per condition. Error bars correspond to 95% confidence intervals. Data from >56 cells per condition were analyzed in each experiment. Right panels show knockdown efficiency of siRNA oligos as detected by Western blot (*, nonspecific immunoreactivity)


HeLa cells stably expressing CHMP4B - eGFP were transfected with siRNAs agains ( C ) CHMP2 48 h post - transfection cells were used for live - cell imaging experiments . LLOMe was added using perfusion system C . CHMP2A depletion accumulates and stabilizes CHMP4B - eGFP positive foci Data information : The quantification graphs represent average CHMP4B foci per cell quantified from three independent live - cell imaging experiments per condition . Error bars correspond to 95 % confidence intervals . Data from > 56 cells per condition were analyzed in each experiment . Right panels show knockdown efficiency of siRNA oligos as detected by Western blot ( * , nonspecific immunoreactivity )

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

HeLa cells stably expressing CHMP4B-eGFP were transfected with siRNAs agains (C) CHMP2 48 h post-transfection cells were used for live-cell imaging experiments. LLOMe was added using perfusion system C. CHMP2A depletion accumulates and stabilizes CHMP4B-eGFP positive foci Data information: The quantification graphs represent average CHMP4B foci per cell quantified from three independent live-cell imaging experiments per condition. Error bars correspond to 95% confidence intervals. Data from >56 cells per condition were analyzed in each experiment. Right panels show knockdown efficiency of siRNA oligos as detected by Western blot (*, nonspecific immunoreactivity)


HeLa cells stably expressing CHMP4B - eGFP were transfected with siRNAs against ( D ) ALI 48 h post - transfection cells were used for live - cell imaging experiments . LLOMe was added using perfusion system D . siRNA mediated depletion of ALIX shows no significant change on dynamics of CHMP4B recruitment Data information : The quantification graphs represent average CHMP4B foci per cell quantified from three independent live - cell imaging experiments per condition . Error bars correspond to 95 % confidence intervals . Data from > 56 cells per condition were analyzed in each experiment . Right panels show knockdown efficiency of siRNA oligos as detected by Western blot ( * , nonspecific immunoreactivity )

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

HeLa cells stably expressing CHMP4B-eGFP were transfected with siRNAs against (D) ALI 48 h post-transfection cells were used for live-cell imaging experiments. LLOMe was added using perfusion system D. siRNA mediated depletion of ALIX shows no significant change on dynamics of CHMP4B recruitment Data information: The quantification graphs represent average CHMP4B foci per cell quantified from three independent live-cell imaging experiments per condition. Error bars correspond to 95% confidence intervals. Data from >56 cells per condition were analyzed in each experiment. Right panels show knockdown efficiency of siRNA oligos as detected by Western blot (*, nonspecific immunoreactivity)


HeLa cells stably expressing CHMP4B - eGFP were transfected with siRNAs against ( E ) both TSG101 and ALIX 48 h post - transfection cells were used for live - cell imaging experiments . LLOMe was added using perfusion system E . Simultaneous depletion of TSG101 and ALIX caused almost no recruitment of CHMP4B upon induction of endolysosomal damage Data information : The quantification graphs represent average CHMP4B foci per cell quantified from three independent live - cell imaging experiments per condition . Error bars correspond to 95 % confidence intervals . Data from > 56 cells per condition were analyzed in each experiment . Right panels show knockdown efficiency of siRNA oligos as detected by Western blot ( * , nonspecific immunoreactivity )

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

HeLa cells stably expressing CHMP4B-eGFP were transfected with siRNAs against (E) both TSG101 and ALIX 48 h post-transfection cells were used for live-cell imaging experiments. LLOMe was added using perfusion system E. Simultaneous depletion of TSG101 and ALIX caused almost no recruitment of CHMP4B upon induction of endolysosomal damage Data information: The quantification graphs represent average CHMP4B foci per cell quantified from three independent live-cell imaging experiments per condition. Error bars correspond to 95% confidence intervals. Data from >56 cells per condition were analyzed in each experiment. Right panels show knockdown efficiency of siRNA oligos as detected by Western blot (*, nonspecific immunoreactivity)


A . HeLa cells stably expressing CHMP4B - eGFP were treated with 250 μM LLOMe and 75 nM Lysotracker DND - 99 and fixed at different time points as indicated . After 10 min of LLOMe treatment CHMP4B is recruited whereas the number of Lysotracker positive foci is reduced . After 30 min , lysosomes gain back functionality ( judging by the increased number of Lysotracker foci ) and appear recovered after 1 h indicating that the ESCRT complex is able to seal the damaged endolysosomal membranes . Representative confocal images for each time point are shown . Scale bars : 5

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

A. HeLa cells stably expressing CHMP4B-eGFP were treated with 250 μM LLOMe and 75 nM Lysotracker DND-99 and fixed at different time points as indicated. After 10 min of LLOMe treatment CHMP4B is recruited whereas the number of Lysotracker positive foci is reduced. After 30 min, lysosomes gain back functionality (judging by the increased number of Lysotracker foci) and appear recovered after 1 h indicating that the ESCRT complex is able to seal the damaged endolysosomal membranes. Representative confocal images for each time point are shown. Scale bars: 5 μm


B . Quantification graph ( > 250 cells per condition from four independent experiments ) showing CHMP4B and Lysotracker positive foci per cell at different time points . Error bars correspond to 95 % confidence

O O O O O O O O O O O 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. Quantification graph (>250 cells per condition from four independent experiments) showing CHMP4B and Lysotracker positive foci per cell at different time points. Error bars correspond to 95% confidence intervals


C . Quantification graph showing dynamics of Lysotracker recovery in control ( siCtrl ) and both TSG101 and ALIX depleted cells . HeLa cells stably expressing CHMP4B - eGFP were co - transfected with siRNAs against TSG101 and ALIX . 48 h post - transfection , cells were pre - treated for 20 min with 75 nM Lysotracker Deep - Red , which was used as a read - out . While the decrease in the number of Lysotracker spots is quickly recovered in siCtrl , simultaneous depletion of ALIX and TSG101 leads to a severe impairment in lysosomal repair . Data from > 40 cells per condition from four independent live - cell imaging experiments are shown . Graph is normalized to the area occupied by the cells . Error bars correspond to 95 % confidence intervals .

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C. Quantification graph showing dynamics of Lysotracker recovery in control (siCtrl) and both TSG101 and ALIX depleted cells. HeLa cells stably expressing CHMP4B-eGFP were co-transfected with siRNAs against TSG101 and ALIX. 48 h post-transfection, cells were pre-treated for 20 min with 75 nM Lysotracker Deep-Red, which was used as a read-out. While the decrease in the number of Lysotracker spots is quickly recovered in siCtrl, simultaneous depletion of ALIX and TSG101 leads to a severe impairment in lysosomal repair. Data from >40 cells per condition from four independent live-cell imaging experiments are shown. Graph is normalized to the area occupied by the cells. Error bars correspond to 95% confidence intervals.


Representative movie montage of a live - cell imaging experiment illustrating the dynamics of CHMP4B - eGFP and mCherry - Galectin3 recruitment at different time points before and after LLOMe treatment . Using the perfusion system , 250 μM LLOMe was added to HeLa cells stably expressing CHMP4B - eGFP and mCherry - Galectin3 and images were acquired every 20 sec for a period of 20 min . As shown in the representative movie montage and the quantification graph , CHMP4B recruitment precedes Galectin3 , which is an established marker for detection of damaged endolysosomal membranes . The quantification graph is normalized to the area occupied by the cells . Data were obtained from > 40 cells per condition from three independent experiments . Error bars correspond to 95 % confidence intervals . Data information : Scale bars : 5 μm .

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

Representative movie montage of a live-cell imaging experiment illustrating the dynamics of CHMP4B-eGFP and mCherry-Galectin3 recruitment at different time points before and after LLOMe treatment. Using the perfusion system, 250 μM LLOMe was added to HeLa cells stably expressing CHMP4B-eGFP and mCherry-Galectin3 and images were acquired every 20 sec for a period of 20 min. As shown in the representative movie montage and the quantification graph, CHMP4B recruitment precedes Galectin3, which is an established marker for detection of damaged endolysosomal membranes. The quantification graph is normalized to the area occupied by the cells. Data were obtained from >40 cells per condition from three independent experiments. Error bars correspond to 95% confidence intervals. Data information: Scale bars: 5 μm.


A . HeLa cells stably expressing CHMP4B - eGFP and mCherry - Galectin3 were incubated with 250 μM LLOMe , fixed at different time points as indicated and stained for ubiquitin . As presented , CHMP4B is recruited before ubiquitin and Galectin3 upon lysosomal membrane damage Data information : Quantification graphs are normalized to the cell number where error bars represent 95 % confidence intervals . Data were obtained from > 150 cells per condition from three independent

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A. HeLa cells stably expressing CHMP4B-eGFP and mCherry-Galectin3 were incubated with 250 μM LLOMe, fixed at different time points as indicated and stained for ubiquitin. As presented, CHMP4B is recruited before ubiquitin and Galectin3 upon lysosomal membrane damage Data information: Quantification graphs are normalized to the cell number where error bars represent 95% confidence intervals. Data were obtained from >150 cells per condition from three independent experiments


B . HeLa cells stably expressing CHMP4B - eGFP were treated as in ( A ) and stained for p62 . CHMP4B is recruited before p62 on the damaged lysosomes . The number of LAMP1 - positive foci appears stable independently of LLOMe treatment Data information : Quantification graphs are normalized to the cell number where error bars represent 95 % confidence intervals . Data were obtained from > 150 cells per condition from three independent

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

B. HeLa cells stably expressing CHMP4B-eGFP were treated as in (A) and stained for p62. CHMP4B is recruited before p62 on the damaged lysosomes. The number of LAMP1-positive foci appears stable independently of LLOMe treatment Data information: Quantification graphs are normalized to the cell number where error bars represent 95% confidence intervals. Data were obtained from >150 cells per condition from three independent experiments


C . Experimental setup as in ( A and B ) where HeLa cells stably expressing CHMP4B - eGFP and mCherry - Galectin3 were stained for LC3 . As shown , CHMP4B is recruited prior to LC3 Data information : Quantification graphs are normalized to the cell number where error bars represent 95 % confidence intervals . Data were obtained from > 150 cells per condition from three independent

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Experimental setup as in (A and B) where HeLa cells stably expressing CHMP4B-eGFP and mCherry-Galectin3 were stained for LC3. As shown, CHMP4B is recruited prior to LC3 Data information: Quantification graphs are normalized to the cell number where error bars represent 95% confidence intervals. Data were obtained from >150 cells per condition from three independent experiments


A . HeLa cells were transfected with different siRNAs as indicated . 48 h post - transfection cells were treated with 250 μM LLOMe for 3 , 6 , 10 hours harvested and stained for Annexin V . Cells were immediately processed for flow cytometry . Already after 3 h of treatment with LLOMe , cells co - transfected with TSG101 + ALIX siRNA showed an increase in apoptotic cell death , whereas TSG101 siRNA transfected cells showed elevated Annexin V staining after 6 h of treatment Data information : Graphs show average data from a set of three independent experiments where > 10 , 000 cells per condition were analyzed . Error bars represent SEM . Statistical significance was determined using one - way ANOVA test . p - values are presented where significant . * p - value ≤ 0 . 05 , * * p - value ≤ 0 . 01 and * * * p - value ≤ 0 .

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

A. HeLa cells were transfected with different siRNAs as indicated. 48 h post-transfection cells were treated with 250 μM LLOMe for 3, 6, 10 hours harvested and stained for Annexin V. Cells were immediately processed for flow cytometry. Already after 3 h of treatment with LLOMe, cells co-transfected with TSG101+ALIX siRNA showed an increase in apoptotic cell death, whereas TSG101 siRNA transfected cells showed elevated Annexin V staining after 6 h of treatment Data information: Graphs show average data from a set of three independent experiments where >10,000 cells per condition were analyzed. Error bars represent SEM. Statistical significance was determined using one-way ANOVA test. p-values are presented where significant. *p-value≤ 0.05, **p-value≤ 0.01 and ***p-value≤ 0.001


B . Experimental set up as in ( A ) with additional propidium iodide ( PI ) staining of transfected HeLa cells Data information : Graphs show average data from a set of three independent experiments where > 10 , 000 cells per condition were analyzed . Error bars represent SEM . Statistical significance was determined using one - way ANOVA test . p - values are presented where significant . * p - value ≤ 0 . 05 , * * p - value ≤ 0 . 01 and * * * p - value ≤ 0 .

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B. Experimental set up as in (A) with additional propidium iodide (PI) staining of transfected HeLa cells Data information: Graphs show average data from a set of three independent experiments where >10,000 cells per condition were analyzed. Error bars represent SEM. Statistical significance was determined using one-way ANOVA test. p-values are presented where significant. *p-value≤ 0.05, **p-value≤ 0.01 and ***p-value≤ 0.001


A . HeLa cells stably expressing CHMP4B - eGFP were transfected with the mCherry - Galectin3 plasmid . 24 h later , cells were infected with WT Coxiella burnetti . Live - cell imaging started 24 h post - infection and several time points are indicated during the next 24 h of observatio Arrows indicate Coxiella - containing vacuole ( DRAQ5 labeling ) becoming positive for CHMP4B - eGFP and mCherry - Galectin3 . Data information : Scale bars : 50

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A. HeLa cells stably expressing CHMP4B-eGFP were transfected with the mCherry-Galectin3 plasmid. 24 h later, cells were infected with WT Coxiella burnetti. Live-cell imaging started 24 h post-infection and several time points are indicated during the next 24 h of observatio Arrows indicate Coxiella-containing vacuole (DRAQ5 labeling) becoming positive for CHMP4B-eGFP and mCherry-Galectin3. Data information: Scale bars: 50 μm


B . Bacterial viability and replication assay upon TSG101 KD . HeLa cells were treated with either siCtrl or with two different siTSG101 for 48 h . Then cells were infected with mCherry - Coxiella burnetii for 48 h before lysis and serial dilutions were made to infect Vero cells . 72 h later , infected cells were fixed , DAPI stained and processed for quantitative image analysis . Between 30 and 40 fields representing more than 9 , 000 cells per condition of three independent experiments were analyzed . Error bars represent SD . Statistical significance was determined using one - way ANOVA test . * * p - value ≤ 0 . 01 and * * * p - value ≤ 0 . 001 .

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B. Bacterial viability and replication assay upon TSG101 KD. HeLa cells were treated with either siCtrl or with two different siTSG101 for 48 h. Then cells were infected with mCherry-Coxiella burnetii for 48 h before lysis and serial dilutions were made to infect Vero cells. 72 h later, infected cells were fixed, DAPI stained and processed for quantitative image analysis. Between 30 and 40 fields representing more than 9,000 cells per condition of three independent experiments were analyzed. Error bars represent SD. Statistical significance was determined using one-way ANOVA test. **p-value≤ 0.01 and ***p-value≤ 0.001.


( A ) EM analysis of KMnO4 - fixed pex3 atg1 and WT cells grown for 16 h on MM - M / G . The inset shows a cluster of vesicles ( enlarged from the boxed region ) .

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(A) EM analysis of KMnO4-fixed pex3 atg1 and WT cells grown for 16 h on MM-M/G. The inset shows a cluster of vesicles (enlarged from the boxed region).


( B ) iEM analysis of pex3 atg1 cells using α - Pex14 antibodies .

(B) iEM analysis of pex3 atg1 cells using α-Pex14 antibodies.


( C and D ) FM images of pex3 atg1 cells producing Pex14 - mCherry and the ER marker BiPN30 - eGFP - HDEL ( C ) , or Pex14 - mGFP complemented with Mitotracker orange staining ( D ) .

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(C and D) FM images of pex3 atg1 cells producing Pex14-mCherry and the ER marker BiPN30-eGFP-HDEL (C), or Pex14-mGFP complemented with Mitotracker orange staining (D).


( E ) Electron tomography analysis of a serial - sectioned pex3 atg1 cell ( a ) containing a perinuclear membrane cluster ( b , arrows ) . ( d - f ) 10 - nm - thin digital slices through the tomogram reconstruction ( viewing direction indicated in c ) revealed vesicles ( black arrows ) and reticular structures ( red arrows ) . The surface - rendered reconstruction in g shows the reticulovesicular structures in 3D viewed at right angles from d . CW , cell wall ; M , mitochondrion ; N , nucleus ; P , peroxisome ; V , vacuole . Bars : ( A ) 500 nm ; ( A , inset ; and B ) 100 nm ; ( C and D ) 2 . 5 µm ; ( E ) 250 nm .

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(E) Electron tomography analysis of a serial-sectioned pex3 atg1 cell (a) containing a perinuclear membrane cluster (b, arrows). (d-f) 10-nm-thin digital slices through the tomogram reconstruction (viewing direction indicated in c) revealed vesicles (black arrows) and reticular structures (red arrows). The surface-rendered reconstruction in g shows the reticulovesicular structures in 3D viewed at right angles from d. CW, cell wall; M, mitochondrion; N, nucleus; P, peroxisome; V, vacuole. Bars: (A) 500 nm; (A, inset; and B) 100 nm; (C and D) 2.5 µm; (E) 250 nm.


( A ) iEM analysis of pex3 cells using α - Pex14 antibodies , identifying structures ( arrows ) in the vicinity of mitochondria . CW , cell wall ; M , mitochondrion . ( B ) Quantification of Pex14 - mGFP spots in pex3 and pex3 atg1 cells .

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(A) iEM analysis of pex3 cells using α-Pex14 antibodies, identifying structures (arrows) in the vicinity of mitochondria. CW, cell wall; M, mitochondrion. (B) Quantification of Pex14-mGFP spots in pex3 and pex3 atg1 cells.


( C ) FM images of pex3 atg1 or pex3 cells , producing Pex14 - mGFP complemented with FM4 - 64 vacuolar staining . The inset ( enlarged from the boxed region ) shows optimized intensities for pex3 cells , highlighting the Pex14 - mGFP spot and vacuolar mGFP .

(C) FM images of pex3 atg1 or pex3 cells, producing Pex14-mGFP complemented with FM4-64 vacuolar staining. The inset (enlarged from the boxed region) shows optimized intensities for pex3 cells, highlighting the Pex14-mGFP spot and vacuolar mGFP.


( D ) WB analysis of cells grown for 16 h on MM - M / G using α - Pex11 or α - Pex14 antibodies . Pyruvate carboxylase 1 ( Pyc1 ) was used as a loading control . Error bars indicate SEM . Bars : ( A ) 100 nm ; ( C ) 1 µm .

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(D) WB analysis of cells grown for 16 h on MM-M/G using α-Pex11 or α-Pex14 antibodies. Pyruvate carboxylase 1 (Pyc1) was used as a loading control. Error bars indicate SEM. Bars: (A) 100 nm; (C) 1 µm.


( A ) FM images of pex3 atg1 cells grown for 16 h on MM - M / G . Cells produced Pex14 - mCherry and C - terminal mGFP fusions of the indicated proteins .

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(A) FM images of pex3 atg1 cells grown for 16 h on MM-M/G. Cells produced Pex14-mCherry and C-terminal mGFP fusions of the indicated proteins.


( B ) WB analysis of WT ( 1 ) and pex3 atg1 cells ( 2 ) , grown for 6 or 16 h on MM - M / G .

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(B) WB analysis of WT (1) and pex3 atg1 cells (2), grown for 6 or 16 h on MM-M/G.


( C ) FM images showing mGFP - fluorescence in pex3 atg1 cells producing Pex14 - mCherry ( control ) or Pex14 - mCherry together with the indicated mGFP fusion protein . Cells were grown for 6 h on MM - M / G .

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(C) FM images showing mGFP-fluorescence in pex3 atg1 cells producing Pex14-mCherry (control) or Pex14-mCherry together with the indicated mGFP fusion protein. Cells were grown for 6 h on MM-M/G.


( D and E ) iEM of pex3 atg1 cells using α - Pex5 ( D ) or α - alcohol oxidase antibodies ( E ) .

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(D and E) iEM of pex3 atg1 cells using α-Pex5 (D) or α-alcohol oxidase antibodies (E).


( F ) Cell fractionation of the two indicated strains ( seperated by the horizontal broken line ) . Postnuclear supernatants ( PNS ) were subjected to differential centrifugation resulting in a 30 , 000 g organelle pellet ( P ) and supernatant fraction ( S ) .

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(F) Cell fractionation of the two indicated strains (seperated by the horizontal broken line). Postnuclear supernatants (PNS) were subjected to differential centrifugation resulting in a 30,000 g organelle pellet (P) and supernatant fraction (S).


( G ) Flotation analysis of the organelle pellet showing the distribution of the indicated proteins in the top ( 1 ) to bottom ( 10 ) fractions .

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(G) Flotation analysis of the organelle pellet showing the distribution of the indicated proteins in the top (1) to bottom (10) fractions.


( H ) Colocalization of Pex8 - GFP and Pex14 - mCherry in pex10 cells .

(H) Colocalization of Pex8-GFP and Pex14-mCherry in pex10 cells.


( A ) FM images of pex3 cells with Pex14 - mCherry upon Pex3 - eGFP reintroduction after shifting cells from MM - Glu with ammonium sulfate to MM - M / G with methylamine .

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(A) FM images of pex3 cells with Pex14-mCherry upon Pex3-eGFP reintroduction after shifting cells from MM-Glu with ammonium sulfate to MM-M/G with methylamine.


( B - E ) Live cell FM images of pex3 atg1 cells upon Pex3 reintroduction . Shown are pex3 atg1 cells producing Pex14 - mCherry and PAMOPEX3 - eGFP ( B ) , PAMOPEX3 . PEX10 - GFP ( C ) , PAMOPEX3 . PMP47 - GFP ( D ) , or PAMOPEX3 . PTEFGFP - SKL ( E ) . Cells were grown similar to the method in A . The arrows in B - D indicate the first detectable GFP signal . Bars , 2 . 5 µm .

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(B-E) Live cell FM images of pex3 atg1 cells upon Pex3 reintroduction. Shown are pex3 atg1 cells producing Pex14-mCherry and PAMOPEX3-eGFP (B), PAMOPEX3.PEX10-GFP (C), PAMOPEX3.PMP47-GFP (D), or PAMOPEX3.PTEFGFP-SKL (E). Cells were grown similar to the method in A. The arrows in B-D indicate the first detectable GFP signal. Bars, 2.5 µm.


( A and B ) WB analysis ( A ) and protein quantification ( B ) in WT ( lane 1 ; blue ) , pex3 atg1 ( lane 2 ; red ) , and pex3 atg1 - PAOXPEX19 ( lane 3 ; green ) cells grown for 6 h on MM - M / G . The protein levels in WT were set to 100 % .

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(A and B) WB analysis (A) and protein quantification (B) in WT (lane 1; blue), pex3 atg1 (lane 2; red), and pex3 atg1-PAOXPEX19 (lane 3; green) cells grown for 6 h on MM-M/G. The protein levels in WT were set to 100%.


( C and D ) FM images ( C ) and quantification ( D ) of Pmp47 - mGFP and Pex10 - mGFP in pex3 atg1 ( 1 ; red ) and pex3 atg1 - PAOXPex19 ( 2 ; green ) cells grown for 6 h on MM - M / G . Control cells in D did not produce mGFP . Significance indications : n . s . , P < 0 . 10 ; * , 0 . 10 > P > 0 . 05 ; * * , 0 . 05 > P > 0 . 01 ; * * * , P < 0 . 01 . Error bars indicate SD .

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(C and D) FM images (C) and quantification (D) of Pmp47-mGFP and Pex10-mGFP in pex3 atg1 (1; red) and pex3 atg1-PAOXPex19 (2; green) cells grown for 6 h on MM-M/G. Control cells in D did not produce mGFP. Significance indications: n.s., P < 0.10; *, 0.10 > P > 0.05; **, 0.05 > P > 0.01; ***, P < 0.01. Error bars indicate SD.


( E and F ) iEM analysis of pex19 ( E ) and pex19 atg1 ( F ) cells using α - Pex14 antibodies .

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(E and F) iEM analysis of pex19 (E) and pex19 atg1 (F) cells using α-Pex14 antibodies.


( G ) EM analysis of KMnO4 - fixed pex3 atg1 pex25 cells grown for 16 h on MM - M / G - showing membrane vesicles ( arrows ) .

(G) EM analysis of KMnO4-fixed pex3 atg1 pex25 cells grown for 16 h on MM-M/G-showing membrane vesicles (arrows).


( H ) FM image of pex3 atg1 pex25 cells producing Pex14 - mGFP . CW , cell wall ; M , mitochondrion ; N , nucleus ; V , vacuole .

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(H) FM image of pex3 atg1 pex25 cells producing Pex14-mGFP. CW, cell wall; M, mitochondrion; N, nucleus; V, vacuole.


Immunostaining from Daam2LacZ / + animals with Aldh1l1 - EGFP reporter indicates that Daam2 ( LacZ + , Red ) is expressed in astrocytes ( Green ) . ( A ) Daam2 expression is observed in multiple brain regions at postnatal day 21 .

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Immunostaining from Daam2LacZ/+ animals with Aldh1l1-EGFP reporter indicates that Daam2 (LacZ+, Red) is expressed in astrocytes (Green). (A) Daam2 expression is observed in multiple brain regions at postnatal day 21.


Immunostaining from Daam2LacZ / + animals with Aldh1l1 - EGFP reporter indicates that Daam2 ( LacZ + , Red ) is expressed in astrocytes ( Green ) . ( B ) Expression of Daam2 in cortex persists throughout development ( P7 to adult ) . C . Quantification of Daam2 - expressing astrocytes ( LacZ + ) with Aldh1l1 - EGFP labeled astrocytes in cortex during development . Data is presented as mean ± SEM . Two brain sections , N = 4 for each age .

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Immunostaining from Daam2LacZ/+ animals with Aldh1l1-EGFP reporter indicates that Daam2 (LacZ+, Red) is expressed in astrocytes (Green). (B) Expression of Daam2 in cortex persists throughout development (P7 to adult). C. Quantification of Daam2-expressing astrocytes (LacZ+) with Aldh1l1-EGFP labeled astrocytes in cortex during development. Data is presented as mean ± SEM. Two brain sections, N=4 for each age.


G . Representative images of astrocytes labeled with Aldh1l1 - EGFP reporter in cortex and OB of Daam2 cHet and cKO mice at postnatal day 28 ( P28 ) . Scale bar : 50 μm . H - K . Overall complexity of astrocytes with Aldh1l1 - EGFP reporter was measured by Sholl analysis ( Two - way ANOVA ) and total process length ( Student ' s t - test ) . Data is presented as mean ± SEM . n = 6 - 10 cells from N = 3 - 5 mice per genotype , * p < 0 . 05 , p * * * * < 0 .

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G. Representative images of astrocytes labeled with Aldh1l1-EGFP reporter in cortex and OB of Daam2 cHet and cKO mice at postnatal day 28 (P28). Scale bar: 50 μm. H-K. Overall complexity of astrocytes with Aldh1l1-EGFP reporter was measured by Sholl analysis (Two-way ANOVA) and total process length (Student's t-test). Data is presented as mean ± SEM. n= 6-10 cells from N=3-5 mice per genotype, *p<0.05, p****<0.0001


A . Representative images of GFAP immunostaining in Daam2 cKO mice compared to controls in cortex and OB at P28 . Scale bar : 20 μm .

A. Representative images of GFAP immunostaining in Daam2 cKO mice compared to controls in cortex and OB at P28. Scale bar: 20 μm.


D . Western blot analysis showed a significant increase in the amount of GFAP protein in Daam2 KO primary astrocytes . N =

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D. Western blot analysis showed a significant increase in the amount of GFAP protein in Daam2 KO primary astrocytes. N=2


F . Overexpression of Daam2 via injection of AAV - GfaABC1D - myc - Daam2 in wild type P1 pups revealed reduced GFAP intensity compared to animals injected with control virus ( AAV - GfaABC1D - GFP ) . AAV - GfaABC1D - myc - Daam2 was injected into Aldh1l1 - GFP reporter mice for morphological complexity analysis and infected cells ( Myc positive ) were compared with non - infected cells . Dotted white line indicates virus - infected cells . Filled triangles and empty triangles indicate astrocytic primary branches with the control and reduced level of GFAP or Aldh1l1 - GFP , respectively . Scale bar : 20 μm .

F. Overexpression of Daam2 via injection of AAV-GfaABC1D-myc-Daam2 in wild type P1 pups revealed reduced GFAP intensity compared to animals injected with control virus (AAV-GfaABC1D-GFP). AAV-GfaABC1D-myc-Daam2 was injected into Aldh1l1-GFP reporter mice for morphological complexity analysis and infected cells (Myc positive) were compared with non-infected cells. Dotted white line indicates virus-infected cells. Filled triangles and empty triangles indicate astrocytic primary branches with the control and reduced level of GFAP or Aldh1l1-GFP, respectively. Scale bar: 20 μm.


E . Schematics of astrocytic calcium imaging and representative images of spontaneous calcium signaling ( green ) of Daam2 cHet and cKO mice injected with AAV - GfaABC1D - GCaMP6f virus in OB . Approximate territory of astrocytes including soma and multiple microdomains is outlined in yellow . Representative traces of GCaMP6f signal in Daam2 cHet and cKO astrocytes are shown in black and red respectively . F - H . Quantification of amplitude ( F ) , frequency ( G ) , and area under curve ( AUC ) ( H ) of ∆ F / F GCaMP6f signal events in OB . Amplitude and AUC of Daam2 - deficient astrocytes are significantly increased in OB . Data is presented as mean ± SEM . Total number of cells is n = 16 , 15 ( F - H ) from N = 3 - 4 mice of each genotype . Student ' s t - test was used for statistics , * p < 0 . 05 .

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E. Schematics of astrocytic calcium imaging and representative images of spontaneous calcium signaling (green) of Daam2 cHet and cKO mice injected with AAV-GfaABC1D-GCaMP6f virus in OB. Approximate territory of astrocytes including soma and multiple microdomains is outlined in yellow. Representative traces of GCaMP6f signal in Daam2 cHet and cKO astrocytes are shown in black and red respectively. F-H. Quantification of amplitude (F), frequency (G), and area under curve (AUC) (H) of ∆F/F GCaMP6f signal events in OB. Amplitude and AUC of Daam2-deficient astrocytes are significantly increased in OB. Data is presented as mean ± SEM. Total number of cells is n=16, 15 (F-H) from N=3-4 mice of each genotype. Student's t-test was used for statistics, *p<0.05.


A - F . ( A , D ) Schematics of whole cell recording in ex vivo brain slices and representative traces of spontaneous EPSC and IPSC in neurons in cortex from Daam2 cHet ( top , black ) and cKO ( bottom , red ) mice . ( B - C , E - F ) Quantification of amplitude and frequency of sEPSC and sIPSC in each genotype . Data is presented as mean ± SEM . Total number of cells is n = 19 , 20 ( B - C ) and 17 , 13 ( E - F ) from N = 7 mice for each genotype . Student ' s t - test was used for comparison , * p < 0 . 05 .

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A-F. (A, D) Schematics of whole cell recording in ex vivo brain slices and representative traces of spontaneous EPSC and IPSC in neurons in cortex from Daam2 cHet (top, black) and cKO (bottom, red) mice. (B-C, E-F) Quantification of amplitude and frequency of sEPSC and sIPSC in each genotype. Data is presented as mean ± SEM. Total number of cells is n=19, 20 (B-C) and 17, 13 (E-F) from N=7 mice for each genotype. Student's t-test was used for comparison, *p<0.05.


A . Astrocyte - specific Daam2 cKO mice show delays in buried food finding . N = 13 , 15 mice per genotype . Data is presented as mean ± SEM . Student ' s t - test was used , * p < 0 . 05 .

A. Astrocyte-specific Daam2 cKO mice show delays in buried food finding. N=13, 15 mice per genotype. Data is presented as mean ± SEM. Student's t-test was used, *p<0.05.