A Electron micrograph of wild - type yeast treated with DTT for 3 h . Inset 1 shows an ER whorl and inset 2 shows ER fragments in macroautophagic bodies ( white arrows ) . Cy , cytosol ; V , vacuole .

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A Electron micrograph of wild-type yeast treated with DTT for 3 h. Inset 1 shows an ER whorl and inset 2 shows ER fragments in macroautophagic bodies (white arrows). Cy, cytosol; V, vacuole.


B Relative activity of cyto - Pho8 ∆ 60 after nitrogen starvation , which reports on non - selective macroautophagy of cytosol . Mean + SEM , n ≥ 3 . WT , wild - type .

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B Relative activity of cyto-Pho8∆60 after nitrogen starvation, which reports on non-selective macroautophagy of cytosol. Mean + SEM, n ≥ 3. WT, wild-type.


Relative activity of the ER - phagy reporter Yop1 - Pho8 ∆ 60 after nitrogen starvation ( C ) Mean + SEM , n ≥ 3 .

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Relative activity of the ER-phagy reporter Yop1-Pho8∆60 after nitrogen starvation (C) Mean + SEM, n ≥ 3.


Relative activity of the ER - phagy reporter Yop1 - Pho8 ∆ 60 after tunicamycin treatment ( D , E ) . Mean + SEM , n ≥ 3 .

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Relative activity of the ER-phagy reporter Yop1-Pho8∆60 after tunicamycin treatment (D, E). Mean + SEM, n ≥ 3.


F , G Growth assays of untreated and tunicamycin - treated WT , ∆ atg39 / 40 , ∆ snf7 and ∆ snf7 ∆ atg39 / 40 cells . Numbers indicate growth relative to WT cells based on the areas under the curves . Mean ± SEM , n = 3 .

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F, G Growth assays of untreated and tunicamycin-treated WT, ∆atg39/40, ∆snf7 and ∆snf7 ∆atg39/40 cells. Numbers indicate growth relative to WT cells based on the areas under the curves. Mean ± SEM, n = 3.


A Fraction of wild - type ( WT ) , ∆ vps23 , ∆ snf7 and ∆ vps4 cells with GFP - Pho8 structures . Mean + SEM , n ≥ 3 .

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A Fraction of wild-type (WT), ∆vps23, ∆snf7 and ∆vps4 cells with GFP-Pho8 structures. Mean + SEM, n ≥ 3.


B Fraction of GFP - Pho8 structures classified as stacks , cytosolic whorls , uptake intermediates or vacuolar whorls . The number of GFP - Pho8 structures in WT , ∆ vps23 , ∆ snf7 and ∆ vps4 cells was 562 , 345 , 367 and 449 , respectively . The table shows the results of Chi - Square tests for independence for all pairs of strains . * * * , highly significant ( P < 0 . 00001 ) ; ns , not significant ( P > 0 . 05 ) .

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B Fraction of GFP-Pho8 structures classified as stacks, cytosolic whorls, uptake intermediates or vacuolar whorls. The number of GFP-Pho8 structures in WT, ∆vps23, ∆snf7 and ∆vps4 cells was 562, 345, 367 and 449, respectively. The table shows the results of Chi-Square tests for independence for all pairs of strains. ***, highly significant (P < 0.00001); ns, not significant (P > 0.05).


C Fraction of stacks classified as peripheral , perinuclear or vacuole - associated . The number of stacks in WT and ∆ snf7 cells was 252 and 351 , respectively . The observed distributions in the two strains were analyzed with a Chi - Square test for independence . * * * , highly significant ( P < 0 . 00001 ) .

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C Fraction of stacks classified as peripheral, perinuclear or vacuole-associated. The number of stacks in WT and ∆snf7 cells was 252 and 351, respectively. The observed distributions in the two strains were analyzed with a Chi-Square test for independence. ***, highly significant (P < 0.00001).


Fluorescence images of a cell expressing non - fluorescent GFP ( Y66F ) - Pho8 , Snf7 - mNeonGreen and the ER marker mCherry - Ubc6 , and stained with the vacuole dye CMAC . Numbers indicate the time in minutes after the start of the image sequence . White arrows indicate a Snf7 - mNeonGreen punctum adjacent to an ER whorl . Black arrow point to a vacuole membrane invagination . In this series , mNeonGreen fluorescence was adjusted differently for different time points to compensate signal decay over time . Scale bar : 2 µm .

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Fluorescence images of a cell expressing non-fluorescent GFP(Y66F)-Pho8, Snf7-mNeonGreen and the ER marker mCherry-Ubc6, and stained with the vacuole dye CMAC. Numbers indicate the time in minutes after the start of the image sequence. White arrows indicate a Snf7-mNeonGreen punctum adjacent to an ER whorl. Black arrow point to a vacuole membrane invagination. In this series, mNeonGreen fluorescence was adjusted differently for different time points to compensate signal decay over time. Scale bar: 2 µm.


Fluorescence images of a cell expressing non - fluorescent GFP ( Y66F ) - Pho8 , Snf7 - mNeonGreen and the ER marker mCherry - Ubc6 , and stained with the vacuole dye CMAC . Numbers indicate the time in minutes after the start of the image sequence . White arrows indicate a Snf7 - mNeonGreen punctum adjacent to an ER whorl . Black arrow point to a vacuole membrane invagination . Panel ( E ) illustrates transient appearance of Snf7 at an ER whorl . Panel ( F ) illustrates orientation of a Snf7 punctum towards the neck of a vacuole membrane invagination . In this series , mNeonGreen fluorescence was adjusted differently for different time points to compensate signal decay over time . Scale bar : 2 µm .

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Fluorescence images of a cell expressing non-fluorescent GFP(Y66F)-Pho8, Snf7-mNeonGreen and the ER marker mCherry-Ubc6, and stained with the vacuole dye CMAC. Numbers indicate the time in minutes after the start of the image sequence. White arrows indicate a Snf7-mNeonGreen punctum adjacent to an ER whorl. Black arrow point to a vacuole membrane invagination. Panel (E) illustrates transient appearance of Snf7 at an ER whorl. Panel (F) illustrates orientation of a Snf7 punctum towards the neck of a vacuole membrane invagination. In this series, mNeonGreen fluorescence was adjusted differently for different time points to compensate signal decay over time. Scale bar: 2 µm.


A Electron micrographs of wild - type and ∆ snf7 cells treated with DTT for 3 h . Cy , cytosol ; V , vacuole . Scale bars : 300 nm .

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A Electron micrographs of wild-type and ∆snf7 cells treated with DTT for 3 h. Cy, cytosol; V, vacuole. Scale bars: 300 nm.


B Quantification of vacuolar whorls for which the uptake process was complete or incomplete . The observed distributions in the two strains were analyzed with a Fisher ' s exact test .

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B Quantification of vacuolar whorls for which the uptake process was complete or incomplete. The observed distributions in the two strains were analyzed with a Fisher's exact test.


C , D Electron micrographs of serial thin sections of a ∆ snf7 cell ( C ) or a wild - type cell ( D ) treated with DTT for 3 h . Note that the whorl appears separated from the cytosol in some sections ( e . g . section 300 nm ) but is still in contact with the cytosol ( see sections 600 nm and 700 nm ) . In the wild - type cell ( D ) , the vacuolar whorl is completely separated from the cytosol . Scale bar : 300 nm .

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C, D Electron micrographs of serial thin sections of a ∆snf7 cell (C) or a wild-type cell (D) treated with DTT for 3 h. Note that the whorl appears separated from the cytosol in some sections (e.g. section 300 nm) but is still in contact with the cytosol (see sections 600 nm and 700 nm). In the wild-type cell (D), the vacuolar whorl is completely separated from the cytosol. Scale bar: 300 nm.


Callus , de novo shoot , and root formation in wild - type ( WT ; Col ) , hag1 - 6 , hag1 - 7 , and 35S : : FLAG : HAG1 hag1 - 7 root explants . Root explants were transferred onto CIM for 1 week ( w ) and then transferred onto fresh CIM , SIM , or RIM for callus , de novo shoot , or root induction , respectively . Scale bar : 1 cm . Graph on the right shows the percentage of explants with shoots formed as scored at 18 days ( d ) on SIM . 36 explants of each genotype were used for scoring .

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Callus, de novo shoot, and root formation in wild-type (WT; Col), hag1-6, hag1-7, and 35S::FLAG:HAG1 hag1-7 root explants. Root explants were transferred onto CIM for 1 week (w) and then transferred onto fresh CIM, SIM, or RIM for callus, de novo shoot, or root induction, respectively. Scale bar: 1 cm. Graph on the right shows the percentage of explants with shoots formed as scored at 18 days (d) on SIM. 36 explants of each genotype were used for scoring.


Shoot regeneration assay using Col and hag1 - 6 root explants on media with different cytokinin ( 2IP ) to auxin ( IAA ) ratios . Col and hag1 - 6 root explants derived from 20 seedlings were first incubated on CIM for 2 weeks and then transferred onto 150 mg / ml IAA - containing media supplemented with 0 , 500 , or 5 , 000 mg / ml 2IP . Pictures were taken at 27 d after transfer onto each media using representative explants . Scale bar : 2 mm .

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Shoot regeneration assay using Col and hag1-6 root explants on media with different cytokinin (2IP) to auxin (IAA) ratios. Col and hag1-6 root explants derived from 20 seedlings were first incubated on CIM for 2 weeks and then transferred onto 150 mg/ml IAA-containing media supplemented with 0, 500, or 5,000 mg/ml 2IP. Pictures were taken at 27 d after transfer onto each media using representative explants. Scale bar: 2 mm.


Root explant - derived calli of Col and hag1 - 6 at 4 w on CIM ( left ) . Scale bar : 1 mm . 4 - w - old calli derived from 30 seedling roots of each genotype were collected to measure callus fresh weight ( right ) .

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Root explant-derived calli of Col and hag1-6 at 4 w on CIM (left). Scale bar: 1 mm. 4-w-old calli derived from 30 seedling roots of each genotype were collected to measure callus fresh weight (right).


Histochemical GUS staining of CYCB1 ; 1 : GUS in Col ( left ) and CYCB1 ; 1 : GUS in hag1 - 6 . Shown are root explants at 4 d on CIM . Scale bar : 1 mm .

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Histochemical GUS staining of CYCB1;1:GUS in Col (left) and CYCB1;1:GUS in hag1-6. Shown are root explants at 4 d on CIM. Scale bar: 1 mm.


Statistic chart of differentially expressed genes ( DEGs ) in Col and hag1 - 6 roots ( R ) , 4 d CIM - incubated root explants ( C ) , 4 d CIM - incubated + 2 d SIM - incubated root explants ( S ) , and 4 d CIM - incubated + 2 w SIM - incubated root explants ( S2W ) . Green : up - regulated genes , log2 ratio ≥ 1 ; red : down - regulated genes , log2 ratio ≤ - 1 ; FDR < 0 . 01 .

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Statistic chart of differentially expressed genes (DEGs) in Col and hag1-6 roots (R), 4 d CIM-incubated root explants (C), 4 d CIM-incubated + 2 d SIM-incubated root explants (S), and 4 d CIM-incubated + 2 w SIM-incubated root explants (S2W). Green: up-regulated genes, log2 ratio ≥ 1; red: down-regulated genes, log2 ratio ≤ -1; FDR < 0.01.


Immunoblot analysis of HAG1 : HA protein . Samples are as described in ( E ) . 1 w CIM - incubated Col root explants ( Col ) were used as negative control . Ponceau staining was performed as loading control .

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Immunoblot analysis of HAG1:HA protein. Samples are as described in (E). 1 w CIM-incubated Col root explants (Col) were used as negative control. Ponceau staining was performed as loading control.


pWOX5 : : GFP - ER expression in Col ( WT ) and hag1 - 6 explants on CIM and SIM . Time points selected for observation are indicated . Cellular outlines were visualized with propidium iodide ( PI ) staining ( red ) .

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pWOX5::GFP-ER expression in Col (WT) and hag1-6 explants on CIM and SIM. Time points selected for observation are indicated. Cellular outlines were visualized with propidium iodide (PI) staining (red).


pSCR : : GFP - ER expression in Col ( WT ) and hag1 - 6 explants on CIM and SIM . Time points selected for observation are indicated . Cellular outlines were visualized with PI staining ( red ) .

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pSCR::GFP-ER expression in Col (WT) and hag1-6 explants on CIM and SIM. Time points selected for observation are indicated. Cellular outlines were visualized with PI staining (red).


pSCR : : GFP - ER expression in Col ( WT ) and hag1 - 6 root tips at various time points on CIM . Time points selected for observation are indicated . Cellular outlines were visualized with PI staining ( red ) .

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pSCR::GFP-ER expression in Col (WT) and hag1-6 root tips at various time points on CIM. Time points selected for observation are indicated. Cellular outlines were visualized with PI staining (red).


WOX5 transcript levels in Col and hag1 - 6 hypocotyl ( D ) and cotyledon ( E ) explants at indicated days on CIM .

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WOX5 transcript levels in Col and hag1-6 hypocotyl (D) and cotyledon (E) explants at indicated days on CIM.


SCR transcript levels in Col and hag1 - 6 hypocotyl ( F ) and cotyledon ( G ) explants at indicated days on CIM .

SCR transcript levels in Col and hag1-6 hypocotyl (F) and cotyledon (G) explants at indicated days on CIM.


H3Ac enrichment at the WOX5 ( A ) , SCR ( B ) , PLT1 ( C ) , and PLT2 ( D ) loci in Col ( upper black diagram ) and hag1 - 6 ( lower red diagram ) calli ( 1 w CIM ) as determined by ChIP seq . p - value cutoff for peak detection using MACS2 was 5e - 2 for WOX5 ( A ) and 1e - 5 for SCR ( B ) , PLT1 ( C ) , and PLT2 ( D ) . ChIP - seq data were visualized using Integrated Genome Browser ( IGB ; http : / / bioviz . org / igb / index . html ) . The X and Y axis represent the genomic position of the corresponding gene and the number of overlapping reads per base pair position , respectively . Schematic representation of each locus is depicted at the bottom . Exons are indicated as boxes whereas intergenic sequences and introns are marked with lines . 5 ' and 3 ' UTRs are represented as gray boxes . Transcription start sites are indicated with arrows .

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H3Ac enrichment at the WOX5 (A), SCR (B), PLT1 (C), and PLT2 (D) loci in Col (upper black diagram) and hag1-6 (lower red diagram) calli (1 w CIM) as determined by ChIP seq. p-value cutoff for peak detection using MACS2 was 5e-2 for WOX5 (A) and 1e-5 for SCR (B), PLT1 (C), and PLT2 (D). ChIP-seq data were visualized using Integrated Genome Browser (IGB; http://bioviz.org/igb/index.html). The X and Y axis represent the genomic position of the corresponding gene and the number of overlapping reads per base pair position, respectively. Schematic representation of each locus is depicted at the bottom. Exons are indicated as boxes whereas intergenic sequences and introns are marked with lines. 5' and 3' UTRs are represented as gray boxes. Transcription start sites are indicated with arrows.


ChIP - qPCR analysis of H3Ac levels at the WOX5 ( E ) , SCR ( F ) , PLT1 ( G ) , and PLT2 ( H ) loci in Col and hag1 - 6 . Root ( R ) , 1 w CIM ( C ) , and 1 w CIM + 2 w SIM ( S ) samples were used for assays .

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ChIP-qPCR analysis of H3Ac levels at the WOX5 (E), SCR (F), PLT1 (G), and PLT2 (H) loci in Col and hag1-6. Root (R), 1 w CIM (C), and 1 w CIM + 2 w SIM (S) samples were used for assays.


Direct targeting of HAG1 : HA protein to WOX5 ( I ) , SCR ( J ) , PLT1 ( K ) , and PLT2 ( L ) chromatin as determined by ChIP - qPCR .

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Direct targeting of HAG1:HA protein to WOX5 (I), SCR (J), PLT1 (K), and PLT2 (L) chromatin as determined by ChIP-qPCR.


Transcript levels of WOX7 ( A ) and WOX14 ( B ) in Col and hag1 - 6 roots ( R ) , 4 d CIM explants ( C ) , 4 d CIM + 2 d SIM explants ( S ) , and 4 d CIM + 2 w SIM explants ( S2W ) . Levels of Col R were set to 1 after normalization with UBQ10 .

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Transcript levels of WOX7 (A) and WOX14 (B) in Col and hag1-6 roots (R), 4 d CIM explants (C), 4 d CIM + 2 d SIM explants (S), and 4 d CIM + 2 w SIM explants (S2W). Levels of Col R were set to 1 after normalization with UBQ10.


De novo shoot regeneration of Col , Nossen ( No ) , wox5 - 3 , wox7 - 1 , wox14 - 1 , and wox5 - 3 wox7 - 1 wox14 - 1 root explants . Col is the WT control for wox5 - 3 and wox7 - 1 , whereas No is the WT control for wox14 - 1 . The picture was taken at 3 w on SIM using representative explants of each genotype . Scale bar : 1 cm . Percentage of explants with shoots was scored at indicated weeks on SIM ( D ) .

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De novo shoot regeneration of Col, Nossen (No), wox5-3, wox7-1, wox14-1, and wox5-3 wox7-1 wox14-1 root explants. Col is the WT control for wox5-3 and wox7-1, whereas No is the WT control for wox14-1. The picture was taken at 3 w on SIM using representative explants of each genotype. Scale bar: 1 cm. Percentage of explants with shoots was scored at indicated weeks on SIM (D).


H3Ac enrichment at the WOX14 locus in Col ( upper black diagram ) or hag1 - 6 ( lower red diagram ) calli as determined by ChIP seq . p - value cutoff for peak detection using MACS2 was 1e - 5 . See Figs 3A - D for schematic .

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H3Ac enrichment at the WOX14 locus in Col (upper black diagram) or hag1-6 (lower red diagram) calli as determined by ChIP seq. p-value cutoff for peak detection using MACS2 was 1e-5. See Figs 3A-D for schematic.


ChIP - qPCR analysis of H3Ac levels at the WOX14 locus of Col and hag1 - 6 . Regions tested are indicated in the schematic in ( E ) . Sample preparation and data presentation were performed as described in Figs 3E - H .

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ChIP-qPCR analysis of H3Ac levels at the WOX14 locus of Col and hag1-6. Regions tested are indicated in the schematic in (E). Sample preparation and data presentation were performed as described in Figs 3E-H.


Direct targeting of HAG1 : HA protein to WOX14 chromatin as determined by ChIP - qPCR .

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Direct targeting of HAG1:HA protein to WOX14 chromatin as determined by ChIP-qPCR.


De novo shoot regeneration using Col and scr mutants ; scr - 3 ( A and B ) and scr - 6 ( A and C ) . Explants with shoots were scored at indicated days on SIM using Col and scr - 3 root explants that had been pre - incubated on CIM for 2 w ( B ) or Col and scr - 6 hypocotyl explants that had been pre - incubated on CIM for 1 w ( C ) . Scale bar : 1 mm .

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De novo shoot regeneration using Col and scr mutants; scr-3 (A and B) and scr-6 (A and C). Explants with shoots were scored at indicated days on SIM using Col and scr-3 root explants that had been pre-incubated on CIM for 2 w (B) or Col and scr-6 hypocotyl explants that had been pre-incubated on CIM for 1 w (C). Scale bar: 1 mm.


De novo shoot regeneration of Col , wox5 - 3 , scr - 3 , and wox5 - 3 scr - 3 . The percentage of root explants with shoots ( E ) and the number of shoots per root explant ( F ) were scored at 15 d on SIM . The explants had been pre - incubated on CIM for 2 w before transfer onto SIM . Scale bar : 1 cm .

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De novo shoot regeneration of Col, wox5-3, scr-3, and wox5-3 scr-3. The percentage of root explants with shoots (E) and the number of shoots per root explant (F) were scored at 15 d on SIM. The explants had been pre-incubated on CIM for 2 w before transfer onto SIM. Scale bar: 1 cm.


Transcript levels of PLT1 ( G ) and PLT2 ( H ) in Col , wox5 - 3 , scr - 3 , and wox5 - 3 scr - 3 roots ( R ) , 1 w CIM explants ( C ) , and 1 w CIM + 2 w SIM explants ( S2W ) as determined by RT - qPCR . Levels of Col R were set to 1 after normalization with UBQ10 .

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

Transcript levels of PLT1 (G) and PLT2 (H) in Col, wox5-3, scr-3, and wox5-3 scr-3 roots (R), 1 w CIM explants (C), and 1 w CIM + 2 w SIM explants (S2W) as determined by RT-qPCR. Levels of Col R were set to 1 after normalization with UBQ10.


Activation of WOX5 by DEX treatment of 35S : : GVG - WOX5 in Col and hag1 - 6 . Root explants of 35S : : GVG - WOX5 and 35S : : GVG - WOX5 hag1 - 6 were transferred onto the indicated combinations of CIM and SIM supplemented with or without 20 μM DEX . DEX was treated for 3 - d - pulse period on CIM or / and SIM ( see Materials and Methods for details and Appendix Fig S11A for schematics of the experimental procedure ) . Morphology of shoots regenerated from 35S : : GVG - WOX5 and 35S : : GVG - WOX5 hag1 - 6 root explants ( A ) . Explants with shoots were scored at 3 w after transfer onto SIM with or without DEX ( B ) .

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 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 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

Activation of WOX5 by DEX treatment of 35S::GVG-WOX5 in Col and hag1-6. Root explants of 35S::GVG-WOX5 and 35S::GVG-WOX5 hag1-6 were transferred onto the indicated combinations of CIM and SIM supplemented with or without 20 μM DEX. DEX was treated for 3-d-pulse period on CIM or/and SIM (see Materials and Methods for details and Appendix Fig S11A for schematics of the experimental procedure). Morphology of shoots regenerated from 35S::GVG-WOX5 and 35S::GVG-WOX5 hag1-6 root explants (A). Explants with shoots were scored at 3 w after transfer onto SIM with or without DEX (B).


Activation of SCR by DEX treatment of UBI : : mCHERRY - GR - SCR in Col and hag1 - 6 .

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Activation of SCR by DEX treatment of UBI::mCHERRY-GR-SCR in Col and hag1-6.


Activation of WOX5 and SCR by DEX treatment of 35S : : GVG - WOX5 UBI : : mCHERRY - GR - SCR in Col and hag1 - 6 .

O O 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

Activation of WOX5 and SCR by DEX treatment of 35S::GVG-WOX5 UBI::mCHERRY-GR-SCR in Col and hag1-6.


A The mRNA expression of FAM189A2 / ENTREP in the primary human mammary epithelium HMEC and human breast cancer cell lines . In qRT - PCR analyses , the expression was normalized to the immortalized normal human mammary epithelium HMEC4tertshp16 . Relative expression ratios shown as mean + SD from three biological replicates .

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

A The mRNA expression of FAM189A2/ENTREP in the primary human mammary epithelium HMEC and human breast cancer cell lines. In qRT-PCR analyses, the expression was normalized to the immortalized normal human mammary epithelium HMEC4tertshp16. Relative expression ratios shown as mean + SD from three biological replicates.


B Relative expression of FAM189A2 / ENTREP in the normal ( white ) and cancer ( green ) tissues of breast ( Curtis Breast ) , lung ( Okayama Lung ) , colorectal ( TCGA Colorectal ) and head and neck ( Estilo Head - Neck ) . Data were downloaded from the Oncomine database P - values obtained by Student ' s t - tests and the number of cases are listed on the top and bottom of figures , respectively . P < 0 . 05 was considered as statistically significant . Box - whisker plot represents the interquartile range ( 25th and 75th percentiles ) as a box and the median as a line . The maximn and minimun values within 1 . 5 x interquartile range are shown as whiskers . Outlier data are plotted as dots . C Relative expression of FAM189A2 / ENTREP in the primary ( green ) and the metastatic sites ( blue ) including lymph node , bone , liver , lung , soft tissues of prostatic cancer ( Grasso Prostate ) ( left ) and in the normal ( white ) , primary ( green ) and metastatic sites ( three cases of sentinel lymph node ) ( blue ) of breast cancer ( TCGA Breast ) ( right ) . Note that only three cases of the metastatic breast cancer were available in the dataset . Data were downloaded from the Oncomine database P - values obtained by Student ' s t - tests and the number of cases are listed on the top and bottom of figures , respectively . P < 0 . 05 was considered as statistically significant . Box - whisker plot represents the interquartile range ( 25th and 75th percentiles ) as a box and the median as a line . The maximn and minimun values within 1 . 5 x interquartile range are shown as whiskers . Outlier data are plotted as dots .

O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Relative expression of FAM189A2/ENTREP in the normal (white) and cancer (green) tissues of breast (Curtis Breast), lung (Okayama Lung), colorectal (TCGA Colorectal) and head and neck (Estilo Head-Neck). Data were downloaded from the Oncomine database P-values obtained by Student's t-tests and the number of cases are listed on the top and bottom of figures, respectively. P < 0.05 was considered as statistically significant. Box-whisker plot represents the interquartile range (25th and 75th percentiles) as a box and the median as a line. The maximn and minimun values within 1.5 x interquartile range are shown as whiskers. Outlier data are plotted as dots. C Relative expression of FAM189A2/ENTREP in the primary (green) and the metastatic sites (blue) including lymph node, bone, liver, lung, soft tissues of prostatic cancer (Grasso Prostate) (left) and in the normal (white), primary (green) and metastatic sites (three cases of sentinel lymph node) (blue) of breast cancer (TCGA Breast) (right). Note that only three cases of the metastatic breast cancer were available in the dataset. Data were downloaded from the Oncomine database P-values obtained by Student's t-tests and the number of cases are listed on the top and bottom of figures, respectively. P < 0.05 was considered as statistically significant. Box-whisker plot represents the interquartile range (25th and 75th percentiles) as a box and the median as a line. The maximn and minimun values within 1.5 x interquartile range are shown as whiskers. Outlier data are plotted as dots.


D - F Relapse - free survival ( RFS ) of breast cancer patients . Data were downloaded from the Kaplan - Meier

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D-F Relapse-free survival (RFS) of breast cancer patients. Data were downloaded from the Kaplan-Meier plotter


B , C The immunoprecipitation analysis . Co - precipitation was impaired by either the WW domain deletion of ITCH or the mutation in the PPxY motifs of ENTREP . Data shown are representative of at least two independent experiments . IP , immunoprecipitation ; IB , immunoblot .

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B, C The immunoprecipitation analysis. Co-precipitation was impaired by either the WW domain deletion of ITCH or the mutation in the PPxY motifs of ENTREP. Data shown are representative of at least two independent experiments. IP, immunoprecipitation; IB, immunoblot.


E Representative images of the proximity ligation assay ( PLA ) using Cos7 cells transfected with indicated vectors . PLA signals , granular dots of green color , were presented with DAPI images . Arrowheads , the plasma membrane . Scale bar , 10mm . F The box - and - whisker plot presentation of the PLA . A minimum of 10 cells per condition were counted from two independent experiments . P - values obtained by Student ' s t - tests and P < 0 . 05 was considered as statistically significant . * P < 0 . 05 . Box - whisker plot represents the interquartile range ( 25th and 75th percentiles ) as a box and the median as a line . The maximn and minimun values within 1 . 5 x interquartile range are shown as whiskers .

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E Representative images of the proximity ligation assay (PLA) using Cos7 cells transfected with indicated vectors. PLA signals, granular dots of green color, were presented with DAPI images. Arrowheads, the plasma membrane. Scale bar, 10mm. F The box-and-whisker plot presentation of the PLA. A minimum of 10 cells per condition were counted from two independent experiments. P-values obtained by Student's t-tests and P < 0.05 was considered as statistically significant. * P < 0.05. Box-whisker plot represents the interquartile range (25th and 75th percentiles) as a box and the median as a line. The maximn and minimun values within 1.5 x interquartile range are shown as whiskers.


A The nickel - pull down assay using myc / HIS - tagged ENTREP along with HA - tagged ubiquitin . Arrows indicate HA - ubiquitin - incorporating ENTREP . Arrowhead indicates ENTREP without ubiquitination . Data shown are representative of three independent experiments .

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A The nickel-pull down assay using myc/HIS-tagged ENTREP along with HA-tagged ubiquitin. Arrows indicate HA-ubiquitin-incorporating ENTREP. Arrowhead indicates ENTREP without ubiquitination. Data shown are representative of three independent experiments.


A The immunoprecipitation analysis . The cytoplasmic ENTREP ( wild or mut1 + 2 mutant ) co - precipitated with EPN1myc in the presence of ITCH wild or C830A . The blot bands were semi - quantified using ImageJ software . ENTREP ratio was caluculated as a ratio of ENTREP in the myc - coprecipitaed sample to that of the total lysate . The ratios shown as mean ± SD from three biological replicates . P - values were obtained by Student ' s t - tests and P < 0 . 005 was considered as statistically significant . No significant difference was observed .

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

A The immunoprecipitation analysis. The cytoplasmic ENTREP (wild or mut1+2 mutant) co-precipitated with EPN1myc in the presence of ITCH wild or C830A. The blot bands were semi-quantified using ImageJ software. ENTREP ratio was caluculated as a ratio of ENTREP in the myc-coprecipitaed sample to that of the total lysate. The ratios shown as mean ± SD from three biological replicates. P-values were obtained by Student's t-tests and P < 0.005 was considered as statistically significant. No significant difference was observed.


B Representative images of the proximity ligation assay ( PLA ) using Cos7 cells transfected with indicated vectors . PLA signals , granular dots of green color , were presented with DAPI images . Scale bar , 10mm . C The box - and - whisker plot presentation of the PLA . A minimum of 10 cells per condition were counted from two independent experiments . P - values obtained by Student ' s t - tests and P < 0 . 05 was considered as statistically significant . * P < 0 . 05 . Box - whisker plot represents the interquartile range ( 25th and 75th percentiles ) as a box and the median as a line . The maximn and minimun values within 1 . 5 x interquartile range are shown as whiskers .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Representative images of the proximity ligation assay (PLA) using Cos7 cells transfected with indicated vectors. PLA signals, granular dots of green color, were presented with DAPI images. Scale bar, 10mm. C The box-and-whisker plot presentation of the PLA. A minimum of 10 cells per condition were counted from two independent experiments. P-values obtained by Student's t-tests and P < 0.05 was considered as statistically significant. * P < 0.05. Box-whisker plot represents the interquartile range (25th and 75th percentiles) as a box and the median as a line. The maximn and minimun values within 1.5 x interquartile range are shown as whiskers.


B The immunoprecipitation analysis . Cytoplasmic ENTREP co - precipitated with either ITCH C830A , delWW12 or delWW34 but not with delWW14 . Data shown are representative of at least two independent experiments . C The immunoprecipitation analysis . HA - CXCR4SS - DsRed hardly co - precipitated with either ITCHC830A or delWW mutants . HA - CXCR4DD - DsRed co - precipitated with ITCH C830A and delWW12 but not with delWW14 and delWW34 . Data shown are representative of at least two independent experiments .

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

B The immunoprecipitation analysis. Cytoplasmic ENTREP co-precipitated with either ITCH C830A, delWW12 or delWW34 but not with delWW14. Data shown are representative of at least two independent experiments. C The immunoprecipitation analysis. HA-CXCR4SS-DsRed hardly co-precipitated with either ITCHC830A or delWW mutants. HA-CXCR4DD-DsRed co-precipitated with ITCH C830A and delWW12 but not with delWW14 and delWW34. Data shown are representative of at least two independent experiments.


F The nickel - pull down assay using indicated vectors along with HISx6 - tagged wild type ubiquitin vector . HA - tagged DsRed was used as a control for HA - CXCR4DD - DsRed . Arrowhead , FLAG - tagged ITCH ( wild or C830A ) ; arrow , FLAG - ENTREP wild cyt . Data shown are representative of at least two independent experiments . G Co - localization of ENTREP and CXCR4 in the endosome . Cos7 cells were transfected with ENTREP - FLAG , CXCR4 - DsRed and Halo - ITCH wild vectors and treated with CXCL12 . ENTREP - FLAG and CXCR4 - DsRed co - localized in the RAB7 - positive endosome . Inserts , the high - magnification images of the marked area indicating the colocalizaion of ENTREP - FLAG , CXCR4 - DsRed and RAB7 . Scale bar 10mm . The image is representative of at least two independent experiments .

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

F The nickel-pull down assay using indicated vectors along with HISx6-tagged wild type ubiquitin vector. HA-tagged DsRed was used as a control for HA-CXCR4DD-DsRed. Arrowhead, FLAG-tagged ITCH (wild or C830A); arrow, FLAG-ENTREP wild cyt. Data shown are representative of at least two independent experiments. G Co-localization of ENTREP and CXCR4 in the endosome. Cos7 cells were transfected with ENTREP-FLAG, CXCR4-DsRed and Halo-ITCH wild vectors and treated with CXCL12. ENTREP-FLAG and CXCR4-DsRed co-localized in the RAB7-positive endosome. Inserts, the high-magnification images of the marked area indicating the colocalizaion of ENTREP-FLAG, CXCR4-DsRed and RAB7. Scale bar 10mm. The image is representative of at least two independent experiments.


C The expression of endogenous CXCR4 in lentivirally - transduced MCF - 7 ENTREP - KO cells . CXCL12 ( + ) and ( - ) indicate the presence and absence of CXCL12 treatment . In CXCL12 - treated ENTREP - EGFP expressing cells , a non - negligible amount of CXCR4 was observed in the cytoplasm , in which CXCR4 was overlapped with ENTREP - EGFP . CXCR4 expression on the cell surface still remained ( arrows ) . Scale bar 10 mm . The images are representative of at least three independet experiments .

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C The expression of endogenous CXCR4 in lentivirally-transduced MCF-7 ENTREP-KO cells. CXCL12 (+) and (-) indicate the presence and absence of CXCL12 treatment. In CXCL12-treated ENTREP-EGFP expressing cells, a non-negligible amount of CXCR4 was observed in the cytoplasm, in which CXCR4 was overlapped with ENTREP-EGFP. CXCR4 expression on the cell surface still remained (arrows). Scale bar 10 mm. The images are representative of at least three independet experiments.


A Dot plot presentation of the chemotaxis analyses using the Boyden chamber . CXCL12 ( + ) and ( - ) indicate the presence and absence of CXCL12 as an attractant in the bottom chambers . ENTREP - knockout enhanced the chemotaxis toward CXCL12 , which was blocked by pre - treatment with AMD3100 ( upper panel ) . The doxycyclin ( DOX ) - induced ENTREP expression suppressed the chemotaxis of mouse 4T1 - Luc cells toward CXCL12 ( lower panel ) . Ratio of migrated cells into the bottom chamber against the applied cells on the top chambers was normalized by the results of non - treated cells ( n = 7 , upper panel , and n = 9 , lower panel , from at least two independent experiments , respectively ) . P - values were obtained by Student ' s t - tests and P < 0 . 005 was considered as statistically significant . Insert , the immunoblot analysis of the DOX - induced ENTREP expression in 4T1 - Luc cells . NS , not significant .

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

A Dot plot presentation of the chemotaxis analyses using the Boyden chamber. CXCL12 (+) and (-) indicate the presence and absence of CXCL12 as an attractant in the bottom chambers. ENTREP-knockout enhanced the chemotaxis toward CXCL12, which was blocked by pre-treatment with AMD3100 (upper panel). The doxycyclin (DOX)-induced ENTREP expression suppressed the chemotaxis of mouse 4T1-Luc cells toward CXCL12 (lower panel). Ratio of migrated cells into the bottom chamber against the applied cells on the top chambers was normalized by the results of non-treated cells (n=7, upper panel, and n=9, lower panel, from at least two independent experiments, respectively). P-values were obtained by Student's t-tests and P < 0.005 was considered as statistically significant. Insert, the immunoblot analysis of the DOX-induced ENTREP expression in 4T1-Luc cells. NS, not significant.


B Dot plot presentation of mammosphere assay ( left panel ) and the images of mammosphere ( right panel ) . Each mark in dot plot presentation indicates the number of mammosphere from 3 , 000 cells per well in an ultra - low attachment plate from two independent experiments . To estimate the effect of CXCL12 / CXCR4 pathway , 100 ng / ml of CXCL12 and 12 μM of AMD3100 were applied as indicated . For statistical analysis , variance was assessed using two - way ANOVA and significance was calculated using Tukey post hoc test correcting for multiple comparison . NS , not significant . Scale bar in the images of mammosphere 100 mm .

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B Dot plot presentation of mammosphere assay (left panel) and the images of mammosphere (right panel). Each mark in dot plot presentation indicates the number of mammosphere from 3,000 cells per well in an ultra-low attachment plate from two independent experiments. To estimate the effect of CXCL12/CXCR4 pathway, 100 ng/ml of CXCL12 and 12 μM of AMD3100 were applied as indicated. For statistical analysis, variance was assessed using two-way ANOVA and significance was calculated using Tukey post hoc test correcting for multiple comparison. NS, not significant. Scale bar in the images of mammosphere 100 mm.


C Co - crystal structure of gephyrin E - domain ( blue ) and bound GlyR ß - loop ( red - PDB code 2FTS ( Kim et al . , 2006 ) ) . The mutated gephyrin Gly375 residue is shown in orange .

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C Co-crystal structure of gephyrin E-domain (blue) and bound GlyR ß-loop (red - PDB code 2FTS (Kim et al., 2006)). The mutated gephyrin Gly375 residue is shown in orange.


A Representative images of GFP - gephyrin and GFP - G375D , expressed in hippocampal neurons at 9 + 4 days in vitro . Inset shows higher magnification . Scale bar = 20 µm . B Relative distribution of cluster density of gephyrin variants on dendrites from transfected neurons . Number of gephyrin clusters in each dendrite is normalized to 20 µm segments .

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A Representative images of GFP-gephyrin and GFP-G375D, expressed in hippocampal neurons at 9 + 4 days in vitro. Inset shows higher magnification. Scale bar = 20 µm.B Relative distribution of cluster density of gephyrin variants on dendrites from transfected neurons. Number of gephyrin clusters in each dendrite is normalized to 20 µm segments.


C Representative segments of dendrites expressing GFP - gephyrin or GFP - G375D and immunostained with the presynaptic marker VGAT . Arrowheads show co - localization of gephyrin and VGAT clusters . Scale bar = 10 µm . D Quantification of co - localization between gephyrin and VGAT clusters ( gephyrin - VGAT 77 . 4 ± 2 . 9 % , G375D - VGAT 30 ± 4 . 4 % ) . 31 GFP - gephyrin and 30 GFP - G375D neurons from three independent cultures were used for quantifications . Results are expressed as mean ±

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C Representative segments of dendrites expressing GFP-gephyrin or GFP-G375D and immunostained with the presynaptic marker VGAT. Arrowheads show co-localization of gephyrin and VGAT clusters. Scale bar = 10 µm.D Quantification of co-localization between gephyrin and VGAT clusters (gephyrin-VGAT 77.4±2.9 %, G375D-VGAT 30±4.4 %). 31 GFP-gephyrin and 30 GFP-G375D neurons from three independent cultures were used for quantifications. Results are expressed as mean ± SEM


A Representative dendritic segment of a neuron expressing GFP - G375D and 3B11 - immunostained endogenous gephyrin ( red ) . B Quantification of density and ( C ) size of endogenous gephyrin clusters in neurons expressing GFP , GFP - G375D or non - transfected neighboring neurons . 20 GFP - G375 and non - transfected neurons and 15 GFP - expressing neurons from three independent cultures were used for quantifications . Results are expressed as mean ± SEM and data were analyzed by t - test .

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A Representative dendritic segment of a neuron expressing GFP-G375D and 3B11-immunostained endogenous gephyrin (red).B Quantification of density and (C) size of endogenous gephyrin clusters in neurons expressing GFP, GFP-G375D or non-transfected neighboring neurons. 20 GFP-G375 and non-transfected neurons and 15 GFP-expressing neurons from three independent cultures were used for quantifications. Results are expressed as mean ± SEM and data were analyzed by t-test.


A Representative neurons expressing myc - tagged gephyrin or G375D and pHluorin - tagged GABAAR γ2 . Surface - expressed γ2 - subunits were immunolabelled with GFP - antibodies in red , and the pHluorin - fluorescence ( total γ2 levels ) are shown in green . B Size and C fluorescence ( fl . ) intensity of surface - expressed γ2 clusters in myc - gephyrin and myc - G375D - expressing dendrites ( n = 20 myc - gephyrin and 21 myc - G375D neurons in B and 18 myc - gephyrin and myc - G375D neurons in C ) D Mean γ2 - pHluorin fluorescence in myc - gephyrin and myc - G375D - expressing dendrites ( n = 20 and 18 myc - gephyrin and myc - G375D expressing neurons ) . E Number of surface γ2 clusters on dendrites normalized to 20 µm segments ( n = 18 myc - gephyrin and myc - G375D expressing neurons ) . At least three independent cultures were used for each experiment . Results are expressed as mean ± SEM ; Data were analyzed by t - test .

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A Representative neurons expressing myc-tagged gephyrin or G375D and pHluorin-tagged GABAAR γ2. Surface-expressed γ2-subunits were immunolabelled with GFP-antibodies in red, and the pHluorin-fluorescence (total γ2 levels) are shown in green.B Size and C fluorescence (fl.) intensity of surface-expressed γ2 clusters in myc-gephyrin and myc-G375D-expressing dendrites (n = 20 myc-gephyrin and 21 myc-G375D neurons in B and 18 myc-gephyrin and myc-G375D neurons in C)D Mean γ2-pHluorin fluorescence in myc-gephyrin and myc-G375D-expressing dendrites (n = 20 and 18 myc-gephyrin and myc-G375D expressing neurons).E Number of surface γ2 clusters on dendrites normalized to 20 µm segments (n = 18 myc-gephyrin and myc-G375D expressing neurons). At least three independent cultures were used for each experiment. Results are expressed as mean ± SEM; Data were analyzed by t-test.


F Representative traces of miniature recordings on cultured hippocampal neurons transfected either with GFP - gephyrin or GFP - G375D . G mIPSC amplitudes from neurons expressing GFP - gephyrin or GFP - G375D . Numbers show analyzed cell - numbers . Data were analyzed by one - way ANOVA and post - hoc Tukey test . H Frequency of the mIPSCs from neurons expressing GFP - gephyrin or GFP - G375D . The horizontal line within the boxes indicates the median frequency for the represented condition ( bottom / top boundary indicates the 25th / 75th percentile ) . Top and bottom error bars indicate the 90th and 10th percentiles , respectively . Analysis by Kruskal - Wallis test followed by post - hoc Mann - Whitney test .

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F Representative traces of miniature recordings on cultured hippocampal neurons transfected either with GFP-gephyrin or GFP-G375D.G mIPSC amplitudes from neurons expressing GFP-gephyrin or GFP-G375D. Numbers show analyzed cell-numbers. Data were analyzed by one-way ANOVA and post-hoc Tukey test.H Frequency of the mIPSCs from neurons expressing GFP-gephyrin or GFP-G375D. The horizontal line within the boxes indicates the median frequency for the represented condition (bottom/top boundary indicates the 25th /75th percentile). Top and bottom error bars indicate the 90th and 10th percentiles, respectively. Analysis by Kruskal-Wallis test followed by post-hoc Mann-Whitney test.


A Western blot of HEK293 cells expressing GFP - tagged gephyrin or G375D using GFP - specific and polyclonal gephyrin antibodies . Note that both gephyrin variants are stably expressed and show no degradation in the cells . Asterisk in the poly - gephyrin blot shows endogenous gephyrin .

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A Western blot of HEK293 cells expressing GFP-tagged gephyrin or G375D using GFP-specific and polyclonal gephyrin antibodies. Note that both gephyrin variants are stably expressed and show no degradation in the cells. Asterisk in the poly-gephyrin blot shows endogenous gephyrin.


B SDS - PAGE of E . coli expressed and purified 6His - gephyrins and CD spectra . Mean residue ellipticity of gephyrin and G375D show profiles with minima at 208 nm and 222 nm .

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B SDS-PAGE of E. coli expressed and purified 6His-gephyrins and CD spectra. Mean residue ellipticity of gephyrin and G375D show profiles with minima at 208 nm and 222 nm.


C Size - exclusion chromatography of purified 6His - gephyrin and isolated E - domain variants . Full - length gephyrins and isolated E - domains were loaded onto a Superose 6 gel filtration column and elution of the protein was monitored by measuring absorbance at 280 nm ( mAU , milliabsorbance units ) .

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C Size-exclusion chromatography of purified 6His-gephyrin and isolated E-domain variants. Full-length gephyrins and isolated E-domains were loaded onto a Superose 6 gel filtration column and elution of the protein was monitored by measuring absorbance at 280 nm (mAU, milliabsorbance units).


D Co - immunoprecipitation of HEK293 cell lysates expressing myc - tagged gephyrin together with GFP - tagged gephyrin or G375D using myc - specific antibodies . Control IgG were used to show specificity of the assay .

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D Co-immunoprecipitation of HEK293 cell lysates expressing myc-tagged gephyrin together with GFP-tagged gephyrin or G375D using myc-specific antibodies. Control IgG were used to show specificity of the assay.


B Representative pulldown - experiment and quantification of 6His - tagged gephyrin and G375D using immobilized α3 - peptide . 6His - tagged gephyrin variants were expressed in E . coli cells , purified to homogeneity and incubated with α3 - peptides immobilized to NeutrAvidin beads . n = 3 , normalized to 6His - gephyrin . 38 . 9 ± 1 . 5 % G375D were pulled down .

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B Representative pulldown-experiment and quantification of 6His-tagged gephyrin and G375D using immobilized α3-peptide. 6His-tagged gephyrin variants were expressed in E. coli cells, purified to homogeneity and incubated with α3-peptides immobilized to NeutrAvidin beads. n = 3, normalized to 6His-gephyrin. 38.9±1.5% G375D were pulled down.


C Representative SPR sensograms of 6His - gephyrin and 6His - G375D binding to immobilized GABAAR α3 - peptide . Response units are plotted against time . Injected gephyrin concentrations were 0 . 5 , 2 , 10 , 25 , 40 µM .

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C Representative SPR sensograms of 6His-gephyrin and 6His-G375D binding to immobilized GABAAR α3-peptide. Response units are plotted against time. Injected gephyrin concentrations were 0.5, 2, 10, 25, 40 µM.


D SPR binding isotherms of a representative experiment as in C . Calculated KD values of 3 . 2 and 10 µM for gephyrin and G375D , respectively , are indicated by vertical lines . The experiment was repeated three times with two independently purified protein batches and two sensor chips .

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D SPR binding isotherms of a representative experiment as in C. Calculated KD values of 3.2 and 10 µM for gephyrin and G375D, respectively, are indicated by vertical lines. The experiment was repeated three times with two independently purified protein batches and two sensor chips.


E Representative ITC binding isotherms of the cytoplasmic loop of the GlyR ß - subunit ( GlyR - ßL , residue 378 - 426 ) and 6His - tagged gephyrin or G375D . The GlyR - ßL - gephyrin binding data ( black ) were fitted in a two - side model , whereas G375D binding could only be fitted to a single binding site . Dissociation constants KD and stoichiometry ( N = number of binding sites ) are given for each binding component . n = 5 from two independently purified protein batches . Results are expressed as mean ± SEM .

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E Representative ITC binding isotherms of the cytoplasmic loop of the GlyR ß-subunit (GlyR-ßL, residue 378-426) and 6His-tagged gephyrin or G375D. The GlyR-ßL-gephyrin binding data (black) were fitted in a two-side model, whereas G375D binding could only be fitted to a single binding site. Dissociation constants KD and stoichiometry (N = number of binding sites) are given for each binding component. n = 5 from two independently purified protein batches. Results are expressed as mean ± SEM.


A Biosynthesis pathway of Moco with all stable intermediates . Gephyrin G - and E - domain catalyze the last two steps as depicted . B In vitro Moco - synthesis assay using 150 pmol purified 6His - tagged gephyrins . D580A is an activity - deficient gephyrin mutation previously identified in Moco - deficient patients ( Reiss et al . , 2011 ) . Assays without the addition of gephyrin ( - gephyrin ) or molybdenum ( - Mo ) served as internal controls of the assay ( see also ( Belaidi Schwarz , 2013 ) ) . C Experiment as in B but with pre - incubation of 6His - gephyrin / G375D in a 1 : 1 ratio to simulate the heterozygous mutation . n = at least 3 with two independently purified protein batches . Results are expressed as mean ± SEM .

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A Biosynthesis pathway of Moco with all stable intermediates. Gephyrin G- and E-domain catalyze the last two steps as depicted.B In vitro Moco-synthesis assay using 150 pmol purified 6His-tagged gephyrins. D580A is an activity-deficient gephyrin mutation previously identified in Moco-deficient patients (Reiss et al., 2011). Assays without the addition of gephyrin (-gephyrin) or molybdenum (-Mo) served as internal controls of the assay (see also (Belaidi Schwarz, 2013)).C Experiment as in B but with pre-incubation of 6His-gephyrin/G375D in a 1:1 ratio to simulate the heterozygous mutation. n = at least 3 with two independently purified protein batches. Results are expressed as mean ± SEM.


( b ) Parkin is recruited selectively to depolarized mitochondria and directs mitophagy . HeLa cells transfected with HA - Parkin were treated with CCCP for the indicated times . Mitochondria were stained by anti - TOM20 ( pseudo coloured ; blue ) and a ΔΨm dependent MitoTracker ( red ) . Parkin was stained with anti - HA ( green ) . Without treatment , mitochondria are intact and stained by both mitochondrial markers , whereas Parkin is equally distributed in the cytoplasm . After 2 h of CCCP treatment , mitochondria are depolarized as shown by the loss of MitoTracker staining . Parkin completely translocates to mitochondria clustering at perinuclear regions . After 24 h of CCCP treatment , massive loss of mitochondria is observed as shown by the disappearance of the mitochondrial marker . Only Parkin - positive cells show mitochondrial clustering and clearance , in contrast to adjacent untransfected cells . Scale bars , 10 μm .

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(b) Parkin is recruited selectively to depolarized mitochondria and directs mitophagy. HeLa cells transfected with HA-Parkin were treated with CCCP for the indicated times. Mitochondria were stained by anti-TOM20 (pseudo coloured; blue) and a ΔΨm dependent MitoTracker (red). Parkin was stained with anti-HA (green). Without treatment, mitochondria are intact and stained by both mitochondrial markers, whereas Parkin is equally distributed in the cytoplasm. After 2 h of CCCP treatment, mitochondria are depolarized as shown by the loss of MitoTracker staining. Parkin completely translocates to mitochondria clustering at perinuclear regions. After 24 h of CCCP treatment, massive loss of mitochondria is observed as shown by the disappearance of the mitochondrial marker. Only Parkin-positive cells show mitochondrial clustering and clearance, in contrast to adjacent untransfected cells. Scale bars, 10 μm.


( c ) Pathogenic Parkin mutations abrogate mitochondrial translocation and / or clearance . HeLa cells transfected with Flag - Parkin wild - type or pathogenic mutants were treated with CCCP and stained with anti - Flag ( green ) , anti - TOM20 ( red ) and Hoechst33342 ( blue ) . Representative merged images are shown . Scale bars , 10 μm . Immunostaining of cells transfected with non - functional cysteine mutants and functional Parkin variants is shown in Supplementary Information , Fig . S1a and S1b , respectively .

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(c) Pathogenic Parkin mutations abrogate mitochondrial translocation and/or clearance. HeLa cells transfected with Flag-Parkin wild-type or pathogenic mutants were treated with CCCP and stained with anti-Flag (green), anti-TOM20 (red) and Hoechst33342 (blue). Representative merged images are shown. Scale bars, 10 μm. Immunostaining of cells transfected with non-functional cysteine mutants and functional Parkin variants is shown in Supplementary Information, Fig. S1a and S1b, respectively.


( d , e ) Based on the mitochondrial effects , Parkin mutants can be classified into functional and non - functional classes . ( d ) Quantification of Parkin translocation to damaged mitochondria after 2 h of CCCP treatment , as defined by complete colocalization of Parkin with the mitochondrial marker ( n = 3 ) . ( e ) Quantification of cells with uncleared mitochondria after 24 h of CCCP treatment , as shown by complete loss of mitochondrial staining ( n = 3 ) . Data represent the mean ± s . d . , * P ≤ 0 . 05 , * * P ≤ 0 . 005 , * * * P ≤ 0 . 0005 compared with wild - type . WT , wild - type .

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(d, e) Based on the mitochondrial effects, Parkin mutants can be classified into functional and non-functional classes. (d) Quantification of Parkin translocation to damaged mitochondria after 2 h of CCCP treatment, as defined by complete colocalization of Parkin with the mitochondrial marker (n = 3). (e) Quantification of cells with uncleared mitochondria after 24 h of CCCP treatment, as shown by complete loss of mitochondrial staining (n = 3). Data represent the mean ± s.d., *P ≤ 0.05, **P ≤ 0.005, ***P ≤ 0.0005 compared with wild-type. WT, wild-type.


Stable Parkin wild - type or pathogenic mutants expressing SH - SY5Y cell lines were induced with doxycycline ( DOX ) for 72 h and treated with CCCP for the indicated times . Cells were stained with anti - Parkin ( green ) , anti - TOM20 ( red ) and Hoechst33342 ( blue ) . ( a - b ) CCCP treatment of stable SH - SY5Y cells for 0 h . Unstimulated control cells ( - DOX ) show relatively low levels of endogenous Parkin , compared to cells with induced expression of Parkin wild - type or K161N , R275W and G430D mutants ( + DOX ) . Immunostaining ( a ) and western blot analysis ( b ) of the induced cells demonstrate comparable levels of Parkin expression in the selected clones . β - Actin served as a loading control .

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Stable Parkin wild-type or pathogenic mutants expressing SH-SY5Y cell lines were induced with doxycycline (DOX) for 72 h and treated with CCCP for the indicated times. Cells were stained with anti-Parkin (green), anti-TOM20 (red) and Hoechst33342 (blue). (a-b) CCCP treatment of stable SH-SY5Y cells for 0 h. Unstimulated control cells (- DOX) show relatively low levels of endogenous Parkin, compared to cells with induced expression of Parkin wild-type or K161N, R275W and G430D mutants (+ DOX). Immunostaining (a) and western blot analysis (b) of the induced cells demonstrate comparable levels of Parkin expression in the selected clones. β-Actin served as a loading control.


( c , d ) After 2 h of CCCP treatment , massive colocalization of wild - type Parkin and mitochondrial clusters can be observed in the induced cells when compared with unstimulated controls . Similarly , Parkin K161N and R275W mutants translocate to mitochondria , whereas G430D does not .

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(c, d) After 2 h of CCCP treatment, massive colocalization of wild-type Parkin and mitochondrial clusters can be observed in the induced cells when compared with unstimulated controls. Similarly, Parkin K161N and R275W mutants translocate to mitochondria, whereas G430D does not.


( e , f ) After 24 h of CCCP treatment , induced wild - type Parkin - expressing cells show full clearance of damaged mitochondria . In contrast , the pathogenic mutants analysed fail to execute mitophagy and , therefore , damaged mitochondria remain uncleared . Representative images of three independent experiments are shown . Scale bars , 10 μm ( a , c and e ) . ( d , f ) Quantifications of the observed mitochondrial effects in Parkin - expressing SH - SY5Y cells ( n = 3 ) . Data represent the mean ± s . d . , * * * P ≤ 0 . 0005 compared with induced wild - type Parkin . WT , wild - type .

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(e, f) After 24 h of CCCP treatment, induced wild-type Parkin-expressing cells show full clearance of damaged mitochondria. In contrast, the pathogenic mutants analysed fail to execute mitophagy and, therefore, damaged mitochondria remain uncleared. Representative images of three independent experiments are shown. Scale bars, 10 μm (a, c and e). (d, f) Quantifications of the observed mitochondrial effects in Parkin-expressing SH-SY5Y cells (n = 3). Data represent the mean ± s.d., ***P ≤ 0.0005 compared with induced wild-type Parkin. WT, wild-type.


( b ) Co - immunoprecipitations of Parkin and PINK1 after mitochondrial depolarization . Endogenous Parkin and PINK1 were co - immunoprecipitated from neuronal SH - SY5Y cells after CCCP treatment with specific antibodies .

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(b) Co-immunoprecipitations of Parkin and PINK1 after mitochondrial depolarization. Endogenous Parkin and PINK1 were co-immunoprecipitated from neuronal SH-SY5Y cells after CCCP treatment with specific antibodies.


( c ) Western blot of PINK1 siRNA - transfected HeLa cells used in d - h demonstrates the knockdown efficiency and specificity of PINK1 silencing .

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(c) Western blot of PINK1 siRNA-transfected HeLa cells used in d-h demonstrates the knockdown efficiency and specificity of PINK1 silencing.


d - h ) PINK1 - specific siRNA abrogates Parkin translocation to mitochondria after 2 h , and mitochondrial clearance after 24 h , of CCCP treatment . This can be rescued by re - transfection with wild - type PINK1 , but not with a kinase - dead mutant or a PINK1 variant lacking the MTS . Control siRNA - or PINK1 siRNA - silenced HeLa cells were transfected with EGFP - Parkin wild - type and PINK1 - V5 wild - type , 3 × KD or ΔMTS mutants . Use of appropriate empty vector controls is depicted . Cells were treated with CCCP and stained with anti - TOM20 ( pseudo coloured ; blue ) and anti - V5 ( red ) to visualize mitochondrial morphology and re - transfected PINK1 , respectively . Wild - type Parkin was visualized by EGFP epifluorescence ( green ) . Parkin - positive cells are surrounded by a line . Representative images of four independent experiments are shown . Scale bars , 10 μm .

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d-h) PINK1-specific siRNA abrogates Parkin translocation to mitochondria after 2 h, and mitochondrial clearance after 24 h, of CCCP treatment. This can be rescued by re-transfection with wild-type PINK1, but not with a kinase-dead mutant or a PINK1 variant lacking the MTS. Control siRNA- or PINK1 siRNA-silenced HeLa cells were transfected with EGFP-Parkin wild-type and PINK1-V5 wild-type, 3×KD or ΔMTS mutants. Use of appropriate empty vector controls is depicted. Cells were treated with CCCP and stained with anti-TOM20 (pseudo coloured; blue) and anti-V5 (red) to visualize mitochondrial morphology and re-transfected PINK1, respectively. Wild-type Parkin was visualized by EGFP epifluorescence (green). Parkin-positive cells are surrounded by a line. Representative images of four independent experiments are shown. Scale bars, 10 μm.


( e ) Wild - type Parkin colocalization with impaired mitochondria after 2 h of CCCP treatment was analysed ( n = 4 ) . ( g ) Quantification of results from cells in e .

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(e) Wild-type Parkin colocalization with impaired mitochondria after 2 h of CCCP treatment was analysed (n = 4). (g) Quantification of results from cells in e.


( f ) Uncleared mitochondria were analysed after 24 h of CCCP treatment ( n = 4 ) . ( h ) Quantification of results from cells in f . Data represent the mean ± s . d . , * * P ≤ 0 . 005 , * * * P ≤ 0 . 0005 compared with control siRNA - transfected cells . Immunostaining of control siRNA - transfected cells at all analysed time points is shown in Supplementary Information , Fig . S2 . WT , wild - type .

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(f) Uncleared mitochondria were analysed after 24 h of CCCP treatment (n = 4). (h) Quantification of results from cells in f. Data represent the mean ± s.d., **P ≤ 0.005, ***P ≤ 0.0005 compared with control siRNA-transfected cells. Immunostaining of control siRNA-transfected cells at all analysed time points is shown in Supplementary Information, Fig. S2. WT, wild-type.


a , b ) Stable Mito - DsRed ( pseudo coloured ; blue ) - expressing HeLa cells were transfected with Flag - Parkin wild - type or pathogenic mutants . Cells were treated with CCCP before immunostaining of Parkin with anti - Flag ( green ) and endogenous poly - ubiquitin with a specific antibody FK1 ( red ) . ( a ) In contrast to pathogenic mutations , wild - type Parkin shows colocalization with poly - ubiquitin after 6 h of CCCP treatment at the site of condensed mitochondria . ( b ) After 24 h of CCCP treatment , K161N and R275W Parkin mutants colocalize with both poly - ubiquitin and mitochondria . In contrast , translocation - deficient Parkin mutants K211N , T240R and G430D lack the mitochondria - associated poly - ubiquitin signal . Representative images are shown . Scale bars , 10 μm . Complementary Parkin of untreated cells and quantification of colocalization of Parkin , mitochondria and poly - ubiquitin upon CCCP treatment are shown in Supplementary Information , Fig . S3a , b and c , respectively .

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

a, b) Stable Mito-DsRed (pseudo coloured; blue)-expressing HeLa cells were transfected with Flag-Parkin wild-type or pathogenic mutants. Cells were treated with CCCP before immunostaining of Parkin with anti-Flag (green) and endogenous poly-ubiquitin with a specific antibody FK1 (red). (a) In contrast to pathogenic mutations, wild-type Parkin shows colocalization with poly-ubiquitin after 6 h of CCCP treatment at the site of condensed mitochondria. (b) After 24 h of CCCP treatment, K161N and R275W Parkin mutants colocalize with both poly-ubiquitin and mitochondria. In contrast, translocation-deficient Parkin mutants K211N, T240R and G430D lack the mitochondria-associated poly-ubiquitin signal. Representative images are shown. Scale bars, 10 μm. Complementary Parkin of untreated cells and quantification of colocalization of Parkin, mitochondria and poly-ubiquitin upon CCCP treatment are shown in Supplementary Information, Fig. S3a, b and c, respectively.


( c , d ) Parkin - directed mitophagy specifically involves linkage of ubiquitin through lysines 27 and 63 . HeLa cells were transfected with EGFP - Parkin wild - type and ubiquitin lysine variants and treated with CCCP before immunostaining with anti - HtrA2 / Omi , as a mitochondrial marker ( pseudo coloured ; blue ) and anti - His ( red ) to detect ectopic ubiquitin . Parkin was visualized by its fusion to EGFP ( green ) . ( c ) Note that only wild - type , K27 and K63 6 × His - tagged ubiquitin variants are colocalized with condensed mitochondria and EGFP - Parkin after 6 h of CCCP treatment . All other ubiquitin mutants interfere with Parkin translocation to mitochondria . ( d ) Only co - expression of K27 or K63 ubiquitin recapitulates mitochondrial clearance similarly to 6 × His - tagged wild - type ubiquitin after 24 h of CCCP treatment . Representative images are shown . Scale bars , 10 μm . Immunostaining of untreated cells and all other ubiquitin variant combinations tested are shown in Supplementary Information , Fig . S4a and b , c , respectively . WT , wild - type ; PolyUb , poly - ubiquitin .

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(c, d) Parkin-directed mitophagy specifically involves linkage of ubiquitin through lysines 27 and 63. HeLa cells were transfected with EGFP-Parkin wild-type and ubiquitin lysine variants and treated with CCCP before immunostaining with anti-HtrA2/Omi, as a mitochondrial marker (pseudo coloured; blue) and anti-His (red) to detect ectopic ubiquitin. Parkin was visualized by its fusion to EGFP (green). (c) Note that only wild-type, K27 and K63 6×His-tagged ubiquitin variants are colocalized with condensed mitochondria and EGFP-Parkin after 6 h of CCCP treatment. All other ubiquitin mutants interfere with Parkin translocation to mitochondria. (d) Only co-expression of K27 or K63 ubiquitin recapitulates mitochondrial clearance similarly to 6×His-tagged wild-type ubiquitin after 24 h of CCCP treatment. Representative images are shown. Scale bars, 10 μm. Immunostaining of untreated cells and all other ubiquitin variant combinations tested are shown in Supplementary Information, Fig. S4a and b, c, respectively. WT, wild-type; PolyUb, poly-ubiquitin.


( a , b ) Stable Mito - DsRed ( pseudo coloured ; blue ) - expressing HeLa cells were transfected with Flag - Parkin wild - type or pathogenic mutants . Cells were treated with CCCP for the indicated times before immunostaining with anti - Flag ( green ) and anti - p62 ( red ) . ( a ) In contrast to the pathogenic mutants , wild - type Parkin colocalizes with p62 at the site of clustered mitochondria , after 6 h of CCCP treatment . ( b ) After 24 h of CCCP treatment , wild - type Parkin - transfected cells show complete loss of mitochondria , whereas K161N and R275W Parkin mutants colocalize with both p62 and mitochondria . In contrast , translocation - compromised mutants K211N , T240R and G430D , lacking the mitochondria - associated poly - ubiquitin signal ( Fig . 4b ) , did not colocalize with p62 . Representative images of four independent experiments are shown . Data from untreated control cells and quantification of colocalization of Parkin , mitochondria and p62 upon CCCP treatment are shown in Supplementary Information , Fig . 5a and b , c , respectively .

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

(a, b) Stable Mito-DsRed (pseudo coloured; blue)-expressing HeLa cells were transfected with Flag-Parkin wild-type or pathogenic mutants. Cells were treated with CCCP for the indicated times before immunostaining with anti-Flag (green) and anti-p62 (red). (a) In contrast to the pathogenic mutants, wild-type Parkin colocalizes with p62 at the site of clustered mitochondria, after 6 h of CCCP treatment. (b) After 24 h of CCCP treatment, wild-type Parkin-transfected cells show complete loss of mitochondria, whereas K161N and R275W Parkin mutants colocalize with both p62 and mitochondria. In contrast, translocation-compromised mutants K211N, T240R and G430D, lacking the mitochondria-associated poly-ubiquitin signal (Fig. 4b), did not colocalize with p62. Representative images of four independent experiments are shown. Data from untreated control cells and quantification of colocalization of Parkin, mitochondria and p62 upon CCCP treatment are shown in Supplementary Information, Fig. 5a and b, c, respectively.


( c , d ) Control siRNA - or p62 siRNA - silenced HeLa cells were transfected with EGFP - Parkin wild - type . Cells were treated with CCCP for the indicated times and stained with anti - TOM20 ( pseudo coloured ; blue ) to visualize mitochondrial morphology and anti - p62 ( red ) to monitor silencing efficiency . Parkin wild - type was visualized by EGFP epifluorescence ( green ) . Parkin - positive cells with cleared mitochondria are surrounded by a line . (

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(c, d) Control siRNA- or p62 siRNA-silenced HeLa cells were transfected with EGFP-Parkin wild-type. Cells were treated with CCCP for the indicated times and stained with anti-TOM20 (pseudo coloured; blue) to visualize mitochondrial morphology and anti-p62 (red) to monitor silencing efficiency. Parkin wild-type was visualized by EGFP epifluorescence (green). Parkin-positive cells with cleared mitochondria are surrounded by a line. (


( c - f ) Knockdown of p62 has no influence on Parkin translocation to mitochondria after 6 h of CCCP treatment ( c - e ) , but completely blocks final clearance after 24 h ( c , d , f ) . ( e , f ) Relative parkin translocation ( e ) and mitochondrial clearance ( f ) after p62 knockdown and CCCP treatment ( n = 4 ) . Data represent the mean ± s . d . , * * * P ≤ 0 . 0005 compared with control siRNA - transfected cells . Representative images of four independent experiments are shown . Scale bars , 10 μm . Immunostainings of control cells are shown in Supplementary Information , Fig . S5d , e .

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(c-f) Knockdown of p62 has no influence on Parkin translocation to mitochondria after 6 h of CCCP treatment (c-e), but completely blocks final clearance after 24 h (c, d, f). (e, f) Relative parkin translocation (e) and mitochondrial clearance (f) after p62 knockdown and CCCP treatment (n = 4). Data represent the mean ± s.d., ***P ≤ 0.0005 compared with control siRNA-transfected cells. Representative images of four independent experiments are shown. Scale bars, 10 μm. Immunostainings of control cells are shown in Supplementary Information, Fig. S5d, e.


( g ) Western blot of siRNA - transfected HeLa cells used in c - f demonstrates the efficiency of p62 silencing . β - Actin probing served as a loading control . WT , wild - type .

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(g) Western blot of siRNA-transfected HeLa cells used in c-f demonstrates the efficiency of p62 silencing. β-Actin probing served as a loading control. WT, wild-type.


( a ) Time - course experiment of HeLa cells transfected with Flag - Parkin wild - type and treated with CCCP for the indicated periods . Total lysates were prepared , and western blots were probed with specific anti - Parkin , anti - PINK1 , anti - TOM20 and anti - VDAC1 antibodies . β - Actin probing served as a loading control . All of the proteins detected are endogenously expressed , except Flag - Parkin .

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(a) Time-course experiment of HeLa cells transfected with Flag-Parkin wild-type and treated with CCCP for the indicated periods. Total lysates were prepared, and western blots were probed with specific anti-Parkin, anti-PINK1, anti-TOM20 and anti-VDAC1 antibodies. β-Actin probing served as a loading control. All of the proteins detected are endogenously expressed, except Flag-Parkin.


( b ) Pathogenic Parkin mutants fail to poly - ubiquitylate endogenous VDAC1 in response to CCCP treatment . HeLa cells were transfected with empty vector ( - ) , Flag - Parkin wild - type or pathogenic mutants along with 6 × His - tagged ubiquitin wild - type for 24 h . Cells were treated with CCCP for 6 h and subsequently protein lysates were prepared in urea buffer . Denaturing conditions of Ni - NTA affinity purifications were used to specifically pulldown only proteins covalently modified with 6xHis - tagged ubiquitin . Western blots of protein lysates and purified Ni - NTA agarose elutions were probed with anti - VDAC1 , anti - TOM20 and anti - Parkin antibodies . Modification of VDAC1 was observed in cells expressing Parkin wild - type and the functional mutant G328E , whereas non - functional mutants did not have this effect . (

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

(b) Pathogenic Parkin mutants fail to poly-ubiquitylate endogenous VDAC1 in response to CCCP treatment. HeLa cells were transfected with empty vector (-), Flag-Parkin wild-type or pathogenic mutants along with 6×His-tagged ubiquitin wild-type for 24 h. Cells were treated with CCCP for 6 h and subsequently protein lysates were prepared in urea buffer. Denaturing conditions of Ni-NTA affinity purifications were used to specifically pulldown only proteins covalently modified with 6xHis-tagged ubiquitin. Western blots of protein lysates and purified Ni-NTA agarose elutions were probed with anti-VDAC1, anti-TOM20 and anti-Parkin antibodies. Modification of VDAC1 was observed in cells expressing Parkin wild-type and the functional mutant G328E, whereas non-functional mutants did not have this effect. (


( c ) In response to CCCP treatment , Parkin catalyses predominantly K27 - linked poly - ubiquitin chains on endogenous VDAC1 and is itself strongly poly - ubiquitylated . HeLa cells were transfected with an empty vector ( - ) or Flag - Parkin wild - type along with 6 × His - tagged ubiquitin variants for 24 h . Treatment of cells , preparation of lysates and Ni - NTA purification were performed as described in c and probed for Parkin , VDAC1 and β - actin . WT , wild - type ; Ub , ubiquitin .

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(c) In response to CCCP treatment, Parkin catalyses predominantly K27-linked poly-ubiquitin chains on endogenous VDAC1 and is itself strongly poly-ubiquitylated. HeLa cells were transfected with an empty vector (-) or Flag-Parkin wild-type along with 6×His-tagged ubiquitin variants for 24 h. Treatment of cells, preparation of lysates and Ni-NTA purification were performed as described in c and probed for Parkin, VDAC1 and β-actin. WT, wild-type; Ub, ubiquitin.


( a ) Western blot analysis of parental SH - SY5Y cells treated with CCCP for the indicated times . Time - course experiments of CCCP - treated neuronal cells reveal increasing modifications of VDAC1 over time . Total lysates were prepared , and western blots were probed with specific anti - Parkin , anti - PINK1 , anti - TOM20 and anti - VDAC1 antibodies . All proteins represent endogenous levels .

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(a) Western blot analysis of parental SH-SY5Y cells treated with CCCP for the indicated times. Time-course experiments of CCCP-treated neuronal cells reveal increasing modifications of VDAC1 over time. Total lysates were prepared, and western blots were probed with specific anti-Parkin, anti-PINK1, anti-TOM20 and anti-VDAC1 antibodies. All proteins represent endogenous levels.


( b ) Immunoprecipitations of Parkin and VDAC1 from parental SH - SY5Y cells confirm ubiquitylation of both proteins . CCCP treatment of neuronal cells increases ubiquitylated species of endogenous Parkin and VDAC1 over time . Using specific antibodies , Parkin and VDAC1 were immunoprecipitated from lysates of parental SH - SY5Y cells at different time points , western blotted and probed with anti - ubiquitin , anti - Parkin and anti - VDAC1 antibodies .

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(b) Immunoprecipitations of Parkin and VDAC1 from parental SH-SY5Y cells confirm ubiquitylation of both proteins. CCCP treatment of neuronal cells increases ubiquitylated species of endogenous Parkin and VDAC1 over time. Using specific antibodies, Parkin and VDAC1 were immunoprecipitated from lysates of parental SH-SY5Y cells at different time points, western blotted and probed with anti-ubiquitin, anti-Parkin and anti-VDAC1 antibodies.


( c ) Pathogenic Parkin mutations fail to poly - ubiquitylate endogenous VDAC1 in response to CCCP treatment . Western blot analysis of a time - course experiment with inducible Parkin SH - SY5Y cells after CCCP treatment . Total lysates were prepared , and western blots were probed with specific anti - Parkin and anti - VDAC1 antibodies . β - Actin probing served as a loading control in a - c . WT , wild - type ; Ub , ubiquitin .

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

(c) Pathogenic Parkin mutations fail to poly-ubiquitylate endogenous VDAC1 in response to CCCP treatment. Western blot analysis of a time-course experiment with inducible Parkin SH-SY5Y cells after CCCP treatment. Total lysates were prepared, and western blots were probed with specific anti-Parkin and anti-VDAC1 antibodies. β-Actin probing served as a loading control in a-c. WT, wild-type; Ub, ubiquitin.


VDAC1 - specific siRNA abrogates Parkin translocation to mitochondria after 2 h , and mitochondrial clearance after 24 h , of CCCP treatment . Both effects can be rescued by re - transfection with wild - type VDAC1 . ( a ) Immunofluorescence and ( b ) western blot of siRNA - transfected HeLa cells used in c - g demonstrate partial silencing of endogenous VDAC1 . ( a ) Silenced cells were stained with anti - HtrA2 / Omi , a mitochondrial marker ( pseudo coloured ; blue ) , and anti - VDAC1 ( red ) antibodies . ( b ) Lysates from silenced cells were subjected to western blot analysis and probed with an anti - VDAC1 antibody , while TOM20 and β - actin probing served as loading controls .

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VDAC1-specific siRNA abrogates Parkin translocation to mitochondria after 2 h, and mitochondrial clearance after 24 h, of CCCP treatment. Both effects can be rescued by re-transfection with wild-type VDAC1. (a) Immunofluorescence and (b) western blot of siRNA-transfected HeLa cells used in c-g demonstrate partial silencing of endogenous VDAC1. (a) Silenced cells were stained with anti-HtrA2/Omi, a mitochondrial marker (pseudo coloured; blue), and anti-VDAC1 (red) antibodies. (b) Lysates from silenced cells were subjected to western blot analysis and probed with an anti-VDAC1 antibody, while TOM20 and β-actin probing served as loading controls.


( c - e ) Control siRNA - or VDAC1 siRNA - silenced HeLa cells were transfected with the indicated combinations of EGFP - Parkin wild - type , VDAC1 - Flag wild - type or appropriate empty vector controls . Cells were treated with CCCP for 2 h and 24 h and stained with anti - HtrA2 / Omi ( pseudo coloured ; blue ) and anti - Flag ( red ) to visualize mitochondrial morphology and re - transfected VDAC1 , respectively . Wild - type Parkin was visualized by EGFP epifluorescence ( green ) . Parkin - positive cells are surrounded by a line . Representative pictures of three independent experiments are shown . Scale bars , 10 μm .

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(c-e) Control siRNA- or VDAC1 siRNA-silenced HeLa cells were transfected with the indicated combinations of EGFP-Parkin wild-type, VDAC1-Flag wild-type or appropriate empty vector controls. Cells were treated with CCCP for 2 h and 24 h and stained with anti-HtrA2/Omi (pseudo coloured; blue) and anti-Flag (red) to visualize mitochondrial morphology and re-transfected VDAC1, respectively. Wild-type Parkin was visualized by EGFP epifluorescence (green). Parkin-positive cells are surrounded by a line. Representative pictures of three independent experiments are shown. Scale bars, 10 μm.


( f , g ) Quantification of d ( f ) and e ( g ) . Parkin wild - type colocalization with impaired mitochondria after 2 h ( f ) and uncleared mitochondria after 24 h ( g ) of CCCP treatment ( n = 3 ) . Data represent the mean ± s . d . , * P ≤ 0 . 05 compared with control siRNA - transfected or with VDAC1 - Flag wild - type - transfected cells .

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(f, g) Quantification of d (f) and e (g). Parkin wild-type colocalization with impaired mitochondria after 2 h (f) and uncleared mitochondria after 24 h (g) of CCCP treatment (n = 3). Data represent the mean ± s.d., *P ≤ 0.05 compared with control siRNA-transfected or with VDAC1-Flag wild-type-transfected cells.


( A ) Acyl - RAC assay of HEK293T cells transfected with the indicated plasmids for 24 h . Data information : EtOH : ethanol ; HAM : hydroxylamine ; 2 - BP : 2 -

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(A) Acyl-RAC assay of HEK293T cells transfected with the indicated plasmids for 24 h. Data information: EtOH: ethanol; HAM: hydroxylamine; 2-BP: 2-bromopalmitate


( B ) Acyl - RAC assay of HEK293T cells transfected with the indicated plasmids for 24 h . Data information : FL : full length ; N : N - terminal domain ; C : C - terminal

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(B) Acyl-RAC assay of HEK293T cells transfected with the indicated plasmids for 24 h. Data information: FL: full length; N: N-terminal domain; C: C-terminal domain


( C ) Click chemistry was applied to detect endogenous cGAS palmitoylation in RAW264 . 7 cells . Data information : PA : palmitic acid ; 17 - ODYA : 17 - octadecanoic acid .

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(C) Click chemistry was applied to detect endogenous cGAS palmitoylation in RAW264.7 cells. Data information: PA: palmitic acid; 17-ODYA: 17-octadecanoic acid.