( A ) Immunofluorescence analysis of GFP - mAtg9 and phospho ‐ myosin regulatory light chain ( MRLC ) in MCF7 / GFP - mAtg9 cells . GFP - mAtg9 was enriched in the trans ‐ Golgi network ( TGN ) labelled with anti ‐ TGN46 antibody and phospho ‐ MRLC localized to cell peripheral in nutrient ‐ rich condition ( DMEM ) . GFP - mAtg9 redistributed and co ‐ localized with phospho ‐ MRLC in starvation condition ( Earle ' s balanced salt solution ; EBSS ) .

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

(A) Immunofluorescence analysis of GFP-mAtg9 and phospho‐myosin regulatory light chain (MRLC) in MCF7/GFP-mAtg9 cells. GFP-mAtg9 was enriched in the trans‐Golgi network (TGN) labelled with anti‐TGN46 antibody and phospho‐MRLC localized to cell peripheral in nutrient‐rich condition (DMEM). GFP-mAtg9 redistributed and co‐localized with phospho‐MRLC in starvation condition (Earle's balanced salt solution; EBSS).


( B ) mAtg9 interacted with non ‐ muscle myosin heavy chain ‐ IIA ( NMHC ‐ IIA ) under starvation conditions . 293T cells ( right panel ) or 293T cells stably infected with lentivirus expressing control ( shLuc ) , UNC ‐ 51 ‐ like kinases ( Ulk1 ) or zipper ‐ interacting protein kinase ( ZIPK ) shRNA ( left panel ) were transfected with V5 ‐ tagged mAtg9 and GFP - tagged NMHC ‐ IIA . At 48 h after transfection , cells were incubated in either serum ‐ containing medium ( DMEM ) or EBSS for 2 h with or without the treatment of 50 μM ML ‐ 7 and blebbistatin ( Blebb ) . Cell lysates were immunoprecipitated with anti ‐ V5 antibody and immunoblotted with indicated antibodies .

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(B) mAtg9 interacted with non‐muscle myosin heavy chain‐IIA (NMHC‐IIA) under starvation conditions. 293T cells (right panel) or 293T cells stably infected with lentivirus expressing control (shLuc), UNC‐51‐like kinases (Ulk1) or zipper‐interacting protein kinase (ZIPK) shRNA (left panel) were transfected with V5‐tagged mAtg9 and GFP-tagged NMHC‐IIA. At 48 h after transfection, cells were incubated in either serum‐containing medium (DMEM) or EBSS for 2 h with or without the treatment of 50 μM ML‐7 and blebbistatin (Blebb). Cell lysates were immunoprecipitated with anti‐V5 antibody and immunoblotted with indicated antibodies.


( C ) MCF7 / GFP - mAtg9 cells were stably infected with lentivirus expressing control ( shLuc ) , ZIPK , or NMHC ‐ IIA shRNA . Under starved conditions , mAtg9 was redistributed from the TGN to a dispersed peripheral pool in control cells ( shLuc ) . Starvation ‐ induced GFP - mAtg9 redistribution was blocked in ZIPK and NMHC ‐ IIA knockdown cells . The images were analysed by quantifying mAtg9 localization in cells for either a TGN ‐ enriched or dispersed pattern . Data are represented as mean ± s . e . of 70 cells . * P0 . 05 , * * P0 . 01 . Bar , 10 μm .

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(C) MCF7/GFP-mAtg9 cells were stably infected with lentivirus expressing control (shLuc), ZIPK, or NMHC‐IIA shRNA. Under starved conditions, mAtg9 was redistributed from the TGN to a dispersed peripheral pool in control cells (shLuc). Starvation‐induced GFP-mAtg9 redistribution was blocked in ZIPK and NMHC‐IIA knockdown cells. The images were analysed by quantifying mAtg9 localization in cells for either a TGN‐enriched or dispersed pattern. Data are represented as mean±s.e. of 70 cells. *P0.05, **P0.01. Bar, 10 μm.


Single - marker association results for the combined non - Jewish and Jewish samples using the Cochran - Mantel - Haenszel ( CMH ) test . Each chromosome is depicted as a different color . The red line indicates the threshold ( P 5 × 10 − 5 ) selected to define regions for evaluation in the replication studies . The P value thresholds for suggestive and significant associations are 3 . 28 × 10 − 6 and 1 . 64 × 10 − 7 , respectively , based on a Bonferroni correction for multiple testing . A modest correction factor ( λ = 1 . 056 ) is necessary to correct for stratification between cases and controls , but it does not modify the current results .

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

Single-marker association results for the combined non-Jewish and Jewish samples using the Cochran-Mantel-Haenszel (CMH) test. Each chromosome is depicted as a different color. The red line indicates the threshold (P 5 × 10−5) selected to define regions for evaluation in the replication studies. The P value thresholds for suggestive and significant associations are 3.28 × 10−6 and 1.64 × 10−7, respectively, based on a Bonferroni correction for multiple testing. A modest correction factor (λ = 1.056) is necessary to correct for stratification between cases and controls, but it does not modify the current results.


( a - c ) We used quantitative real - time PCR to determine the expression patterns of ATG5 ( a ) , ATG7 ( b ) and ATG16L1 ( c ) in various human immune and epithelial cell lines . ATG5 was broadly expressed in the cell lines studied ( a ) , with between 1 . 5 - and fourfold variations in expression level across the cell lines . ATG7 expression ( b ) was broadly comparable to that of ATG5 , with the exception of THP1 cells , which showed 35 - fold higher expression than SW480 cells . ATG16L1 expression ( c ) was also broadly similar to that of ATG5 and ATG7 ( except for ATG7 expression in THP1 cells ) , with moderate variations across the cell lines tested ( 1 . 5 - to 3 . 5 - fold ) .

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(a-c) We used quantitative real-time PCR to determine the expression patterns of ATG5 (a), ATG7 (b) and ATG16L1 (c) in various human immune and epithelial cell lines. ATG5 was broadly expressed in the cell lines studied (a), with between 1.5- and fourfold variations in expression level across the cell lines. ATG7 expression (b) was broadly comparable to that of ATG5, with the exception of THP1 cells, which showed 35-fold higher expression than SW480 cells. ATG16L1 expression (c) was also broadly similar to that of ATG5 and ATG7 (except for ATG7 expression in THP1 cells), with moderate variations across the cell lines tested (1.5- to 3.5-fold).


( d ) Expression of ATG16L1 in human primary immune cells . In a resting human immune cell RNA panel ( Clontech ) , ATG16L1 showed a clear peak of expression in the T cell compartment , with CD4 + and CD8 + cells expressing high levels of transcript ( 15 - to 20 - fold more than control samples ) . We detected modest levels of ATG16L1 in CD19 + cells and low levels in CD14 + monocytes and unfractionated mononuclear cells . In all cases , we performed real - time quantitative RT - PCR reactions in duplicate and plotted the means ; error bars represent 1 s . d . We normalized expression levels by comparison with GAPDH controls and plotted arbitrary relative expression units , where the SW480 or placental RNA expression equals 1 ( for cell lines or primary cells , respectively ) . We isolated all RNAs from resting cells in the absence of stimulation or activation .

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(d) Expression of ATG16L1 in human primary immune cells. In a resting human immune cell RNA panel (Clontech), ATG16L1 showed a clear peak of expression in the T cell compartment, with CD4+ and CD8+ cells expressing high levels of transcript (15- to 20-fold more than control samples). We detected modest levels of ATG16L1 in CD19+ cells and low levels in CD14+ monocytes and unfractionated mononuclear cells. In all cases, we performed real-time quantitative RT-PCR reactions in duplicate and plotted the means; error bars represent 1 s.d. We normalized expression levels by comparison with GAPDH controls and plotted arbitrary relative expression units, where the SW480 or placental RNA expression equals 1 (for cell lines or primary cells, respectively). We isolated all RNAs from resting cells in the absence of stimulation or activation.


( a ) We achieved specific siRNA knockdown of overexpressed , Flag - tagged ATG16L1 in HEK293 cells within 48 h of transfection with oligonucleotide duplexes . Flag - ATG16L1 was undetectable by protein blotting after treatment with specific siRNA constructs , but expression was maintained with control duplexes .

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(a) We achieved specific siRNA knockdown of overexpressed, Flag-tagged ATG16L1 in HEK293 cells within 48 h of transfection with oligonucleotide duplexes. Flag-ATG16L1 was undetectable by protein blotting after treatment with specific siRNA constructs, but expression was maintained with control duplexes.


( b ) Endogenous ATG16L1 mRNA knockdown ( mean relative abundance ± s . d . ) by siRNA 2 in HeLa cells within 48 h of transfection , as assessed by real - time quantitative RT - PCR normalized to GAPDH . Compared with control duplex , siRNA 2 yielded an 89 % reduction in transcripts . RT - PCR was performed in triplicate ; results represent two independent experiments .

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(b) Endogenous ATG16L1 mRNA knockdown (mean relative abundance ± s.d.) by siRNA 2 in HeLa cells within 48 h of transfection, as assessed by real-time quantitative RT-PCR normalized to GAPDH. Compared with control duplex, siRNA 2 yielded an 89% reduction in transcripts. RT-PCR was performed in triplicate; results represent two independent experiments.


c ) Knockdown of ATG16L1 prevented effective autophagy of S . typhimurium in HeLa cells . Forty - eight hours after cotransfection with control siRNA or duplex 2 and LC3 - GFP plasmid , HeLa cells were infected for 1 h with Salmonella typhimurium SL1344 , fixed and examined microscopically . Mean percentages ( ± s . e . m . ) of bacteria per cell encapsulated by LC3 + membranes ( autophagosomes ) are shown . Bacterial counts were pooled from two separate experiments ; each counted a minimum of 100 infected cells . Significance was assessed by two - tailed Students t - test , assuming unequal variances .

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c) Knockdown of ATG16L1 prevented effective autophagy of S. typhimurium in HeLa cells. Forty-eight hours after cotransfection with control siRNA or duplex 2 and LC3-GFP plasmid, HeLa cells were infected for 1 h with Salmonella typhimurium SL1344, fixed and examined microscopically. Mean percentages (± s.e.m.) of bacteria per cell encapsulated by LC3+ membranes (autophagosomes) are shown. Bacterial counts were pooled from two separate experiments; each counted a minimum of 100 infected cells. Significance was assessed by two-tailed Students t-test, assuming unequal variances.


( d ) Representative images of intracellular S . typhimurium show normal autophagic encapsulation by LC3 + membranes in control siRNA - treated cells but loss of autophagic targeting after ATG16L1 knockdown . Channels are separated to show both the LC3 - GFP fusion protein ( autophagosomal marker ) and S . typhimurium SL1344 dsRed2 ( SL1344 ) , with a merged image in the final column . Rows 2 and 4 show magnified views of boxed areas in rows 1 and 3 , respectively . Images were obtained using confocal microscopy and are a flat projection of z stacks taken throughout the plane of the infected cell . Scale bars = 5 μm .

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(d) Representative images of intracellular S. typhimurium show normal autophagic encapsulation by LC3+ membranes in control siRNA-treated cells but loss of autophagic targeting after ATG16L1 knockdown. Channels are separated to show both the LC3-GFP fusion protein (autophagosomal marker) and S. typhimurium SL1344 dsRed2 (SL1344), with a merged image in the final column. Rows 2 and 4 show magnified views of boxed areas in rows 1 and 3, respectively. Images were obtained using confocal microscopy and are a flat projection of z stacks taken throughout the plane of the infected cell. Scale bars = 5 μm.


B , C , Analysis of GFP transfer after transplantation of Nrl : : GFP donor photoreceptors into wildtype C57BL / 6J ( B ) and Nrl - / - ( C ) recipient retinas after 14 ( wt , n = 9 ; Nrl - / - , n = 7 ) and 21 ( wt , n = 6 ; Nrl - / - , n = 4 ) and 90 ( wt , n = 5 ; Nrl - / - , n = 4 ) days . Tissue is stained for GFP and nuclei ( Hoechst ) . In this and all following figures , donor cells ( D ) are located above , and acceptor cells with GFP ( A ) are located below the outer limiting membrane ( white dashed line ) , which delimits the apical border of the outer nuclear layer ( ONL ) . In wildtype recipients , donor cells are typically located above the outer segment region ( between the grey dashed lines ) . In Nrl - / - recipients , donor cells are located in the inner segment ( IS ) region . Plotted is the ratio of the numbers of GFP + photoreceptors in the ONL ( acceptor photoreceptors ) and GFP + donor photoreceptors in the subretinal space ( SRS ) over time .

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B, C, Analysis of GFP transfer after transplantation of Nrl::GFP donor photoreceptors into wildtype C57BL/6J (B) and Nrl-/- (C) recipient retinas after 14 (wt, n=9; Nrl-/-, n=7) and 21 (wt, n=6; Nrl-/-, n=4) and 90 (wt, n=5; Nrl-/-, n=4) days. Tissue is stained for GFP and nuclei (Hoechst). In this and all following figures, donor cells (D) are located above, and acceptor cells with GFP (A) are located below the outer limiting membrane (white dashed line), which delimits the apical border of the outer nuclear layer (ONL). In wildtype recipients, donor cells are typically located above the outer segment region (between the grey dashed lines). In Nrl-/- recipients, donor cells are located in the inner segment (IS) region. Plotted is the ratio of the numbers of GFP+ photoreceptors in the ONL (acceptor photoreceptors) and GFP+ donor photoreceptors in the subretinal space (SRS) over time.


F , Co - transplanted P3 - 4 Crx : : Cre and P3 - 5 ROSAmT / mG donor photoreceptors into C57BL / 6J recipient retinas show Cre reporter gene induction in donor cells located in the SRS 14 days post - transplantation .

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F, Co-transplanted P3-4 Crx::Cre and P3-5 ROSAmT/mG donor photoreceptors into C57BL/6J recipient retinas show Cre reporter gene induction in donor cells located in the SRS 14 days post-transplantation.


A - C , Combined FISH and IHF to detect GFP ( A , B ) and GNAT1 ( C ) 21 days post - transplantation of Nrl : : GFP photoreceptors into C57BL / 6J ( A ) and Nrl - / - ( B , C ) recipients .

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A-C, Combined FISH and IHF to detect GFP (A, B) and GNAT1 (C) 21 days post-transplantation of Nrl::GFP photoreceptors into C57BL/6J (A) and Nrl-/- (B, C) recipients.


F - K , FISH and IHF for GFP and GFP transcripts in C57BL / 6J recipients transplanted with lentiviral - infected donor cells expressing cytoplasmic GFP ( Lenti - GFP ) ( F , I ) and nuclear - localized GFP ( Lenti - nls - GFP ) ( G , J ) . H , MT index for cytosolic ( Lenti - GFP , n = 5 ) and nuclear localized ( Lenti - nls - GFP , n = 3 ) . K , Quantification of transcripts in donor and acceptor photoreceptors . ( Lenti - GFP , n = 6 images from 3 transplanted animals ; Lenti - nls - GFP , n = 8 images from 4 transplanted animals ) .

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F-K, FISH and IHF for GFP and GFP transcripts in C57BL/6J recipients transplanted with lentiviral-infected donor cells expressing cytoplasmic GFP (Lenti -GFP) (F, I) and nuclear-localized GFP (Lenti-nls-GFP) (G, J). H, MT index for cytosolic (Lenti-GFP, n=5) and nuclear localized (Lenti-nls-GFP, n=3). K, Quantification of transcripts in donor and acceptor photoreceptors. (Lenti-GFP, n=6 images from 3 transplanted animals; Lenti-nls-GFP, n=8 images from 4 transplanted animals).


B - D ' ' , Higher magnification of regions in A ( indicated by yellow dashed boxes ) showing colocalization of ATP5B and Mito - DsRed - labelled mitochondria in the outer segments ( B ) , soma ( C ) , and the axon ( D ) of acceptor photoreceptors .

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B-D'', Higher magnification of regions in A (indicated by yellow dashed boxes) showing colocalization of ATP5B and Mito-DsRed-labelled mitochondria in the outer segments (B), soma (C), and the axon (D) of acceptor photoreceptors.


F , G , C57BL / 6J cells with mitochondria labelled with MitoTrackerTM - Green ( MTG ) or MitoTrackerTM - Red ( MTR ) exchange mitochondria after 3 DIV ( 0 DIV , n = 6 ; 3 DIV , n = 6 ) . White dashed line delimitates both cell somas and the protrusion connecting them ( F ) . No exchange is observed when the two photoreceptor populations are separated by a transwell ( n = 3 ) , nor in co - cultures of live MTG - labelled and lysed MTR - labelled cells ( n = 3 ) ( G ) .

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F, G, C57BL/6J cells with mitochondria labelled with MitoTrackerTM-Green (MTG) or MitoTrackerTM-Red (MTR) exchange mitochondria after 3 DIV (0 DIV, n=6; 3 DIV, n=6). White dashed line delimitates both cell somas and the protrusion connecting them (F). No exchange is observed when the two photoreceptor populations are separated by a transwell (n=3), nor in co-cultures of live MTG-labelled and lysed MTR-labelled cells (n=3) (G).


A , Maximum intensity projection of a confocal micrograph of a cryostat section from a Nrl - / - recipient retina 21 days after transplantation with Nrl : : GFP donor photoreceptors , showing protrusions ( pink arrows ) connecting donor and acceptor photoreceptors .

A, Maximum intensity projection of a confocal micrograph of a cryostat section from a Nrl-/- recipient retina 21 days after transplantation with Nrl::GFP donor photoreceptors, showing protrusions (pink arrows) connecting donor and acceptor photoreceptors.


D , E , Left : 3D reconstruction of two - photon ( D ) and light sheet microscopy images of intact cleared ROSAmT / mG eye showing that GFP + - donor and - acceptor photoreceptors are in the same area . D , Right : A high magnification image showing GFP + - donor and - acceptor photoreceptors connected by protrusions ( pink arrows ) . E , Right : In detail , donor and acceptor photoreceptors also appear connected through cell protrusions ( pink arrow ) .

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D, E, Left: 3D reconstruction of two-photon (D) and light sheet microscopy images of intact cleared ROSAmT/mG eye showing that GFP+-donor and -acceptor photoreceptors are in the same area. D, Right: A high magnification image showing GFP+-donor and -acceptor photoreceptors connected by protrusions (pink arrows). E, Right: In detail, donor and acceptor photoreceptors also appear connected through cell protrusions (pink arrow).


A , Confocal image shows a Lenti - GFP infected donor photoreceptor ( yellow arrow ) connected to an acceptor photoreceptor . The donor protrusion ( pink arrow ) co - localizes with α - tubulin but not the outer segment marker GNAT1 .

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A, Confocal image shows a Lenti-GFP infected donor photoreceptor (yellow arrow) connected to an acceptor photoreceptor. The donor protrusion (pink arrow) co-localizes with α-tubulin but not the outer segment marker GNAT1.


B , Nrl : : GFP donor photoreceptors transplanted in a Prphr2 - / - recipient retina exhibit polarized Prphr2 expression ( yellow arrow ) on the apical side of the donor photoreceptor . Basal protrusions ( pink arrows ) indicate donor protrusions that contact the host retina .

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B, Nrl::GFP donor photoreceptors transplanted in a Prphr2-/- recipient retina exhibit polarized Prphr2 expression (yellow arrow) on the apical side of the donor photoreceptor. Basal protrusions (pink arrows) indicate donor protrusions that contact the host retina.


C , D , Expression of β - tubulin ( C ) , SV2 ( D ) and GFP in the protrusions connecting Lenti - GFP - infected C57BL / 6J donor photoreceptors with acceptor photoreceptors . Pink arrows point to the protrusions connecting donor and acceptor cells .

C, D, Expression of β-tubulin (C), SV2 (D) and GFP in the protrusions connecting Lenti-GFP-infected C57BL/6J donor photoreceptors with acceptor photoreceptors. Pink arrows point to the protrusions connecting donor and acceptor cells.


A , B , Examples of live Nrl : : GFP and C57BL / 6J retinal cultures showing actin + ( SirActin ) and GFP + ( A ) and tubulin + ( ViaFluor ) and MitoTracker Green + ( MTG ) protrusions ( B ) connecting photoreceptors . DIC , differential interference contrast microscopy .

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A, B, Examples of live Nrl::GFP and C57BL/6J retinal cultures showing actin+ (SirActin) and GFP+ (A) and tubulin+ (ViaFluor) and MitoTracker Green+ (MTG) protrusions (B) connecting photoreceptors. DIC, differential interference contrast microscopy.


E , Time lapse imaging showing movement of MitoTracker Green + puncta in photoreceptors connected by a protrusion . White arrows point MTG + puncta inside of the protrusion during the time lapse imaging .

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E, Time lapse imaging showing movement of MitoTracker Green+ puncta in photoreceptors connected by a protrusion. White arrows point MTG+ puncta inside of the protrusion during the time lapse imaging.


C , Confocal microscopy image of cryostat sections of C57BL / 6J recipient retinas 21 days after transplant of C57BL / J6 photoreceptors infected with empty vector ( n = 5 ) , RHOA overexpression ( RHOA OE ) ( n = 7 ) or RAC1 dominant negative ( RAC1 DN ) ( n = 7 ) lentiviruses . Quantification of MT in retinas transplanted with control , RHOA and RAC1 DN lentiviruses .

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C, Confocal microscopy image of cryostat sections of C57BL/6J recipient retinas 21 days after transplant of C57BL/J6 photoreceptors infected with empty vector (n=5), RHOA overexpression (RHOA OE) (n=7) or RAC1 dominant negative (RAC1 DN) (n=7) lentiviruses. Quantification of MT in retinas transplanted with control, RHOA and RAC1 DN lentiviruses.


D , GFP staining of whole - mounted retinas after transplantation with control , RHOA and RAC1 DN lentivirus infected donor cells ( n = 4 per group ) . Quantification shows a significant reduction in the average protrusion length per cell and in the total protrusion length per cell after RHOA OE and RAC1 DN infection compared to the empty vector control .

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D, GFP staining of whole-mounted retinas after transplantation with control, RHOA and RAC1 DN lentivirus infected donor cells (n=4 per group). Quantification shows a significant reduction in the average protrusion length per cell and in the total protrusion length per cell after RHOA OE and RAC1 DN infection compared to the empty vector control.


Cell counting experiments analyzing the response to trastuzumab ( 50 nM , 5 days ) of naïve BT474 as well as the resistant BTRH pool and the BTRH # 10 clone .

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Cell counting experiments analyzing the response to trastuzumab (50 nM, 5 days) of naïve BT474 as well as the resistant BTRH pool and the BTRH#10 clone.


Western blot analyses of the expression of HER2 , pHER2 , HER3 and pHER3 in BT474 , BTRH and BTRH # 10 cells . HER2 and HER3 were immunoprecipitated from 100 or 500 μg of cell extracts , respectively , and blots probed with anti - HER2 , anti - HER3 or anti - phosphotyrosine . Calnexin was used as a loading control . Numbers below the blots show the quantitation of the signal of each band , referred to BT474 cells ( intensity of band in BT474 cells = 1 ) .

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Western blot analyses of the expression of HER2, pHER2, HER3 and pHER3 in BT474, BTRH and BTRH#10 cells. HER2 and HER3 were immunoprecipitated from 100 or 500 μg of cell extracts, respectively, and blots probed with anti-HER2, anti-HER3 or anti-phosphotyrosine. Calnexin was used as a loading control. Numbers below the blots show the quantitation of the signal of each band, referred to BT474 cells (intensity of band in BT474 cells=1).


Analyses of cell surface levels of HER2 and HER3 in BT474 and BTRH cells by surface immunoprecipitation .

Analyses of cell surface levels of HER2 and HER3 in BT474 and BTRH cells by surface immunoprecipitation.


Response of BT474 , BTRH and BTRH # 10 to EV20 / MMAF . Cells treated with the indicated doses of EV20 / MMAF for 5 days were counted .

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Response of BT474, BTRH and BTRH#10 to EV20/MMAF. Cells treated with the indicated doses of EV20/MMAF for 5 days were counted.


Action of the nude EV20 antibody or EV20 / MMAF ( 10 nM each , 5 days ) on BT474 , BTRH and BTRH # 10 cells .

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Action of the nude EV20 antibody or EV20/MMAF (10 nM each, 5 days) on BT474, BTRH and BTRH#10 cells.


Comparison of the effect of MMAF and EV20 / MMAF on the proliferation of BT474 and BTRH cells after five days of treatment with the drugs .

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Comparison of the effect of MMAF and EV20/MMAF on the proliferation of BT474 and BTRH cells after five days of treatment with the drugs.


Response to trastuzumab ( 50 nM , 5 days ) of BT474 , BTRH , patient derived cells ( PDX118 ) and their trastuzumab - resistant derivatives TR1 and TR2 .

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Response to trastuzumab (50 nM, 5 days) of BT474, BTRH, patient derived cells (PDX118) and their trastuzumab-resistant derivatives TR1 and TR2.


HER2 , HER3 and pHER3 levels in BT474 , BTRH , PDX118 , TR1 and TR2 . pHER3 levels were analyzed by a specific anti - pHER3 antibody .

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HER2, HER3 and pHER3 levels in BT474, BTRH, PDX118, TR1 and TR2. pHER3 levels were analyzed by a specific anti-pHER3 antibody.


Effect of EV20 / MMAF on BT474 , BTRH , PDX118 , TR1 and TR2 . Cells were counted after 5 days of treatment with the indicated doses of EV20 / MMAF .

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Effect of EV20/MMAF on BT474, BTRH, PDX118, TR1 and TR2. Cells were counted after 5 days of treatment with the indicated doses of EV20/MMAF.


Effect of trastuzumab ( 50 nM , 5 days ) in HER2 positive breast cancer cell lines ( SKBR3 , BT474 , BTRH , MDA - MB - 361 , HCC1419 , HCC1569 and HCC1954 ) . The blue line indicates the threshold ( established from BTRH ) used to consider a cell line resistant to trastuzumab .

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Effect of trastuzumab (50 nM, 5 days) in HER2 positive breast cancer cell lines (SKBR3, BT474, BTRH, MDA-MB-361, HCC1419, HCC1569 and HCC1954). The blue line indicates the threshold (established from BTRH) used to consider a cell line resistant to trastuzumab.


Analyses of HER2 , HER3 and pHER3 in the cells used in ( D ) . Numbers below the blots show the quantitation of the signal of each band , referred to SKBR3 cells ( intensity of band in SKBR3 cells = 1 ) . pHER3 levels were analyzed by a specific anti - pHER3 antibody .

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Analyses of HER2, HER3 and pHER3 in the cells used in (D). Numbers below the blots show the quantitation of the signal of each band, referred to SKBR3 cells (intensity of band in SKBR3 cells=1). pHER3 levels were analyzed by a specific anti-pHER3 antibody.


Effect of EV20 / MMAF on the cell lines used in ( D ) . Cells were counted after 5 days of treatment with the indicated doses of EV20 / MMAF .

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Effect of EV20/MMAF on the cell lines used in (D). Cells were counted after 5 days of treatment with the indicated doses of EV20/MMAF.


Knockdown of HER3 in BT474 cells . Control and HER3 - targeted short hairpin sequences were transduced in BT474 cells and HER3 , pHER3 and pAKT levels analyzed by Western . Levels of pHER3 were analyzed by a specific anti - pHER3 antibody .

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Knockdown of HER3 in BT474 cells. Control and HER3-targeted short hairpin sequences were transduced in BT474 cells and HER3, pHER3 and pAKT levels analyzed by Western. Levels of pHER3 were analyzed by a specific anti-pHER3 antibody.


Effect of HER3 knockdown on the antiproliferative action of EV20 / MMAF analyzed by cell counting after 5 days of treatment with the ADC .

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Effect of HER3 knockdown on the antiproliferative action of EV20/MMAF analyzed by cell counting after 5 days of treatment with the ADC.


Internalization of EV20 / MMAF ( 10 nM ) at indicated times in BT474 and BTRH cells . Scale bar : 20 µm . HER3 staining : red ; DAPI : blue .

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Internalization of EV20/MMAF (10 nM) at indicated times in BT474 and BTRH cells. Scale bar: 20 µm. HER3 staining: red; DAPI: blue.


Internalization of biotin - S - S - EV20 / MMAF in BT474 and BTRH cells . These cell lines were pulsed with 10 nM of biotin - S - S - EV20 / MMAF for 1 hour at 4 ° C and chased at 37 ° C at the indicated times . Subsequently , surface accessible biotin was cleaved , cells were lysed , and extracts precipitated with streptavidin - sepharose . EV20 / MMAF was analyzed by Western blotting using anti - human - HRP . Quantitative plot of the Western studies shown in ( B ) . Data are presented as the mean + SD of two independent experiments .

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Internalization of biotin-S-S-EV20/MMAF in BT474 and BTRH cells. These cell lines were pulsed with 10 nM of biotin-S-S-EV20/MMAF for 1 hour at 4°C and chased at 37°C at the indicated times. Subsequently, surface accessible biotin was cleaved, cells were lysed, and extracts precipitated with streptavidin-sepharose. EV20/MMAF was analyzed by Western blotting using anti-human-HRP. Quantitative plot of the Western studies shown in (B). Data are presented as the mean + SD of two independent experiments.


Co - localization ( yellow ) between EV20 / MMAF and LAMP1 at the indicated incubation times with EV20 / MMAF in BT474 and BTRH cells . Scale bar : 5 µm . HER3 staining : red ; LAMP1 : green , DAPI : blue .

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Co-localization (yellow) between EV20/MMAF and LAMP1 at the indicated incubation times with EV20/MMAF in BT474 and BTRH cells. Scale bar: 5 µm. HER3 staining: red; LAMP1: green, DAPI: blue.


Western blot analyses of the effect of EV20 / MMAF ( 10 nM ) on the levels of HER2 and HER3 in BT474 and BTRH cells at the indicated incubation times with the ADC . The same Western was probed for HER2 or HER3 expression using two differently labelled species - specific secondary fluorescent antibodies . Quantitative representation of the Western studies shown in ( E ) .

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Western blot analyses of the effect of EV20/MMAF (10 nM) on the levels of HER2 and HER3 in BT474 and BTRH cells at the indicated incubation times with the ADC. The same Western was probed for HER2 or HER3 expression using two differently labelled species-specific secondary fluorescent antibodies. Quantitative representation of the Western studies shown in (E).


Cell cycle analysis by flow cytometry of BT474 and BTRH cells ( 50 , 000 events ) treated with EV20 / MMAF ( 10 nM ) for the indicated times .

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Cell cycle analysis by flow cytometry of BT474 and BTRH cells (50,000 events) treated with EV20/MMAF (10 nM) for the indicated times.


BT474 and BTRH cells were treated for the indicated days with EV20 / MMAF ( 10 nM ) , lysed and the amount of different proteins analyzed by Western . Numbers below the blots show the quantitation of the signal of each band , referred to time 0 .

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BT474 and BTRH cells were treated for the indicated days with EV20/MMAF (10 nM), lysed and the amount of different proteins analyzed by Western. Numbers below the blots show the quantitation of the signal of each band, referred to time 0.


Action of EV20 / MMAF on mitotic spindle formation . BT474 and BTRH cells were treated ( 10 nM , 48 hours ) , fixed , stained for β - tubulin ( green ) and DAPI ( blue ) . Scale bar : 7 . 5 μm .

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Action of EV20/MMAF on mitotic spindle formation. BT474 and BTRH cells were treated (10 nM, 48 hours), fixed, stained for β-tubulin (green) and DAPI (blue). Scale bar: 7.5 μm.


Quantitative analysis of mitotic cells with abnormal spindles treated or not with EV20 / MMAF ( 10 nM , 48 hours ) in BT474 and BTRH cells . Bars represent the mean + SD of two independent experiments , calculated as follows : ( number of mitotic cells with abnormal spindles / total number of mitotic cells ) × 100 ( % ) .

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Quantitative analysis of mitotic cells with abnormal spindles treated or not with EV20/MMAF (10 nM, 48 hours) in BT474 and BTRH cells. Bars represent the mean + SD of two independent experiments, calculated as follows: (number of mitotic cells with abnormal spindles/total number of mitotic cells) ×100 (%).


Cell death induction by EV20 / MMAF ( 10 nM ) analyzed by double annexin V and PI staining in BT474 and BTRH cells ( 50 , 000 events ) .

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Cell death induction by EV20/MMAF (10 nM) analyzed by double annexin V and PI staining in BT474 and BTRH cells (50,000 events).


Time - course analysis of the effect of EV20 / MMAF ( 10 nM ) on cleaved - caspase - 3 , analyzed by Western blotting in BT474 and BTRH cells . GAPDH was used as a loading control . Samples with intensity values of 1 were used as reference .

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Time-course analysis of the effect of EV20/MMAF (10 nM) on cleaved-caspase-3, analyzed by Western blotting in BT474 and BTRH cells. GAPDH was used as a loading control. Samples with intensity values of 1 were used as reference.


Fluorimetric analyses of caspase - 3 activity in BT474 and BTRH cells treated with 10 nM EV20 / MMAF . Data show the mean + SD of triplicates of an experiment that was repeated twice .

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Fluorimetric analyses of caspase-3 activity in BT474 and BTRH cells treated with 10 nM EV20/MMAF. Data show the mean + SD of triplicates of an experiment that was repeated twice.


In vivo effect of EV20 ( 10 mg / kg , n = 4 ) , untreated control ( PBS , n = 4 ) and EV20 / MMAF ( 3 . 3 mg / kg , n = 5 ; 10 mg / kg , n = 4 ) on tumor growth in mice injected with BTRH cells . Mice were treated when tumors reached a volume of 500 mm3 . Data are represented as mean + SEM .

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In vivo effect of EV20 (10 mg/kg, n=4), untreated control (PBS, n=4) and EV20/MMAF (3.3 mg/kg, n=5; 10 mg/kg, n=4) on tumor growth in mice injected with BTRH cells. Mice were treated when tumors reached a volume of 500 mm3. Data are represented as mean + SEM.


In vivo effect of single - dose EV20 ( 10 mg / kg , n = 3 ) untreated control ( PBS , n = 4 ) , single - dose EV20 / MMAF ( 10 mg / kg , n = 4 ) or weekly trastuzumab ( 30 mg / kg , n = 4 ) on tumor growth in mice injected with BTRH # 10 cells . Data are represented as mean + SEM . In vivo effect of single - dose EV20 ( 10 mg / kg , n = 4 ) , untreated control ( PBS , n = 4 ) , single - dose EV20 / MMAF ( 10 mg / kg , n = 4 ) or weekly trastuzumab ( 30 mg / kg , n = 4 ) on tumor growth in mice injected with HCC1954 cells . Data are represented as mean + SEM .

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In vivo effect of single-dose EV20 (10 mg/kg, n=3) untreated control (PBS, n=4), single-dose EV20/MMAF (10 mg/kg, n=4) or weekly trastuzumab (30 mg/kg, n=4) on tumor growth in mice injected with BTRH#10 cells. Data are represented as mean + SEM. In vivo effect of single-dose EV20 (10 mg/kg, n=4), untreated control (PBS, n=4), single-dose EV20/MMAF (10 mg/kg, n=4) or weekly trastuzumab (30 mg/kg, n=4) on tumor growth in mice injected with HCC1954 cells. Data are represented as mean + SEM.


Analysis of pHistone H3 , HER3 , pHER3 and cleaved caspase - 3 in tumors of mice injected with BTRH and treated ( T ) or not ( C ) with EV20 / MMAF ( 3 . 3 mg / kg for 24 hours or two weeks ) . Numbers below the blots show the quantitation of the signal of each band , referred to that of a control untreated tumor .

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Analysis of pHistone H3, HER3, pHER3 and cleaved caspase-3 in tumors of mice injected with BTRH and treated (T) or not (C) with EV20/MMAF (3.3 mg/kg for 24 hours or two weeks). Numbers below the blots show the quantitation of the signal of each band, referred to that of a control untreated tumor.


Long - term monitoring of mice injected with BTRH cells and treated with EV20 / MMAF ( 3 . 3 mg / kg , n = 5 ; 10 mg / kg , n = 4 ) . Where indicated , mice were treated with EV20 / MMAF . Data were represented as mean + SD of the different mice groups .

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Long-term monitoring of mice injected with BTRH cells and treated with EV20/MMAF (3.3 mg/kg, n=5; 10 mg/kg, n=4). Where indicated, mice were treated with EV20/MMAF. Data were represented as mean + SD of the different mice groups.


Dose - response to EV20 / MMAF after 5 days of treatment in BT474 cells resistant to neratinib ( BTRN # 5 and BTRN # 24 )

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Dose-response to EV20/MMAF after 5 days of treatment in BT474 cells resistant to neratinib (BTRN#5 and BTRN#24)


Levels of HER2 , pHER2 , HER3 , pHER3 , pAKT and calnexin of ( B ) cells resistant to

Levels of HER2, pHER2, HER3, pHER3, pAKT and calnexin of (B) cells resistant to neratinib


Dose - response to EV20 / MMAF after 5 days of treatment in BT474 cells resistant to lapatinib ( BTRL , BTRL # 109 and BTRL # 3 )

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Dose-response to EV20/MMAF after 5 days of treatment in BT474 cells resistant to lapatinib (BTRL, BTRL#109 and BTRL#3)


Levels of HER2 , pHER2 , HER3 , pHER3 , pAKT and calnexin of cells resistant to

Levels of HER2, pHER2, HER3, pHER3, pAKT and calnexin of cells resistant to lapatinib


Dose - response to EV20 / MMAF after 5 days of treatment in BT474 cells resistant to T - DM1 ( BT - TDM1R # 1 and BT - TDM1R # 6 ) .

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Dose-response to EV20/MMAF after 5 days of treatment in BT474 cells resistant to T-DM1 (BT-TDM1R#1 and BT-TDM1R#6).


Levels of HER2 , pHER2 , HER3 , pHER3 , pAKT and calnexin of cells resistant to TDM1 .

Levels of HER2, pHER2, HER3, pHER3, pAKT and calnexin of cells resistant to TDM1.


B Representative TIRF - M of DCP1 - GFP ( DCP1pro : DCP1 - GFP transgene ) in the lateral PM of epidermal cells showing the transient attachment of DCP1 - positive puncta to the PM . Yellow arrowheads denote a PB that shows motility at the PM focal plane ; blue arrowheads show a PB that transiently localizes to the PM . Scale bars : 2 μm . The corresponding kymographs are shown to the right . Right : distribution of immobile and mobile DCP1 molecules relative to the motility log ( D ) value of - 0 . 75 ( threshold ; see Material and methods for details ) , in NS or HS conditions ( D , diffusion coefficient ) . Inset : individual trajectories of mobile DCP1 - GFP in NS and HS conditions ( 500 frames , n = 120 ) , showing a combination of directional and Brownian motion for both NS / HS . The green arrowheads denote the beginning of the NS and HS tracks for DCP1 - GFP .

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B Representative TIRF-M of DCP1-GFP (DCP1pro:DCP1-GFP transgene) in the lateral PM of epidermal cells showing the transient attachment of DCP1-positive puncta to the PM. Yellow arrowheads denote a PB that shows motility at the PM focal plane; blue arrowheads show a PB that transiently localizes to the PM. Scale bars: 2 μm. The corresponding kymographs are shown to the right. Right: distribution of immobile and mobile DCP1 molecules relative to the motility log(D) value of -0.75 (threshold; see Material and methods for details), in NS or HS conditions (D, diffusion coefficient). Inset: individual trajectories of mobile DCP1-GFP in NS and HS conditions (500 frames, n = 120), showing a combination of directional and Brownian motion for both NS/HS. The green arrowheads denote the beginning of the NS and HS tracks for DCP1-GFP.


C Representative confocal micrographs from lines co - expressing RPS5apro : HF - mScarlet - DCP1 and PIN2pro : PIN2 - GFP ( epidermal cells , region 3 , Scale bars : 5 μm ) . Bottom : polarity index of DCP1 in root meristematic cells ( compared to propidium iodide ( PI ) and tubulin staining of root cells ; N , biological replicates = 3 roots , n = 13 cells ) . Polarity index calculated as the ratio of average of apical and basal VS lateral side of fluorescence signal intensity of the root epidemies cells . The arrowhead in PIN2 indicates the cell plate or PM in DCP1 . Right : representative confocal micrographs showing that PM localization is independent of the promoter used ( DCP1pro , 35Spro ; region 2 , epidermal cells , or RPS5apro on the lower right ) . The details from the inset show increased localization at the cell edge ( discussed later ) . mSc , mScarlet .

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C Representative confocal micrographs from lines co-expressing RPS5apro:HF-mScarlet-DCP1 and PIN2pro:PIN2-GFP (epidermal cells, region 3, Scale bars: 5 μm). Bottom: polarity index of DCP1 in root meristematic cells (compared to propidium iodide (PI) and tubulin staining of root cells; N, biological replicates = 3 roots, n = 13 cells). Polarity index calculated as the ratio of average of apical and basal VS lateral side of fluorescence signal intensity of the root epidemies cells. The arrowhead in PIN2 indicates the cell plate or PM in DCP1. Right: representative confocal micrographs showing that PM localization is independent of the promoter used (DCP1pro, 35Spro; region 2, epidermal cells, or RPS5apro on the lower right). The details from the inset show increased localization at the cell edge (discussed later). mSc, mScarlet.


B Representative confocal micrograph of DCP1 - GFP ( DCP1pro : DCP1 - GFP , transgene ) in root meristematic cells under NS / HS conditions ( region 2 ) . Scale bars , 5 μm . Note the depletion of DCP1 from the PM upon HS , but the increased edge / vertex signal ( yellow arrowheads denote the vertex signal ) . Right : DCP1 signal intensity at the PM or edge / vertex ( N , biological replicates = 3 , n ( pooled data of 3 biological replicates ) = 18 - 23 PMs or edges / vertices ) .

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B Representative confocal micrograph of DCP1-GFP (DCP1pro:DCP1-GFP, transgene) in root meristematic cells under NS/HS conditions (region 2). Scale bars, 5 μm. Note the depletion of DCP1 from the PM upon HS, but the increased edge/vertex signal (yellow arrowheads denote the vertex signal). Right: DCP1 signal intensity at the PM or edge/vertex (N, biological replicates = 3, n (pooled data of 3 biological replicates) = 18-23 PMs or edges/vertices).


C Representative confocal images showing fusion ( coarsening ) , fission , and growth of PBs ( DCP1 - positive ) at the PM ( region 3 ) . Right : states of PBs ( dynamic : fusion and fission and non - dynamic : stable ; N , biological replicates = 2 , n ( pooled data of 3 biological replicates ) = 6 - 8 PBs ) . As a cautionary note , the " stable " PBs may not show dynamicity in the imaging time used ( ~ 3 - 5 min ) but later , may do .

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C Representative confocal images showing fusion (coarsening), fission, and growth of PBs (DCP1-positive) at the PM (region 3). Right: states of PBs (dynamic: fusion and fission and non-dynamic: stable; N, biological replicates = 2, n (pooled data of 3 biological replicates) = 6-8 PBs). As a cautionary note, the "stable" PBs may not show dynamicity in the imaging time used (~3-5 min) but later, may do.


Confocal micrographs showing single optical sections PLA - assays producing signal that resembles spots . The antibodies used were anti - FLAG / anti - GFP detecting the HF - mScarlet - DCP1 / DCP2 - YFP , respectively ( RPS5apro : HF - mScarlet - DCP1 and 35Spro : DCP2 - YFP ) . Inset 1 : magnification showing the colocalization of PLA spots with DCP1 or DCP2 signals ( colocalization and Pearson ' s correlation coefficient PCC value for the spots shown ) . The dotted white line in the PLA channel corresponds to the PM plane . Inset 2 : positive PLA signal for DCP1 and DCP2 in the nucleus . " n1 - n3 " correspond to nuclei regions ( green circles ) . On the right , note the PLA spot nearby the nucleus ( " detail of PLA signal " ) . The chart shows the quantification of PLA spots per cell at puncta ( cytoplasm ) or on the PM ( N , biological replicates = 3 , n ( pooled data of 3 biological replicates ) = 16 - 33 cells ) . As a cautionary technical note , the cytoplasmic , nuclear or PM " spots " do not connote physiologically relevant puncta , condensates or PM clusters . Scale bar : 20 μm / 10 μm for the insets .

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

Confocal micrographs showing single optical sections PLA-assays producing signal that resembles spots. The antibodies used were anti-FLAG/anti-GFP detecting the HF-mScarlet-DCP1/ DCP2-YFP, respectively (RPS5apro:HF-mScarlet-DCP1 and 35Spro:DCP2-YFP). Inset 1: magnification showing the colocalization of PLA spots with DCP1 or DCP2 signals (colocalization and Pearson's correlation coefficient PCC value for the spots shown). The dotted white line in the PLA channel corresponds to the PM plane. Inset 2: positive PLA signal for DCP1 and DCP2 in the nucleus. "n1-n3" correspond to nuclei regions (green circles). On the right, note the PLA spot nearby the nucleus ("detail of PLA signal"). The chart shows the quantification of PLA spots per cell at puncta (cytoplasm) or on the PM (N, biological replicates = 3, n (pooled data of 3 biological replicates) = 16-33 cells). As a cautionary technical note, the cytoplasmic, nuclear or PM "spots" do not connote physiologically relevant puncta, condensates or PM clusters. Scale bar: 20 μm / 10 μm for the insets.


B Representative confocal micrographs with acceptor photobleaching - FRET efficiency between SCAR2 - mCherry ( up ) or BRK1 - mRuby ( down ) and DCP1 - GFP ( epidermal cells , regions 1 and 3 ) . Right : normalized FRET efficiency between SCAR2 or BRK1 and DCP1 , respectively , among the different developmental root regions ( N , biological replicates = 2 , n = 16 cells ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 confocal micrographs with acceptor photobleaching-FRET efficiency between SCAR2-mCherry (up) or BRK1-mRuby (down) and DCP1-GFP (epidermal cells, regions 1 and 3). Right: normalized FRET efficiency between SCAR2 or BRK1 and DCP1, respectively, among the different developmental root regions (N, biological replicates = 2, n = 16 cells).


D Representative confocal micrographs showing DCP1 localization detected by α - DCP1 in the wild type ( WT ) or the scar1 scar2 scar3 scar4 ( scar1234 ) quadruple mutant or in live - cell imaging of mScarlet - DCP1 ( RPS5apro : HF - mScarlet - DCP1 ) in WT or brk1 mutant ( bottom right ; epidermal cells , region 3 for α - DCP1 and 2 for live - cell imaging ) . The arrowheads denote the lack of robust DCP1 localization in scar1234 at the edge / vertex . Small panels ( insets ) at right show details corresponding to the regions delineated by dashed lines , where arrowhead denotes the edge signal of DCP1 in WT ; scale bars , 1 μm . Bottom : percentage of cells with proper edge / vertex localization and quantification of PB numbers ( DCP1 - foci ; N , biological replicates = 3 , n ( pooled data of 3 biological replicates ) = 18 - 35 cells ) .

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

D Representative confocal micrographs showing DCP1 localization detected by α-DCP1 in the wild type (WT) or the scar1 scar2 scar3 scar4 (scar1234) quadruple mutant or in live-cell imaging of mScarlet-DCP1 (RPS5apro:HF-mScarlet-DCP1) in WT or brk1 mutant (bottom right; epidermal cells, region 3 for α-DCP1 and 2 for live-cell imaging). The arrowheads denote the lack of robust DCP1 localization in scar1234 at the edge/vertex. Small panels (insets) at right show details corresponding to the regions delineated by dashed lines, where arrowhead denotes the edge signal of DCP1 in WT; scale bars, 1 μm. Bottom: percentage of cells with proper edge/vertex localization and quantification of PB numbers (DCP1-foci; N, biological replicates = 3, n (pooled data of 3 biological replicates) = 18-35 cells).


A Representative confocal micrographs showing colocalization between DCP1 or two DCP1 phosphovariants with LifeAct - mCherry in lines co - expressing DCP1pro : DCP1 - GFP ( or variants ) and UBQ10pro : LifeAct - mCherry ( cell edges are indicated by yellow arrowheads ) . Right : relative signal intensity of actin at cell edges / vertices ( normalized to the PM ) in epidermal cells ( N , biological replicates = 3 , n = 15 - 30 , regions 2 - 3 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 confocal micrographs showing colocalization between DCP1 or two DCP1 phosphovariants with LifeAct-mCherry in lines co-expressing DCP1pro:DCP1-GFP (or variants) and UBQ10pro:LifeAct-mCherry (cell edges are indicated by yellow arrowheads). Right: relative signal intensity of actin at cell edges/vertices (normalized to the PM) in epidermal cells (N, biological replicates = 3, n = 15-30, regions 2-3).


B Representative confocal micrographs showing actin localization in WT , dcp1 - 1 , dcp1 - 3 , scar1234 , dcp2 - 1 , dcp5 and pat1 upon phalloidin staining and graph ( right ) indicating the percentage of cells in region 3 with an accumulation of actin at edges in various genotypes ( N , biological replicates = 3 , n = 7 - 9 roots , bars show means + s . d . ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 confocal micrographs showing actin localization in WT, dcp1-1, dcp1-3, scar1234, dcp2-1, dcp5 and pat1 upon phalloidin staining and graph (right) indicating the percentage of cells in region 3 with an accumulation of actin at edges in various genotypes (N, biological replicates = 3, n = 7-9 roots, bars show means + s.d.).


A SE - FRET efficiency between DCP1 - GFP or its phosphovariants with SCAR2 - mCherry ( among the three different root regions ; mainly epidermal cells ) . Scale bar : 50 μm . The arrowhead denotes high FRET efficiency at edges / vertices of region 3 . Right : signal quantification of SE - FRET efficiency between the indicated combinations at the epidermis of 3 regions ( N , biological replicates = 6 , n ( pooled data of 3 biological replicates ) = 10 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 SE-FRET efficiency between DCP1-GFP or its phosphovariants with SCAR2-mCherry (among the three different root regions; mainly epidermal cells). Scale bar: 50 μm. The arrowhead denotes high FRET efficiency at edges/vertices of region 3. Right: signal quantification of SE-FRET efficiency between the indicated combinations at the epidermis of 3 regions (N, biological replicates = 6, n (pooled data of 3 biological replicates) = 10).


C Representative confocal micrographs showing SCAR2 - mCherry localization in WT and dcp1 - 3 mutant , respectively ( root region 2 , epidermal cells ) and quantification of edge / vertex with SCAR2 a confined signal ( N , biological replicates = 3 , n = 5 - 8 cells ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Representative confocal micrographs showing SCAR2-mCherry localization in WT and dcp1-3 mutant, respectively (root region 2, epidermal cells) and quantification of edge/vertex with SCAR2 a confined signal (N, biological replicates = 3, n = 5-8 cells).


D Representative confocal micrographs showing the colocalization between DCP1 - GFP or phosphovariants and SCAR2 - mCherry in root meristematic cells ( root region 2 , epidermal cells ) . Scale bars : 10 μm . The insets show details of colocalization ; the white arrowhead denotes the expansion of the SCAR2 / DCP1 domain , while the yellow arrowheads the restricted edge / vertex SCAR2 / DCP1 domains . Scale bars : 3 μm . The graph indicates the relative signal intensity for the indicated combinations ( as Pearson ' s correlation coefficient ; N , biological replicates = 3 , n = 5 at edges / vertices : spread edges were not considered in calculations ) .

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

D Representative confocal micrographs showing the colocalization between DCP1-GFP or phosphovariants and SCAR2-mCherry in root meristematic cells (root region 2, epidermal cells). Scale bars: 10 μm. The insets show details of colocalization; the white arrowhead denotes the expansion of the SCAR2/DCP1 domain, while the yellow arrowheads the restricted edge/vertex SCAR2/DCP1 domains. Scale bars: 3 μm. The graph indicates the relative signal intensity for the indicated combinations (as Pearson's correlation coefficient; N, biological replicates = 3, n = 5 at edges/vertices: spread edges were not considered in calculations).


A Representative images showing the phenotypes of dcp1 mutants and mutants in other PB core components or SCAR - WAVE components ( 5 - day - old seedlings ) . The arrowheads show the growth defects of homozygous dcp1 - 1 or dcp2 - 1 mutants ( denoted - / - ; heterozygous denoted dcp1 - 1 / DCP1 ; details are also shown ) . Lower : graph showing relative root length ( N , biological replicates = 3 , n ( pooled data of 3 biological replicates ) = 3 - 4 roots , bars show means + s . d ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 images showing the phenotypes of dcp1 mutants and mutants in other PB core components or SCAR-WAVE components (5-day-old seedlings). The arrowheads show the growth defects of homozygous dcp1-1 or dcp2-1 mutants (denoted -/-; heterozygous denoted dcp1-1/DCP1; details are also shown). Lower: graph showing relative root length (N, biological replicates = 3, n (pooled data of 3 biological replicates) = 3-4 roots, bars show means + s.d).


B DCP1 regulates cell expansion anisotropy . Representative confocal micrographs showing FM4 - 64 staining of the WT , dcp1 - 1 and dcp2 - 1 mutants ( 2 μM , 10 min ) . Scale bars , 20 μm . Right : percentage of isotropic cells per root meristem ( % , epidermal cells ) in each genotype ( N , biological replicates = 3 , n ( pooled data of 3 biological replicates ) = 3 - 5 roots , bars show means ± s . d ) . Examples of isotropic or anisotropic cells are shown , along with the developmental axis offset at the x - and y - axes .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 DCP1 regulates cell expansion anisotropy. Representative confocal micrographs showing FM4-64 staining of the WT, dcp1-1 and dcp2-1 mutants (2 μM, 10 min). Scale bars, 20 μm. Right: percentage of isotropic cells per root meristem (%, epidermal cells) in each genotype (N, biological replicates = 3, n (pooled data of 3 biological replicates) = 3-5 roots, bars show means ± s.d). Examples of isotropic or anisotropic cells are shown, along with the developmental axis offset at the x- and y-axes.


( c ) Shown are the responses of the glucose sensor promoter ( PgluA7 ) , a negative control lacking the CRP operator ( PgluA7 * ) , and a constitutive promoter ( BBa _ J23101 ) to the presence ( + ) and absence ( - ) of

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

(c) Shown are the responses of the glucose sensor promoter (PgluA7), a negative control lacking the CRP operator (PgluA7*), and a constitutive promoter (BBa_J23101) to the presence (+) and absence (-) of glucose


( d ) Shown are the responses of the oxygen sensor promoter ( PfnrF8 ) , a negative control lacking the FNR operator ( PfnrF8 * ) , and a constitutive promoter ( BBa _ J23101 ) to the presence ( + ) and absence ( - ) of

(d) Shown are the responses of the oxygen sensor promoter (PfnrF8), a negative control lacking the FNR operator (PfnrF8*), and a constitutive promoter (BBa_J23101) to the presence (+) and absence (-) of oxygen


Shown are the schematics and responses for the glucose sensor The response functions ( center ) are shown for each sensor ( black circles ) compared to promoter variants where the operators are removed ( PgluA7 * , PfnrF8 * ) ( open circles ) text ) . Representative cytometry florescence distributions for Figures

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

Shown are the schematics and responses for the glucose sensor The response functions (center) are shown for each sensor (black circles) compared to promoter variants where the operators are removed (PgluA7*, PfnrF8*) (open circles) text). Representative cytometry florescence distributions for Figures 1f


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

Shown are the schematics and responses for th oxygen sensors Horizontal error bars in the PfnrF8 response reflect one standard deviation of three DO measurements Representative cytometry florescence distributions for Figure 1g


Shown are the schematics and responses for th acetate sensors For the acetate sensor , the response of the sensor is shown in unmodified E . coli MG1655 with glnL intact ( open diamonds ) . The dynamics of induction are shown ( right graph ) where cells are induced at the time indicated by the dashed line ( see text ) . Representative cytometry florescence distributions for Figure 1h are shown

O O O O O O O O B-CONTROLLED_VAR 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

Shown are the schematics and responses for th acetate sensors For the acetate sensor, the response of the sensor is shown in unmodified E. coli MG1655 with glnL intact (open diamonds). The dynamics of induction are shown (right graph) where cells are induced at the time indicated by the dashed line (see text). Representative cytometry florescence distributions for Figure 1h are shown i


The responses of the glucose sensors during growth in shake flasks are shown


The responses of th e , oxygen sensors during growth in shake flasks are

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

The responses of th e, oxygen sensors during growth in shake flasks are shown


The responses of th acetate sensors during growth in shake flasks are shown


( e ) The responses of a 3 - input , 2 - output circuit are shown . ( f ) Shown are the circuit ( left ) and genetic diagram ( right ) of the circuit corresponding to part e . In the genetic diagram , the dashed line and * indicates a second copy of the PgluA7 promoter that drives rfp expression and is repressed by PhlF via an immediately downstream PhlF operator . ( g ) The response of the circuit in parts e and f to different combinations of stimuli under the same conditions as Figure 1 ( Methods ) . The ( + ) and ( - ) indicate whether the output promoter of each sensor is active under those conditions . Bars where the circuit is predicted to be on are shown in grey and white when predicted to be

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

(e) The responses of a 3-input, 2-output circuit are shown. (f) Shown are the circuit (left) and genetic diagram (right) of the circuit corresponding to part e. In the genetic diagram, the dashed line and * indicates a second copy of the PgluA7 promoter that drives rfp expression and is repressed by PhlF via an immediately downstream PhlF operator. (g) The response of the circuit in parts e and f to different combinations of stimuli under the same conditions as Figure 1 (Methods). The (+) and (-) indicate whether the output promoter of each sensor is active under those conditions. Bars where the circuit is predicted to be on are shown in grey and white when predicted to be off


( h ) The response of a 3 - input 1 - output circuit is shown . ( i ) Shown are the circuit ( left ) and genetic diagram ( right ) of the circuit corresponding to part h . ( j ) The response of the circuit in parts h and i to different combinations of stimuli under the same conditions as Figure 1 ( Methods ) . Representative cytometry florescence distributions for Figure 2a / b / c and Figure 2e / g / h / j are shown in Appendix Figures S7 and S10 , respectively . Error bars represent one standard deviation of three independent experiments done on different

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

(h) The response of a 3-input 1-output circuit is shown. (i) Shown are the circuit (left) and genetic diagram (right) of the circuit corresponding to part h. (j) The response of the circuit in parts h and i to different combinations of stimuli under the same conditions as Figure 1 (Methods). Representative cytometry florescence distributions for Figure 2a/b/c and Figure 2e/g/h/j are shown in Appendix Figures S7 and S10, respectively. Error bars represent one standard deviation of three independent experiments done on different days


( b ) Reduction of fluorescence of RFP by the different mechanisms of repression after 18 hours of growth ( Methods ) . The inducers are either 1 mM IPTG ( sRNA ) or 25 μM DAPG ( sgRNA , mf - LON ) . sgRNA and mf - LON are co - transcribed on a single transcript , processed by ribozymes ( Appendix Figure S13 ) . ( c ) The dynamics of repression by each of the mechanisms is shown . Empty circles are uninduced and black circles are induced ( 1 mM IPTG or 25 µM DAPG ) at the 2 - hour time point ( dashed line ) ( Methods )

(b) Reduction of fluorescence of RFP by the different mechanisms of repression after 18 hours of growth (Methods). The inducers are either 1 mM IPTG (sRNA) or 25 μM DAPG (sgRNA, mf-LON). sgRNA and mf-LON are co-transcribed on a single transcript, processed by ribozymes (Appendix Figure S13). (c) The dynamics of repression by each of the mechanisms is shown. Empty circles are uninduced and black circles are induced (1 mM IPTG or 25 µM DAPG) at the 2-hour time point (dashed line) (Methods)


( f ) The impact on acetate production for a double mutant where poxB is complemented on a BAC ( Methods ) . Note that complementation causes a ~ 3 - fold reduction in acetate compared to WT in part d . N - ter and E170 refer to the location of the SuMMV tag . ( g ) Reduction of acetate production by the expression of SuMMV from an DAPG - inducible promoter ( - no inducer , + 25 µM ) . The PoxB variant with the degron inserted at position E170 is either carried on a BAC or at its native location in the

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 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 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

(f) The impact on acetate production for a double mutant where poxB is complemented on a BAC (Methods). Note that complementation causes a ~3-fold reduction in acetate compared to WT in part d. N-ter and E170 refer to the location of the SuMMV tag. (g) Reduction of acetate production by the expression of SuMMV from an DAPG-inducible promoter (- no inducer, + 25 µM). The PoxB variant with the degron inserted at position E170 is either carried on a BAC or at its native location in the genome


( b ) The transcription of poxB over time ( E . coli MG1655 ∆ glnL ) , as calculated from RNA - seq data ( Methods ) . The colored bars indicate the times at which the glucose / acetate sensors will be on based on metabolite measurements . It should be noted that these are right - shifted compared to Figure 2 due to slower growth of the tested

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O 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) The transcription of poxB over time (E. coli MG1655∆glnL), as calculated from RNA-seq data (Methods). The colored bars indicate the times at which the glucose/acetate sensors will be on based on metabolite measurements. It should be noted that these are right-shifted compared to Figure 2 due to slower growth of the tested strains


( c ) The production of acetate is shown over time for E . coli MG1655 ∆ glnL ∆ pta poxB : : E170 ( black ) as compared to the same strain containing the complete circuit ( green ) . A version of the circuit in which the sgRNA is targeted to rfp ( not present in the system ) and is tested in MG1655 ∆ glnL ∆ pta containing an untagged poxB as a control ( red ) . Empty circles connected by the dashed line represent MG1655 ∆ glnL ∆ pta ∆

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

(c) The production of acetate is shown over time for E. coli MG1655∆glnL∆pta poxB::E170 (black) as compared to the same strain containing the complete circuit (green). A version of the circuit in which the sgRNA is targeted to rfp (not present in the system) and is tested in MG1655∆glnL∆pta containing an untagged poxB as a control (red). Empty circles connected by the dashed line represent MG1655∆glnL∆pta∆poxB


( e ) The transcription of pta over time ( E . coli MG1655 ∆ glnL ) , as calculated from RNA - seq data ( Methods ) . The colored bars indicate the times at which the glucose / oxygen sensors will be on based on metabolite

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

(e) The transcription of pta over time (E. coli MG1655∆glnL), as calculated from RNA-seq data (Methods). The colored bars indicate the times at which the glucose/oxygen sensors will be on based on metabolite measurements


( f ) The production of acetate is shown over time for E . coli MG1655 ∆ glnL ∆ poxB pta : : pdt3 as compared to a strain containing the complete circuit ( green ) tested in same strain . A version of the circuit in which the sgRNA is targeted to rfp ( not present in the system ) and is tested in MG1655 ∆ glnL ∆ poxB containing an untagged pta as a control ( red ) Empty circles connected by the dashed line represent MG1655 ∆ glnL ∆ pta ∆

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

(f) The production of acetate is shown over time for E. coli MG1655∆glnL∆poxB pta::pdt3 as compared to a strain containing the complete circuit (green) tested in same strain. A version of the circuit in which the sgRNA is targeted to rfp (not present in the system) and is tested in MG1655∆glnL∆poxB containing an untagged pta as a control (red) Empty circles connected by the dashed line represent MG1655∆glnL∆pta∆poxB


Interaction analysis for MIA40 and AK2 . Stable inducible cell lines that express either different variants of MIA40 - Strep ( wildtype and a redox inactive mutant ( MIA40C53S , C55S ) , ( Fischer et al . , 2013 ) or AK2 - HA were treated with N - ethyl maleimide ( NEM ) to stop thiol - disulfide exchange processes and lysed under denaturing conditions . Lysates were analysed by subsequent affinity purification against Strep ( MIA40 ) and immunoblotting . Like classical MIA40 substrates , AK2 interacted with the active site of MIA40 ( lane 6 ) . 0 . 5 % input was loaded as control .

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

Interaction analysis for MIA40 and AK2. Stable inducible cell lines that express either different variants of MIA40-Strep (wildtype and a redox inactive mutant (MIA40C53S,C55S), (Fischer et al., 2013) or AK2-HA were treated with N-ethyl maleimide (NEM) to stop thiol-disulfide exchange processes and lysed under denaturing conditions. Lysates were analysed by subsequent affinity purification against Strep (MIA40) and immunoblotting. Like classical MIA40 substrates, AK2 interacted with the active site of MIA40 (lane 6). 0.5% input was loaded as control.


Redox state analysis of endogenous AK2 by inverse redox shift assay . HEK293 cells were lysed by TCA precipitation after NEM treatment , lysates were reduced using TCEP and then thiols modified using the alkylating agent mmPEG12 . With this " inverse " method , previously oxidized cysteine thiols are modified with mmPEG12 , which results in a slower migration on SDS - PAGE . Treatment of intact cells with diamide and consequently modification of all cysteines served as control . Likewise , absence of NEM treatment served as ' max ' control . Treatment lysates with NEM instead of mmPEG12 served as minimal shift ( min ) control . A , B , isoforms of AK2 . Both isoforms of AK2 are semi - oxidized in intact cells .

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Redox state analysis of endogenous AK2 by inverse redox shift assay. HEK293 cells were lysed by TCA precipitation after NEM treatment, lysates were reduced using TCEP and then thiols modified using the alkylating agent mmPEG12. With this "inverse" method, previously oxidized cysteine thiols are modified with mmPEG12, which results in a slower migration on SDS-PAGE. Treatment of intact cells with diamide and consequently modification of all cysteines served as control. Likewise, absence of NEM treatment served as 'max' control. Treatment lysates with NEM instead of mmPEG12 served as minimal shift (min) control. A, B, isoforms of AK2. Both isoforms of AK2 are semi-oxidized in intact cells.


Oxidation kinetics experiment to investigate maturation of endogenous AK2 . HEK293 cells were pulse labeled for 10 min with [ 35S ] - methionine and chased with cold methionine for the indicated times . The chase was stopped by TCA precipitation , and then the lysate was treated with mmPEG12 to determine the AK2 redox states , followed by IP against AK2 . Eluates were analyzed by Tris - Tricine - PAGE and autoradiography . With this " direct " redox state determination method reduced AK2 was modified with either three ( isoform B ) or four ( isoform A ) mmPEG12 . Semi - oxidized AK2 was modified with only one ( " B " ) or two ( " A " ) mmPEG12 , respectively . Unmodified and fully reduced cell lysates served as controls . Endogenous AK2 becomes oxidized with a half time of less than 20 min .

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Oxidation kinetics experiment to investigate maturation of endogenous AK2. HEK293 cells were pulse labeled for 10 min with [35S]-methionine and chased with cold methionine for the indicated times. The chase was stopped by TCA precipitation, and then the lysate was treated with mmPEG12 to determine the AK2 redox states, followed by IP against AK2. Eluates were analyzed by Tris-Tricine-PAGE and autoradiography. With this "direct" redox state determination method reduced AK2 was modified with either three (isoform B) or four (isoform A) mmPEG12. Semi-oxidized AK2 was modified with only one ("B") or two ("A") mmPEG12, respectively. Unmodified and fully reduced cell lysates served as controls. Endogenous AK2 becomes oxidized with a half time of less than 20 min.


Oxidation kinetics experiment to investigate the influence of MIA40 on AK2 maturation . Experiment was performed as described in ( D ) , except that 72 h before experiments cells were transfected with control siRNA or siRNA directed against MIA40 . Endogenous AK2 oxidation depends on MIA40 . Reported values are the mean of 3 independent experiments ; error bars represent ± SD . Student ' s t - test was performed . * represents p < 0 . 05 , * * represents p < 0 . 01 and * * * represents p < 0 . 001 .

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Oxidation kinetics experiment to investigate the influence of MIA40 on AK2 maturation. Experiment was performed as described in (D), except that 72 h before experiments cells were transfected with control siRNA or siRNA directed against MIA40. Endogenous AK2 oxidation depends on MIA40. Reported values are the mean of 3 independent experiments; error bars represent ±SD. Student's t-test was performed. * represents p<0.05, ** represents p<0.01 and *** represents p<0.001.


Influence of AK2 cysteines on the AK2 redox state . Cells expressing single cysteine to serine mutants of AK2 - HA ( isoform A ) were lysed after NEM treatment , and analyzed by inverse redox shift assay using mmPEG12 , SDS - PAGE and immunoblot against the HA tag . AK2C42S and AK2C92S are present in their completely reduced state indicating presence of a disulfide bond between both cysteines .

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Influence of AK2 cysteines on the AK2 redox state. Cells expressing single cysteine to serine mutants of AK2-HA (isoform A) were lysed after NEM treatment, and analyzed by inverse redox shift assay using mmPEG12, SDS-PAGE and immunoblot against the HA tag. AK2C42S and AK2C92S are present in their completely reduced state indicating presence of a disulfide bond between both cysteines.


Influence of AK2 cysteines on cellular localization . Cells expressing single cysteine to serine mutants of AK2 - HA ( isoform A ) were fixed and analyzed by immunofluorescence . The colocalization of AK2 - HA variants with Mitotracker was quantified using Cellprofiler ( Pearson ' s correlation coefficient ) . C42 and C92 are critical for mitochondrial localization of AK2 . The C40S mutant exhibits significantly decreased colocalization with mitochondria . A Kruskall - Wallis rank sum test / Dunn - test was performed . Numbers of analysed cells are indicated in the plot . * * * represents p - value < 0 . 001 compared to wild - type . The black horizontal line represents the median . Data information : Scale bars : 10 μm .

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Influence of AK2 cysteines on cellular localization. Cells expressing single cysteine to serine mutants of AK2-HA (isoform A) were fixed and analyzed by immunofluorescence. The colocalization of AK2-HA variants with Mitotracker was quantified using Cellprofiler (Pearson's correlation coefficient). C42 and C92 are critical for mitochondrial localization of AK2. The C40S mutant exhibits significantly decreased colocalization with mitochondria. A Kruskall-Wallis rank sum test/ Dunn-test was performed. Numbers of analysed cells are indicated in the plot. *** represents p-value <0.001 compared to wild-type. The black horizontal line represents the median. Data information: Scale bars: 10 μm.


Steady state levels of endogenous AK2 in presence and absence of MIA40 . 72 h before experiments , HeLa cells were transfected with control siRNA or siRNAs directed against MIA40 or ALR . Cell lysates were analysed by immunoblot against the indicated proteins . MIA40 depletion led to a strong decrease in AK2 and ALR levels but not of the control proteins . ALR depletion did not affect AK2 levels . Reported values are the mean of 3 independent experiments ; error bars represent ± SD . Student ' s t - test was performed . * represents p < 0 . 05 , .

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Steady state levels of endogenous AK2 in presence and absence of MIA40. 72 h before experiments, HeLa cells were transfected with control siRNA or siRNAs directed against MIA40 or ALR. Cell lysates were analysed by immunoblot against the indicated proteins. MIA40 depletion led to a strong decrease in AK2 and ALR levels but not of the control proteins. ALR depletion did not affect AK2 levels. Reported values are the mean of 3 independent experiments; error bars represent ±SD. Student's t-test was performed. * represents p<0.05,.


Stability of endogenous AK2 in the presence and absence of MIA40 . 72 h before experiments HeLa , cells were transfected with control siRNA or siRNAs directed against MIA40 . Cells were pulse labeled for 10 min with [ 35S ] - methionine and chased with cold methionine for the indicated times . The chase was stopped by cell lysis , and AK2 was isolated by denaturing immunoprecipitation . Eluates were analyzed by SDS - PAGE and autoradiography . Stability of endogenous AK2 is significantly decreased upon depletion of MIA40 . Reported values are the mean of 3 independent experiments ; error bars represent ± SD . Student ' s t - test was performed ; * represents p - value < 0 . 05 compared to wild - type .

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Stability of endogenous AK2 in the presence and absence of MIA40. 72 h before experiments HeLa, cells were transfected with control siRNA or siRNAs directed against MIA40. Cells were pulse labeled for 10 min with [35S]-methionine and chased with cold methionine for the indicated times. The chase was stopped by cell lysis, and AK2 was isolated by denaturing immunoprecipitation. Eluates were analyzed by SDS-PAGE and autoradiography. Stability of endogenous AK2 is significantly decreased upon depletion of MIA40. Reported values are the mean of 3 independent experiments; error bars represent ±SD. Student's t-test was performed; * represents p-value <0.05 compared to wild-type.


Stability of cytosolic AK2 - HA cysteine variant . Experiment was performed as in ( B ) except that cells expressing AK2C40S , C42S , C92S - HA were analysed by IP against HA . AK2C40S , C42S , C92S - HA is unstable but becomes stabilized by application of the proteasomal inhibitor MG132 . Reported values are the mean of 3 independent experiments ; error bars represent ± SD . Student ' s t - test was performed . * represents p < 0 . 05 , .

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Stability of cytosolic AK2-HA cysteine variant. Experiment was performed as in (B) except that cells expressing AK2C40S,C42S,C92S-HA were analysed by IP against HA. AK2C40S,C42S,C92S-HA is unstable but becomes stabilized by application of the proteasomal inhibitor MG132. Reported values are the mean of 3 independent experiments; error bars represent ±SD. Student's t-test was performed. * represents p<0.05,.


Steady state levels of AK2 - HA cysteine variants . AK2C40S , C42S , C92S - HA and AK2WT - HA were expressed in the presence or absence of proteasomal inhibitor MG132 . Protein levels at steady state were analysed by immunoblot . All variants are stabilized by proteasomal inhibition . Reported values are the mean of 3 ( AK2WT - HA ) or 5 ( AK2C40S , C42S , C92S - HA ) independent experiments ; error bars represent ± SD . ANOVA / Tukey ' s post - hoc test was performed . * * * represents p < 0 . 001 .

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Steady state levels of AK2-HA cysteine variants. AK2C40S,C42S,C92S-HA and AK2WT-HA were expressed in the presence or absence of proteasomal inhibitor MG132. Protein levels at steady state were analysed by immunoblot. All variants are stabilized by proteasomal inhibition. Reported values are the mean of 3 (AK2WT-HA) or 5 (AK2C40S,C42S,C92S-HA) independent experiments; error bars represent ±SD. ANOVA/Tukey's post-hoc test was performed. *** represents p<0.001.


Steady state levels of AK2 - HA cysteine variant . 72 h before experiments HEK293 cells were transfected with control siRNA or siRNAs directed against the proteasomal subunits PSMB3 and PSMC5 . AK2C40S , C42S , C92S - HA was stabilized by decreased proteasome levels . Reported values are the mean of 4 independent experiments ; error bars represent ± SD . Student ' s t - test was performed . * represents p < 0 . 05 .

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Steady state levels of AK2-HA cysteine variant. 72 h before experiments HEK293 cells were transfected with control siRNA or siRNAs directed against the proteasomal subunits PSMB3 and PSMC5. AK2C40S,C42S,C92S-HA was stabilized by decreased proteasome levels. Reported values are the mean of 4 independent experiments; error bars represent ±SD. Student's t-test was performed. * represents p<0.05.


Steady state levels of AK2C40S , C42S , C92S - HA in presence and absence of DPP8 and DPP9 . 72 h before experiments , HEK293 cells stably expressing AK2C40S , C42S , C92S - HA were transfected with control siRNA or siRNAs directed against DPP8 or / and DPP9 . Cell lysates were analysed by immunoblot against the indicated proteins . DPP9 depletion led to an increase in AK2C40S , C42S , C92S - HA levels , while DPP8 depletion did not . Combined depletion of DPP8 and DPP9 increased AK2C40S , C42S , C92S - HA levels even further . This indicates that DPP9 is the main peptidase and DPP8 can serve as its backup . White and black arrow heads indicate the endogenous and HA - tagged AK2 , respectively . The gray arrow head indicates the DPP9 band . Reported values are the mean of 3 independent experiments ; error bars represent ± SD . ANOVA / Tukey ' s post - hoc test was performed . * represents p < 0 . 05 , , # # represent p < 0 . 01 and * * * , # # # represent p < 0 . 001 .

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Steady state levels of AK2C40S,C42S,C92S-HA in presence and absence of DPP8 and DPP9. 72 h before experiments, HEK293 cells stably expressing AK2C40S,C42S,C92S-HA were transfected with control siRNA or siRNAs directed against DPP8 or/and DPP9. Cell lysates were analysed by immunoblot against the indicated proteins. DPP9 depletion led to an increase in AK2C40S,C42S,C92S-HA levels, while DPP8 depletion did not. Combined depletion of DPP8 and DPP9 increased AK2C40S,C42S,C92S-HA levels even further. This indicates that DPP9 is the main peptidase and DPP8 can serve as its backup. White and black arrow heads indicate the endogenous and HA-tagged AK2, respectively. The gray arrow head indicates the DPP9 band. Reported values are the mean of 3 independent experiments; error bars represent ±SD. ANOVA/Tukey's post-hoc test was performed. * represents p<0.05, ,## represent p<0.01 and ***,### represent p<0.001.


Steady state levels of AK2C40S , C42S , C92S - HA upon incubation with the DPP9 inhibitor 1G244 ( Wu et al . , 2009 ) . As ( B ) except that cells were treated for 16 h with inhibitor or DMSO as control . AK2C40S , C42S , C92S - HA levels were strongly increased upon DPP8 / 9 inhibition . Reported values are the mean of 4 independent experiments ; error bars represent ± SD . Student ' s t - test was performed . * * * represents p < 0 . 001 .

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Steady state levels of AK2C40S,C42S,C92S-HA upon incubation with the DPP9 inhibitor 1G244 (Wu et al., 2009). As (B) except that cells were treated for 16 h with inhibitor or DMSO as control. AK2C40S,C42S,C92S-HA levels were strongly increased upon DPP8/9 inhibition. Reported values are the mean of 4 independent experiments; error bars represent ±SD. Student's t-test was performed. *** represents p<0.001.


Steady state levels of endogenous AK2 in presence and absence of DPP9 and MIA40 . 72 h before experiments HeLa cells were transfected with control siRNA or siRNAs directed against MIA40 and / or DPP9 . Cell lysates were analysed by immunoblot against the indicated proteins . If import of endogenous AK2 was prevented by depletion of MIA40 , then concomitant depletion of DPPs increased its levels . Strikingly , depletion of DPP9 alone also increased the levels of endogenous AK2 . Reported values are the mean of 3 independent experiments ; error bars represent ± SD . ANOVA / Tukey ' s post - hoc test was performed . * * represents p < 0 . 01 .

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Steady state levels of endogenous AK2 in presence and absence of DPP9 and MIA40. 72 h before experiments HeLa cells were transfected with control siRNA or siRNAs directed against MIA40 and/or DPP9. Cell lysates were analysed by immunoblot against the indicated proteins. If import of endogenous AK2 was prevented by depletion of MIA40, then concomitant depletion of DPPs increased its levels. Strikingly, depletion of DPP9 alone also increased the levels of endogenous AK2. Reported values are the mean of 3 independent experiments; error bars represent ±SD. ANOVA/Tukey's post-hoc test was performed. ** represents p<0.01.


Steady state levels of endogenous AK2 upon incubation with the DPP9 inhibitor 1G244 . As ( D ) except that cells were treated for 3 or 5 days with inhibitor or DMSO as control . Endogenous AK2 levels were increased upon DPP8 / 9 inhibition . Reported values are the mean of 5 ( 3 d ) and 6 ( 5 d ) independent experiments ; error bars represent ± SD . Student ' s t - test was performed . * represents p < 0 . 05 , and * * * represents p < 0 . 001 .

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Steady state levels of endogenous AK2 upon incubation with the DPP9 inhibitor 1G244. As (D) except that cells were treated for 3 or 5 days with inhibitor or DMSO as control. Endogenous AK2 levels were increased upon DPP8/9 inhibition. Reported values are the mean of 5 (3 d) and 6 (5 d) independent experiments; error bars represent ±SD. Student's t-test was performed. * represents p<0.05, and *** represents p<0.001.


Steady state levels of AK2 - HA variants with mutated N - termini upon incubation with either 1G244 or vehicle control . HEK293 cells stably expressing AK2 - HA variants were lysed and subjected to immunoblot against the indicated proteins . Levels of DPP8 / 9 - insensitive AK2 variants are strongly increased . Reported values are the mean of 4 independent experiments ; error bars represent ± SD . ANOVA / Tukey ' s post - hoc test was performed . * * represents p < 0 . 01 and # # # , * * * represents p < 0 . 001 . Asterisk denominate statistical comparison with respective control , and hash tag represents comparison to N - WT .

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Steady state levels of AK2-HA variants with mutated N-termini upon incubation with either 1G244 or vehicle control. HEK293 cells stably expressing AK2-HA variants were lysed and subjected to immunoblot against the indicated proteins. Levels of DPP8/9-insensitive AK2 variants are strongly increased. Reported values are the mean of 4 independent experiments; error bars represent ±SD. ANOVA/Tukey's post-hoc test was performed. ** represents p<0.01 and ###, *** represents p<0.001. Asterisk denominate statistical comparison with respective control, and hash tag represents comparison to N-WT.