Luciferase with specific of locus bound by T around L # 1 L # 3 as shown in ( A ) . Relative luciferase activities of low T - inducible ZHBTc4 ESCs stimulated as indicated are shown with SDs . ( three independent experiments with two technical replicates ) .

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Luciferase assay with the specific sequence of the locus bound by T around L#1 and L#3 as shown in (A). Relative luciferase activities of low T-inducible ZHBTc4 ESCs stimulated as indicated are shown with SDs. (three independent experiments with two technical replicates).


Expression of Blimp1 Prdm14 measured by qPCR in PGCLCs derived from indicated ESC lines . ∆ Ct values from average Ct values of Arbp Ppia are shown ( log2 scale , mean value of two independent experiments with two technical replicates ) .

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Expression of Blimp1 and Prdm14 measured by qPCR in PGCLCs derived from indicated ESC lines. The ∆Ct values from the average Ct values of Arbp and Ppia are shown (log2 scale, the mean value of two independent experiments with two technical replicates).


Top : Oct4 - GFP ( green ) reporter expression ( left ) , Blimp1 - RFP ( red ) Oct4 - GFP ( BROG ) reporter expression ( right ) in gonads of E13 . 5 generated by 4N complementation with indicated ESC lines . Scale bar : 100um . Bottom : of Oct4 - GFP reporter - positive PGCs ( left ) BROG reporter - positive PGCs ( right ) measured by FACS . Blue circles , red squares , green triangles represent of gonadal PGCs in first , second , third independent mutant , respectively , of examined locus ( L # 1 - 5 ) .

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Top: Oct4-GFP (green) reporter expression (left), and Blimp1-RFP (red) and Oct4-GFP (BROG) reporter expression (right) in the gonads of E13.5 embryos generated by 4N complementation with the indicated ESC lines. Scale bar: 100um. Bottom: percentage of Oct4-GFP reporter-positive PGCs (left) and BROG reporter-positive PGCs (right) measured by FACS analysis. Blue circles, red squares, and green triangles represent the percentage of gonadal PGCs in the first, second, and third independent mutant line, respectively, of the examined locus (L#1-5).


High constitutive expression of CXCR4 in of SW1417 CRC as measured by flow cytometry .

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High and constitutive expression of CXCR4 in the membrane of SW1417 CRC cells as measured by flow cytometry.


Lack of human SDF - 1α release from cultured SW1417 CRC , as measured by ELISA , whereas human control 1BR3 . G fibroblasts express high SDF - 1α , after 48h or 72h of growth in culture . ( N = 2 experiment in duplicate )

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Lack of human SDF-1α release from cultured SW1417 CRC cells, as measured by ELISA, whereas human control 1BR3.G fibroblasts express high SDF-1α levels, after 48h or 72h of growth in culture. (N=2 experiment in duplicate)


Nanoconjugate internalization in CXCR4 overexpressing ( CXCR4 + ) SW1417 CRC after 1 hour exposure at 1µM , as measured by emission using flow cytometry . ( N = 3 experiments in duplicate ) Significant difference at p = 0 . 002 between T22 - GFP - H6 - FdU AMD3100 + T22 - GFP - H6 - FdU

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Nanoconjugate internalization in CXCR4 overexpressing (CXCR4+) SW1417 CRC cells after 1 hour exposure at 1µM, as measured by fluorescence emission using flow cytometry. (N=3 experiments in duplicate) Significant difference at p= 0.002 between T22-GFP-H6-FdU and AMD3100+T22-GFP-H6-FdU groups


Intracellular trafficking of T22 - GFP - H6 - FdU in CXCR4 + SW1417 by confocal microscopy after exposure at 1µM for 24 h . green corresponds to GFP containing nanoconjugates red corresponds to plasma membranes stained with a red dye ( CellMask ™ ) , whereas nucleus was stained in blue with hoescht . insets show detail of intracellular localization of nanostructured , entities , in an isosurface representation within a three - dimensional volumetric x - y - z data

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Intracellular trafficking of T22-GFP-H6-FdU in CXCR4+ SW1417 cells by confocal microscopy after exposure at 1µM for 24 h. The green staining corresponds to GFP containing nanoconjugates and the red staining corresponds to plasma cell membranes stained with a red dye (CellMask™), whereas cell nucleus was stained in blue with hoescht. The insets show detail of the intracellular localization of nanostructured, fluorescent entities, in an isosurface representation within a three-dimensional volumetric x-y-z data field


Linearized T22 - GFP - H6 - FdU dose - response trend representation compared with unconjugated free oligo - FdU exposure . Antitumor effect was measured as CXCR4 + SW1417 viability by MTT after 72 h exposure as described concentrations . ( N = 3 experiments in duplicate )

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Linearized T22-GFP-H6-FdU dose-response trend line representation compared with unconjugated free oligo-FdU exposure. Antitumor effect was measured as CXCR4+ SW1417 cell viability by MTT after 72 h exposure as the described concentrations. (N=3 experiments in duplicate)


Reduction of viability determined by optical microscope of SW1417 exposed to 1µM T22 - GFP - H6 - FdU for 48 h , as compared to T22 - GFP - H6 or free oligo - FdU . ( N = 3 experiments in duplicate ; Scale bar , 100µm ) .

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Reduction of cell viability determined by optical microscope images of SW1417 cells exposed to 1µM T22-GFP-H6-FdU for 48 h, as compared to T22-GFP-H6 or free oligo-FdU. (N=3 experiments in duplicate; Scale bar, 100µm).


Selective T22 - GFP - H6 - FdU nanoconjugate biodistribution in subcutaneous CXCR4 + SW1417 CRC tissue 5h after a 100µg single intravenous dose , as measured by emission using IVIS Spectrum 200 ( N = 5 / group ) . Biodistribution is similar to that achieved by T22 - GFP - H6 targeting vector undetectable after Buffer or free oligo - FdU treatment . ( N = 5 mice / group )

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Selective T22-GFP-H6-FdU nanoconjugate biodistribution in subcutaneous CXCR4+ SW1417 CRC tumor tissue 5h after a 100µg single intravenous dose, as measured by fluorescence emission using IVIS Spectrum 200 (N=5/group). Biodistribution is similar to that achieved by the T22-GFP-H6 targeting vector and undetectable after Buffer or free oligo-FdU treatment. (N=5 mice/group)


Co - localization ( yellow merged ) of T22 - GFP - H6 - FdU ( green ) CXCR4 receptor ( red ) release of T22 - GFP - H6 - FdU into cytosol in CXCR4 + 5h after a 100ug dose of nanoconjugate , as measured by dual anti - GFP / anti - CXCR4 immunofluorescence ( IF ) . DAPI ( blue ) . emission was measured in green red channels using Image J software expressed as mean ( A ) ± s . e . m ( µm2 ) ( N = 10 , 2 fields x 5 mice ; 200X ) . Note significant ( P = 0 . 003 ) increase in occupied by green dots ( nanoconjugate realeased to cytosol ) in T22 - GFP - H6 - FdU - treated tumors , compared to free oligo - FdU - treated control tissues . Scale bar , 50 µm .

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Co-localization (yellow merged) of the T22-GFP-H6-FdU (green) and the CXCR4 receptor (red) and release of T22-GFP-H6-FdU into the cytosol in CXCR4+ tumor cells 5h after a 100ug dose of nanoconjugate, as measured by dual anti-GFP/anti-CXCR4 immunofluorescence (IF). DAPI (blue nuclear staining). Fluorescence emission was measured in the green and red channels using the Image J software and expressed as mean Area (A)±s.e.m (µm2) (N=10, 2 tumor fields x 5 mice; 200X). Note the significant (P=0.003) increase in the area occupied by the green dots (nanoconjugate realeased to the cell cytosol) in T22-GFP-H6-FdU-treated tumors, compared to free oligo-FdU-treated control tissues. Scale bar, 50 µm.


Administration of CXCR4 antagonist AMD3100 completely blocks T22 - GFP - H6 - FdU biodistribution , as measured by emission . is not detected in Buffer or free oligo - FdU controls ( N = 5 fields / group ) .

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Administration of the CXCR4 antagonist AMD3100 completely blocks T22-GFP-H6-FdU tumor biodistribution, as measured by fluorescence emission. Fluorescence is not detected in Buffer or free oligo-FdU controls (N=5 tumor fields/group).


uptake of T22 - GFP - H6 - FdU observed in CXCR4 + SW1417 tissues is almost completely blocked by prior AMD3100 administration , as quantified using anti - GFP H - score . ( N = 5 fields / group ) .

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The uptake of T22-GFP-H6-FdU observed in CXCR4+ SW1417 tumor tissues is almost completely blocked by prior AMD3100 administration, as quantified using the anti-GFP IHC H-score. (N=5 tumor fields/group).


Representative of T22 - GFP - H6 - FdU uptake AMD3100 competition by anti - GFP inmunostaining , which quantitation is reported in panel E . Scale bar , 50

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Representative images of T22-GFP-H6-FdU uptake and AMD3100 competition by anti-GFP inmunostaining, which quantitation is reported in panel E. Scale bar, 50 µm


Representative of CXCR4 overexpression in subcutaneous tissue , showing similar CXCR4 among compared groups ( N = 5 / group ; Buffer , T22 - GFP - H6 - FdU , T22 - GFP - H6 free oligo - FdU ) before treatment ( upper panels ) . Representative of double - strand break induction caspase 3 activation ( measured with anti - ɣH2AX or anti - caspase 3 by ) 5h post - administration ( middle panels ) . Apoptotic induction ( Hoescht , 24h post - administration , lower panels ) . Note higher number of positive for DSBs , Caspase 3 activation apoptosis induction in T22 - GFP - H6 - FdU as compared to free oligo - FdU . Black or arrows indicate dead . Quantitation of number of containing DSBs or active Caspase - 3 in - stained sections 5h post - treatment number of condensated or disaggregated nuclei ( by Hoescht ) 24h post - treatment in sections of 10 high - powder fields ( 400x magnification ) using D software . ( N = 50 ; 10 fields / mice ; 5 mice / group ) . Data expressed as mean ± s . e . m .

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Representative images of CXCR4 overexpression in subcutaneous tumor tissue, showing similar CXCR4 levels among compared groups (N=5/group; Buffer, T22-GFP-H6-FdU, T22-GFP-H6 and free oligo-FdU) before treatment (upper panels). Representative images of DNA double-strand break induction and caspase 3 activation (measured with anti-ɣH2AX or anti-cleaved caspase 3 by IHC) 5h post-administration (middle panels). Apoptotic induction (Hoescht staining, 24h post-administration, lower panels). Note the higher number of cells positive for DSBs, Caspase 3 activation and apoptosis induction in the T22-GFP-H6-FdU as compared to free oligo-FdU. Black or white arrows indicate dead cells. Quantitation of the number of cells containing DSBs or active Caspase-3 in IHC-stained tumor sections 5h post-treatment and the number of condensated or disaggregated nuclei (by Hoescht staining) 24h post-treatment in tumor sections of 10 high-powder fields (400x magnification) using the Cell D software. (N=50; 10 tumor fields/mice; 5 mice/group). Data expressed as mean±s.e.m.


T22 - GFP - H6 - FdU depletes CXCR4 + cancer from SW1417 CRC tissue after a 100 µg single dose administration . Note reduction in CXCR4 + fraction in 24h after injection , their almost complete elimination at 48h re - emergence of CXCR4 + 72h post - administration , using anti - CXCR4 . In contrast , CXCR4 + cancer fraction ( CXCR4 + CCF ) in tissue remains constant along time after free oligo - FdU treatment . three day time - lapse for CXCR4 + re - appearance defines dosage interval used in a repeated dose schedule of nanoconjugate administration in experiments to evaluate its antimetastatic effect . ( N = 5 : 5 mice / group ; 1 samples / mouse ) . Scale bar , 50 µm . Data expressed as mean ± s . e .

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T22-GFP-H6-FdU depletes CXCR4+ cancer cells from SW1417 CRC tumor tissue after a 100 µg single dose administration. Note the reduction in CXCR4+ cell fraction in the tumor 24h after injection, their almost complete elimination at 48h and the re-emergence of CXCR4+ cells 72h post-administration, using anti-CXCR4 IHC. In contrast, the CXCR4+ cancer cell fraction (CXCR4+ CCF) in tumor tissue remains constant along time after free oligo-FdU treatment. The three day time-lapse for CXCR4+ tumor cell re-appearance defines the dosage interval used in a repeated dose schedule of nanoconjugate administration in the experiments to evaluate its antimetastatic effect. (N=5: 5 mice/group; 1 samples/mouse). Scale bar, 50 µm. Data expressed as mean±s.e.m


Significant reduction in number of spheroid formed ( C . optical microscope ) their bioluminescence emission ( D , IVIS Spectrum 200 ) , generated by 1x106 disaggregated ( cultured in conditioned media low - adhesion plates ) , obtained from CXCR4 + luciferase + SW1417 subcutaneous tumors , 24h after 100µg T22 - GFP - H6 - FdU intravenous doses , for 2 consecutive days , as compared to Buffer - treated or free oligo - FdU - treated mice . D . Quantitation of bioluminescent signal ( BLI ) expressed as Average Radiant , obtained using IVIS spectrum 200 equipment ( N = 2 plates / group ) .

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Significant reduction in the number of spheroid formed (C. optical microscope) and their bioluminescence emission (D, IVIS Spectrum 200), generated by 1x106 disaggregated cells (cultured in stem cell conditioned media and low-adhesion plates), obtained from CXCR4+ luciferase+ SW1417 subcutaneous tumors, 24h after 100µg T22-GFP-H6-FdU intravenous doses, for 2 consecutive days, as compared to Buffer-treated or free oligo-FdU-treated mice. D. Quantitation of the bioluminescent signal (BLI) expressed as Average Radiant Intensity, obtained using the IVIS spectrum 200 equipment (N=2 plates/group).


Reduction in number of formed spheroids ( optical microscope ) generated by 1x106 disaggregated ( cultured in conditioned media low - adhesion plates ) obtained from CXCR4 + M5 subcutaneous tumors , 24h after 100µg T22 - GFP - H6 - FdU intravenous doses , for 2 consecutive days , as compared to Buffer - treated or free oligo - FdU - treated mice . ( N = 8 ; 2 mice / group ; 4 plates / mouse ) . ( Scale bar , 100µm ) .

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Reduction in the number of formed spheroids (optical microscope) generated by 1x106 disaggregated cells (cultured in stem cell conditioned media and low-adhesion plates) obtained from CXCR4+ M5 subcutaneous tumors, 24h after 100µg T22-GFP-H6-FdU intravenous doses, for 2 consecutive days, as compared to Buffer-treated or free oligo-FdU-treated mice. (N=8; 2 mice/group; 4 plates/mouse). (Scale bar, 100µm).


Reduction in re - initiation capacity after subcutaneous inoculation of 5x106 in NSG mice ( N = 4 tumors / group ) derived from disaggregated obtained from SC tumors 10 days after administration of 100µg T22 - GFP - H6 - FdU intravenous doses , for 2 consecutive days , as compared to Free oligo - FdU - treated or Buffer - treated mice . Recording of number of positive tumors . ( N = 4 ; 2 mice / group , 2 injection points / mouse ) .

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Reduction in tumor re-initiation capacity after subcutaneous inoculation of 5x106 cells in NSG mice (N=4 tumors/group) derived from disaggregated tumor cells obtained from SC tumors 10 days after administration of 100µg T22-GFP-H6-FdU intravenous doses, for 2 consecutive days , as compared to Free oligo-FdU-treated or Buffer-treated mice. Recording of the number and size of positive tumors. (N=4; 2 mice/group, 2 injection points/mouse).


T22 - GFP - H6 - FdU - induced reduction in number of intravasated emboli in peri - tumoral vessels of M5 - orthotopic ( E . optical ; F . emboli number quantitation ) reduction of CXCR4 expression in these emboli ( G ) , treated 7 days after implantation with 100µg T22 - GFP - H6 - FdU intravenous doses , for 2 consecutive days , as compared to Buffer - treated or free oligo - FdU - treated mice . emboli counting in 10 high powder field at 200x magnification in H & E stained sections from each . ( N = 5 / group ) .

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T22-GFP-H6-FdU-induced reduction in the number of intravasated tumor emboli in peri-tumoral vessels of the M5-orthotopic primary tumor (E. optical images; F. emboli number quantitation) and reduction of CXCR4 expression in these emboli (G), treated 7 days after tumor cell implantation with 100µg T22-GFP-H6-FdU intravenous doses, for 2 consecutive days, as compared to Buffer-treated or free oligo-FdU-treated mice. Tumor emboli counting in 10 high powder field at 200x magnification in H&E stained sections from each tumor. (N=5/group).


T22 - GFP - H6 - FdU prevents metastases in CXCR4 + - derived M5 model by potently reducing total mean number of liver , lung peritoneal mets , as recorded in H & E stained histology sections at end of treatment , in comparison to free oligo - FdU o Buffer - treatment . In contrast , number of LN mets is not reduced after T22 - GFP - H6 - FdU or free oligo - FdU administration ( N = 6 mice per Buffer group ; N = 7 mice per Free oligo - FdU group N = 8 mice per T22 - GFP - H6 - FdU group ; 3 samples / mouse ) . Data expressed as mean ± s . e . m .

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T22-GFP-H6-FdU prevents metastases in the CXCR4+ patient-derived M5 model by potently reducing the total and mean number of liver, lung and peritoneal mets, as recorded in H&E stained histology sections at the end of treatment, in comparison to free oligo-FdU o Buffer-treatment. In contrast, the number of LN mets is not reduced after T22-GFP-H6-FdU or free oligo-FdU administration (N=6 mice per Buffer group; N=7 mice per Free oligo-FdU group and N=8 mice per T22-GFP-H6-FdU group; 3 samples/mouse). Data expressed as mean±s.e.m.


T22 - GFP - H6 - FdU induces a higher reduction of CXCR4 + cancer fraction ( CXCR4 + CCF ) in liver , lung peritoneal metastatic tissue , at end of treatment , than free oligo - FdU , as measured by anti - CXCR4 . In contrast , T22 - GFP - H6 - FdU or free oligo - FdU does not reduce CXCR4 + CCF in LN mets or tissue after therapy . ( N = 6 mice per Buffer group ; N = 7 mice per Free oligo - FdU group N = 8 mice per T22 - GFP - H6 - FdU group ; 3 samples / mouse ) . Data expressed as mean ± s . e .

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T22-GFP-H6-FdU induces a higher reduction of CXCR4+ cancer cell fraction (CXCR4+ CCF) in liver, lung and peritoneal metastatic tissue, at the end of treatment, than free oligo-FdU, as measured by anti-CXCR4 IHC. In contrast, T22-GFP-H6-FdU or free oligo-FdU does not reduce the CXCR4+ CCF in LN mets or primary tumor tissue after therapy. (N=6 mice per Buffer group; N=7 mice per Free oligo-FdU group and N=8 mice per T22-GFP-H6-FdU group; 3 samples/mouse). Data expressed as mean±s.e.m


Representative CXCR4 of reduction in CXCR4 + CCF induced by T22 - GFP - H6 - FdU ( or its absence in free oligo - FdU mice ) at end of treatment , in M5 - derived CRC model , which quantitation is reported in panel B . In M5 model , highest reduction in foci number occurs in liver metastases , which show highest reduction in CXCR4 + CCF .

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Representative CXCR4 IHC images of the reduction in CXCR4+ CCF induced by T22-GFP-H6-FdU (or its absence in free oligo-FdU mice) at the end of treatment, in the M5 patient-derived CRC model, which quantitation is reported in panel B. In the M5 model, the highest reduction in foci number and size occurs in liver metastases, which show the highest reduction in CXCR4+ CCF.


Undetectable T22 - GFP - H6 - FdU emitted in normal tissues , except for a transient accumulation 5h after a 100μg dose in liver , which disappears at 24 h . Liver emitted is transient significantly lower than one registered in tissue . / Liver = 7 . 5 ( see in Fig 2B , which was registered in same experiment ) ( N = 5 mice / group ) . Scale bar , 1 cm .

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Undetectable T22-GFP-H6-FdU emitted fluorescence in normal tissues, except for a transient accumulation 5h after a 100μg dose in the liver, which disappears at 24 h. Liver emitted fluorescence is transient and significantly lower than the one registered in tumor tissue. Tumor/Liver ratio= 7.5 (see tumor intensity in Fig 2B, which was registered in the same experiment) (N=5 mice/group). Scale bar, 1 cm.


Representative depicting level of double strand break ( DSB ) induction in histologically normal bone marrow 5h after treatment , as measured by anti - ɣ - H2AX , which is higher in free oligo - FdU - treated mice than in T22 - GFP - H6 - FdU ( P = 0 . 047 ) . Low level of containing DSBs in histologically normal kidney after T22 - GFP - H6 - FdU or free oligo - FdU treatment , a finding occurring in normal tissues analysed . ( N = 50 , 5 mice / group ; 10 fields / mouse ) .

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Representative images depicting the level of DNA double strand break (DSB) induction in histologically normal bone marrow 5h after treatment, as measured by anti-ɣ-H2AX, which is higher in free oligo-FdU-treated mice than in T22-GFP-H6-FdU (P=0.047). Low level of cells containing DSBs in histologically normal kidney after T22-GFP-H6-FdU or free oligo-FdU treatment, a finding occurring in all normal tissues analysed. (N=50, 5 mice/group; 10 fields/mouse).


Representative showing lack of histopathological alterations in H & E stained tissue or apoptotic induction in H & E stained samples of CXCR4 + ( bone marrow ) CXCR4 - ( brain , kidney , liver , lung heart ) normal tissues 24h after administration of a 100 μg dose of T22 - GFP - H6 - FdU or an equimolecular dose of free oligo - FdU ( N = 5 / group ) . Note that transient nanoconjugate distribution to liver or damage induced in bone marrow do not lead to cytoxicity on these no - tissues . ( N = 50 , 5 mice / group ; 10 fields / mouse )

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Representative images showing lack of histopathological alterations in H&E stained tissue or apoptotic induction in H&E stained samples of CXCR4+ (bone marrow) and CXCR4- (brain, kidney, liver, lung and heart) normal tissues 24h after the administration of a 100 μg dose of T22-GFP-H6-FdU or an equimolecular dose of free oligo-FdU (N=5/group). Note that the transient nanoconjugate distribution to liver or the DNA damage induced in bone marrow do not lead to cytoxicity on these no-tumor tissues. (N=50, 5 mice/group; 10 fields/mouse)


Lack of differences in among groups registered along time in SW1417 - derived CCR model regression of metastases protocol ( N = 10 mice / group ) . Lack of differences in mouse among groups registered along time in SW1417 - derived model prevention of metastasis protocol ( Buffer ( N = 11 ; Free oligo - FdU ( N = 12 ) , T22 - GFP - H6 - FdU ( N = 12 ) ) . Lack of differences in mouse among groups registered along time in M5 - derived model prevention of metastasis protocol ( Buffer ( N = 6 ) ; Free oligo - FdU ( N = 17 ) ; T22 - GFP - H6 - FdU ( N = 8 ) . Scale bar , 100

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Lack of differences in body weight among groups registered along time in the SW1417-derived CCR model and the regression of metastases protocol (N=10 mice/group). Lack of differences in body mouse weight among groups registered along time in the SW1417 cell line-derived model and the prevention of metastasis protocol (Buffer (N=11; Free oligo-FdU (N=12), T22-GFP-H6-FdU (N=12)). Lack of differences in body mouse weight among groups registered along time in the M5 patient-derived model and the prevention of metastasis protocol (Buffer (N=6); Free oligo-FdU (N=17); T22-GFP-H6-FdU (N=8). Scale bar, 100 μm


A . SDS - PAGE gel showing purified holoElongator used as input for gel filtration . bands marked with asterisks presumably correspond to different phosphorylation states of Elp1 that have been previously described [ 34 ] .

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A. SDS-PAGE gel showing the purified holoElongator complex used as input for gel filtration. Protein bands marked with asterisks presumably correspond to different phosphorylation states of Elp1 that have been previously described [34].


B . Representative negative stain - EM field of holoElongator . Particles in side tilted views are highlighted . Scale bar , 50 nm .

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B. Representative negative stain-EM field of the holoElongator complex. Particles in side and tilted views are highlighted. Scale bar, 50 nm.


C . Reference - free class averages back - projections of holoElongator model . Scale bar , 20 nm .

C. Reference-free class averages and back-projections of the holoElongator model. Scale bar, 20 nm.


D . EM reconstruction of holoElongator at 31 Å resolution . E . Fitting of Elp456 crystal structure into ring density of holoElongator reconstruction . F . Fitting of Elp1 CTD into holoElongator reconstruction .

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D. EM reconstruction of the holoElongator complex at 31 Å resolution.E. Fitting of the Elp456 crystal structure into the ring density of the holoElongator reconstruction.F. Fitting of the Elp1 CTD into the holoElongator reconstruction.


A . SDS - PAGE gel showing purified Elp123 sub - used as input for gel filtration . bands marked with asterisks presumably correspond to different phosphorylation states of Elp1 .

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A. SDS-PAGE gel showing the purified Elp123 sub-complex used as input for gel filtration. Protein bands marked with asterisks presumably correspond to different phosphorylation states of Elp1.


B . Representative negative stain - EM field of Elp123 sub - . Particles in side top views are highlighted . Scale bar , 50 nm .

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B. Representative negative stain-EM field of the Elp123 sub-complex. Particles in side and top views are highlighted. Scale bar, 50 nm.


C . Reference - free class averages back - projections of Elp123 model . Scale bar , 20 nm .

C. Reference-free class averages and back-projections of the Elp123 model. Scale bar, 20 nm.


D . EM reconstruction of Elp123 sub - at 27 Å resolution . E . Fitting of Elp1 CTD into Elp123 reconstruction . F . Fitting of Elp2 crystal structure in difference density generated by subtracting partial Elp123 reconstruction from complete Elp123 reconstruction .

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D. EM reconstruction of the Elp123 sub-complex at 27 Å resolution.E. Fitting of the Elp1 CTD into the Elp123 reconstruction.F. Fitting of the Elp2 crystal structure in the difference density generated by subtracting the partial Elp123 reconstruction from the complete Elp123 reconstruction.


A. The model of the Elp123 sub-complex.


B . Crosslinks supporting orientation of Elp3 ( left ) Elp2 ( right ) . crosslinks are shown separately to highlight crosslinks linking from Elp3 Elp2 to other subunits , respectively .

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B. Crosslinks supporting the orientation of Elp3 (left) and Elp2 (right). The crosslinks are shown separately to highlight crosslinks linking from Elp3 and Elp2 to other subunits, respectively.


D . Crosslinks supporting orientation of Elp456 hexamer .

D. Crosslinks supporting the orientation of the Elp456 hexamer.


A . GST - pull down assays of purified GST - tagged Elp3 SAM domain ( aa72 - 389 ) KAT domain ( aa348 - 557 ) with untagged full Elp1 Elp451 - 2706 . GSH resin were used as input controls . Lower panel shows 5 % of input upper panel shows bound fractions . samples were analyzed by SDS - PAGE stained by Coomassie . Identities of respective are indicated on right .

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A. GST-pull down assays of purified GST-tagged Elp3 SAM domain (aa72-389) and KAT domain (aa348-557) with untagged full length Elp1 and Elp451-2706. GSH resin and proteins were used as input controls. Lower panel shows 5% of the input and upper panel shows bound fractions. All samples were analyzed by SDS-PAGE and stained by Coomassie. Identities of respective proteins are indicated on the right.


B . same as A , but using only GST - tagged Elp3 SAM domain with combinations variants of Elp1 , Elp2 , Elp456 .

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B. same as A, but using only GST-tagged Elp3 SAM domain with combinations and variants of Elp1, Elp2, and Elp456.


C . same as A using GST - tagged full Elp1 truncations deletions of Elp456 .

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C. same as A using GST-tagged full length Elp1 and truncations and deletions of Elp456.


D . same as A using GST - tagged C - terminally truncated Elp1 in combination with truncated versions of Elp4 .

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D. same as A using GST-tagged C-terminally truncated Elp1 in combination with truncated versions of Elp4.


B . Same as A but tilted view to highlight active site cavity ( left ) . Close up view of active site in which known tRNA binding sites a region of Elp2 important for tRNA modification activity are highlighted . structures of subunits are shown in cartoon surface representation .

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B. Same as A but tilted view to highlight the active site cavity (left). Close up view of the active site in which all known tRNA binding sites and a region of Elp2 important for tRNA modification activity are highlighted. Individual structures of the subunits are shown in cartoon and surface representation.


C . Close up view on localization of Elp3 orientation of its KAT SAM domain in relation to Elp1 Elp2 .

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C. Close up view on the localization of Elp3 and the orientation of its KAT and SAM domain in relation to Elp1 and Elp2.


D . Close up view on relative spatial orientation of conserved basic patch in Elp2 potential interaction site between Elp456 a region of Elp2 important for tRNA modification activity .

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D. Close up view on the relative spatial orientation of the conserved basic patch in Elp2 and the potential interaction site between Elp456 and a region of Elp2 important for tRNA modification activity.


A . Representative pseudocolor of PC6 transfected with EYFP - Parkin ( green ) mitochondrially targeted CFP ( red ) with without PINK1 Miro1 shRNA Miro2 shRNA . Zoomed show translocation of EYFP - Parkin to mitochondria in PINK1 - overexpressing PC6

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A. Representative pseudocolor images of PC6 cells transfected with EYFP-Parkin (green) and mitochondrially targeted CFP (red) with and without PINK1 and Miro1 shRNA and Miro2 shRNA. Zoomed images show translocation of EYFP-Parkin to mitochondria in PINK1-overexpressing PC6 cells


B . of EYFP - Parkin translocation to mitochondria . of PC6 with EYFP - Parkin translocated to mitochondria in response to PINK1 overexpression is lower in Miro shRNAs - expressing groups when compared with scrambled shRNA - expressing group ( scr shRNA ) . * * * * P < 0 . 0001 , n = 6 dishes per group , 20 fields per dish , one - way

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B. Quantification of EYFP-Parkin translocation to mitochondria. The percentage of PC6 cells with EYFP-Parkin translocated to mitochondria in response to PINK1 overexpression is lower in the Miro shRNAs-expressing groups when compared with the scrambled shRNA-expressing group (scr shRNA). ****P < 0.0001, n = 6 dishes per group, 20 fields per dish, one-way ANOVA


C . kinetics of EYFP - Parkin translocation is different in PC6 transfected with scrambled or Miro shRNAs . were plated in separate compartments of same dish , enabling us to visualize Parkin translocation simultaneously over 3 h under similar conditions in response to antimycin with oligomycin treatment ( A & O , both 10 µM ) . spatial heterogeneity of EYFP - Parkin ( coefficient of variation of of pixels ) was estimated for for each time point ( n = 39 - 47 ; P < 0 . 0001 difference between curves P < 0 . 0001 , two - way ANOVA ; lines show mean , dashed shows )

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C. The kinetics of EYFP-Parkin translocation is different in PC6 cells transfected with scrambled or Miro shRNAs. Cells were plated in separate compartments of the same dish, enabling us to visualize Parkin translocation simultaneously over 3 h under similar conditions in response to antimycin with oligomycin treatment (A&O, both 10 µM). The spatial heterogeneity of EYFP-Parkin (coefficient of variation of the intensity of individual pixels) was estimated for individual cells for each time point (n = 39-47 cells; P < 0.0001 difference between the curves P < 0.0001, two-way ANOVA; lines show mean, dashed area shows SEM)


D . Miro shRNAs mitigate effect of A & O on EYFP - Parkin translocation . PC6 transfected with EYFP - Parkin with without Miro shRNAs were treated with DMSO or A & O ( both 10 µM ) for 3h . figure demonstrates that of with Parkin translocated to mitochondria was lower in Miro shRNAs - expressing group when compared with scrambled shRNA - expressing group . * * * * P < 0 . 0001 , n = 4 dishes , 20 fields per dish , one - way

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D. Miro shRNAs mitigate the effect of A&O on EYFP-Parkin translocation. PC6 cells transfected with EYFP-Parkin with and without Miro shRNAs were treated with DMSO or A&O (both 10 µM) for 3h. The figure demonstrates that the percentage of cells with Parkin translocated to mitochondria was lower in the Miro shRNAs-expressing group when compared with the scrambled shRNA-expressing group. ****P < 0.0001, n = 4 dishes, 20 fields per dish, one-way ANOVA


E . Miro shRNAs inhibit PINK1 - induced mitophagy in PC6 . figure shows changes in excitation spectra of mitochondrially targeted pH - sensitive Keima ( of 561 nm / 458nm intensities ) . * * * * P < 0 . 0001 , n = 80 from 4 dishes , Kruskal - Wallis

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E. Miro shRNAs inhibit PINK1-induced mitophagy in PC6 cells. The figure shows the changes in the excitation spectra of mitochondrially targeted pH-sensitive Keima (ratio of 561 nm/458nm fluorescence intensities). ****P < 0.0001, n = 80 cells from 4 dishes, Kruskal-Wallis test


F . Miro shRNAs inhibit basal mitophagy in cortical neurons expressing mitochondrially targeted Keima . Mitochondria in lysosomes ( acidic pH ) were counted manually , by a blinded observer ( * * P < 0 . 01 , n = 9 dishes , 9 fields per dish , from 2 independent experiments , one - way ANOVA )

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F. Miro shRNAs inhibit basal mitophagy in primary cortical neurons expressing mitochondrially targeted Keima. Mitochondria in lysosomes (acidic pH) were counted manually, by a blinded observer (**P < 0.01, n = 9 dishes, 9 fields per dish, from 2 independent experiments, one-way ANOVA)


G . Miro shRNAs decrease number of mitochondria co - localised with autophagosome marker EGFP - LC3B in cortical neurons ( * * P < 0 . 01 , n = 5 dishes , 9 fields per dish , one - way ANOVA ) .

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G. Miro shRNAs decrease the number of mitochondria co-localised with the autophagosome marker EGFP-LC3B in primary cortical neurons (**P < 0.01, n = 5 dishes, 9 fields per dish, one-way ANOVA).


A . pattern of Parkin translocation caused by Miro1 overexpression is different from A & O - induced translocation . figure illustrates - like EYFP - Parkin on mitochondria in antimycin oligomycin - treated ( both 10 µM for 3h ) PC6 ( middle panels ) , whereas in Miro1 - overexpressing , EYFP - Parkin appears along rod - shaped mitochondria visualised with CFP - mito ( lower panels ) . merged panels present EYFP - Parkin ( green ) CFP - mito ( red )

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A. The pattern of Parkin translocation caused by Miro1 overexpression is different from A&O-induced translocation. The figure illustrates puncta-like EYFP-Parkin on mitochondria in antimycin and oligomycin-treated (both 10 µM for 3h) PC6 cells (middle panels), whereas in Miro1-overexpressing cells, EYFP-Parkin appears along rod-shaped mitochondria visualised with CFP-mito (lower panels). The merged panels present EYFP-Parkin (green) and CFP-mito (red)


B . Overexpression of myc - Miro1 untagged Miro2 , but not myc - Miro2 , induce EYFP - Parkin translocation to mitochondria . * * * * P < 0 . 0001 vs control group , n = 5 - 6 dishes , 20 fields per dish , one - way ANOVA or t -

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B. Overexpression of myc-Miro1 and untagged Miro2, but not myc-Miro2, induce EYFP-Parkin translocation to mitochondria. ****P < 0.0001 vs control group, n = 5-6 dishes, 20 fields per dish, one-way ANOVA or t-test


C . of EYFP - Parkin signal heterogeneity in PC6 demonstrates that Miro1 overexpression induces Parkin translocation from cytosol to mitochondria , which is significantly weaker than PINK1 overexpression - or A & O ( 3 h ) - induced translocation . * * P < 0 . 01 * * * * P < 0 . 0001 vs control group , n = 18 - 32 from 3 dishes per group , Kruskal - Wallis

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C. Quantification of EYFP-Parkin signal heterogeneity in PC6 cells demonstrates that Miro1 overexpression induces Parkin translocation from the cytosol to mitochondria, which is significantly weaker than PINK1 overexpression- or A&O (3 h)-induced translocation. **P < 0.01 and ****P < 0.0001 vs control group, n = 18-32 cells from 3 dishes per group, Kruskal-Wallis test


Miro1 overexpression does not cause mitochondrial depolarization ( D ) Representative image of Miro1 EYFP - Parkin - transfected stained with TMRE . merged panels present EYFP - Parkin ( green ) TMRE ( red )

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Miro1 overexpression does not cause mitochondrial depolarization (D) Representative image of Miro1 and EYFP-Parkin-transfected cells stained with TMRE. The merged panels present EYFP-Parkin (green) and TMRE (red)


Miro1 overexpression does not cause mitochondrial depolarization ( E ) Relative TMRE quantified in neurons transfected with Miro1 ( detected by CFP - mito co - transfection ) ; control group is non - transfected in same image . relative TMRE signal was slightly increased in Miro1 overexpressing neurons but unaffected in Miro shRNAs - expressing neurons . Note that FCCP treatment ( 3 μM for 15 min ) almost completely abolished TMRE . * * * * P < 0 . 0001 vs control group , n = 94 - 186 from 4 dishes per group , Kruskal - Wallis

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Miro1 overexpression does not cause mitochondrial depolarization (E) Relative TMRE intensity quantified in neurons transfected with Miro1 (detected by CFP-mito co-transfection); the control group is non-transfected cells in the same image. The relative TMRE signal was slightly increased in Miro1 overexpressing neurons but unaffected in Miro shRNAs-expressing neurons. Note that FCCP treatment (3 μM for 15 min) almost completely abolished TMRE fluorescence. ****P < 0.0001 vs control group, n = 94-186 cells from 4 dishes per group, Kruskal-Wallis test


F . Co - expression of HA - Parkin with Miro1 does not suppress mitochondrial motility in axons of cortical neurons . Overexpression of Miro1 alone increased motion time in both directions as well as relative mitochondrial velocity . Co - transfection of Parkin did not affect significantly motility parameters . Data are presented as Tukey boxplot . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 * * * * P < 0 . 0001 , n = 689 - 846 mitochondria from 39 - 44 axons per group pooled from at least from 10 dishes from 4 independent experiments , Kruskal - Wallis

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F. Co-expression of HA-Parkin with Miro1 does not suppress mitochondrial motility in axons of cortical neurons. Overexpression of Miro1 alone increased the motion time in both directions as well as the relative mitochondrial velocity. Co-transfection of Parkin did not affect significantly the motility parameters. Data are presented as Tukey boxplot. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001, n = 689-846 individual mitochondria from 39-44 axons per group pooled from at least from 10 individual dishes from 4 independent experiments, Kruskal-Wallis test


G . Parkin overexpression does not induce degradation of myc - Miro1 in control conditions . Representative Western blot image of myc - Miro1 expression in HEK . Treatment with A & O ( both 15 µM ) for 3h in absence of MG132 led to slight degradation of myc - Miro1 . Co - expression of EYFP - Parkin did not induce degradation of Miro1 although an additional band above Miro1 band was present . However , Parkin co - expression enhanced Miro1 degradation when were treated with A & O . * P < 0 . 05 * * * * P < 0 . 0001 , ns : not significant , n = 4 independent experiments , one way ANOVA . Note that only main band of myc - Miro1 at 80 kDa was analysed that YFP was used to compensate in groups not expressing EYFP -

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G. Parkin overexpression does not induce degradation of myc-Miro1 in control conditions. Representative Western blot image and analysis of myc-Miro1 expression in HEK cells. Treatment with A&O (both 15 µM) for 3h in the absence of MG132 led to slight degradation of myc-Miro1. Co-expression of EYFP-Parkin did not induce degradation of Miro1 although an additional band above the Miro1 band was present. However, Parkin co-expression enhanced Miro1 degradation when cells were treated with A&O. *P < 0.05 and ****P < 0.0001, ns: not significant, n = 4 independent experiments, one way ANOVA. Note that only the main band of myc-Miro1 at 80 kDa was analysed and that YFP was used to compensate in groups not expressing EYFP-Parkin


H . EYFP - Parkin co - immunoprecipitates with overexpressed myc - Miro1 , both in DMSO - or A & O ( both 15 µM for 3h ) treated HEK

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H. EYFP-Parkin co-immunoprecipitates with overexpressed myc-Miro1, both in DMSO- or A&O (both 15 µM for 3h) treated HEK cells


I . Endogenous Parkin co - immunoprecipitates with endogenous Miro1 . Miro1 was immunoprecipitated from non - transfected non - treated HEK using mouse monoclonal anti - Miro1 antibody immunoblotted for endogenous Parkin .

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I. Endogenous Parkin co-immunoprecipitates with endogenous Miro1. Miro1 was immunoprecipitated from non-transfected and non-treated HEK cells using mouse monoclonal anti-Miro1 antibody and immunoblotted for endogenous Parkin.


A . Miro1 induces EYFP - Parkin translocation in presence of PINK1 shRNA . PC6 were transfected with EYFP - Parkin Miro1 or treated for 3h with A & O ( both 10 µM ) in presence of scrambled shRNA or PINK1 shRNA . Miro1 induced Parkin translocation also in presence of PINK1 shRNA while A & O treatment did not . * * * * P < 0 . 0001 compared with respective control group , n = 5 - 6 dishes , 20 fields per dish , one - way

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A. Miro1 induces EYFP-Parkin translocation in the presence of PINK1 shRNA. PC6 cells were transfected with EYFP-Parkin and Miro1 or treated for 3h with A&O (both 10 µM) in the presence of scrambled shRNA or PINK1 shRNA. Miro1 induced Parkin translocation also in the presence of PINK1 shRNA while A&O treatment did not. ****P < 0.0001 compared with the respective control group, n = 5-6 dishes, 20 fields per dish, one-way ANOVA


B . Miro1 induces EYFP - Parkin translocation in PINK1 - / - MEFs . MEFs obtained from PINK1 + / + - / - mice were transfected with EYFP - Parkin Miro1 or treated with A & O ( both 10 µM for 3h ) . Miro1 induced Parkin translocation in both MEF lines but A & O only in PINK1 + / + MEFs . * * * * P < 0 . 0001 compared with respective control group , n = 6 dishes , one - way

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B. Miro1 induces EYFP-Parkin translocation in PINK1 -/- MEFs. MEFs obtained from PINK1 +/+ and -/- mice were transfected with EYFP-Parkin and Miro1 or treated with A&O (both 10 µM for 3h). Miro1 induced Parkin translocation in both MEF lines but A&O only in PINK1 +/+ MEFs. ****P < 0.0001 compared with respective control group, n = 6 dishes, one-way ANOVA


C . Mutated EYFP - Parkin is recruited to mitochondria in response to Miro1 overexpression . PC6 were transfected with wild type , T240R or C431N EYFP - Parkin Miro1 or treated for 3 hours with A & O ( both 10 µM ) . Miro1 induced translocation of EYFP - Parkin mutants whereas A & O treatment was not able to induce equivalent translocation of Parkin mutants . * * P < 0 . 01 * * * * P < 0 . 0001 compared with respective control group , n = 3 - 6 dishes , 20 fields per dish , one - way

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C. Mutated EYFP-Parkin is recruited to mitochondria in response to Miro1 overexpression. PC6 cells were transfected with wild type, T240R or C431N EYFP-Parkin and Miro1 or treated for 3 hours with A&O (both 10 µM). Miro1 induced translocation of all EYFP-Parkin mutants whereas A&O treatment was not able to induce equivalent translocation of Parkin mutants. **P < 0.01 and ****P < 0.0001 compared with respective control group, n = 3-6 dishes, 20 fields per dish, one-way ANOVA


D . Parkin mutants T240R C431N interact with Miro1 both in DMSO - or A & O - treated ( both 15 µM for 3h ) HEK . Note that higher molecular bands of Miro1 were observed only in WT Parkin - expressing groups .

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D. Parkin mutants T240R and C431N interact with Miro1 both in DMSO- or A&O-treated (both 15 µM for 3h) HEK cells. Note that the higher molecular weight bands of Miro1 were observed only in WT Parkin-expressing groups.


A . Miro1 K572R mutant interacts with HA - Parkin similarly to Miro1 WT . HEK overexpressing myc - Miro1 WT or K572R mutant were treated with DMSO ( control ) or A & O ( both 15 µM ) for 3 hrs . Myc - Miro1 was immunoprecipitated probed for myc - Miro1 HA - Parkin . Note diminished degradation of Miro1 K572R in input additional band above Miro1 WT in IP , which is missing in case of K572R

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A. Miro1 K572R mutant interacts with HA-Parkin similarly to Miro1 WT. HEK cells overexpressing myc-Miro1 WT or K572R mutant were treated with DMSO (control) or A&O (both 15 µM) for 3 hrs. Myc-Miro1 was immunoprecipitated and probed for myc-Miro1 and HA-Parkin. Note the diminished degradation of Miro1 K572R in input and the additional band above Miro1 WT in the IP, which is missing in the case of the K572R mutant


B . Miro1 K572R ubiquitination by Parkin is reduced . HEK overexpressing HA - ubiquitin , myc - Miro1 or myc - Miro1 K572R EYFP - Parkin were treated with DMSO ( control ) or A & O ( both 15 µM ) for 3 hrs . HA - ubiquitin was immunoprecipitated probes were immunoblotted for ubiquitin - HA myc -

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B. Miro1 K572R ubiquitination by Parkin is reduced. HEK cells overexpressing HA-ubiquitin, myc-Miro1 or myc-Miro1 K572R and EYFP-Parkin were treated with DMSO (control) or A&O (both 15 µM) for 3 hrs. HA-ubiquitin was immunoprecipitated and probes were immunoblotted for ubiquitin-HA and myc-Miro1


C . Representative of PC6 overexpressing Miro1 K572R or Miro1 WT together with EYFP - Parkin . K572R mutant induces Parkin translocation similar to WT in basal conditions but not after treatment with A & O ( both 7 . 5 µM ) for 3 hours . Note spatial heterogeneity values shown in

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C. Representative images of PC6 cells overexpressing Miro1 K572R or Miro1 WT together with EYFP-Parkin. K572R mutant induces Parkin translocation similar to WT in basal conditions but not after treatment with A&O (both 7.5 µM) for 3 hours. Note the spatial heterogeneity values shown in the images


D . of EYFP - Parkin signal heterogeneity in PC6 demonstrates that WT Miro1 overexpression amplifies Parkin translocation in response A & O ( both 7 . 5 µM ) treatment for 3 hours , but K572R mutation of Miro1 abolishes this effect . * * * P < 0 . 001 * * * * P < 0 . 0001 , n = 152 - 172 from 3 dishes per group , Kruskal - Wallis . Data are presented as means ± .

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D. Quantification of EYFP-Parkin signal heterogeneity in PC6 cells demonstrates that WT Miro1 overexpression amplifies Parkin translocation in response A&O (both 7.5 µM) treatment for 3 hours, but K572R mutation of Miro1 abolishes this effect. ***P < 0.001 and ****P < 0.0001, n = 152-172 cells from 3 dishes per group, Kruskal-Wallis test. Data are presented as means ± SEM.


A . A Ca2 + - insensitive mutant of Miro1 ( Miro1 - EF ) interacts with Parkin . HEK overexpressing myc - Miro1 or myc - Miro1 - EF EYFP - Parkin were treated with DMSO ( control ) or A & O ( both 15 µM ) for 3 hrs . Myc - Miro1 was immunoprecipitated probes were immunoblotted for Miro1 ( red ) Parkin ( green ) . high - contrast grayscale image from green channel of this blot shows that endogenous Parkin co - immunoprecipitated with Miro1 in conditions . bar chart shows of co - immunoprecipitated Parkin EYFP from 4 different blots normalised to total EYFP - Parkin signal ( * P < 0 . 05 * * P < 0 . 01 , repeated measures

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A. A Ca2+-insensitive mutant of Miro1 (Miro1-EF) interacts with Parkin. HEK cells overexpressing myc-Miro1 or myc-Miro1-EF and EYFP-Parkin were treated with DMSO (control) or A&O (both 15 µM) for 3 hrs. Myc-Miro1 was immunoprecipitated and probes were immunoblotted for Miro1 (red) and Parkin (green). The high-contrast grayscale image from the green channel of this blot shows that endogenous Parkin co-immunoprecipitated with Miro1 in all conditions. The bar chart shows the quantification of co-immunoprecipitated Parkin EYFP from 4 different blots normalised to total EYFP-Parkin signal (*P < 0.05 and **P < 0.01, repeated measures ANOVA


B . Miro1 - EF is less susceptible to ubiquitination by Parkin . HEK overexpressing HA - ubiquitin , myc - Miro1 or myc - Miro1 - EF EYFP - Parkin were treated with DMSO ( control ) or A & O ( both 15 µM ) for 3 hrs . HA - ubiquitin was immunoprecipitated probes were immunoblotted for ubiquitin ( red ) , Miro1 ( green ) Parkin ( green ; representative are from same experimental material on separate SDS - PAGE - WB membranes ) . high - contrast grayscale shows green channel of this blot , bar chart shows of co - immunoprecipitated Miro from 3 different blots ( * * P < 0 . 01 * * * P < 0 . 001 , repeated measures ANOVA ) . Note that EYFP - Parkin co - immunoprecipitated with ubiquitin only after A & O

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B. Miro1-EF is less susceptible to ubiquitination by Parkin. HEK cells overexpressing HA-ubiquitin, myc-Miro1 or myc-Miro1-EF and EYFP-Parkin were treated with DMSO (control) or A&O (both 15 µM) for 3 hrs. HA-ubiquitin was immunoprecipitated and probes were immunoblotted for ubiquitin (red), Miro1 (green) and Parkin (green; representative images are from the same experimental material on separate SDS-PAGE-WB membranes). The high-contrast grayscale shows the green channel of this blot, and the bar chart shows quantification of co-immunoprecipitated Miro from 3 different blots (**P < 0.01 and ***P < 0.001, repeated measures ANOVA). Note that EYFP-Parkin co-immunoprecipitated with ubiquitin only after A&O treatment


C . Mutation of Miro1 EF - hands diminishes Miro1 ubiquitination with S65 - phosphorylated ubiquitin . HEK were transfected with EYFP - Parkin with either myc - Miro1 - WT or myc - Miro1 - EF with or without PINK1 - V5 . were treated with either DMSO or A & O ( both 10 µM ) for 5 hrs with exception of PINK1 - V5 group . Myc - Miro1 was immunoprecipitated probes were immunoblotted for Miro1 ( red ) , S65 - Phosphoubiquitin PINK1 - V5 ( green ; weak bands marked with asterisk are nonspecific ) .

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C. Mutation of Miro1 EF-hands diminishes Miro1 ubiquitination with S65-phosphorylated ubiquitin. HEK cells were transfected with EYFP-Parkin with either myc-Miro1-WT or myc-Miro1-EF with or without PINK1-V5. Cells were treated with either DMSO or A&O (both 10 µM) for 5 hrs with the exception of the PINK1-V5 group. Myc-Miro1 was immunoprecipitated and probes were immunoblotted for Miro1 (red), S65-Phosphoubiquitin and PINK1-V5 (green; the weak bands marked with the asterisk are nonspecific).


A . Kinetics of EYFP - Parkin translocation in response to A & O is different in control Miro1 - EF - expressing PC6 . inset shows how different compartments of same dish were transfected with EYFP - Parkin control plasmid or Miro1 - EF followed over 3 hrs using a confocal microscope after addition of A & O ( both 10 µM ) . graph shows average spatial heterogeneity of EYFP - Parkin , reflecting of mitochondrial - to - Parkin over time ( n = 40 - 43 , difference between curves P < 0 . 0001 , two - way ANOVA ; lines show mean , dashed shows )

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A. Kinetics of EYFP-Parkin translocation in response to A&O is different in control and Miro1-EF-expressing PC6 cells. The inset shows how different compartments of the same dish were transfected with EYFP-Parkin and control plasmid or Miro1-EF and followed over 3 hrs using a confocal microscope after addition of A&O (both 10 µM). The graph shows the average spatial heterogeneity of EYFP-Parkin fluorescence, reflecting the ratio of mitochondrial-to-cytosolic Parkin over time (n = 40-43 cells, difference between the curves P < 0.0001, two-way ANOVA; lines show mean, dashed area shows SEM)


of where EYFP - Parkin translocates in response to A & O ( both 10 µM , B is decreased in Miro1 - EF - expressing PC6 . * * P < 0 . 01 , * * * * P < 0 . 0001 compared with indicated group ( n = 3 - 6 dishes , 20 fields per dish , one way ANOVA

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The percentage of cells where EYFP-Parkin translocates in response to A&O (both 10 µM, B is decreased in Miro1-EF-expressing PC6 cells. **P < 0.01, ****P < 0.0001 compared with the indicated group (n = 3-6 dishes, 20 fields per dish, one way ANOVA for


of where EYFP - Parkin translocates in response t PINK1 overexpression ( C ) is decreased in Miro1 - EF - expressing PC6 . * * P < 0 . 01 , * * * * P < 0 . 0001 compared with indicated group ( n = 3 - 6 dishes , 20 fields per dis Welch " s ANOVA

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The percentage of cells where EYFP-Parkin translocates in response t PINK1 overexpression (C) is decreased in Miro1-EF-expressing PC6 cells. **P < 0.01, ****P < 0.0001 compared with the indicated group (n = 3-6 dishes, 20 fields per dis Welch"s ANOVA f


D . Time course showing - induced EYFP - Parkin accumulation in irradiated of neuronal soma of rat cortical neurons . were transfected with CFP - mito EYFP - Parkin visualized using a confocal microscope . Mitochondria were irradiated in a 0 . 9 µm x 0 . 9 µm - frame using a 405 - nm ( 25 iterations using 100 % power , pixel time 164 µs )

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D. Time course showing laser-induced EYFP-Parkin accumulation in the irradiated area of the neuronal soma of primary rat cortical neurons. Cells were transfected with CFP-mito and EYFP-Parkin and visualized using a confocal microscope. Mitochondria were irradiated in a 0.9 µm x 0.9 µm-frame using a 405-nm laser line (25 iterations using 100% laser power, pixel time 164 µs)


E , F Miro1 - EF delays reduces irradiation - induced EYFP - Parkin translocation . ( E ) time course of translocation strength in control Miro1 - EF - expressing neurons . Difference between curves P = 0 . 0016 , two - way ANOVA . ( F ) Comparison at 60 - min time point ( * P < 0 . 05 , n = 6 dishes , at least 9 neurons per dish , t - ) .

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E, F Miro1-EF delays and reduces irradiation-induced EYFP-Parkin translocation. (E) The time course of translocation strength in control and Miro1-EF-expressing neurons. Difference between the curves P = 0.0016, two-way ANOVA. (F) Comparison at the 60-min time point (*P < 0.05, n = 6 dishes, at least 9 neurons per dish, t-test).


A . EYFP - Parkin translocation requires Ca2 + . PC6 plated in separated compartments of same dish maintained during experiment in Krebs containing 1 mM Ca2 + or in Ca2 + free Krebs containing 5 μM BAPTA - AM , were treated with A & O ( both 10 μM ) . ( left panels ) demonstrate that EYFP - Parkin translocation was significantly weaker at zero Ca2 + at 195 min after A & O treatment ( compared with 15 min ; note spatial heterogeneity values shown in photos ) . Spatial heterogeneity ( right panel ) demonstrated that Parkin translocation was significantly decelerated in zero Ca2 + conditions ( n = 25 - 27 ; difference between curves P < 0 . 0001 , two - way ANOVA ; lines show mean , dashed shows )

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A. EYFP-Parkin translocation requires Ca2+. PC6 cells plated in separated compartments of the same dish maintained during the experiment in Krebs containing 1 mM Ca2+ or in Ca2+ free Krebs containing 5 μM BAPTA-AM, were treated with A&O (both 10 μM). Images (left panels) demonstrate that EYFP-Parkin translocation was significantly weaker at zero Ca2+ at 195 min after A&O treatment (compared with 15 min; note the spatial heterogeneity values shown in photos). Spatial heterogeneity analysis (right panel) demonstrated that Parkin translocation was significantly decelerated in zero Ca2+ conditions (n = 25-27 cells; difference between the curves P < 0.0001, two-way ANOVA; lines show mean, dashed area shows SEM)


B . EYFP - Parkin translocation accelerates in presence of L - type Ca2 + channel agonist Bay K 8644 ( 5 μM ; n = 19 - 21 ; difference between curves p < 0 . 0001 , two - way ANOVA ; lines show mean , dashed shows )

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B. EYFP-Parkin translocation accelerates in the presence of the L-type Ca2+ channel agonist Bay K 8644 (5 μM; n = 19-21 cells; difference between the curves p < 0.0001, two-way ANOVA; lines show mean, dashed area shows SEM)


Treatment of neurons for 10 min with 100 μM glutamate / 10 μM glycine led to excessive Ca2 + entry into cytosol , visualised by increased GCaMP7b

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Treatment of neurons for 10 min with 100 μM glutamate/10 μM glycine led to excessive Ca2+ entry into the cytosol, visualised by increased cytosolic GCaMP7b fluorescence


Treatment of neurons for 10 min with 100 μM glutamate / 10 μM glycine led to excessive Ca2 + entry into cytosol Glutamate - induced Ca2 + entry was not affected by Miro1 WT or Miro1 EF overexpression ; n = 31 - 48 neurons per group ; ns , Kruskal - Wallis )

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Treatment of neurons for 10 min with 100 μM glutamate/10 μM glycine led to excessive Ca2+ entry into the cytosol Glutamate-induced Ca2+ entry was not affected by Miro1 WT or Miro1 EF overexpression; n = 31-48 neurons per group; ns, Kruskal-Wallis test)


E , F Glutamate treatment ( 100 μM glutamate / 10 μM glycine for 10 min ) led to appearance of a few Parkin - positive strong co - localising with mitochondrial marker ( arrow ) , imaged 2 . 5 h after treatment ( E ) . Further demonstrated that expression of Miro1 - EF mutant decreased Miro1 WT increased number of neurons with EYFP - Parkin - positive mitochondria following glutamate treatment ( F ; n = 6 - 10 ; * * P < 0 . 01 * * * * P < 0 . 0001 , one - way ANOVA )

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E, F Glutamate treatment (100 μM glutamate/10 μM glycine for 10 min) led to the appearance of a few Parkin-positive strong puncta co-localising with mitochondrial marker (white arrow), imaged 2.5 h after treatment (E). Further analysis demonstrated that the expression of Miro1-EF mutant decreased and Miro1 WT increased the number of neurons with EYFP-Parkin-positive mitochondria following glutamate treatment (F; n = 6-10; **P < 0.01 and ****P < 0.0001, one-way ANOVA)


G . Glutamate treatment ( 100 μM glutamate / 10 μM glycine for 10 min ) was associated with an increased number of mitochondrial co - localisation with autophagosome marker EGFP - LC3B in neuronal soma at 5 - 6 h later , which was not observed in Miro1 - EF - expressing neurons ( n = 8 dishes ; * P < 0 . 05 , one - way ANOVA )

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G. Glutamate treatment (100 μM glutamate/10 μM glycine for 10 min) was associated with an increased number of mitochondrial co-localisation with the autophagosome marker EGFP-LC3B in neuronal soma at 5-6 h later, which was not observed in Miro1-EF-expressing neurons (n = 8 dishes; *P < 0.05, one-way ANOVA)


H . Glutamate treatment ( 30 μM glutamate 1 μM glycine for 10 min ) was also associated with a significant loss of mitochondrial density in axons at 6 h later , which was reversed by Miro1 - EF but not by Miro1 WT ( n = 60 - 90 axons from 6 - 9 dishes ; * * P < 0 . 01 , * * * P < 0 . 001 * * * * P < 0 . 0001 , Kruskal - Wallis )

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H. Glutamate treatment (30 μM glutamate and 1 μM glycine for 10 min) was also associated with a significant loss of mitochondrial density in axons at 6 h later, which was reversed by Miro1-EF but not by Miro1 WT (n = 60-90 axons from 6-9 dishes; **P < 0.01, ***P < 0.001 and ****P < 0.0001, Kruskal-Wallis test)


I . Glutamate treatment ( 100 μM glutamate 10 μM glycine for 10 min ) led to a decline , at 6 - 8 h , in ATP measured using ATP / ADP sensor Perceval HR , which was reversed by Miro1 - EF but not by Miro1 WT ( n = 59 - 60 neurons from 6 dishes ; * * P < 0 . 01 , Welch " s

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I. Glutamate treatment (100 μM glutamate and 10 μM glycine for 10 min) led to a decline, at 6-8 h, in cytosolic ATP measured using ATP/ADP sensor Perceval HR, which was reversed by Miro1-EF but not by Miro1 WT (n = 59-60 neurons from 6 dishes; **P < 0.01, Welch"s A


J . Glutamate ( 100 μM glutamate 10 μM glycine for 10 min ) induced excitotoxicity ( 24 h after treatment ) that was partially reversed by Miro1 - EF but not by Miro1 WT . Data is presented as a of control neuron ( n = 10 dishes ; * * * P < 0 . 001 , repeated measures ANOVA ) .

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J. Glutamate (100 μM glutamate and 10 μM glycine for 10 min) induced excitotoxicity (24 h after treatment) that was partially reversed by Miro1-EF but not by Miro1 WT. Data is presented as a percentage of control neuron count (n = 10 dishes; ***P < 0.001, repeated measures ANOVA).


A microfluidic device for quantitative recovery time measurements . Each PDMS - fabricated chip consists of six independent channels , one of which is shown , from top view ( top row ) . Green circles indicate media inflow ; black circle indicates outflow . are manually loaded in inflow ports are trapped in culturing chambers ( gray circles ) , height of which ensures that are imaged in a monolayer ( bottom row ) . Growth conditions ( e . g . antibiotic dose profile ) are controlled via programmable syringe pumps . ( bottom row ) show representative growth of a monolayer of E . coli BW25113 constitutively expressing GFP over four hours .

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A microfluidic device for quantitative recovery time measurements. Each PDMS-fabricated chip consists of six independent channels, one of which is shown, from top view (top row). Green circles indicate media inflow; black circle indicates outflow. Bacteria are manually loaded in inflow ports and are trapped in individual culturing chambers (gray circles), the height of which ensures that bacteria are imaged in a monolayer (bottom row). Growth conditions (e.g. antibiotic dose profile) are controlled via programmable syringe pumps. Fluorescent images (bottom row) show representative growth of a monolayer of E. coli BW25113 cells constitutively expressing GFP over four hours.


Representative time series data showing dose - dependent population recovery in response to transient streptomycin treatment . Here , time zero corresponds to end of treatment ( 120 minutes ) . Trajectories show mean standard deviation for five replicates ; colors indicate increasing antibiotic ( 2 , 4 , 6 , 8 , 10 , 12 μg / mL ) . values are normalized to those at time zero . Dotted indicates a 2 - fold increase , corresponding to recovery time for each population .

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Representative time series fluorescence data showing dose-dependent population recovery in response to transient streptomycin treatment. Here, time zero corresponds to the end of treatment (120 minutes). Trajectories show mean and standard deviation for five replicates; colors indicate increasing antibiotic concentration (2, 4, 6, 8, 10, 12 μg/mL). Fluorescence values are normalized to those at time zero. Dotted line indicates a 2-fold increase, corresponding to the recovery time for each population.


Recovery time increases with total antibiotic exposure . Inset shows recovery time as a function of dose duration for increasing streptomycin concentrations ( as in panel C ) . When plotted against total antibiotic exposure ( calculated as ) , recovery time values collapse onto a single exponential function ( black , R2 = 0 . 83 ) . Recovery time was measured in response to streptomycin concencentrations of 0 , 2 , 4 , 6 , 8 , 10 , 12 μg / mL , treatment durations 30 , 60 , 90 , 120 minutes .

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Recovery time increases with total antibiotic exposure. Inset shows recovery time as a function of dose duration for increasing streptomycin concentrations (as in panel C). When plotted against total antibiotic exposure (calculated as ), all recovery time values collapse onto a single exponential function (black line, R2 = 0.83). Recovery time was measured in response to streptomycin concencentrations of 0, 2, 4, 6, 8, 10, and 12 μg/mL, and treatment durations 30, 60, 90, and 120 minutes.


Streptomycin treatment ( closed data points , R2 = 0 . 83 ) results in significantly longer recovery times than chloramphenicol treatment ( open data points ) . Here , difference between two responses is statistically significant ( p < 0 . 05 by ANOVA ) .

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Streptomycin treatment (closed data points, R2 = 0.83) results in significantly longer recovery times than chloramphenicol treatment (open data points). Here, the difference between two responses is statistically significant (p<0.05 by ANOVA).


addition of efflux pump inhibitor ( CCCP ) ( closed data points , R2 = 0 . 91 ) increases population recovery time in response to chloramphenicol treatment ( open data points , R2 = 0 . 80 ) . Here , CCCP was added at subinhibitory concentrations ( 3μg / mL ) ; in absence of antibiotic treatment , CCCP alone did not inhibit population recovery . CCCP - mediated increase in recovery time is statistically significant ( p < 0 . 01 ) .

The addition of efflux pump inhibitor (CCCP) (closed data points, R2 = 0.91) increases population recovery time in response to chloramphenicol treatment (open data points, R2 = 0.80). Here, CCCP was added at subinhibitory concentrations (3μg/mL); in the absence of antibiotic treatment, CCCP alone did not inhibit population recovery. The CCCP-mediated increase in recovery time is statistically significant (p < 0.01).


ribosome synthesis rate was increased by increasing of casamino in media from 0 . 01 % w / v ( closed data points ) to 0 . 05 % w / v ( open data points ) . Faster synthesis resulted in lower recovery times ( p < 0 . 001 ) in response to streptomycin treatment . In each of these panels , color scheme indicates increasing antibiotic , as in Figure 1C .

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The ribosome synthesis rate was increased by increasing the concentration of the casamino acids in the media from 0.01% w/v (closed data points) to 0.05% w/v (open data points). Faster synthesis resulted in lower recovery times (p<0.001) in response to streptomycin treatment. In each of these panels, the color scheme indicates increasing antibiotic concentration, as in Figure 1C.


Inhibiting antibiotic efflux with CCCP significantly increased recovery time for that induced rapid target degradation involved a positive feedback loop . are as follows : penicillin G ( PenG ) , spectinomycin ( Spec ) , gentamicin ( Gent ) , streptomycin ( Str ) , chloramphenicol ( Cm ) , tetracyclin ( Tet ) , carbenicillin ( Carb ) . Antibiotic concentrations used have been scaled by their respective IC50 values ( solid points open circles show response with no efflux inhibition 2μg / mL CCCP , respectively ) . Colors correspond to motifs of action shown in Figure 4A .

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Inhibiting antibiotic efflux with CCCP significantly increased recovery time for antibiotics that induced rapid target degradation and involved a positive feedback loop. Antibiotics are as follows: penicillin G (PenG), spectinomycin (Spec), gentamicin (Gent), streptomycin (Str), chloramphenicol (Cm), tetracyclin (Tet), and carbenicillin (Carb). Antibiotic concentrations used have been scaled by their respective IC50 values (solid points and open circles show response with no efflux inhibition and 2μg/mL CCCP, respectively). Colors correspond to the motifs of action shown in Figure 4A.


a , Silver of LC3 complexes purified from U2OS expressing ZZ - Flag - LC3 in normal medium or Earle ' s balanced salt solution ( EBSS ) for 2 h . Asterisks mark centriolar satellite .

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a, Silver staining of LC3 complexes purified from U2OS cells expressing ZZ-Flag-LC3 in normal medium or Earle's balanced salt solution (EBSS) for 2 h. Asterisks mark centriolar satellite proteins.


b - d , Co - immunoprecipitation ( IP ) of OFD1 with LC3 , LC3 with PCM1 , or OFD1 with PCM1 in HEK293T . HA , haemagglutinin .

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b-d, Co-immunoprecipitation (IP) of OFD1 with LC3, LC3 with PCM1, or OFD1 with PCM1 in HEK293T cells. HA, haemagglutinin.


e , Western blotting of PCM1 OFD1 in control or PCM1 knockdown ( KD ) HEK293T .

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e, Western blotting analysis of PCM1 and OFD1 protein levels in control or PCM1 knockdown (KD) HEK293T cells.


f , Co - immunoprecipitation of OFD1 with LC3 in control or PCM1 KD HEK293T . IP efficiency is calculated as of immunoprecipitated OFD1 / LC3 .

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f, Co-immunoprecipitation of OFD1 with LC3 in control or PCM1 KD HEK293T cells. IP efficiency is calculated as the ratio of immunoprecipitated OFD1/LC3.


g , h , Western blotting of of indicated in MEFs with indicated genotypes in normal medium or subjected to 24 hserum starvation ( SS ) . Quantified OFD1 level was normalized with alpha - tubulin . Similar results were observed in three independent experiments .

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g, h, Western blotting analysis of protein levels of indicated proteins in MEFs with indicated genotypes in normal medium or subjected to 24 hserum starvation (SS). Quantified OFD1 level was normalized with alpha-tubulin. Similar results were observed in three independent experiments.


g , h , Western blotting of of indicated in MEFs with indicated genotypes in normal medium or subjected to 24 h serum starvation ( SS ) , 50 nM bafilomycin A1 ( Baf ) , 20 µM chloroquine ( CQ ) or 1 µM MG132 . Quantified OFD1 level was normalized with alpha - tubulin . Similar results were observed in three independent experiments .

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g, h, Western blotting analysis of protein levels of indicated proteins in MEFs with indicated genotypes in normal medium or subjected to 24 h serum starvation (SS), 50 nM bafilomycin A1 (Baf), 20 µM chloroquine (CQ) or 1 µM MG132. Quantified OFD1 level was normalized with alpha-tubulin. Similar results were observed in three independent experiments.


a , Representative confocal of endogenous OFD1 with cilium marker acetylated tubulin ( Ac - tubulin ) , or centriole marker γ - tubulin in hTERT - RPE1 in normal medium ( Un ) or subjected to 24 hserum starvation ( SS ) .

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a, Representative confocal images of endogenous OFD1 puncta with cilium marker acetylated tubulin (Ac-tubulin), or centriole marker γ-tubulin in hTERT-RPE1 cells in normal medium (Un) or subjected to 24 hserum starvation (SS).


b , Representative confocal of endogenous OFD1 acetylated tubulin in Atg5 + / + Atg5 − / − MEFs subjected to 24 hserum starvation . a , b , Data shown represent mean ± s . d . of with centriolar satelliteOFD1 for 100 per well in triplicate samples . * * * P 0 . 001 , two - unpaired student ' s t - . Similar results were observed in three independent experiments .

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b, Representative confocal images of endogenous OFD1 and acetylated tubulin in Atg5+/+ and Atg5−/− MEFs subjected to 24 hserum starvation. a, b, Data shown represent mean ± s.d. percentage of cells with centriolar satelliteOFD1 for 100 cells per well in triplicate samples. ***P 0.001, two-tailed unpaired student's t-test. Similar results were observed in three independent experiments.


a , b , Representative confocal of cilium marker acetylated tubulin from MEFs with indicated genotype subjected to 24 h serum starvation . Data shown represent mean ± s . d . of with cilia or of cilia for 500 or 100 per well , respectively , in triplicate samples . * * * P 0 . 001 , two - unpaired student ' s t - . Similar results were observed in three independent experiments . Un , untreated ; SS , serum - starved .

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a, b, Representative confocal images of cilium marker acetylated tubulin from MEFs with indicated genotype subjected to 24 h serum starvation. Data shown represent mean ± s.d. percentage of cells with primary cilia or length of the cilia for 500 cells or 100 cells per well, respectively, in triplicate samples. ***P  0.001, two-tailed unpaired student's t-test. Similar results were observed in three independent experiments. Un, untreated; SS, serum-starved.


a , Western blotting of OFD1 in control or OFD1 knockdown MCF7 ( C - 19 ) , quantified OFD1 level was normalized with alpha - tubulin .

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a, Western blotting analysis of OFD1 protein levels in control or OFD1 knockdown MCF7 (C-19) cells, quantified OFD1 level was normalized with alpha-tubulin.


b , of of with cilia in MCF7 or C19 subjected to 72 h serum starvation . Data shown represent mean ± s . d . of with cilia for 500 per well in triplicate samples . * * * P 0 . 001 , two - unpaired student ' s t - .

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b, Quantification of percentage of cells with primary cilia in MCF7 or C19 cells subjected to 72 h serum starvation. Data shown represent mean ± s.d. percentage of cells with primary cilia for 500 cells per well in triplicate samples. ***P 0.001, two-tailed unpaired student's t-test.


c , of of cilia in C19 subject to 48 h or 72 h serum starvation .

c, Quantification of length of primary cilia in C19 cells subject to 48 h or 72 h serum starvation.


d , e , Representative confocal of cilia with variable formed in MCF7 or C19 subject to 72 h serum starvation . Data shown represent 100 per well in triplicate samples .

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d, e, Representative confocal images of primary cilia with variable length formed in MCF7 or C19 cells subject to 72 h serum starvation. Data shown represent 100 cells per well in triplicate samples.


d , e , Representative confocal of cilia with variable formed in C19 subject to 72 hserum starvation . Data shown represent 100 per well in triplicate samples .

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d, e, Representative confocal images of primary cilia with variable length formed in C19 cells subject to 72 hserum starvation. Data shown represent 100 cells per well in triplicate samples.


f , Scanning electron microscope of cilia ( marked by arrows ) formed in C - 19 subject to 72 h serum starvation cycling Atg5 + / + MEFs in normal medium for 24 h . Similar results were observed in three independent experiments .

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f, Scanning electron microscope analysis of primary cilia (marked by arrows) formed in C-19 cells subject to 72 h serum starvation and cycling Atg5+/+ MEFs in normal medium for 24 h. Similar results were observed in three independent experiments.


( A ) PI3Kγ is expressed by human hepatocytes infiltrating immune in biopsies from with minimal to mild inflammatory activity . Triangles point to immune ( clusters ) , including some neutrophils known to express PI3Kγ highly . In negative control , antibody was replaced by an equal volume buffer . number of included , gender , diagnosis are summarized

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(A) PI3Kγ is expressed by human hepatocytes and infiltrating immune cells in biopsies from patients with minimal to mild inflammatory activity. Triangles point to immune cells (clusters), including some neutrophils known to express PI3Kγ highly. In the negative control, the primary antibody was replaced by an equal volume buffer. The number of included patients, gender, and diagnosis are summarized


( A ) PI3Kγ is expressed by human hepatocytes infiltrating immune in biopsies from with minimal to mild inflammatory activity . Triangles point to immune ( clusters ) , including some neutrophils known to express PI3Kγ highly . In negative control , antibody was replaced by an equal volume buffer . number of included , gender , diagnosis are

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( B ) PI3Kγ expression in human hepatocytes from 20 ( ♂ ) or ( ♀ ) donor pools ( HEP , DP20 ) , but not non - parenchymal ( NPC ) . Isolated human leukocytes from healthy volunteers ( LEU ) served as positive controls . characterization of donor pools is summarized

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(B) PI3Kγ expression in human primary hepatocytes from 20 male (♂) or female (♀) donor pools (HEP, DP20), but not non-parenchymal cells (NPC). Isolated human leukocytes from healthy volunteers (LEU) served as positive controls. The characterization of the donor pools is summarized


( B ) PI3Kγ expression in human hepatocytes from 20 ( ♂ ) or ( ♀ ) donor pools ( HEP , DP20 ) , but not non - parenchymal ( NPC ) . Isolated human leukocytes from healthy volunteers ( LEU ) served as positive controls . characterization of donor pools is

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( D ) Survival of WT , PI3Kγ null ( left ) liver - specific PI3Kγ knockout mice ( PI3Kγ floxtg / tg x AlbCre ( tg ) / tg , middle ) or systemic application of PI3Kγ inhibitor , AS605240 ( right ) in a model of polymicrobial sepsis induced by peritoneal contamination with a human stool suspension . numbers : B6 : 86 ( % : 52 , % : 48 ) , B6 - Pi3kcgko / ko : 59 ( % : 51 , % 49 ) , B6 - Pi3kcgflox / flox x AlbCre ( tg ) / tg : 25 ( % : 68 , % 32 ) , FVB / N Vehicle : 25 ( % : 44 , % 56 ) , FVB / N AS : 43 ( % : 44 , % 56 )

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(D) Survival of WT, PI3Kγ null (left) and liver-specific PI3Kγ knockout mice (PI3Kγ floxtg/tg x AlbCre(tg)/tg, middle) or systemic application of the PI3Kγ inhibitor, AS605240 (right) in a model of polymicrobial sepsis induced by peritoneal contamination with a human stool suspension. Animal numbers: B6: 86 (%male: 52, %female: 48), B6-Pi3kcgko/ko: 59 (%male: 51, %female 49), B6-Pi3kcgflox/flox x AlbCre(tg)/tg: 25 (%male: 68, %female 32), FVB/N Vehicle: 25 (%male: 44, %female 56), FVB/N AS: 43 (%male: 44, %female 56)


( C ) Uptake characteristics of T - LipoAS by human hepatocytes demonstrate competitive inhibition by cyclosporin A - energy dependence , confirming active uptake by organic anion transporters ( OATPs ) . data points are depicted mean ± SD from two batches of human hepatocytes in quadruples .

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(C) Uptake characteristics of T-LipoAS by primary human hepatocytes demonstrate competitive inhibition by cyclosporin A- and energy dependence, confirming active uptake by organic anion transporters (OATPs). The data points are depicted mean ± SD from two batches of primary human hepatocytes in quadruples.


( F ) T - LipoAS effectively inhibits fMLP - stimulated PI3Kγ , reflected by inhibiting AKT S473 phosphorylation activity in hepatocytes from three different batchtes . mean pAKT / AKT standard deviation ( SD ) were calculated for experiments separately .

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(F) T-LipoAS effectively inhibits fMLP-stimulated PI3Kγ, reflected by inhibiting AKT S473 phosphorylation activity in hepatocytes from three different batchtes. The mean pAKT/AKT ratio and standard deviation (SD) were calculated for all experiments separately.


( G ) Episcopic imaging of mice treated with T - LipoAS for 15 or 60 min relative of targeting moiety DY - 635 in different compartments .

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(G) Episcopic imaging of mice treated with T-LipoAS for 15 or 60 min and relative concentration of the targeting moiety DY-635 in the different compartments.


( B Pro - anti - inflammatory cytokines were analyzed in EDTA - plasma from peritoneal contamination infection ( PCI ) sham . # p < 0 . 05 against sham , * p < 0 . 05 as indicated ; Kruskal - Wallis ANOVA with controlled false - discovery rates ( Benjamini - Hochberg procedure ) .

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(B Pro- anti-inflammatory cytokines were analyzed in EDTA-plasma from peritoneal contamination and infection (PCI) and sham animals. #p<0.05 against sham, *p<0.05 as indicated; Kruskal-Wallis ANOVA with controlled false-discovery rates (Benjamini-Hochberg procedure).


C ) anti - inflammatory cytokines were analyzed in EDTA - plasma from peritoneal contamination infection ( PCI ) sham . # p < 0 . 05 against sham , * p < 0 . 05 as indicated ; Kruskal - Wallis ANOVA with controlled false - discovery rates ( Benjamini - Hochberg procedure ) .

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C) anti-inflammatory cytokines were analyzed in EDTA-plasma from peritoneal contamination and infection (PCI) and sham animals. #p<0.05 against sham, *p<0.05 as indicated; Kruskal-Wallis ANOVA with controlled false-discovery rates (Benjamini-Hochberg procedure).


( F , G ) of ( F ) neutrophils ( G ) monocytes in 2 mL peritoneal lavage .

(F,G) Concentration of (F) neutrophils and (G) monocytes in 2 mL peritoneal lavage.


( A ) Intravital microscopy of elimination of CDFDA , a substrate subject to excretory elimination by hepatocytes via Mrp - 2 , allows direct visualization of excretory function in sham or septic ( PCI ) treated with vehicle or a hepatocyte - directed inhibitor of PI3Kγ ( T - LipoAS ) .

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(A) Intravital microscopy of elimination of CDFDA, a fluorescent substrate subject to excretory elimination by hepatocytes via Mrp-2, allows direct visualization of excretory function in sham or septic (PCI) animals treated with vehicle or a hepatocyte-directed inhibitor of PI3Kγ (T-LipoAS).