( D ) More viral shedding in ZIKV - infected ISG20 - / - Sw . 71 culture supernatant . Supernatant from ZIKV - infected Sw . 71 ISG20 - / - Sw . 71 were collected plaque was performed using Vero . A representative plaque picture is presented . Note that more plaques were formed in Vero by incubating with ZIKV - infected ISG20 - / - Sw . 71 culture supernatant . Scale bar = 130μm . ( E ) Plaques were counted plaque forming - unit ( pfu ) was calculated for comparison of viral titers . Data represent as mean ± ; n = 3 biological replicates . * p < 0 . 05 by Student ' s t - .

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(D) More viral shedding in ZIKV-infected ISG20-/- Sw.71 cell culture supernatant. Supernatant from ZIKV-infected Sw.71 and ISG20-/- Sw.71 cells were collected and plaque assay was performed using Vero cells. A representative plaque assay picture is presented. Note that more plaques were formed in Vero cells by incubating with ZIKV-infected ISG20-/- Sw.71 cell culture supernatant. Scale bar=130μm. (E) Plaques were counted and plaque forming-unit (pfu) ratio was calculated for the comparison of the viral titers. Data represent as mean ± SEM; n=3 biological replicates. *p < 0.05 by Student's t-test.


( B ) ISGs mRNA expression stimulated by IFNβ in Sw . 71 ISG20 - / - Sw . 71 trophoblast . Sw . 71 ISG20 - / - Sw . 71 were treated with 30 ng / ml IFNβ for 24 h , was collected for determining ISGs gene expression by qRT - PCR . Data represent as mean ± ; n = 3 biological replicates ; * p < 0 . 05 , * * p < 0 . 01 by Student ' s t - against control .

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(B) ISGs mRNA expression stimulated by IFNβ in Sw.71 and ISG20-/- Sw.71 trophoblast cells. Sw.71 and ISG20-/- Sw.71 cells were treated with 30 ng/ml IFNβ for 24 h, and RNA was collected for determining the ISGs gene expression by qRT-PCR. Data represent as mean ± SEM; n=3 biological replicates; *p < 0.05, **p < 0.01 by Student's t-test against control.


( D ) IFNβ pre - treatment significantly prevented trophoblast from ZIKV infection , however , this protection was evidently attenuated due to lack of ISG20 . Sw . 71 ISG20 - / - Sw . 71 were pre - treated with or without 30 ng / ml IFNβ for 24 h , followed by ZIKV infection ( MOI = 2 ) for 1 h refreshed with regular media for 24 h , was collected to determine viral titers by qRT - PCR . Data represent as mean ± ; n = 4 biological replicates ; * p < 0 . 05 by Student ' s t - .

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(D) IFNβ pre-treatment significantly prevented trophoblast cells from ZIKV infection, however, this protection was evidently attenuated due to lack of ISG20. Sw.71 and ISG20-/- Sw.71 cells were pre-treated with or without 30 ng/ml IFNβ for 24 h, followed by ZIKV infection (MOI=2) for 1 h and refreshed with regular media for 24 h, and RNA was collected to determine the viral titers by qRT-PCR. Data represent as mean ± SEM; n=4 biological replicates; *p < 0.05 by Student's t-test.


( D ) ISG20 secretion in positive clone supernatant significantly decreased ZIKV infection in trophoblast . Supernatants from negative positive clones were collected added to ISG20 - / - Sw . 71 trophoblast together with ZIKA ( MOI = 2 ) for 1 h , followed by refreshing with new growth media for 48 h . was then collected to determine viral titers by qRT - PCR . Data represent as mean ± ; n = 3 biological replicates ; * * p < 0 . 01 by Student ' s t - .

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(D) ISG20 secretion in the positive clone supernatant significantly decreased ZIKV infection in trophoblast cells. Supernatants from negative and positive clones were collected and added to ISG20-/- Sw.71 trophoblast cells together with ZIKA virus (MOI=2) for 1 h, followed by refreshing with new growth media for 48 h. RNA was then collected to determine viral titers by qRT-PCR. Data represent as mean ± SEM; n=3 biological replicates; **p < 0.01 by Student's t-test.


( E ) Recombinant ISG20 - Fc degrades ZIKA viral HSV2 viral . 50ng purified viral ( ZIKV ) or ( HSV - 2 ) were incubated with increasing concentrations of rISG20 - Fc ( 5 , 50 , 500 ng ) in presence of RNase inhibitor for 90min at 37 ° C followed by of viral titers by qRT - PCR , agarose gel was used to evaluate degradation by electrophoresis . representative picture of agarose gel is presented . rISG20 - Fc was able to degrade both ZIKV HSV - 2 in a dose dependent manner ; however , rISG20 - Fc was more efficient in degrading viral than . Data represent as mean ± ; n = 3 biological replicates ; * * p < 0 . 01 . * * * p < 0 . 001 * * * * p < 0 . 0001 by One - way ANOVA .

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(E) Recombinant ISG20-Fc degrades ZIKA viral RNA and HSV2 viral DNA. 50ng purified viral RNA (ZIKV) or DNA (HSV-2) were incubated with increasing concentrations of rISG20-Fc (5, 50, 500 ng) in the presence of RNase inhibitor for 90min at 37°C followed by quantification of viral titers by qRT-PCR, and agarose gel was used to evaluate the RNA degradation by electrophoresis. The representative picture of agarose gel is presented. rISG20-Fc was able to degrade both ZIKV RNA and HSV-2 DNA in a dose dependent manner; however, rISG20-Fc was more efficient in degrading viral RNA than DNA. Data represent as mean ± SEM; n=3 biological replicates; **p < 0.01. ***p < 0.001 and ****p < 0.0001 by One-way ANOVA.


( B ) Macroscopic evaluation of pregnant uterus . Compare to reabsorptions in ZIKV - infected mice , rISG20 - Fc treatment rescued this phenotype by decreasing pregnancy loss ( resorption ) .

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(B) Macroscopic evaluation of the pregnant uterus and fetus. Compare to the fetal death and reabsorptions in ZIKV-infected mice, rISG20-Fc treatment rescued this phenotype by decreasing pregnancy loss (resorption).


( A ) rISG20 - Fc treatment alleviates maternal viral burden . Note that rISG20 - Fc treatment significantly decreased maternal serum viral titers . Data represent as mean ± ; n = 3 mice for control PBS group , n = 4 mice for treatment groups ; * p < 0 . 05 by Student ' s t - . ( B ) rISG20 - Fc treatment reduces ZIKV titers in brain . Although there was no difference of ZIKV titer in placenta , at side , there was a significant decrease of viral titer in brain , suggesting rISG20 - Fc can block viral transmission from placenta to . Data represent as mean ± ; n = 4 for each group , 3 - 4 placentas / brains from every mouse were analyzed ; * p < 0 . 05 by Student ' s t - . (

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(A) rISG20-Fc treatment alleviates maternal viral burden. Note that rISG20-Fc treatment significantly decreased the maternal serum viral titers. Data represent as mean ± SEM; n=3 mice for control PBS group, and n=4 mice for treatment groups; *p < 0.05 by Student's t-test. (B) rISG20-Fc treatment reduces ZIKV titers in the fetal brain. Although there was no difference of ZIKV titer in the placenta, at the fetal side, there was a significant decrease of viral titer in the fetal brain, suggesting rISG20-Fc can block viral transmission from placenta to fetus. Data represent as mean ± SEM; n=4 for each group, and 3-4 placentas/fetal brains from every mouse were analyzed; *p < 0.05 by Student's t-test. (


( C ) rISG20 - Fc treatment decreases Eotaxin significantly in maternal circulation . Data represent as mean ± ; n = 2 for PBS control group , n = 3 for every treatment group ; * p < 0 . 05 by One - way ANOVA ; difference not significant for IL - 9 IFNγ by One - way ANOVA .

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(C) rISG20-Fc treatment decreases Eotaxin significantly in maternal circulation. Data represent as mean ± SEM; n=2 for PBS control group, and n=3 for every treatment group; *p < 0.05 by One-way ANOVA; difference not significant for IL-9 and IFNγ by One-way ANOVA.


( A ) RT - qPCR of early passage RMS tumoroid models shows positivity for at least one gene used in standard - of - care pathology ( DES , MYOG , or MYOD1 ) . Conventional RMS lines ( RD RH30 ) were used as positive controls , while two Synovial Sarcoma ( SS000DAZ SS077 ) tumoroid models were used as negative controls . Gene expression was normalized to expression of a house - keeping gene human reference ( HREF ) via ΔΔCq method . Each tumoroid was measured once with four technical replicates with error bars representing standard deviation of said technical replicates .

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(A) RT-qPCR of early passage RMS tumoroid models shows positivity for at least one gene used in standard-of-care pathology analysis (DES, MYOG, or MYOD1). Conventional RMS cell lines (RD and RH30) were used as positive controls, while two Synovial Sarcoma (SS000DAZ and SS077) tumoroid models were used as negative controls. Gene expression was normalized to the expression of a house-keeping gene and human reference RNA (HREF) via the ΔΔCq method. Each tumoroid line was measured once with four technical replicates with the error bars representing the standard deviation of said technical replicates.


( C ) Morphological ( via H & E ) immunohistochemical ( ) comparison of RMS tumors derived RMS tumoroid models shows retained marker ( Desmin , Myogenin MYOD1 ) expression . Scale bars equal 200 µm ( RMS012 , RMS102 ) or 100 µm ( RMS000HQC ) .

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(C) Morphological (via H&E) and immunohistochemical (IHC) comparison of RMS tumors and derived RMS tumoroid models shows retained marker protein (Desmin, Myogenin and MYOD1) expression and cellular morphology. Scale bars equal 200 µm (RMS012, RMS102) or 100 µm (RMS000HQC).


( A ) Copy number frequency plots of RMS tumors ( upper row ) derived RMS tumoroid models ( lower row ) divided by fusion - status ( columns ) . Chromosomes are annotated on x - axis from left to right while y - axis shows of samples in this group carrying a gain ( red ) or loss ( blue ) in this genomic region .

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(A) Copy number frequency plots of RMS tumors (upper row) and derived RMS tumoroid models (lower row) divided by fusion-status (columns). Chromosomes are annotated on the x-axis from left to right while the y-axis shows the percentage of samples in this group carrying a gain (red) or loss (blue) in this genomic region.


( B ) Contribution of somatic mutational signatures per tumoroid model . SBS = single base substitution , TMZ = temozolomide , T = , O = tumoroid .

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(B) Contribution of somatic mutational signatures per tumor and tumoroid model. SBS = single base substitution, TMZ = temozolomide, T = tumor, O = tumoroid.


( C ) Table depicting pathogenic single - nucleotide variants ( SNVs ) in RMS tumors ( T ) tumoroid models ( O ) . Circle color indicates SNV type while circle indicates variant allele fraction ( VAF ) . Vertical dotted lines separate samples derived from . Highlighted are genes previously reported for this RMS subtype .

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(C) Table depicting pathogenic single-nucleotide variants (SNVs) in RMS tumors (T) and tumoroid models (O). Circle color indicates SNV type while circle size indicates variant allele fraction (VAF). Vertical dotted lines separate samples derived from individual patients. Highlighted are genes previously reported for this RMS subtype.


( D ) Correlogram of bulk mRNA sequencing expression profiles of pediatric kidney tumors ( controls ) as well as RMS tumoroid models RMS tumors . CCRCC = Clear Renal Carcinoma , CMN = Congenital Mesoblastic Nephroma , WT = Wilms , Cor = correlation .

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(D) Correlogram of bulk mRNA sequencing expression profiles of pediatric kidney tumors (controls) as well as RMS tumoroid models and RMS tumors. CCRCC = Clear Cell Renal Cell Carcinoma, CMN = Congenital Mesoblastic Nephroma, WT = Wilms Tumor, Cor = correlation.


( A ) Clustered of viability measurements per RMS tumoroid model ( x axis ) drug ( y axis ) , showing Under Curve ( AUC ) after treatment of for 120 h with a dose - range of 0 . 1 nM to 10 µM . Low AUC ( red ) indicates high drug efficacy while high AUC ( blue ) indicates low drug efficacy . Annotated clusters of ( 1 ) MEK / ERK γ - secretase inhibitors showing specific efficacy in RMS tumoroid models without ( RMS007 , RMS012 , RMS444 ) or low ( RMS000HQC ) fusion transcript expression , ( 2 ) drugs that show broad efficacy across RMS tumoroid models .

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(A) Clustered heatmap of viability measurements per RMS tumoroid model (x axis) and drug (y axis), showing the Area Under the Curve (AUC) after treatment of the cells for 120 h with a dose-range of 0.1 nM to 10 µM. Low AUC (red) indicates high drug efficacy while high AUC (blue) indicates low drug efficacy. Annotated clusters of (1) MEK/ERK and γ-secretase inhibitors showing specific efficacy in RMS tumoroid models without (RMS007, RMS012, RMS444) or low (RMS000HQC) fusion transcript expression, and (2) drugs that show broad efficacy across RMS tumoroid models.


( B ) Western Blot of TP53 wildtype ( WT ) knockout ( KO ) RMS tumoroid RMS012 . Histone 3 ( H3 ) served as loading control .

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(B) Western Blot analysis of TP53 wildtype (WT) and knockout (KO) RMS tumoroid line RMS012. Histone 3 (H3) served as loading control.


( C ) Dose - response curve of TP53 WT KO treated with Chk - 1 inhibitor prexasertib . Thin lines with numbers indicate biological replicates ( n = 3 ) while thick lines indicate fitted lines over replicates . statistical significance of differences in fitted IC50 values between WT KO were obtained using a two - sided t - ( p = 0 . 008 ) .

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(C) Dose-response curve of TP53 WT and KO cells treated with the Chk-1 inhibitor prexasertib. Thin lines with numbers indicate individual biological replicates (n = 3) while thick lines indicate fitted lines over all replicates. The statistical significance of the differences in fitted IC50 values between WT and KO were obtained using a two-sided t-test (p = 0.008).


( C - J ) panels show Drosophila 3rd instar larval eye imaginal discs with eyeless - Flippase - induced clones of homozygous for EMC3Δ4 labelled by absence of ubiGFP ( green ) . red channel shows ELAV , which labels nuclei of photoreceptor . UAS constructs were expressed under control of GMR - GAL4 . ( C ) Expression of UAS - Rh1 ( 4C5 , in blue ) ( D ) Na + K + ATPase ( A5 - C , in blue ) is strongly reduced in EMC3Δ4 homozygous mutant . ( E ) Expression of UAS - Xport - A - HA ( anti - HA , in blue ) ( F ) UAS - fan - HA ( anti - HA , in blue ) is strongly reduced in EMC3Δ4 homozygous mutant . ( G ) Expression of UAS - rtv - HA ( anti - HA , in blue ) ( H ) hid ( anti - hid , in blue ) is increased in EMC3Δ4 homozygous mutant . ( I - J ) Normal expression of two TA in EMC3Δ4 homozygous mutant . ( I ) UAS - CG8814 - HA ( anti - HA , in blue ) is a TA containing a TMD of low hydrophobicity ( J ) UAS - Syx13 - HA is a TA with high hydrophobicity TMD . Scale bars represent 10 μm .

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(C-J) All panels show Drosophila 3rd instar larval eye imaginal discs with eyeless-Flippase-induced clones of cells homozygous for EMC3Δ4 labelled by the absence of ubiGFP (green). The red channel shows ELAV, which labels the nuclei of photoreceptor cells. All UAS constructs were expressed under the control of GMR-GAL4. (C) Expression of UAS-Rh1 (4C5, in blue) and (D) Na+K+ATPase (A5-C, in blue) is strongly reduced in EMC3Δ4 homozygous mutant cells. (E) Expression of UAS-Xport-A-HA (anti-HA, in blue) and (F) UAS-fan-HA (anti-HA, in blue) is strongly reduced in EMC3Δ4 homozygous mutant cells. (G) Expression of UAS-rtv-HA (anti-HA, in blue) and (H) hid (anti-hid, in blue) is increased in EMC3Δ4 homozygous mutant cells. (I-J) Normal expression of two TA proteins in EMC3Δ4 homozygous mutant cells. (I) UAS-CG8814-HA (anti-HA, in blue) is a TA protein containing a TMD of low hydrophobicity (J) UAS-Syx13-HA is a TA protein with high hydrophobicity TMD. Scale bars represent 10 μm.


( B - D ) - Immunostaining of 3rd instar larval eye imaginal discs with eyeless - Flippase - induced clones of homozygous for EMC3Δ4 , labelled by absence of ubiGFP ( green ) shows reduced expression of ( B ) UAS - HA - Xport - A ( anti - HA , in blue ) but normal expression of ( C ) UAS - HA - Xport - A4L ( anti - HA , in blue ) or ( D ) UAS - Xport - A4L - HA ( anti - HA , in blue ) in EMC3 mutant homozygous . ELAV ( red in B C ) marks photoreceptor Na + K + ATPase ( red in D ) acts as a positive control for presence of EMC3Δ4 homozygous mutant clones . UAS constructs were expressed under control of GMR - GAL4 . Scale bars represent 10 μm .

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(B-D) - Immunostaining of 3rd instar larval eye imaginal discs with eyeless-Flippase-induced clones of cells homozygous for EMC3Δ4, labelled by the absence of ubiGFP (green) shows reduced expression of (B) UAS-HA-Xport-A (anti-HA, in blue) but normal expression of (C) UAS-HA-Xport-A4L (anti-HA, in blue) or (D) UAS-Xport-A4L-HA (anti-HA, in blue) in EMC3 mutant homozygous cells. ELAV (red in B and C) marks photoreceptor cells and Na+K+ATPase (red in D) acts as a positive control for the presence of EMC3Δ4 homozygous mutant clones. The UAS constructs were expressed under the control of GMR-GAL4. Scale bars represent 10 μm.


( E - F ) Immunostaining of mosaic adult retina with eyeless - Flippase - induced clones of homozygous for EMC3e02662 , labelled by absence of RFP ( red ) , show loss of ( E ) UAS - HA - Xport - A ( anti - HA , in blue ) in EMC3 mutant homozygous . ( F ) Expression of UAS - HA - Xport - A4L ( anti - HA , in blue ) is normal in EMC3e02662 homozygous mutant clones . rhabdomeres are stained for actin ( Phalloidin , in green ) . Scale bars represent 10 μm .

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(E-F) Immunostaining of mosaic adult retina with eyeless-Flippase-induced clones of cells homozygous for EMC3e02662, labelled by the absence of RFP (red), show loss of (E) UAS-HA-Xport-A (anti-HA, in blue) in EMC3 mutant homozygous cells. (F) Expression of UAS-HA-Xport-A4L (anti-HA, in blue) is normal in EMC3e02662 homozygous mutant clones. The rhabdomeres are stained for actin (Phalloidin, in green). Scale bars represent 10 μm.


( B - D ) Histograms of flow cytometry data monitoring in indicated U2OS lines for each construct . " EMC5KO + EV " indicates a knockout of EMC5 stably harbouring empty vector , while ' ' EMC5KO + EMC5 " indicates EMC5KO rescued by inducible re - expression of a stably integrated EMC5 . ( E - G ) Histograms of flow cytometry data monitoring in indicated U2OS lines for each construct . " EMC6KO + EV " indicates a knockout of EMC6 stably harbouring empty vector , while ' ' EMC6KO + EMC6 " indicates EMC6KO rescued by re - expression of a stably integrated EMC6 .

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(B-D) Histograms of flow cytometry data monitoring the fluorescence protein ratio in the indicated U2OS cell lines for each construct. "EMC5KO+EV" indicates a knockout of EMC5 stably harbouring empty vector, while ''EMC5KO+EMC5" indicates EMC5KO cells rescued by inducible re-expression of a stably integrated EMC5. (E-G) Histograms of flow cytometry data monitoring the fluorescence protein ratio in the indicated U2OS cell lines for each construct. "EMC6KO+EV" indicates a knockout of EMC6 stably harbouring empty vector, while ''EMC6KO+EMC6" indicates EMC6KO cells rescued by re-expression of a stably integrated EMC6.


( C - G ) ( C - D , F ) Western - Blot of WT EMC6KO ( 6KO ) U2OS lines transiently transfected with opsin - tagged constructs probed for HA . When indicated , denatured samples were treated with PNGase to confirm glycosylation . Glycosylated shift upwards in gel are indicated by arrows (  ) . ( E , G ) Western - Blot of WT EMC6KO ( 6KO ) U2OS lines transiently transfected with opsin - tagged constructs probed for Actin EMC6 .

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(C-G) (C-D, F) Western-Blot of WT and EMC6KO (6KO) U2OS cell lines transiently transfected with the opsin-tagged constructs probed for HA. When indicated, denatured samples were treated with PNGase to confirm glycosylation. Glycosylated proteins shift upwards in the gel and are indicated by arrows (). (E, G) Western-Blot of WT and EMC6KO (6KO) U2OS cell lines transiently transfected with the opsin-tagged constructs probed for Actin and EMC6.


( A - B ) Immunostaining of 3rd instar larval eye imaginal discs with eyeless - Flippase - induced clones of homozygous for EMC3Δ4 , labelled by absence of ubiGFP ( green ) show loss of ( A ) UAS - Xport - A - HA ( anti - HA , in blue ) UAS - Rh1 ( 4C5 , in red ) . ( B ) Expression of UAS - Xport - A4L ( anti - HA , in blue ) UAS - Rh1 ( 4C5 , in red ) is observed in EMC3Δ4 homozygous . UAS constructs were expressed under control of GMR - GAL4 . Scale bars represent 10 μm .

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(A-B) Immunostaining of 3rd instar larval eye imaginal discs with eyeless-Flippase-induced clones of cells homozygous for EMC3Δ4, labelled by the absence of ubiGFP (green) show loss of (A) UAS-Xport-A-HA (anti-HA, in blue) and UAS-Rh1 (4C5, in red). (B) Expression of UAS-Xport-A4L (anti-HA, in blue) and UAS-Rh1 (4C5, in red) is observed in EMC3Δ4 homozygous cells. The UAS constructs were expressed under the control of GMR-GAL4. Scale bars represent 10 μm.


( C - D ) Immunostaining of mosaic adult retinas with eyeless - Flippase - induced clones of homozygous for EMC3e02662 , labelled by absence of RFP ( red ) , show loss of ( C ) TRP ( Mab83F6 , in blue ) when UAS - HA - Xport - A is expressed . ( D ) Expression of TRP ( Mab83F6 , in blue ) is observed in EMC3e02662 homozygous , when HA - Xport - A4L is expressed . rhabdomeres are stained for actin ( Phalloidin , in green ) . UAS constructs were expressed under control of Rh1 - GAL4 . Scale bars represent 10 μm .

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

(C-D) Immunostaining of mosaic adult retinas with eyeless-Flippase-induced clones of cells homozygous for EMC3e02662, labelled by the absence of RFP (red), show loss of (C) TRP (Mab83F6, in blue) when UAS-HA-Xport-A is expressed. (D) Expression of TRP (Mab83F6, in blue) is observed in EMC3e02662 homozygous cells, when HA-Xport-A4L is expressed. The rhabdomeres are stained for actin (Phalloidin, in green). The UAS constructs were expressed under the control of Rh1-GAL4. Scale bars represent 10 μm.


( E - G ) Western blot of fly heads labelled for V5 , TRP , Rh1 , Xport - A ( Xp - A ) tubulin . ( E ) Immunoblot of fly heads expressing Rh1Gal4 ( driver ) ( left ) or Rh1 TMD1 - V5 ( right ) in Xport - A heterozygous ( Xport - A1 / TM6B ) or homozygous mutant flies ( Xport - A1 / Xport - A1 ) . Extracts were untreated ( - ) or treated ( + ) with PNGase F enzyme ( lanes 2 , 4 , 6 8 ) . For Rh1Gal4 , 4 fly heads were loaded per well , while for Rh1 TMD1 - V5 , 8 fly heads were loaded per well . ( F ) Immunoblot of fly heads expressing Rh1 TMD1 - 3 - V5 in Xport - A heterozygous ( Xport - A1 / TM6B ) or homozygous mutant flies ( Xport - A1 / Xport - A1 ) . Extracts were untreated ( - ) or treated ( + ) with PNGase F enzyme ( lanes 2 4 ) . Eight fly heads were loaded per lane . ( G ) Immunoblot of fly heads expressing Rh1 TMD1 - 5 - V5 in Xport - A heterozygote ( Xport - A1 / TM6B ) or homozygous mutant flies ( Xport - A1 / Xport - A1 ) . Extracts were untreated ( - ) or treated ( + ) with either Endo - H ( lanes 2 5 ) or PNGase F enzyme ( lanes 3 6 ) . Each lane contains approximately 2 , 5 fly heads .

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

(E-G) Western blot of fly heads labelled for V5, TRP, Rh1, Xport-A (Xp-A) and tubulin. (E) Immunoblot of fly heads expressing Rh1Gal4 (driver) (left) or Rh1 TMD1-V5 (right) in Xport-A heterozygous (Xport-A1/TM6B) or homozygous mutant flies (Xport-A1/Xport-A1). Extracts were untreated (-) or treated (+) with PNGase F enzyme (lanes 2, 4, 6 and 8). For Rh1Gal4, 4 fly heads were loaded per well, while for Rh1 TMD1-V5, 8 fly heads were loaded per well. (F) Immunoblot of fly heads expressing Rh1 TMD1-3-V5 in Xport-A heterozygous (Xport-A1/TM6B) or homozygous mutant flies (Xport-A1/Xport-A1). Extracts were untreated (-) or treated (+) with PNGase F enzyme (lanes 2 and 4). Eight fly heads were loaded per lane. (G) Immunoblot of fly heads expressing Rh1 TMD1-5-V5 in Xport-A heterozygote (Xport-A1/TM6B) or homozygous mutant flies (Xport-A1/Xport-A1). Extracts were untreated (-) or treated (+) with either Endo-H (lanes 2 and 5) or PNGase F enzyme (lanes 3 and 6). Each lane contains approximately 2,5 fly heads.


. ( b ) Conjugation of LC3 two other mammalian homologues ( GL1 GL2 ) with phosphatidylethanolamine ( PE ) was performed as described in ref . . Complete reactions contain 1 . 5 μM Atg7 , 2 . 5 μM Atg3 , 8 - 12 μM LC3 / GL1 / GL2 , 2 mM lipid ( 30 nm liposomes composed of 10 mol % bl - PI , 55 mol % DOPE , 0 . 15 mol % Rh - DOPE 34 . 85 mol % POPC ; see Methods for full definitions ) , 1 mM dithiothreitol 1 mM ATP were run at 30 ° C for 90 min . Each reaction was run on a 12 % SDS - PAGE gel visualized by Coomassie blue stain . mobility of lipidated is faster than corresponding non - lipidated forms . Intermediates in reaction are also detectable , including Atg7 - GRL1 conjugate , Atg3 - GRL1 conjugate a modified form of GRL1 , GRL2 LC3 that is labelled simply as GRL1 / GRL2 / LC3 - intermediate is probably adenylated form of each LC3 - related .

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

. (b) Conjugation of LC3 and two other mammalian homologues (GL1 and GL2) with phosphatidylethanolamine (PE) was performed as described in ref. . Complete reactions contain 1.5 μM Atg7, 2.5 μM Atg3, 8-12 μM LC3/GL1/GL2, 2 mM lipid (30 nm liposomes composed of 10 mol% bl-PI, 55 mol% DOPE, 0.15 mol% Rh-DOPE and 34.85 mol% POPC; see Methods for full definitions), 1 mM dithiothreitol and 1 mM ATP and were run at 30 °C for 90 min. Each reaction was run on a 12% SDS-PAGE gel and visualized by Coomassie blue stain. The mobility of the lipidated protein is faster than the corresponding non-lipidated forms. Intermediates in the reaction are also detectable, including the Atg7-GRL1 conjugate, the Atg3-GRL1 conjugate and a modified form of GRL1, GRL2 and LC3 that is labelled simply as GRL1/GRL2/LC3-intermediate and is probably the adenylated form of each LC3-related protein.


( c ) lipidation reactions are highly dependent on molar of DOPE in liposomes . Reactions are as in b , but run on 400 nm liposomes . Liposomes are composed of indicated amount of DOPE , 10 mol % bl - PI , 0 . 15 mol % Rh - DOPE POPC .

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

(c) The lipidation reactions are highly dependent on the molar percentage of DOPE in the liposomes. Reactions are as in b, but run on 400 nm liposomes. Liposomes are composed of the indicated amount of DOPE, 10 mol% bl-PI, 0.15 mol% Rh-DOPE and POPC.


( d ) Completed reactions in c were subjected to density gradient flotation to isolate only lipidated material . Floated samples were run on SDS - PAGE imaged by Coomassie . For c d , samples were prepared at same time from same materials run simultaneously on multiple gels , as indicated by vertical lines . Uncropped versions of gels are included in Supplementary Fig . 7 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Completed reactions in c were subjected to density gradient flotation to isolate only the lipidated material. Floated samples were run on SDS-PAGE and imaged by Coomassie. For c and d, the samples were prepared at the same time from the same materials and run simultaneously on multiple gels, as indicated by the vertical lines. Uncropped versions of all gels are included in Supplementary Fig. 7.


( b ) Filling defects with inverted cone - shaped lipids inhibits lipidation . In vitro coupling reactions of GL1 were run on sonicated liposomes containing 30 mol % DOPE ( 2 mM total lipid ) as in Fig . 1 . To determine whether inverted cones influenced reaction , increasing amounts of stearoyl - CoA were added immediately before initiating reaction with ATP . is of n = 3 samples ; error bars represent standard deviation . P values represent a comparison with samples without stearoyl - CoA . * * P 0 . 1 ; * P 0 . 05 .

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(b) Filling the defects with inverted cone-shaped lipids inhibits lipidation. In vitro coupling reactions of GL1 were run on sonicated liposomes containing 30 mol% DOPE (2 mM total lipid) as in Fig. 1. To determine whether inverted cones influenced the reaction, increasing amounts of stearoyl-CoA were added immediately before initiating the reaction with ATP. Quantification is of n = 3 samples; error bars represent standard deviation. P values represent a comparison with samples without stearoyl-CoA. **P 0.1; *P 0.05.


( c ) Altering shape of PE to reduce local defects inhibits lipidation reaction . In vitro coupling reactions of GL1 were run on liposomes of three different sizes ( 400 nm 50 nm extruded liposomes sonicated liposomes ) of three different lipid compositions . Lipid compositions include 30 mol % DPPE , DSPE or DOPE ( see Methods for full definitions ) , 10 % bl - PI , POPC . Total lipid = 2 mM . is of n = 3 samples ; error bars represent standard deviation . extent of lipidation in b c is plotted as a of total GL1 as determined by densitometry . P values represent comparison with most active liposomes ( sonicated DOPE ) . * * P 0 . 1 ; * P 0 . 05 .

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

(c) Altering the shape of PE to reduce local membrane defects inhibits the lipidation reaction. In vitro coupling reactions of GL1 were run on liposomes of three different sizes (400 nm and 50 nm extruded liposomes and sonicated liposomes) and of three different lipid compositions. Lipid compositions include 30 mol% DPPE, DSPE or DOPE (see Methods for full definitions), 10% bl-PI, and POPC. Total lipid = 2 mM. Quantification is of n = 3 samples; error bars represent standard deviation. The extent of lipidation in b and c is plotted as a percentage of total GL1 as determined by densitometry. P values represent comparison with most active liposomes (sonicated DOPE). **P 0.1; *P 0.05.


( b ) To determine impact of curvature on lipidation , we compared liposomes with unphysiologically high surface densities of PE ( 55 mol % ) as have been used in other Atg8 or LC3 lipidation publications ( for example , refs , , ) , with liposomes having highest densities of liposomes observed in mammalian organelle membranes ( 30 mol % ) . At physiologically relevant concentrations of liposomes ( 30 mol % ) , lipidation becomes curvature dependent . In vitro coupling reactions were performed as in Fig . 1 , except that liposomes were made by extrusion through membranes with different pore diameters ( as indicated ) . Actual diameters of these liposomes were determined by dynamic scattering ( Supplementary Table 1 ) . Top : lipidation of GL2 , GL2 PE proceeds efficiently with relatively little curvature dependence on liposomes composed of 55 % PE . However , when liposomes is reduced to 30 % , flat liposomes are no longer suitable for lipidation , while highly curved liposomes ( 100 nm diameter or less ) continue to couple effectively . Bottom : to confirm that this curvature dependence reveals lipidated product not intermediate , 30 % reactions were each run on Nycodenz flotation gradients . Only lipidated product is recovered at top of gradient .

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

(b) To determine the impact of curvature on lipidation, we compared liposomes with unphysiologically high surface densities of PE (55 mol%) as have been used in other Atg8 or LC3 lipidation publications (for example, refs , , ), with liposomes having the highest densities of liposomes observed in mammalian organelle membranes (30 mol%). At physiologically relevant concentrations of liposomes (30 mol%), lipidation becomes membrane curvature dependent. In vitro coupling reactions were performed as in Fig. 1, except that liposomes were made by extrusion through membranes with different pore diameters (as indicated). Actual diameters of these liposomes were determined by dynamic light scattering (Supplementary Table 1). Top: the lipidation of GL2, GL2 and PE proceeds efficiently and with relatively little curvature dependence on liposomes composed of 55% PE. However, when the liposomes is reduced to 30%, flat liposomes are no longer suitable for lipidation, while highly curved liposomes (100 nm diameter or less) continue to couple effectively. Bottom: to confirm that this curvature dependence reveals the lipidated product and not the intermediate, the 30% reactions were each run on Nycodenz flotation gradients. Only the lipidated product is recovered at the top of the gradient.


( c ) lipidation reaction is largely insensitive to PE concentrations when run on highly curved liposomes . Coupling of GL1 LC3 to PE was assessed on liposomes of two sizes ( 400 nm , black squares ; sonicated , red circles ) varying molar of DOPE . extent of lipidation is determined as in Fig . 2 . ( n = 2 for 30 55 % PE , n = 4 for other points , error bars represent standard deviation . ) P values were determined by comparing sonicated with 400 nm samples at each PE . * * P 0 . 1 ; * P 0 . 01 . Uncropped versions of gels are included in Supplementary Fig . 7 .

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

(c) The lipidation reaction is largely insensitive to PE concentrations when run on highly curved liposomes. Coupling of GL1 and LC3 to PE was assessed on liposomes of two sizes (400 nm, black squares; sonicated, red circles) and varying molar percentage of DOPE. The extent of lipidation is determined as in Fig. 2. (n = 2 for 30 and 55% PE, n = 4 for all the other points, and error bars represent standard deviation.) P values were determined by comparing sonicated with 400 nm samples at each PE concentration. **P 0.1; *P 0.01. Uncropped versions of all gels are included in Supplementary Fig. 7.


( a ) accumulation of Atg3 - GL1 conjugate depends on extent of curvature . In vitro GL1 coupling reactions were run as in Fig . 1 . reactions were visualized by immunoblot against Atg3 ( top ) by Coomassie of GRL1 ( bottom ) . accumulation of Atg3 - GL1 is inversely related to formation of lipidated GRL1 , suggesting that - curvature - dependent step may involve Atg3 recognition of surface .

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(a) The accumulation of the Atg3-GL1 conjugate depends on the extent of membrane curvature. In vitro GL1 coupling reactions were run as in Fig. 1. The reactions were visualized by immunoblot against Atg3 (top) and by Coomassie staining of GRL1 (bottom). The accumulation of Atg3-GL1 is inversely related to the formation of the lipidated GRL1, suggesting that the membrane-curvature-dependent step may involve Atg3 recognition of the membrane surface.


( b ) Atg3 binding to liposomes . Atg3 ( 10 μM ) was incubated at 30 ° C for 90 min with liposomes containing 0 , 30 or 55 mol % DOPE ( 5 mM lipids ) of varying sizes ( extrusion dimensions are shown on figure ; actual final sizes were determined by dynamic scattering ( Supplementary Table 1 ) ) . liposome - associated Atg3 was recovered by Nycodenz density gradient centrifugation analysed by SDS - PAGE .

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

(b) Atg3 binding to liposomes. Atg3 (10 μM) was incubated at 30 °C for 90 min with liposomes containing 0, 30 or 55 mol% DOPE (5 mM lipids) and of varying sizes (extrusion membrane dimensions are shown on the figure; actual final sizes were determined by dynamic light scattering (Supplementary Table 1)). The liposome-associated Atg3 was recovered by Nycodenz density gradient centrifugation and analysed by SDS-PAGE.


( c ) of liposome - associated Atg3 . Densitometric plot of amount of Atg3 recovered in top fraction in flotation assays as in b . Error bars indicate s . e . m . from three independent experiments . P values calculated as compared with putative negative control ( 0 % PE , 400 nm ) . * P 0 . 05 . Uncropped versions of gels are included in Supplementary Fig . 7 .

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(c) Quantification of liposome-associated Atg3. Densitometric plot of the amount of Atg3 recovered in the top fraction in flotation assays as in b. Error bars indicate s.e.m. from three independent experiments. P values calculated as compared with putative negative control (0% PE, 400 nm). *P 0.05. Uncropped versions of all gels are included in Supplementary Fig. 7.


( b ) of reaction efficiency for point mutants of amphipathic helix . GL1 lipidation reactions were run on 400 nm sonicated liposomes ( 30 mol % DOPE ) with indicated Atg3 mutants . extent of GL1 - PE formation ( top gel ) was determined from densitometry is plotted as of total GL1 ( bar graph ) . ability to form Atg3 - GL1 conjugate was also assessed for each mutant ( bottom immunoblot ) . Note that this conjugate forms in every case , indicating that no mutant inhibited a step upstream in reaction . Further , this conjugate specifically accumulates in reactions where lipidation is impaired . In both panels , samples were prepared at same time from same materials run simultaneously on multiple gels , as indicated by lines .

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

(b) Analysis of reaction efficiency for point mutants of the amphipathic helix. GL1 lipidation reactions were run on 400 nm and sonicated liposomes (30 mol% DOPE) with the indicated Atg3 mutants. The extent of GL1-PE formation (top gel) was determined from densitometry and is plotted as percentage of total GL1 (bar graph). The ability to form the Atg3-GL1 conjugate was also assessed for each mutant (bottom immunoblot). Note that this conjugate forms in every case, indicating that no mutant inhibited a step upstream in the reaction. Further, this conjugate specifically accumulates in reactions where lipidation is impaired. In both panels, the samples were prepared at the same time from the same materials and run simultaneously on multiple gels, as indicated by the lines.


( c ) Curvature dependence of K11L mutant binding to liposomes compared with wild - type Atg3 . binding reaction was performed as in Fig . 4b on 30 DOPE % liposomes . F , float - up ; T , total input .

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(c) Curvature dependence of K11L mutant binding to liposomes compared with wild-type Atg3. The binding reaction was performed as in Fig. 4b on 30 DOPE% liposomes. F, float-up; T, total input.


( d ) Lys 11 represents a tunable node through which curvature dependence of lipidation reaction can be controlled . Lipidation assays were run as in b . Total GL1 lipidation was assessed from reactions with wild - type Atg3 or mutant forms of Atg3 in which position Lys 11 was changed as indicated . Lipidation efficiency on low PE / low curvature liposomes ( 30 % PE , 400 nm ) increases with increasing hydrophobicity of new acid , while liposomes with high PE ( 55 % ) or high curvature ( sonicated ) are good substrates for active forms of Atg3 . Left : Coomassie gels of lipidation reactions . Right : densitometry of Coomassie - stained gels revealed of GL1 - PE to sum of three GL1 species as in Fig . 2 . grey bars are sonicated liposomes ; grey bars are 400 nm extruded liposomes . For b , c , P values were calculated between sonicated 400 nm samples of same mutant . For d , P values represent comparison with WT on same liposome composition . For panels , n = 3 independent experiments , error bars represent standard deviation * * P 0 . 05 ; * P 0 . 01 . Uncropped gels in Supplementary Fig . 7 .

O O O O O 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 B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR 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 B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Lys 11 represents a tunable node through which curvature dependence of the lipidation reaction can be controlled. Lipidation assays were run as in b. Total GL1 lipidation was assessed from reactions with wild-type Atg3 or mutant forms of Atg3 in which position Lys 11 was changed as indicated. Lipidation efficiency on low PE/low curvature liposomes (30% PE, 400 nm) increases with increasing hydrophobicity of the new amino acid, while liposomes with high PE (55%) or high curvature (sonicated) are good substrates for all active forms of Atg3. Left: Coomassie gels of individual lipidation reactions. Right: densitometry of Coomassie-stained gels revealed the ratio of GL1-PE to the sum of all three GL1 species as in Fig. 2. Light grey bars are sonicated liposomes; dark grey bars are 400 nm extruded liposomes. For b,c, P values were calculated between sonicated and 400 nm samples of the same mutant. For d, P values represent comparison with WT protein on the same liposome size and composition. For all panels, n = 3 independent experiments, error bars represent standard deviation and **P 0.05; *P 0.01. Uncropped gels in Supplementary Fig. 7.


Atg3 - knockout MEFs ( Atg3 − / − ) are incapable of forming LC3 - II do not accumulate LC3 . Instead , they accumulate autophagic intermediates that are positive for Atg16 . To determine whether amphipathic helix is critical for in vivo function , Atg3 with wild - type or mutant forms of amphipathic helix was introduced into Atg3 − / − MEFs by lentiviral infection rescue of three was tested . ( a ) Immunoblot of LC3 - II , GL2 - II GABARAP - II formation . GABARAP lipidation is significantly observed only in presence of bafilomycin A1 ( BafA1 : 100 nM ) . Asterisk indicates a nonspecific band recognized by Atg3 antibody .

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Atg3-knockout MEFs (Atg3−/−) are incapable of forming LC3-II and do not accumulate LC3 puncta. Instead, they accumulate autophagic intermediates that are positive for Atg16. To determine whether the amphipathic helix is critical for in vivo function, Atg3 with wild-type or mutant forms of the amphipathic helix was introduced into Atg3−/− MEFs by lentiviral infection and rescue of the three phenotypes was tested. (a) Immunoblot analysis of LC3-II, GL2-II and GABARAP-II formation. GABARAP lipidation is significantly observed only in the presence of bafilomycin A1 (BafA1: 100 nM). Asterisk indicates a nonspecific band recognized by the Atg3 antibody.


( b ) were infected as in a transduced were selected with puromycin for 1 - 2 days before starvation immunolabelling with anti - LC3B antibody . Representative are shown in

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(b) Cells were infected as in a and transduced cells were selected with puromycin for 1-2 days before starvation and immunolabelling with the anti-LC3B antibody. Representative images are shown in b


( c ) number of LC3 per was counted manually from 22 - 96 per replicate . Raw data can be found in Supplementary Fig . 5 . P values represent difference from vector negative control . n = 2 - 5 independent experiments ; precise n values number of per experiment are listed in Methods .

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(c) The number of LC3 puncta per cell was counted manually from 22-96 cells per replicate. Raw data can be found in Supplementary Fig. 5. P values represent difference from vector negative control. n = 2-5 independent experiments; the precise n values and number of cells per experiment are listed in the Methods.


d ) as in b were kept in full media before being subjected to fixation immunolabelling with anti - Atg16L antibody . Atg16L as a fraction of total was obtained as described in Methods . This was plotted as arbitrary units . Grey dashed indicates value for Atg3 − / − rescued with wild - type Atg3 . n = 2 ( for V15K ) or 3 ( for others ) independent experiments ; precise n values number of fields visualized per experiment are listed in Methods . Raw data can be found in Supplementary Fig . 4 . Atg3 variants that support lipidation of LC3 family in vitro ( Fig . 5 ) are indicated with black bars , while those that do not support lipidation are indicated with bars . P values represent difference from wild - type rescue . * * P 0 . 1 ; * P 0 . 05 . Error bars in c , d represent s . d . for samples of n > 2 . Uncropped versions of gels are included in Supplementary Fig . 7 .

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

d) Cells as in b were kept in full media before being subjected to fixation and immunolabelling with the anti-Atg16L antibody. Atg16L puncta area as a fraction of total cellular area was obtained as described in Methods. This ratio was plotted as arbitrary units. Grey dashed line indicates the value for Atg3−/− cells rescued with wild-type Atg3. n = 2 (for V15K) or 3 (for all others) independent experiments; the precise n values and number of fields visualized per experiment are listed in the Methods. Raw data can be found in Supplementary Fig. 4. Atg3 variants that support lipidation of LC3 family proteins in vitro (Fig. 5) are indicated with black bars, while those that do not support lipidation are indicated with white bars. P values represent difference from wild-type rescue. **P 0.1; *P 0.05. Error bars in c,d represent s.d. for all samples of n > 2. Uncropped versions of all gels are included in Supplementary Fig. 7.


E Reproducibility of 2 independent experiments - correlation of observed changes in expression . Blue indicates linear fit with shaded areas as confidence intervals .

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E Reproducibility of 2 independent experiments - correlation of observed changes in expression. Blue line indicates the linear fit with shaded areas as confidence intervals.


A Number of target genes identified using differential expression for each assayed TF . * genes identified as essential for self - renewal of Hoxb8 - FL either in ( Basilico et al , 2020 ) .

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A Number of target genes identified using differential expression for each assayed TF. *genes identified as essential for self-renewal of Hoxb8-FL cells either in (Basilico et al, 2020).


A A representation of pairwise degree of overlap in targets ( of circle ) correlation in gene expression changes ( colour ) among overlapping targets for indicated TF perturbations . Red indicates positive correlations blue negative correlations .

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A A representation of the pairwise degree of overlap in targets (size of the circle) and correlation in gene expression changes (colour) among overlapping targets for indicated TF perturbations. Red indicates positive correlations and blue negative correlations.


D Identification of target gene modules - groups of genes with common regulatory patterns by TFs . Colour indicates fold change ( adjusted for significance ) of gene expression following each TF perturbation . Rows ( perturbed TFs ) columns ( target genes ) are hierarchically clustered . 44 modules for target genes are shown . Below examples . Selected modules with highlighted overall regulation pattern example genes listed below .

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D Identification of target gene modules - groups of genes with common regulatory patterns by TFs. Colour indicates the fold change (adjusted for significance) of gene expression following each TF perturbation. Rows (perturbed TFs) and columns (target genes) are hierarchically clustered. 44 modules for target genes are shown. Below examples. Selected modules with highlighted overall regulation pattern and example genes listed below.


D Changes in expression for genes co - regulated by separate Cebpa / Meis1 / Spi1 Hoxb8 perturbations ( FDR < 0 . 1 | log2 ( FoldChange ) | > 0 . 2 ) . interaction row indicates changes beyond simple additive effect ( = additive effect ) . Each gene was annotated with an interaction class ( int . class ) as explained in ( C ) , using | log2 ( FoldChange ) | > 0 . 2 ) threshold to assign change direction . E Fractions of genes in each interaction class showed in ( D ) .

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D Changes in expression for genes co-regulated by separate Cebpa/Meis1/Spi1 and Hoxb8 perturbations (FDR <0.1 and |log2(FoldChange)| > 0.2). The interaction row indicates changes beyond simple additive effect (white = additive effect). Each gene was annotated with an interaction class (int. class) as explained in (C), using the |log2(FoldChange)| > 0.2) threshold to assign change direction. E Fractions of genes in each interaction class showed in (D).


A Dice scores summarising overlaps among peaks identified for 14 ChIP experiments in Hoxb8 - FL .

A Dice scores summarising overlaps among peaks identified for 14 ChIP experiments in Hoxb8-FL cells.


B Comparison of genes identified as differentially expressed following TF loss with mapped ChIP peaks in a corresponding experiment . Enrichment over a random value p - value ( on hypergeometric ) are shown ( bars corresponding to non - significant enrichment values are semi - transparent ) . was performed separately for genes up - down - regulated .

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B Comparison of genes identified as differentially expressed following TF loss with mapped ChIP peaks in a corresponding experiment. Enrichment over a random value and the p-value (based on the hypergeometric test) are shown (bars corresponding to non-significant enrichment values are semi-transparent). Analysis was performed separately for genes up- and down-regulated.


C , D Genomic views of relevant TF binding to Cebpa Gata3 loci ( supporting direct regulatory interactions identified in Figs 3A , B , C ) . red arrow indicates a putative enhancer element , which is bound by Ebf1 flanked by AcK27 - rich regions .

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C, D Genomic views of relevant TF binding to Cebpa and Gata3 loci (supporting direct regulatory interactions identified in Figs 3A,B,C). The red arrow indicates a putative enhancer element, which is bound by Ebf1 and flanked by AcK27-rich regions.


C Correlation in gene expression changes following Ebf1 Gata3 loss in Hoxb8 - FL . Each quadrant corresponds to a set of target genes with a common regulation by Gata3 / Ebf1 - I - inhibition / inhibition , II - activation / inhibition , III - activation / activation , IV - inhibition / activation . For each group sums of scaled expression values in mouse LK / LSK or human BMMC landscapes is plotted to highlight types with highest overall expression . For landscape annotation see Fig 7B andE . . Changes in expression for example genes are provided in Appendix Fig 2G - J . Blue indicates linear fit with shaded areas as confidence intervals .

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C Correlation in gene expression changes following Ebf1 and Gata3 loss in Hoxb8-FL cells. Each quadrant corresponds to a set of target genes with a common regulation by Gata3/Ebf1 - I - inhibition/inhibition, II - activation/inhibition, III - activation/activation, IV - inhibition/activation. For each group the sums of scaled expression values in mouse LK/LSK or human BMMC landscapes is plotted to highlight cell types with the highest overall expression. For landscape annotation see Fig 7B andE.. Changes in expression for example genes are provided in Appendix Fig 2G-J. Blue line indicates the linear fit with shaded areas as confidence intervals.


E Correlation in expression changes after 24h following Ebf1 loss in Hoxb8 - FL re - expression of Ebf1 in Ebf1 - / - pre - pro B ( Li et al , 2018 ) . Blue indicates linear fit with shaded areas as confidence intervals .

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E Correlation in expression changes after 24h following Ebf1 loss in Hoxb8-FL cells and re-expression of Ebf1 in Ebf1-/- pre-pro B cells (Li et al, 2018). Blue line indicates the linear fit with shaded areas as confidence intervals.


F , G Expression of key marker genes ( F ) Ebf1 , Pax5 , Gata3 , Cebpa factors ( G ) along pseudotime corresponding to B - differentiation trajectory in human foetal liver , data from ( Popescu et al , 2019 ) .

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F, G Expression of the key marker genes (F) and Ebf1, Pax5, Gata3, Cebpa factors (G) along pseudotime corresponding to the B-cell differentiation trajectory in human foetal liver cells, data from (Popescu et al, 2019).


D . Relative FCR activities in roots of wild - type photoreceptor mutants grown under with either Fe - sufficient ( 20 µM , + Fe ) or Fe - deficient ( 2 µM , - Fe ) conditions for 7 d .

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D. Relative FCR activities in the roots of wild-type plants and photoreceptor mutants grown under white light with either Fe-sufficient (20 µM, +Fe) or Fe-deficient (2 µM, -Fe) conditions for 7 d.


D - F . Fe contents of whole , FER expression in roots relative FCR activities in roots of grown under Fe - sufficient ( 20 µM , + Fe ) or Fe - deficient ( 2 µM , - Fe ) conditions for 7 d . were exposed to high ( H - , 2 . 0 ) or low ( L - , 0 . 5 ) red ( R ) to far red ( FR ) ratios .

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D-F. Fe contents of whole plants, FER expression in roots and relative FCR activities in the roots of plants grown under Fe-sufficient (20 µM, +Fe) or Fe-deficient (2 µM, -Fe) conditions for 7 d. Plants were exposed to high (H-, 2.0) or low (L-, 0.5) red (R) to far red (FR) ratios.


C . Accumulation of HY5 in leaves roots of reciprocal grafts of phyB WT grown under ( 200 µmol m - 2s - 1 ) for 7 d . Rubisco β - Actin were used as loading controls for leaves roots in western blot , respectively . values shown above each lane indicate relative abundance of HY5 . D . Accumulation of HY5 in roots of WT phyB under high ( H , 2 . 0 ) or low of ( L , 0 . 5 ) red ( R ) to far red ( FR ) conditions . β - Actin was used as loading controls in western blot . values shown above each lane indicate relative abundance of HY5 .

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C. Accumulation of HY5 protein in leaves and roots of reciprocal grafts of phyB and WT plants grown under white light (200 µmol m-2s-1) for 7 d. Rubisco and β-Actin were used as loading controls for leaves and roots in the western blot analysis, respectively. The values shown above each lane indicate the relative abundance of the HY5 protein. D. Accumulation of HY5 protein in the roots of WT and phyB plants under high (H, 2.0) or low ratio of (L, 0.5) red (R) to far red (FR) light conditions. β-Actin was used as loading controls in the western blot analysis. The values shown above each lane indicate the relative abundance of the HY5 protein.


D . Fe content of whole . WT hy5 were grown under high ( H ) or low ratios of ( L ) red ( R ) to far red ( FR ) conditions with either Fe - sufficient ( 20 µM , + Fe ) or Fe - deficient ( 2 µM , - Fe ) conditions for 7 d .

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D. Fe content of whole plants. WT and hy5 plants were grown under high (H) or low ratios of (L) red (R) to far red (FR) light conditions with either Fe-sufficient (20 µM, +Fe) or Fe-deficient (2 µM, -Fe) conditions for 7 d.


E . FER expression in roots . WT hy5 were grown under high ( H ) or low ratios of ( L ) red ( R ) to far red ( FR ) conditions with either Fe - sufficient ( 20 µM , + Fe ) or Fe - deficient ( 2 µM , - Fe ) conditions for 7 d .

E. FER expression in roots. WT and hy5 plants were grown under high (H) or low ratios of (L) red (R) to far red (FR) light conditions with either Fe-sufficient (20 µM, +Fe) or Fe-deficient (2 µM, -Fe) conditions for 7 d.


E . Direct binding of HY5 to FER promoter analyzed using ChIP - qPCR in 35S - HY5 - 3HA - overexpressing ( HY5 - OE ) tomato . WT HY5 - OE were exposed to ( 200 µmol m - 2s - 1 ) grown under Fe - deficient ( 2 µM , - Fe ) conditions for 7 d . Input was isolated from root samples at 7 d . epitope - tagged HY5 - was immunoprecipitated with an anti - HA antibody . A control reaction was processed side - by - side using mouse IgG . Input - ChIP - samples were quantified by RT - qPCR using primers specific for promoter exon fragment of FER gene as indicated in ( D ) . ChIP results are presented as of input .

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E. Direct binding of HY5 to the FER promoter analyzed using ChIP-qPCR in 35S-HY5-3HA-overexpressing (HY5-OE) tomato plants. WT and HY5-OE plants were exposed to white light (200 µmol m-2s-1) grown under Fe-deficient (2 µM, -Fe) conditions for 7 d. Input chromatin was isolated from root samples at 7 d. The epitope-tagged HY5-chromatin complex was immunoprecipitated with an anti-HA antibody. A control reaction was processed side-by-side using mouse IgG. Input- and ChIP-DNA samples were quantified by RT-qPCR using primers specific for the promoter and exon fragment of the FER gene as indicated in (D). The ChIP results are presented as percentage of the input DNA.


A - C . Fe content of whole ( A ) photochemical efficiencies of PSI PSII [ Y ( I ) Y ( II ) ] ( B C ) in young leaves of grafted tomato with grafts between WT / WT WT / fer grown under H - R / FR ( 2 . 0 ) or L - R / FR ( 0 . 5 ) conditions for 7 d .

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A-C. Fe content of whole plants (A) and the photochemical efficiencies of PSI and PSII [Y(I) and Y(II)] (B and C) in young leaves of grafted tomato plants with grafts between WT/WT and WT/fer grown under H-R/FR (2.0) or L-R/FR (0.5) conditions for 7 d.


D - F . Transcript of FER , FRO1 IRT1 in roots of grafted tomato with grafts between WT / WT WT / fer grown under H - R / FR ( 2 . 0 ) or L - R / FR ( 0 . 5 ) conditions for 7 d .

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D-F. Transcript levels of FER, FRO1 and IRT1 in the roots of grafted tomato plants with grafts between WT/WT and WT/fer grown under H-R/FR (2.0) or L-R/FR (0.5) conditions for 7 d.


B . abundance of FER transcripts in roots of WT phyB grown under 20 μM Fe - EDTA in nutrient solution in a growth room .

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B. The abundance of FER transcripts in the roots of WT and phyB plants grown under 20 μM Fe-EDTA in nutrient solution in a growth room.


D . Fe content in fruits of WT phyB . were grown in soil in a greenhouse .

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D. Fe content in the fruits of WT and phyB plants. Plants were grown in soil in a greenhouse.


1D 1H - NMR spectra of a metabolite mix metabolites contained therein . Identification of single compound peaks from 1D 1H - NMR spectra of a metabolite mix . Compound detection is exemplified by showing sections of 1D 1H - NMR spectra of ATP , IMP , proline ( PRO ) , malate ( MAL ) , shikimate ( SKM ) methionine ( MET ) .

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1D 1H-NMR spectra of a metabolite mix and the individual metabolites contained therein. Identification of single compound peaks from 1D 1H-NMR spectra of a metabolite mix. Compound detection is exemplified by showing sections of the 1D 1H-NMR spectra of ATP, IMP, proline (PRO), malate (MAL), shikimate (SKM) and methionine (MET).


Relative activity of NADP + - dependent glucose - 6 - phosphate dehydrogenase in presence of potential regulators , error bars represent s . e . m . . regulators were tested at two concentrations in four distinct replicates ( n = 4 , ATP : 5mM , 18 mM ; GTP : 2 mM , 10 mM ; IMP AMP : 1 mM , 5 mM ) , asterisk denotes significant inhibition ( 18 mM ATP : p - value = 0 . 029 , 10 mM GTP : p - value = 0 . 025 , one - t - ) .

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Relative activity of NADP+-dependent glucose-6-phosphate dehydrogenase in the presence of potential regulators, error bars represent the s.e.m.. All regulators were tested at two concentrations in four distinct replicates (n=4, ATP: 5mM, 18 mM; GTP: 2 mM, 10 mM; IMP and AMP: 1 mM, 5 mM), the asterisk denotes significant inhibition (18 mM ATP: p-value = 0.029, 10 mM GTP: p-value = 0.025, one-tailed t-test).


Relative activity of phosphate acetyltransferase in presence of phenylpyruvate tryptophan , error bars represent s . e . m . . regulators were tested at three concentrations in three distinct replicates ( n = 3 , phenylpyruvate : 0 . 1 , 1 , 5 mM , L - tryptophan : 0 . 1 , 1 , 4 . 4 mM ) .

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Relative activity of phosphate acetyltransferase in the presence of phenylpyruvate and tryptophan, error bars represent the s.e.m.. All regulators were tested at three concentrations in three distinct replicates (n=3, phenylpyruvate: 0.1, 1, 5 mM, L-tryptophan: 0.1, 1, 4.4 mM).


Relative activity of fructose - bisphosphate aldolase class II in presence of 5 mM of potential regulators , except hypoxanthine , which was tested at 3 . 22 mM . regulators were tested in three distinct replicates ( n = 3 ) , error bars represent s . e . m . . asterisk denotes significant activation ( 5 mM 3PG : p - value = 0 . 025 , 5 mM ATP : p - value = 0 . 024 , 5 mM PEP : p - value = 0 . 046 , one - t - ) .

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Relative activity of fructose-bisphosphate aldolase class II in the presence of 5 mM of potential regulators, except hypoxanthine, which was tested at 3.22 mM. All regulators were tested in three distinct replicates (n=3), error bars represent the s.e.m.. The asterisk denotes significant activation (5 mM 3PG: p-value = 0.025, 5 mM ATP: p-value = 0.024, 5 mM PEP: p-value = 0.046, one-tailed t-test).


B C99 - Bpa constructs remain essentially cleavage - competent . Numbers denote relative γ - secretase cleavage efficiencies of C99 - Bpa substrates as calculated from AICD / C99 - Bpa ratios relative to that of WT C99 , which was set to 1 . Inefficiently substrates ( cleavage efficiency below 20 % ) were mostly found for Bpa substitutions in extramembraneous domains . Specificity of substrate cleavage was confirmed by inhibition of AICD formation in presence of L - 685 , 458 . Residues of TMD are highlighted in orange here elsewhere where appropriate . C Values obtained in ( B ) were additionally plotted . Yellow indicates cleavage efficiency of WT C99 , which was set to 1 . ECD , extracellular domain ; ICD , intracellular domain .

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B C99-Bpa constructs remain essentially cleavage-competent. Numbers denote the relative γ-secretase cleavage efficiencies of C99-Bpa substrates as calculated from the AICD/C99-Bpa ratios relative to that of WT C99, which was set to 1. Inefficiently cleaved substrates (cleavage efficiency below 20%) were mostly found for Bpa substitutions in extramembraneous domains. Specificity of substrate cleavage was confirmed by inhibition of AICD formation in the presence of L-685,458. Residues of the TMD are highlighted in orange here and elsewhere where appropriate.C Values obtained in (B) were additionally plotted. Yellow line indicates the cleavage efficiency of WT C99, which was set to 1. ECD, extracellular domain; ICD, intracellular domain.


B Identification of C99 interaction sites with γ - secretase . C99 - Bpa substrates were irradiated with UV in presence of CHAPSO - solubilized γ - secretase . Crosslinked substrates were captured by Ni - NTA affinity pulldown bound subunits were identified by a ~ 10 kDa molecular increase compared to input . Results are shown for major substrate crosslinking residues . Crosslink formation was not observed in absence of UV irradiation or in presence of Triton X - 100 , which dissociates γ - secretase , proving crosslink specificity . Bracket indicates molecular range of putative C99 - APH - 1 crosslink bands . APH - 1aL , long splice variant of APH - 1a .

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B Identification of C99 interaction sites with γ-secretase. C99-Bpa substrates were irradiated with UV light in the presence of CHAPSO-solubilized γ-secretase. Crosslinked substrates were captured by Ni-NTA affinity pulldown and bound subunits were identified by a ~10 kDa molecular weight increase compared to the input. Results are shown for the major substrate crosslinking residues. Crosslink formation was not observed in the absence of UV irradiation or in the presence of Triton X-100, which dissociates the γ-secretase complex, proving crosslink specificity. Bracket indicates the molecular weight range of putative C99-APH-1 crosslink bands. APH-1aL, long splice variant of APH-1a.


C Excess amounts of parental C99 WT substrate compete crosslinking of C99 - Bpa substrates to γ - secretase . Single double asterisks indicate antibody crossreactivities to C99 monomer dimer , respectively .

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C Excess amounts of parental C99 WT substrate compete crosslinking of C99-Bpa substrates to γ-secretase. Single and double asterisks indicate antibody crossreactivities to C99 monomer and dimer, respectively.


Quantitative crosslinking revealed V44 , L49 , M51 L52 as major interactions sites of C99 with γ - secretase with high crosslinking efficiencies ranging from ~ 2 - 8 % of input . While PS1 NTF was bound by C99 over nearly whole range of residues , only very few residues bound to PS1 CTF . Several residues in substrate N - terminal extracellular domain bound to NCT . PEN - 2 - binding residues clustered in - flanking extracellular intracellular domains of C99 .

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Quantitative crosslinking analysis revealed V44, L49, M51 and L52 as major interactions sites of C99 with γ-secretase with high crosslinking efficiencies ranging from ~2-8% of input. While the PS1 NTF was bound by C99 over nearly the whole range of residues, only very few residues bound to PS1 CTF. Several residues in the substrate N-terminal extracellular domain bound to NCT. PEN-2-binding residues clustered in the membrane-flanking extracellular and intracellular domains of C99.


A Inhibition of substrate crosslinking by GSIs identifies substrate residues binding in active site . L , L - 685 , 458 ; D , DAPT .

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A Inhibition of substrate crosslinking by the GSIs identifies substrate residues binding in the active site. L, L-685,458; D, DAPT.


B Preventing access to active site by GSIs identifies substrate residues interacting with exosites by their increased labeling .

B Preventing access to the active site by GSIs identifies substrate residues interacting with the exosites by their increased labeling intensity.


A PS1 FAD mutations display little or no effect on substrate interactions at exosites . Asterisks indicate antibody crossreactivities to C99 monomer .

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A PS1 FAD mutations display little or no effect on substrate interactions at the exosites. Asterisks indicate antibody crossreactivities to C99 monomer.


B PS1 FAD mutations alter substrate binding to PS1 NTF in γ - secretase cleavage domain . weak PS1 A246E FAD mutation alters substrate interactions predominantly at V49 L52 , whereas highly pathogenic PS1 L166P FAD mutation causes more broad changes over whole cleavage domain . Arrows denote changes in binding of residues . Corresponding quantitation of substrate binding is shown below immunoblots . Bars denote mean ± S . E . ( n = 3 ) . Yellow indicates level of substrate binding to PS1 WT , which was set to 1 . Blue red bars indicate increases or decreases in binding , respectively .

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B PS1 FAD mutations alter substrate binding to the PS1 NTF in the γ-secretase cleavage domain. The weak PS1 A246E FAD mutation alters substrate interactions predominantly at V49 and L52, whereas the highly pathogenic PS1 L166P FAD mutation causes more broad changes over the whole cleavage domain. Arrows denote changes in binding of individual residues. Corresponding quantitation of substrate binding is shown below the immunoblots. Bars denote the mean ± S.E. (n = 3). Yellow line indicates level of substrate binding to PS1 WT, which was set to 1. Blue and red bars indicate increases or decreases in binding, respectively.


C Both mutants decrease crosslinking to PS1 CTF .

C Both mutants decrease crosslinking to the PS1 CTF.


B Substrates bound to exosites in PS1 NTF at 4 ° C can be in 37 ° C chase incubation in a L - 685 , 458 - dependent manner . Note that , although many of these substrates crosslinked only weakly at exosites , their cleavage in 37 ° C chase proved that these reflected functional substrate - binding states . C Substrates crosslinking in exosites to NCT PEN - 2 cannot be .

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B Substrates bound to exosites in the PS1 NTF at 4°C can be cleaved in the 37°C chase incubation in a L-685,458-dependent manner. Note that, although many of these substrates crosslinked only weakly at the exosites, their cleavage in the 37°C chase proved that these reflected functional substrate-binding states.C Substrates crosslinking in the exosites to NCT and PEN-2 cannot be cleaved.


D , E Substrates can be when crosslinked to PS1 NTF but not when crosslinked to NCT ( D ) or PEN - 2 ( E ) .

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D, E Substrates can be cleaved when crosslinked to PS1 NTF but not when crosslinked to NCT (D) or PEN-2 (E).


F Excess amounts of Notch1 substrate N102 - FmH compete with exosite - binding of C99 . See Fig EV4 for competition of C99 binding in active site region .

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F Excess amounts of Notch1 substrate N102-FmH compete with exosite-binding of C99. See Fig EV4 for competition of C99 binding in the active site region.


B Representative confocal microscopy of human PBMCs stained with DAPI ( arrows ) co - cultured with previously labeled UC - MSC with MitoTracker - Green ( top image ) specific antibody for human CD90 ( bottom image ) .

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B Representative confocal microscopy of human PBMCs stained with DAPI (white arrows) co-cultured with previously labeled UC-MSC with MitoTracker-Green (top image) and specific antibody for human CD90 (bottom image).


C of mitochondrial transfer ( MitoT ) on total human CD45 + after 24 hrs co - culture with MitoTracker - Green labeled bone marrow ( BM ) , menstrual ( Mens ) or umbilical cord ( UC ) - MSCs ( MSC : PBMC 1 : 25 ) ( n = 3 biological replicates , with 3 different donors of BM , Mens UC - MSCs ) .

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C Percentage of mitochondrial transfer (MitoT) on total human CD45+ cells after 24 hrs co-culture with MitoTracker-Green labeled bone marrow (BM), menstrual (Mens) or umbilical cord (UC)-MSCs (MSC:PBMC ratio 1:25) (n=3 biological replicates, with 3 different donors of BM, Mens and UC-MSCs).


D FACS of MitoT on T CD3 + ( n = 4 ) , B CD19 + ( n = 4 ) natural killer ( NK ) CD56 + ( n = 3 ) after 24 hrs co - culture with MitoGreen labeled UC - MSCs ( 1 : 25 ) ( Biological replicates , with different donors of UC - MSCs ) .

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D FACS analysis of MitoT on T CD3+ (n=4), B CD19+ (n=4) and natural killer (NK) CD56+ (n=3) cells after 24 hrs co-culture with MitoGreen labeled UC-MSCs (ratio 1:25) (Biological replicates, with different donors of UC-MSCs).


E FACS of MitoT on CD3 + CD4 + or CD3 + CD8 + T populations after 24 hrs co - culture with labeled UC - MSCs ( 1 : 25 ) ( n = 3 biological replicates , with 3 different donors of UC - MSCs ) .

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E FACS analysis of MitoT on CD3+CD4+ or CD3+CD8+ T cell populations after 24 hrs co-culture with labeled UC-MSCs (ratio 1:25) (n=3 biological replicates, with 3 different donors of UC-MSCs).


F Representative FACS plots of MitoT on CD45 + CD3 + T at increasing amounts of UC - MSC .

F Representative FACS plots of MitoT on CD45+CD3+ T cells at increasing amounts of UC-MSC.


G of MitoT on CD45 + CD3 + human after 24 hrs co - culture with increasing ratios of UC - MSC : PBMCs ( n = 5 biological replicates , with 5 different UC - MSCs 5 different PBMCs ) .

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G Percentage of MitoT on CD45+CD3+ human cells after 24 hrs co-culture with increasing ratios of UC-MSC:PBMCs (n=5 biological replicates, with 5 different UC-MSCs and 5 different PBMCs).


H FACS of MitoT on CD45 + CD3 + T after short times of co - culture with UC - MSCs at two different temperatures , at a 1 : 25 ( n = 3 biological replicates , with 3 different UC - MSCs 3 different PBMCs ) .

H FACS analysis of MitoT on CD45+CD3+ T cells after short times of co-culture with UC-MSCs at two different temperatures, at a ratio 1:25 (n=3 biological replicates, with 3 different UC-MSCs and 3 different PBMCs).


I Mean ( MFI ) of MitoTracker - Green + UC - MSCs at baseline or after 72 hours PBMC co - culture . Representative FACS plot ( left panel ) average of MFI ( n = 4 biological replicates , with 4 different UC - MSCs ) ( right panel ) .

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I Mean fluorescence intensity (MFI) of MitoTracker-Green+ UC-MSCs at baseline or after 72 hours PBMC co-culture. Representative FACS plot (left panel) and average of MFI (n=4 biological replicates, with 4 different UC-MSCs) (right panel).


J Confocal microscopy of human PBMCs after co - culture with MitoTracker - Green stained UC - MSCs . showed MSC - mitochondria ( green ) inside CD3 + T ( red ) .

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J Confocal microscopy of human PBMCs after co-culture with MitoTracker-Green stained UC-MSCs. Images showed MSC-mitochondria (green) inside the CD3+ T cells (red).


K qPCR of human β2 - microglobulin ( B2M ) , human specific mitochondrial ( Mito ) mouse specific ( β - actin ) gene expression in UC - MSCs , mouse derived spleen or FACS sorted CD45 + mouse after 24 hrs co - culture with UC - MSCs ( n = 3 biological replicates ) . n / d = not detected .

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K qPCR analysis of human β2-microglobulin (B2M), human specific mitochondrial (Mito) and mouse specific (β-actin) gene expression levels in UC-MSCs, mouse derived spleen cells or FACS sorted CD45+ mouse cells after 24 hrs co-culture with UC-MSCs (n=3 biological replicates). n/d = not detected.


M FACS of in vivo MitoT from UC - MSCs to Balb / c mouse mesenteric lymph node spleen , 24 hours after intraperitoneal transplantation of 5 * 106 MitoTracker - labeled MSCs or without ( control ) ( n = 1 ) .

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M FACS analysis of in vivo MitoT from UC-MSCs to Balb/c mouse mesenteric lymph node and spleen cells, 24 hours after intraperitoneal transplantation of 5*106 MitoTracker-labeled MSCs or without cells (control) (n=1).


B Confocal microscopy of human CD3 + T with endogenous MT - labeled with MitoTracker Red CMXRos ( arrows , left panel ) or after mitoception with MitoTracker Green labeled - MT from UC - MSCs ( asterisk , right panel ) .

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B Confocal microscopy images of human CD3+ T cells with endogenous MT-labeled with MitoTracker Red CMXRos (white arrows, left panel) or after mitoception with MitoTracker Green labeled-MT from UC-MSCs (white asterisk, right panel).


C FACS of MitoT on CD3 + human T after mitoception with increasing amounts of UC - MSC MT corresponding to same number ratios used in co - culture experiments ( n = 4 biological replicates , with 4 different donors of UC - MSCs ) . Graph shows mean ± statistical by Student ' s t - .

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C FACS analysis of MitoT on CD3+ human T cells after mitoception with increasing amounts of UC-MSC MT corresponding to the same cell number ratios used in co-culture experiments (n=4 biological replicates, with 4 different donors of UC-MSCs). Graph shows mean ± SEM and statistical analysis by Student's t-test.


D Representative transmission electron microscopy of FACS - sorted human CD3 + MitoTneg CD3 + MitoTpos following mitoception with UC - MSC MT . Red arrows show human mitochondria .

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D Representative transmission electron microscopy images of FACS-sorted human CD3+MitoTneg and CD3+MitoTpos cells following mitoception with UC-MSC MT. Red arrows show human mitochondria.


of human MT in sorted CD3 + MitoTneg MitoTpos after mitoception with UC - MSCs , by transmission electron microscopy ( n = 25 independent analyzed per group ) . Graph shows mean ± statistical by Student ' s t - .

Quantification of human MT in sorted CD3+ MitoTneg and MitoTpos cells after mitoception with UC-MSCs, by transmission electron microscopy (n=25 independent cells analyzed per group). Graph shows mean ± SEM and statistical analysis by Student's t-test.


B Volcano plot derived from FPKM values showing DE genes ( p < 0 . 05 , Student ' s t ) in sorted MitoTneg MitoTpos CD3 + ( from 4 different donors of PBMCs ) .

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B Volcano plot analysis derived from FPKM values showing DE genes (p<0.05, Student's t test) in sorted MitoTneg and MitoTpos CD3+ cells (from 4 different donors of PBMCs).


C depiction of - seq of significantly ( p < 0 . 01 , Student ' s t ) DE coding genes from sorted MitoTpos MitoTneg CD3 + ( from 4 different donors of PBMCs ) .

C Heatmap depiction of RNA-seq analysis of significantly (p<0.01, Student's t test) DE coding genes from sorted MitoTpos and MitoTneg CD3+ cells (from 4 different donors of PBMCs).


E Differentially expressed genes from significantly enriched pathways listed in figure 2D . Highlighted in red are key markers of Treg phenotype .

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E Differentially expressed genes from the significantly enriched pathways listed in figure 2D. Highlighted in red are key markers of Treg phenotype.


F qRT - PCR of genes related to T activation after mitoception with UC - MSC MT ( n = 4 biological replicates , with 4 different donors of PBMCs ) . graph depicts fold expression in CD3 + MitoTpos relative to CD3 + MitoTneg , which are set at 1 . Graph shows mean ± statistical by Student ' s t - ( * p < 0 . 05 ) .

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F qRT-PCR analysis of genes related to T cell activation after mitoception with UC-MSC MT (n=4 biological replicates, with 4 different donors of PBMCs). The graph depicts fold expression in CD3+MitoTpos cells relative to CD3+MitoTneg cells, which are set at 1. Graph shows mean ± SEM and statistical analysis by Student's t-test (*p<0.05).


A Representative bright - field microscopy of FACS - sorted CD4 + naïve MitoTneg ( upper panel ) MitoTpos ( lower panel ) after 5 days in culture with plain media ( no cytokines added ) . Red arrows are indicative of activation / proliferation clusters .

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A Representative bright-field microscopy images of FACS-sorted CD4+ naïve MitoTneg (upper panel) and MitoTpos (lower panel) cells after 5 days in culture with plain media (no cytokines added). Red arrows are indicative of activation/proliferation clusters.


B Representative FACS plots of Treg ( CD127lowCD25 + FoxP3 + ) populations after T differentiation on media with or without cytokines antibody supplements ( anti - CD3 , IL - 2 TGF - b1 ) .

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B Representative FACS plots of Treg (CD127lowCD25+FoxP3+) populations after T cell differentiation on media with or without cytokines and antibody supplements (anti-CD3, IL-2 and TGF-b1).


C FACS of Treg induction after 5 - 7 days differentiation of FACS - sorted CD4 + naïve MitoTneg MitoTpos in plain ( n = 5 ) or differentiation media ( n = 6 ) ( Biological replicates , with different donors of PBMCs ) .

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C FACS analysis of Treg induction after 5-7 days differentiation of FACS-sorted CD4+ naïve MitoTneg and MitoTpos cells in plain (n=5) or differentiation media (n=6) (Biological replicates, with different donors of PBMCs).


D ICOS expression on FACS - sorted CD4 + naïve MitoTneg MitoTpos after 5 - 7 days in culture with differentiation media . Representative FACS plot ( left panel ) bar graph ( right panel ) of ICOS high population ( n = 4 biological replicates ) .

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D ICOS expression on FACS-sorted CD4+ naïve MitoTneg and MitoTpos cells after 5-7 days in culture with differentiation media. Representative FACS plot (left panel) and bar graph (right panel) of ICOS high population (n=4 biological replicates).


E CCR7 expression on sorted CD4 + naïve MitoTneg MitoTpos after 5 - 7 days in culture with differentiation media . Representative FACS plot ( left panel ) bar graph ( right panel ) of CCR7 + population ( n = 4 biological replicates ) .

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E CCR7 expression on sorted CD4+ naïve MitoTneg and MitoTpos cells after 5-7 days in culture with differentiation media. Representative FACS plot (left panel) and bar graph (right panel) of CCR7+ population (n=4 biological replicates).


F FACS of Th1 ( CD4 + IFNγ + ) induction after 5 - 7 days differentiation of FACS - sorted CD4 + naïve MitoTneg MitoTpos in plain media ( n = 3 biological replicates ) . n / s = not significant .

F FACS analysis of Th1 (CD4+IFNγ+) induction after 5-7 days differentiation of FACS-sorted CD4+ naïve MitoTneg and MitoTpos cells in plain media (n=3 biological replicates). n/s= not significant.


Representative proliferation FACS histogram of CTV - stained PBMC co - cultured with MitoTneg or MitoTpos ( 1 : 1 ) after 5 - 7 days of CD4 + naïve activation with Treg induction / differentiation media ( anti - CD3 , IL - 2 TGF - b1 ) . PBMC alone represents positive control of proliferation PBMC co - culture with MitoTneg correspond to inhibition control with induced Treg .

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Representative proliferation FACS histogram of CTV-stained PBMC co-cultured with MitoTneg or MitoTpos cells (ratio 1:1) after 5-7 days of CD4+ naïve cell activation with Treg induction/differentiation media (anti-CD3, IL-2 and TGF-b1). PBMC alone represents positive control of proliferation and PBMC co-culture with MitoTneg correspond to the inhibition control with induced Treg cells.


C of immunosuppression of CTV - stained PBMC co - cultured with MitoTneg or MitoTpos after 5 - 7 days of CD4 + naïve activation with differentiation media ( MitoT : PBMC ) ( n = 3 biological replicates , with different donors of PBMCs ) . Graph shows mean ± statistical by Student ' s t - .

C Percentage of immunosuppression of CTV-stained PBMC co-cultured with MitoTneg or MitoTpos cells after 5-7 days of CD4+ naïve cell activation with differentiation media (ratio MitoT cells:PBMC) (n=3 biological replicates, with different donors of PBMCs). Graph shows mean ± SEM and statistical analysis by Student's t-test.