Figure 4percentage of mAb - positive live Pb - PfCSP sporozoites ( C ) for the junction cross - reactive mAb 1961 , C - linker - specific mAb 3764 and the control mAbs 2A10 and mGO53 at the indicated concentrations ( n = 3 - 4 ) . Representative mean fluorescence intensity ( MFI ) ( D ) for the junction cross - reactive mAb 1961 , C - linker - specific mAb 3764 and the control mAbs 2A10 and mGO53 at the indicated concentrations ( n = 3 - 4 ) . ( E ) Pf sporozoite hepatocyte traversal inhibitory capacity of the indicated C - CSP reactive , C - CSP cross - reactive ( 1961 ) , and control ( 2A10 , mGO53 and anti - NANP 317 mAbs at the indicated concentrations ( n = 3 - 6 ) . ( F ) Capacity of the indicated passively - transferred antibodies ( 100 μg ) to protect mice from parasitemia after the bite of three PbPfCSP ( mCherry ) - infected mosquitoes ( n = 10 for mAb 317 , n = 8 for mAb 1961 , and n = 9 for mAb 1710 Data show the percentage of parasite - free mice in two independent experiments ( only mice with detectable serum IgG concentrations of the indicated mAbs at the time of the challenge are shown Groups marked with the same letter were not statistically significantly

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Figure 4percentage of mAb-positive live Pb-PfCSP sporozoites (C) for the junction cross-reactive mAb 1961, C-linker-specific mAb 3764 and the control mAbs 2A10 and mGO53 at the indicated concentrations (n=3-4).Representative mean fluorescence intensity (MFI) (D) for the junction cross-reactive mAb 1961, C-linker-specific mAb 3764 and the control mAbs 2A10 and mGO53 at the indicated concentrations (n=3-4).(E) Pf sporozoite hepatocyte traversal inhibitory capacity of the indicated C-CSP reactive, C-CSP cross-reactive (1961), and control (2A10, mGO53 and anti-NANP 317 mAbs at the indicated concentrations (n=3-6).(F) Capacity of the indicated passively-transferred antibodies (100 μg) to protect mice from parasitemia after the bite of three PbPfCSP(mCherry)-infected mosquitoes (n=10 for mAb 317 , n=8 for mAb 1961, and n=9 for mAb 1710 Data show the percentage of parasite-free mice in two independent experiments (only mice with detectable serum IgG concentrations of the indicated mAbs at the time of the challenge are shown Groups marked with the same letter were not statistically significantly different


Figure 5 ( A ) Crystal structure showing the variable regions of the C - linker specific mAb 3764 and P2 , with the mAb 3764 heavy chain illustrated in green and the kappa chain in blue . HCDR1 , 2 and 3 ( dark , pea and light green , respectively ) as well as KCDR1 and 3 ( dark teal and aqua , respectively ) contribute to epitope recognition , but KCDR2 ( teal ) does not . The central DPN core ( deep blue ) of the C - CSP peptide ( magenta ) interacts with HCDR2 and 3 similarly to other VH3 - 33 antibodies ( inset I ) , forming hydrogen bonds ( H - bonds , shown with black dotted lines ) with HCDR2 and 3 . C - CSP residues M285 ( inset II ) , N287 ( inset III ) and R291 ( inset IV ) contribute substantial buried surface area ( BSA ) to the complex . ( II ) M285 does not contribute any H - bonds , but does contribute to BSA . N287 ( III ) and R291 ( IV ) are responsible for hydrogen bonding with HCDR1 or HCDR3 , KCDR1 and KCDR3 respectively .

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Figure 5(A) Crystal structure showing the variable regions of the C-linker specific mAb 3764 and P2, with the mAb 3764 heavy chain illustrated in green and the kappa chain in blue. HCDR1, 2 and 3 (dark, pea and light green, respectively) as well as KCDR1 and 3 (dark teal and aqua, respectively) contribute to epitope recognition, but KCDR2 (teal) does not. The central DPN core (deep blue) of the C-CSP peptide (magenta) interacts with HCDR2 and 3 similarly to other VH3-33 antibodies (inset I), forming hydrogen bonds (H-bonds, shown with black dotted lines) with HCDR2 and 3. C-CSP residues M285 (inset II), N287 (inset III) and R291 (inset IV) contribute substantial buried surface area (BSA) to the complex. (II) M285 does not contribute any H-bonds, but does contribute to BSA. N287 (III) and R291 (IV) are responsible for hydrogen bonding with HCDR1 or HCDR3, KCDR1 and KCDR3 respectively.


Figure 2C Percentage of BrdU positive cb9Δmyc cells , in the presence or absence ( 24h after removal ) of doxycycline . Data are presented as mean ± SD ; n = 3 . Two - tailed Student t - test was used to compare between two groups and expressed as p - values . D Immunoblot analysis of c - myc - / - HO15 . 19 rat fibroblasts infected with retroviral vectors expressing the indicated Myc proteins . Parental TGR1 cells serve as control for endogenous levels of the MycWT protein . F Cumulative cell counts for the same cells as in ( D ) . Data are presented as mean ± SD ; n = 3 ( biological replicates ) . Two - tailed Student t - test was used to compare between two groups and expressed as p - values .

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Figure 2C Percentage of BrdU positive cb9Δmyc cells, in the presence or absence (24h after removal) of doxycycline. Data are presented as mean ± SD; n = 3. Two-tailed Student t-test was used to compare between two groups and expressed as p-values.D Immunoblot analysis of c-myc-/- HO15.19 rat fibroblasts infected with retroviral vectors expressing the indicated Myc proteins. Parental TGR1 cells serve as control for endogenous levels of the MycWT protein.F Cumulative cell counts for the same cells as in (D). Data are presented as mean ± SD; n = 3 (biological replicates). Two-tailed Student t-test was used to compare between two groups and expressed as p-values.


Figure 3C ChIP - seq intensities for peaks ( promoters + distal ) containing the indicated motifs in an interval of ± 100 bp around their summit . For most accurate quantitation , intensities were computed within ± 50 bp from the summit . p - values were calculated using Wilcoxon ' s test . The central band in the boxplot represents the median of the data , boxes the lower and upper quartiles ( 25 % and 75 % ) and whiskers the minimum and maximum values .

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Figure 3C ChIP-seq intensities for peaks (promoters + distal) containing the indicated motifs in an interval of ±100 bp around their summit. For most accurate quantitation, intensities were computed within ± 50 bp from the summit. p-values were calculated using Wilcoxon's test. The central band in the boxplot represents the median of the data, boxes the lower and upper quartiles (25% and 75%) and whiskers the minimum and maximum values.


Figure 4A 3T9 fibroblasts expressing the indicated MycER proteins and control cells ( EV ) were treated with OHT ( 4h ) and subjected to biochemical fractionation in three independent experiments . The quantity of MycER protein in the different fractions was quantified by western blotting ( shown in Fig . EV4A ) and the relative proportion in the various fractions plotted as average and standard deviation .

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Figure 4A 3T9 fibroblasts expressing the indicated MycER proteins and control cells (EV) were treated with OHT (4h) and subjected to biochemical fractionation in three independent experiments. The quantity of MycER protein in the different fractions was quantified by western blotting (shown in Fig. EV4A) and the relative proportion in the various fractions plotted as average and standard deviation.


Figure 5F Percentage of promoters with the indicated DNA motifs under the ChIP - seq peak ( ± 100 bp from the peak summit ) within each regulatory class ( no DEG , UP or DOWN ) for either MycERWT ( left ) or MycERHEA ( right ) . p - values calculated with Fisher ' s exact test .

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Figure 5F Percentage of promoters with the indicated DNA motifs under the ChIP-seq peak (±100 bp from the peak summit) within each regulatory class (no DEG, UP or DOWN) for either MycERWT (left) or MycERHEA (right). p-values calculated with Fisher's exact test.


Figure 1A . 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 the holoElongator complex . Particles in side and tilted views are highlighted . Scale bar , 50 nm . C . Reference - free class averages and back - projections of the holoElongator model . Scale bar , 20 nm . 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 .

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Figure 1A. 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 the holoElongator complex. Particles in side and tilted views are highlighted. Scale bar, 50 nm.C. Reference-free class averages and back-projections of the holoElongator model. Scale bar, 20 nm.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.


Figure 2A . 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 the Elp123 sub - complex . Particles in side and top views are highlighted . Scale bar , 50 nm . C . Reference - free class averages and back - projections of the Elp123 model . Scale bar , 20 nm . 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 .

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Figure 2A. 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 the Elp123 sub-complex. Particles in side and top views are highlighted. Scale bar, 50 nm.C. Reference-free class averages and back-projections of the Elp123 model. Scale bar, 20 nm.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.


Figure 4A . The model of the Elp123 sub - complex . 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 . C . The model of holoElongator . D . Crosslinks supporting the orientation of the Elp456 hexamer .

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Figure 4A. The model of the Elp123 sub-complex.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.C. The model of holoElongator.D. Crosslinks supporting the orientation of the Elp456 hexamer.


Figure 5A . 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 and variants of Elp1 , Elp2 , and Elp456 . 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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Figure 5A. 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 and variants of Elp1, Elp2, and Elp456.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.


Figure 6B . 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 the localization of Elp3 and the orientation of its KAT and SAM domain in relation to Elp1 and Elp2 . 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 .

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Figure 6B. 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 the localization of Elp3 and the orientation of its KAT and SAM domain in relation to Elp1 and Elp2.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.


Figure 1A . GAPDH smFISH and IF analysis using anti - G3BP antibody ( G3BP ) and anti - Dcp1b antibody ( DCP1 ) in A549 cells ( RL - WT ) or A549 RL - KO cells either mock transfected ( mock ) or transfected with poly ( I : C ) ( PIC ) for 5 hours . Scale bar 10 microns . B . Graph of the fraction of G3BP foci with different volumes in PIC treated RL - WT and RL - KO cells . C . Number of DCP1 foci per cell in PIC treated RL - WT and RL - KO cells relative to the number in mock treated cells . D . Average volume of individual Dcp1 foci in mock and PIC treated RL - WT and RL - KO cells .

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Figure 1A. GAPDH smFISH and IF analysis using anti-G3BP antibody (G3BP) and anti-Dcp1b antibody (DCP1) in A549 cells (RL-WT) or A549 RL-KO cells either mock transfected (mock) or transfected with poly(I:C) (PIC) for 5 hours. Scale bar 10 microns. B. Graph of the fraction of G3BP foci with different volumes in PIC treated RL-WT and RL-KO cells. C. Number of DCP1 foci per cell in PIC treated RL-WT and RL-KO cells relative to the number in mock treated cells. D. Average volume of individual Dcp1 foci in mock and PIC treated RL-WT and RL-KO cells.


Figure 2A . Western analysis of nuclear ( N ) or cytoplasmic ( C ) fractions from whole cell lysates ( W ) from A549 cells ( RL WT ) , RNase L knock out A549 cells ( RL KO ) , or A549 cells with either wild - type RNase L ( NLS - RL - WT ) or RNase L - R667A ( NLS - RL - CM ) fused to a nuclear localization signal sequence . Short and long exposure with anti - RL antibody . GAPDH protein used as cytoplasmic marker . Histone H3 protein used as nuclear marker . B . Graph depicting the fraction of RNase L found in the nucleus or cytoplasm . Mean and SEM of two experiments with individual experiment values plotted . C . FISH with probes to GAPDH mRNA or oligo ( dT ) to detect poly ( A ) + RNA in A549 cells without or with either NLS - RL - WT or NLS - RL - CM either mock transfected or transfected with poly ( I : C ) for 4 hours . Scale bar 5 micron . D . Graph depicting median and the value of the intensity of oligo ( dT ) fluorescence in individual nuclei divided by nuclear volume in the indicated cell types either mock transfected or transfected with poly ( I : C ) for 4 hours . At least 43 nuclei analyzed per condition .

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Figure 2A. Western analysis of nuclear (N) or cytoplasmic (C) fractions from whole cell lysates (W) from A549 cells (RL WT), RNase L knock out A549 cells (RL KO), or A549 cells with either wild-type RNase L (NLS-RL-WT) or RNase L-R667A (NLS-RL-CM) fused to a nuclear localization signal sequence. Short and long exposure with anti-RL antibody. GAPDH protein used as cytoplasmic marker. Histone H3 protein used as nuclear marker. B. Graph depicting the fraction of RNase L found in the nucleus or cytoplasm. Mean and SEM of two experiments with individual experiment values plotted.C. FISH with probes to GAPDH mRNA or oligo(dT) to detect poly(A)+ RNA in A549 cells without or with either NLS-RL-WT or NLS-RL-CM either mock transfected or transfected with poly(I:C) for 4 hours. Scale bar 5 micron.D. Graph depicting median and the value of the intensity of oligo(dT) fluorescence in individual nuclei divided by nuclear volume in the indicated cell types either mock transfected or transfected with poly(I:C) for 4 hours. At least 43 nuclei analyzed per condition.


Figure 3Analysis of nuclear RNA granules in A549 cells with RNase L targeted to the nucleus ( NLS - RL - WT ) either mock transfected or treated with poly ( I : C ) ( PIC ) for 4 to 5 hours . A . FISH analysis to detect poly ( A ) + RNA and nucleolar - localized snoRD3A RNA . IF analysis to detect nucleolar granular component protein nucleophosmin ( NPM1 ) . B . Graph of the mean and value of the average volume of individual snoRD3A foci in nuclei . 17 nuclei mock treated cells , 31 nuclei PIC treated cells . C . Fraction of nuclei with NPM1 protein enriched in ring structures classified as granular component assemblies or dispersed in nucleoplasm . 17 nuclei mock treated cells , 31 nuclei PIC treated cells . Analysis of nuclear RNA granules in A549 cells with RNase L targeted to the nucleus ( NLS - RL - WT ) either mock transfected or treated with poly ( I : C ) ( PIC ) for 4 to 5 hours . D . IF analysis to detect dense fibrillar component protein , ribosome processing factor 1 ( RPF1 ) . E . Graph of the mean and value of the average volume of individual snoRD3A foci in nuclei . 43 nuclei mock treated cells , 69 nuclei PIC treated cells . F . Graph of the mean and value of the average volume of RPF1 foci in 43 mock treated cells and 69 nuclei PIC treated cells . Analysis of nuclear RNA granules in A549 cells with RNase L targeted to the nucleus ( NLS - RL - WT ) either mock transfected or treated with poly ( I : C ) ( PIC ) for 4 to 5 hours . G . FISH analysis to detect poly ( A ) + RNA and nuclear speckle - localized MALAT1 RNA and IF analysis with anti - sc35 antibody to detect nuclear speckle protein SRRM2 . H . Graph of the mean and value of the intensity of MALAT1 signal divided by the nuclear volume of individual nuclei . 51 nuclei mock treated cells , 56 nuclei PIC treated cells . I . Graph of the median and value of the volume of individual SRRM2 foci in nuclei . Mock 1526 SRRM2 foci in 51 nuclei . PIC 136 SRRM2 foci in 20 nuclei that contain < 10 SRRM2 foci . Analysis of nuclear RNA granules in A549 cells with RNase L targeted to the nucleus ( NLS - RL - WT ) either mock transfected or treated with poly ( I : C ) ( PIC ) for 4 to 5 hours . J . FISH analysis to detect poly ( A ) + RNA and IF analysis to detect Cajal body protein coilin . K . Graph depicting the fraction of nuclei in mock and PIC treated cells with decreased oligo ( dT ) with different distributions of coilin protein . 90 nuclei in mock treated cells , 192 nuclei in PIC treated cells .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3Analysis of nuclear RNA granules in A549 cells with RNase L targeted to the nucleus (NLS-RL-WT) either mock transfected or treated with poly(I:C) (PIC) for 4 to 5 hours. A. FISH analysis to detect poly(A)+ RNA and nucleolar-localized snoRD3A RNA. IF analysis to detect nucleolar granular component protein nucleophosmin (NPM1). B. Graph of the mean and value of the average volume of individual snoRD3A foci in nuclei. 17 nuclei mock treated cells, 31 nuclei PIC treated cells. C. Fraction of nuclei with NPM1 protein enriched in ring structures classified as granular component assemblies or dispersed in nucleoplasm. 17 nuclei mock treated cells, 31 nuclei PIC treated cells.Analysis of nuclear RNA granules in A549 cells with RNase L targeted to the nucleus (NLS-RL-WT) either mock transfected or treated with poly(I:C) (PIC) for 4 to 5 hours. D. IF analysis to detect dense fibrillar component protein, ribosome processing factor 1 (RPF1). E. Graph of the mean and value of the average volume of individual snoRD3A foci in nuclei. 43 nuclei mock treated cells, 69 nuclei PIC treated cells. F. Graph of the mean and value of the average volume of RPF1 foci in 43 mock treated cells and 69 nuclei PIC treated cells.Analysis of nuclear RNA granules in A549 cells with RNase L targeted to the nucleus (NLS-RL-WT) either mock transfected or treated with poly(I:C) (PIC) for 4 to 5 hours. G. FISH analysis to detect poly(A)+ RNA and nuclear speckle-localized MALAT1 RNA and IF analysis with anti-sc35 antibody to detect nuclear speckle protein SRRM2. H. Graph of the mean and value of the intensity of MALAT1 signal divided by the nuclear volume of individual nuclei. 51 nuclei mock treated cells, 56 nuclei PIC treated cells. I. Graph of the median and value of the volume of individual SRRM2 foci in nuclei. Mock 1526 SRRM2 foci in 51 nuclei. PIC 136 SRRM2 foci in 20 nuclei that contain <10 SRRM2 foci.Analysis of nuclear RNA granules in A549 cells with RNase L targeted to the nucleus (NLS-RL-WT) either mock transfected or treated with poly(I:C) (PIC) for 4 to 5 hours. J. FISH analysis to detect poly(A)+ RNA and IF analysis to detect Cajal body protein coilin. K. Graph depicting the fraction of nuclei in mock and PIC treated cells with decreased oligo(dT) with different distributions of coilin protein. 90 nuclei in mock treated cells, 192 nuclei in PIC treated cells.


Figure 5Analysis of A549 cells with NLS - RL - WT or NLS - RL - CM either mock transfected or treated with poly ( I : C ) ( PIC ) for 5 hours . A . FISH analysis to detect poly ( A ) + RNA and IF analysis against FUS protein . Images are a single 0 . 2 micron Z section . Analysis of A549 cells with NLS - RL - WT or NLS - RL - CM either mock transfected or treated with poly ( I : C ) ( PIC ) for 5 hours . B . Graph depicting the fraction of nuclei with FUS dispersed in the nucleoplasm or in foci in cells with reduced nuclear oligo ( dT ) signal . All the nuclei ( total ) in which nuclear RNA was degraded ( N = 64 ) or the same nuclei classified as to whether or not they contained residual poly ( A ) foci . Nuclei with poly ( A ) foci ( N = 14 ) . Nuclei without poly ( A ) foci ( N = 50 ) . Analysis of A549 cells with NLS - RL - WT either mock transfected or treated with poly ( I : C ) ( PIC ) for 5 hours . C . FISH analysis to detect poly ( A ) + RNA and IF analysis against mediator complex protein , MED1 , to detect super - enhancer condensates . D . Number of MED1 foci in mock and PIC treated NLS - RL - WT cells . E . Average volume of individual MED1 foci in mock and PIC treated NLS - RL - WT cells . Analysis of A549 cells with NLS - RL - WT either mock transfected or treated with poly ( I : C ) ( PIC ) for 5 hours . F . FISH analysis to detect poly ( A ) + RNA and IF analysis against BRD4 , to detect super - enhancer condensates . G . Number of BRD4 foci in mock and PIC treated NLS - RL - WT cells . H . Average volume of individual BRD4 foci in mock and PIC treated NLS - RL - WT cells .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5Analysis of A549 cells with NLS-RL-WT or NLS-RL-CM either mock transfected or treated with poly(I:C) (PIC) for 5 hours. A. FISH analysis to detect poly(A)+ RNA and IF analysis against FUS protein. Images are a single 0.2 micron Z section.Analysis of A549 cells with NLS-RL-WT or NLS-RL-CM either mock transfected or treated with poly(I:C) (PIC) for 5 hours. B. Graph depicting the fraction of nuclei with FUS dispersed in the nucleoplasm or in foci in cells with reduced nuclear oligo(dT) signal. All the nuclei (total) in which nuclear RNA was degraded (N=64) or the same nuclei classified as to whether or not they contained residual poly(A) foci. Nuclei with poly(A) foci (N=14). Nuclei without poly(A) foci (N=50).Analysis of A549 cells with NLS-RL-WT either mock transfected or treated with poly(I:C) (PIC) for 5 hours. C. FISH analysis to detect poly(A)+ RNA and IF analysis against mediator complex protein, MED1, to detect super-enhancer condensates. D. Number of MED1 foci in mock and PIC treated NLS-RL-WT cells. E. Average volume of individual MED1 foci in mock and PIC treated NLS-RL-WT cells.Analysis of A549 cells with NLS-RL-WT either mock transfected or treated with poly(I:C) (PIC) for 5 hours. F. FISH analysis to detect poly(A)+ RNA and IF analysis against BRD4, to detect super-enhancer condensates. G. Number of BRD4 foci in mock and PIC treated NLS-RL-WT cells. H. Average volume of individual BRD4 foci in mock and PIC treated NLS-RL-WT cells.


Figure 1 . Select 2D class averages of the particles that were used to calculate the 2019 - nCoV S reconstruction ( left ) . Side and top views of the prefusion structure of the 2019 - nCoV S protein with a single RBD in the " up " conformation ( right ) . The two RBD " down " protomers are shown as cryo - EM density in either white or gray and the RBD " up " protomer is shown in ribbons , colored corresponding to the schematic in Fig 1A .

O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1.Select 2D class averages of the particles that were used to calculate the 2019-nCoV S reconstruction (left). Side and top views of the prefusion structure of the 2019-nCoV S protein with a single RBD in the "up" conformation (right). The two RBD "down" protomers are shown as cryo-EM density in either white or gray and the RBD "up" protomer is shown in ribbons, colored corresponding to the schematic in Fig 1A.


Figure 3 . SPR sensorgram showing the binding kinetics for human ACE2 and immobilized 2019 - nCoV S . Data are shown as black lines and the best fit of the data to a 1 : 1 binding model is shown in red . Negative - stain EM 2D class averages of 2019 - nCoV S bound by ACE2 . Averages have been rotated so that ACE2 is positioned above the 2019 - nCoV S protein with respect to the viral membrane . A cartoon depicting the ACE2 - bound 2019 - nCoV S protein is shown ( right ) with ACE2 in blue and S protein monomers colored tan , pink and green .

O O O B-PANEL_START O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3.SPR sensorgram showing the binding kinetics for human ACE2 and immobilized 2019-nCoV S. Data are shown as black lines and the best fit of the data to a 1:1 binding model is shown in red.Negative-stain EM 2D class averages of 2019-nCoV S bound by ACE2. Averages have been rotated so that ACE2 is positioned above the 2019-nCoV S protein with respect to the viral membrane. A cartoon depicting the ACE2-bound 2019-nCoV S protein is shown (right) with ACE2 in blue and S protein monomers colored tan, pink and green.


Figure 4 . A biolayer interferometry sensorgram that shows binding to ACE2 by the 2019 - nCoV RBD - SD1 . Binding data are shown as a black line and the best fit of the data to a 1 : 1 binding model is shown in red . Biolayer interferometry to measure cross - reactivity of the SARS - CoV RBD - directed antibodies S230 , m396 and 80R . Sensortips with immobilized antibodies were dipped into wells containing 2019 - nCoV RBD - SD1 and the resulting data are shown as a black line .

O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4.A biolayer interferometry sensorgram that shows binding to ACE2 by the 2019-nCoV RBD-SD1. Binding data are shown as a black line and the best fit of the data to a 1:1 binding model is shown in red.Biolayer interferometry to measure cross-reactivity of the SARS-CoV RBD-directed antibodies S230, m396 and 80R. Sensortips with immobilized antibodies were dipped into wells containing 2019-nCoV RBD-SD1 and the resulting data are shown as a black line.


Figure 1Clustering of metabolome data in patients and controls ; PLS - DA plots ; VIP score plots of top 15 metabolites ; volcano plots of all metabolites in blood of IOSCA ( A ) Data information Colours in VIP score and volcano plots indicate the same most relevant and / or significantly changed metabolites among all patients groups . C3 , component 3 CDCA , chenodeoxycholic acidClustering of metabolome data in patients and controls ; PLS - DA plots ; VIP score plots of top 15 metabolites ; volcano plots of all metabolites in blood of MIRAS ( B ) Data information : a Significantly changed metabolites outside the FDR cut - off . b Metabolites not significantly changed between patients and controls . Colours in VIP score and volcano plots indicate the same most relevant and / or significantly changed metabolites among all patients groups . C3 , component 3 CDCA , chenodeoxycholic acidClustering of metabolome data in patients and controls ; PLS - DA plots ; VIP score plots of top 15 metabolites ; volcano plots of all metabolites in blood of PEO ( C ) Data information : Colours in VIP score and volcano plots indicate the same most relevant and / or significantly changed metabolites among all patients groups . C3 , component 3 ; CDCA , chenodeoxycholic acid ; GABA , γ - aminobutyric acid ; OH - Kyn , 3 - hydroxy - DL - kynurenineA - D Clustering of metabolome data in patients and controls ; PLS - DA plots ; VIP score plots of top 15 metabolites ; volcano plots of all metabolites in blood of MELAS / MIDD ( D ) . Data information : Colours in VIP score and volcano plots indicate the same most relevant and / or significantly changed metabolites among all patients groups . C3 , component 3 HIAA , 5 - Hydroxyindole - 3 - acetic acid ; SDMA , symmetric dimethylarginine ; TCA , taurocholic acid .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1Clustering of metabolome data in patients and controls; PLS-DA plots; VIP score plots of top 15 metabolites; volcano plots of all metabolites in blood of IOSCA (A) Data information Colours in VIP score and volcano plots indicate the same most relevant and/or significantly changed metabolites among all patients groups. C3, component 3 CDCA, chenodeoxycholic acidClustering of metabolome data in patients and controls; PLS-DA plots; VIP score plots of top 15 metabolites; volcano plots of all metabolites in blood of MIRAS (B) Data information: a Significantly changed metabolites outside the FDR cut-off. b Metabolites not significantly changed between patients and controls. Colours in VIP score and volcano plots indicate the same most relevant and/or significantly changed metabolites among all patients groups. C3, component 3 CDCA, chenodeoxycholic acidClustering of metabolome data in patients and controls; PLS-DA plots; VIP score plots of top 15 metabolites; volcano plots of all metabolites in blood of PEO (C) Data information: Colours in VIP score and volcano plots indicate the same most relevant and/or significantly changed metabolites among all patients groups. C3, component 3; CDCA, chenodeoxycholic acid; GABA, γ-aminobutyric acid; OH-Kyn, 3-hydroxy-DL-kynurenineA - D Clustering of metabolome data in patients and controls; PLS-DA plots; VIP score plots of top 15 metabolites; volcano plots of all metabolites in blood of MELAS/MIDD (D). Data information: Colours in VIP score and volcano plots indicate the same most relevant and/or significantly changed metabolites among all patients groups. C3, component 3 HIAA, 5-Hydroxyindole-3-acetic acid; SDMA, symmetric dimethylarginine; TCA, taurocholic acid.


Figure 2Clustering of metabolome data in patients and controls ; PLS - DA plots ; VIP score plots of top 15 metabolites ; volcano plots of all metabolites in blood of IBM ( A ) Colours in VIP score and volcano plots indicate the same most relevant and / or significantly changed metabolites among all patients groups . C3 , component 3 ; CDCA , chenodeoxycholic acid ; OH - Kyn , 3 - hydroxy - DL - kynurenine SDMA , symmetric dimethylarginine . Clustering of metabolome data in patients and controls ; PLS - DA plots ; VIP score plots of top 15 metabolites ; volcano plots of all metabolites in blood of NMD patients ( B ) Data information : a Significantly changed metabolites outside the FDR cut - off . b Metabolites not significantly changed between patients and controls . Colours in VIP score and volcano plots indicate the same most relevant and / or significantly changed metabolites among all patients groups . C3 , component 3 ; CDCA , chenodeoxycholic acidClustering of metabolome data in patients and controls ; PLS - DA plots ; VIP score plots of top 15 metabolites ; volcano plots of all metabolites in blood of MIRAS carriers ( C ) . Data information Colours in VIP score and volcano plots indicate the same most relevant and / or significantly changed metabolites among all patients groups . cAMP , cyclic AMP ; C3 , component 3 ; GABA , γ - aminobutyric acid HIAA , 5 - Hydroxyindole - 3 - acetic acid ; IMP , inosine monophosphate ; OH - Kyn , 3 - hydroxy - DL - kynurenine ; OH - Trp , hydroxytryptophan ; SDMA , symmetric dimethylarginine .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2Clustering of metabolome data in patients and controls; PLS-DA plots; VIP score plots of top 15 metabolites; volcano plots of all metabolites in blood of IBM (A) Colours in VIP score and volcano plots indicate the same most relevant and/or significantly changed metabolites among all patients groups. C3, component 3; CDCA, chenodeoxycholic acid; OH-Kyn, 3-hydroxy-DL-kynurenine SDMA, symmetric dimethylarginine.Clustering of metabolome data in patients and controls; PLS-DA plots; VIP score plots of top 15 metabolites; volcano plots of all metabolites in blood of NMD patients (B) Data information: a Significantly changed metabolites outside the FDR cut-off. b Metabolites not significantly changed between patients and controls. Colours in VIP score and volcano plots indicate the same most relevant and/or significantly changed metabolites among all patients groups. C3, component 3; CDCA, chenodeoxycholic acidClustering of metabolome data in patients and controls; PLS-DA plots; VIP score plots of top 15 metabolites; volcano plots of all metabolites in blood of MIRAS carriers (C). Data information Colours in VIP score and volcano plots indicate the same most relevant and/or significantly changed metabolites among all patients groups. cAMP, cyclic AMP; C3, component 3; GABA, γ-aminobutyric acid HIAA, 5-Hydroxyindole-3-acetic acid; IMP, inosine monophosphate; OH-Kyn, 3-hydroxy-DL-kynurenine; OH-Trp, hydroxytryptophan; SDMA, symmetric dimethylarginine.


Figure 3A Relative values of single metabolites and creatine / creatinine ratios in blood of primary MD , IBM and NMD patients , and MIRAS carriers compared to controls . Data information : All data represent mean ± SD . For individual metabolites : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( two sample T - test ) . For creatine / creatinine ( cr / crn ) ratio : * P = 0 . 022 , * * P = 0 . 005 , * * * P = 0 . 0001 ( Kruskal - Wallis test with Dunn ´ s multiple comparisons test or Mann - Whitney test ) . B Relative values of single metabolites in blood of adult IOSCA ( marked " IOSCA " ) patients and one IOSCA child patient compared to controls . Data information : All data represent mean ± SD .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A Relative values of single metabolites and creatine/creatinine ratios in blood of primary MD, IBM and NMD patients, and MIRAS carriers compared to controls. Data information: All data represent mean ± SD. For individual metabolites: *P < 0.05, **P < 0.01, ***P < 0.001 (two sample T-test). For creatine/creatinine (cr/crn) ratio: *P = 0.022, **P = 0.005, ***P = 0.0001 (Kruskal-Wallis test with Dunn´s multiple comparisons test or Mann-Whitney test).B Relative values of single metabolites in blood of adult IOSCA (marked "IOSCA") patients and one IOSCA child patient compared to controls. Data information: All data represent mean ± SD.


Figure 4Metabolomes of muscle of MIRAS ( A ) patients ; PLS - DA plots ; VIP score plots of top 15 metabolites ; volcano plots of all metabolites . Data information : a Significantly changed metabolites outside the FDR cut - off . b Metabolites not significantly changed between patients and controls . AMP , adenosine monophosphate NAD , nicotinamide adenine dinucleotide TCA , taurocholic acidMetabolomes of muscle of PEO ( B ) patients ; PLS - DA plots ; VIP score plots of top 15 metabolites ; volcano plots of all metabolites . Data information : a Significantly changed metabolites outside the FDR cut - off . b Metabolites not significantly changed between patients and controls . car . , carnitine NAD , nicotinamide adenine dinucleotideD Relative values of metabolites in muscle of MIRAS , PEO and MELAS / MIDD patients compared to controls . All data represent mean ± SD . * P = 0 . 031 , * * P = 0 . 008 ( two sample T - test ) . UDP , uridine diphosphate .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4Metabolomes of muscle of MIRAS (A) patients; PLS-DA plots; VIP score plots of top 15 metabolites; volcano plots of all metabolites. Data information: a Significantly changed metabolites outside the FDR cut-off. b Metabolites not significantly changed between patients and controls. AMP, adenosine monophosphate NAD, nicotinamide adenine dinucleotide TCA, taurocholic acidMetabolomes of muscle of PEO (B) patients; PLS-DA plots; VIP score plots of top 15 metabolites; volcano plots of all metabolites. Data information: a Significantly changed metabolites outside the FDR cut-off. b Metabolites not significantly changed between patients and controls. car., carnitine NAD, nicotinamide adenine dinucleotideD Relative values of metabolites in muscle of MIRAS, PEO and MELAS/MIDD patients compared to controls. All data represent mean ± SD. *P = 0.031, **P = 0.008 (two sample T-test). UDP, uridine diphosphate.


Figure 6A Separate specificity and sensitivity ( ROC ) of sorbitol , alanine , myoinositol and cystathionine ( left ) and conventional blood biomarkers lactate and pyruvate , and cytokine FGF21 ( right ) in blood of MIRAS , PEO and MELAS / MIDD patients ( n = 20 ) compared to controls ( n = 30 ) . Data information : ROC analysis : AUC of sorbitol 0 . 81 ( 95 % CI 0 . 68 - 0 . 94 , P = 0 . 0003 ) , alanine 0 . 81 ( 95 % CI 0 . 66 - 0 . 94 , P = 0 . 0003 ) , myoinositol 0 . 79 ( 95 % CI 0 . 66 - 0 . 91 , P = 0 . 0007 ) and cystathionine 0 . 78 ( 95 % CI 0 . 65 - 0 . 91 , P = 0 . 001 ) . AUC of conventional biomarkers : FGF21 0 . 87 ( 95 % CI 0 . 74 - 0 . 99 , P = 0 . 0001 ) , lactate 0 . 86 ( 95 % CI 0 . 76 - 0 . 97 , P = 0 . 0001 ) and pyruvate 0 . 78 ( 95 % CI 0 . 64 - 0 . 93 , P = 0 . 0017 ) . B Combined ' multi - biomarker ' value of sorbitol / alanine / myoinositol / cystathionine ; ROC - analysis ( left ) ; mean centroids MD , IBM and NMD patients , and MIRAS carriers compared to controls ( right ) . Data information : ROC analysis AUC of ' multi - biomarker ' 0 . 94 ( 95 % CI 0 . 89 - 0 . 995 , P = 0 . 0001 ) . Mean centroid data represent mean ± SD . * * P < 0 . 01 , * * * P < 0 . 001 ( 1 - way ANOVA with Dunnett ´ s multiple comparison test ) .

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Figure 6A Separate specificity and sensitivity (ROC) of sorbitol, alanine, myoinositol and cystathionine (left) and conventional blood biomarkers lactate and pyruvate, and cytokine FGF21 (right) in blood of MIRAS, PEO and MELAS/MIDD patients (n = 20) compared to controls (n = 30). Data information: ROC analysis: AUC of sorbitol 0.81 (95% CI 0.68 - 0.94, P = 0.0003), alanine 0.81 (95% CI 0.66 - 0.94, P = 0.0003), myoinositol 0.79 (95% CI 0.66 - 0.91, P = 0.0007) and cystathionine 0.78 (95% CI 0.65 - 0.91, P = 0.001). AUC of conventional biomarkers: FGF21 0.87 (95% CI 0.74 - 0.99, P = 0.0001), lactate 0.86 (95% CI 0.76 - 0.97, P = 0.0001) and pyruvate 0.78 (95% CI 0.64 - 0.93, P = 0.0017).B Combined 'multi-biomarker' value of sorbitol/alanine/myoinositol/cystathionine; ROC-analysis (left); mean centroids MD, IBM and NMD patients, and MIRAS carriers compared to controls (right). Data information: ROC analysis AUC of 'multi-biomarker' 0.94 (95% CI 0.89 - 0.995, P = 0.0001). Mean centroid data represent mean ± SD. **P < 0.01, ***P < 0.001 (1-way ANOVA with Dunnett´s multiple comparison test).


Figure 1A Tissue distribution of RIPK2 determined by anti - FLAG immunoprecipitation . Organ homogenates from WT ( wild - type ) and KI ( homozygous FLAG - RIPK2 ) mice were subjected to anti - FLAG immunoprecipitation and immunoblotting . B Inflammatory signaling in wild - type ( WT / WT ) , RIPK2 CRISPR KO ( KO / KO ) and FLAG - RIPK2 heterozygous ( WT / KI ) and homozygous ( KI / KI ) BMDMs . BMDMs were primed with IFNy , stimulated with MDP for indicated times and analysed by immunoblotting . C Cytokine production of RIPK2 CRISPR KO ( KO ) , wild - type ( WT ) and FLAG - RIPK2 heterozygous ( WT / KI ) and homozygous ( KI / KI ) BMDMs in response to MDP . BMDMs were left untreated or treated with IFNy or IFNy and MDP over - night and cytokines were measured by ELISA . N = 5 - 8 mice . Shown is average ± SEM . ns = P > 0 . 05 ; * * = P ≤ 0 . 01 ; * * * * = P ≤ 0 . 0001 ; two - way ANOVA . D Serum cytokines in RIPK2 CRISPR KO ( KO ) , wild - type ( WT ) and FLAG - RIPK2 heterozygous ( WT / KI ) or homozygous ( KI / KI ) mice after i . p . MDP administration . Mice were injected i . p . with PBS or MDP , sacrificed after 4h and serum cytokines were measured by ELISA . N = 3 - 6 mice . Shown is average ± SEM . ns = P > 0 . 05 ; * * = P ≤ 0 . 01 ; two - way ANOVA .

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Figure 1A Tissue distribution of RIPK2 determined by anti-FLAG immunoprecipitation. Organ homogenates from WT (wild-type) and KI (homozygous FLAG-RIPK2) mice were subjected to anti-FLAG immunoprecipitation and immunoblotting.B Inflammatory signaling in wild-type (WT/WT), RIPK2 CRISPR KO (KO/KO) and FLAG-RIPK2 heterozygous (WT/KI) and homozygous (KI/KI) BMDMs. BMDMs were primed with IFNy, stimulated with MDP for indicated times and analysed by immunoblotting.C Cytokine production of RIPK2 CRISPR KO (KO), wild-type (WT) and FLAG-RIPK2 heterozygous (WT/KI) and homozygous (KI/KI) BMDMs in response to MDP. BMDMs were left untreated or treated with IFNy or IFNy and MDP over-night and cytokines were measured by ELISA. N = 5-8 mice. Shown is average ± SEM. ns = P > 0.05; ** = P ≤ 0.01; **** = P ≤ 0.0001; two-way ANOVA.D Serum cytokines in RIPK2 CRISPR KO (KO), wild-type (WT) and FLAG-RIPK2 heterozygous (WT/KI) or homozygous (KI/KI) mice after i.p. MDP administration. Mice were injected i.p. with PBS or MDP, sacrificed after 4h and serum cytokines were measured by ELISA. N = 3-6 mice. Shown is average ± SEM. ns = P > 0.05; ** = P ≤ 0.01; two-way ANOVA.


Figure 2A Two step enrichment to isolate ubiquitinated RIPK2 from BMDMs . Protein lysates from FLAG - RIPK2 BMDMs ( A ) were sequentially subjected to ubiquitin enrichment ( UBA , B ) and FLAG pulldown ( C ) prior to protein elution and subsequent mass spectrometry analysis .

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Figure 2A Two step enrichment to isolate ubiquitinated RIPK2 from BMDMs. Protein lysates from FLAG-RIPK2 BMDMs (A) were sequentially subjected to ubiquitin enrichment (UBA, B) and FLAG pulldown (C) prior to protein elution and subsequent mass spectrometry analysis.


Figure 3A Activation of the NF - κB pathway by RIPK2 Lysine - and phosphosite mutants . RIPK2 - deficient THP - 1 cells reconstituted with wild - type RIPK2 or RIPK2 mutants were stimulated with L18 - MDP , harvested at indicated time points and activation of the NF - κB pathway was analyzed by immunoblotting . B IL - 8 production of wild - type THP - 1 and RIPK2 - deficient THP - 1 cells reconstituted with wild - type RIPK2 or RIPK2 mutants and stimulated with L18 - MDP was assessed by ELISA . N = 4 - 8 experiments . Shown is average ± SEM . * = P ≤ 0 . 05 ; two - way ANOVA . C ) RIPK2 ubiquitination determined by UBA pulldown in RIPK2 - deficient cells reconstituted with wild - type or mutant RIPK2 after stimulation with L18 - MDP . D ) Detection of K63 - and M1 - linked ubiquitin chains by UBA pulldown or pulldown with ubiquitin chain type - specific antibodies in RIPK2 - deficient cells reconstituted with RIPK2 K209R after stimulation with L18 - MDP .

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Figure 3A Activation of the NF-κB pathway by RIPK2 Lysine- and phosphosite mutants. RIPK2-deficient THP-1 cells reconstituted with wild-type RIPK2 or RIPK2 mutants were stimulated with L18-MDP, harvested at indicated time points and activation of the NF-κB pathway was analyzed by immunoblotting.B IL-8 production of wild-type THP-1 and RIPK2-deficient THP-1 cells reconstituted with wild-type RIPK2 or RIPK2 mutants and stimulated with L18-MDP was assessed by ELISA. N = 4-8 experiments. Shown is average ± SEM. * = P ≤ 0.05; two-way ANOVA.C) RIPK2 ubiquitination determined by UBA pulldown in RIPK2-deficient cells reconstituted with wild-type or mutant RIPK2 after stimulation with L18-MDP.D) Detection of K63- and M1-linked ubiquitin chains by UBA pulldown or pulldown with ubiquitin chain type-specific antibodies in RIPK2-deficient cells reconstituted with RIPK2 K209R after stimulation with L18-MDP.


Figure 4C Activation of the NF - κB pathway by RIPK2 pocket mutants . RIPK2 - deficient THP - 1 cells were reconstituted with wild - type RIPK2 or RIPK2 mutants , stimulated with L18 - MDP , harvested at indicated time points and analyzed by immunoblotting . D IL - 8 production of RIPK2 - deficient THP - 1cells reconstituted with wild - type RIPK2 or RIPK2 mutants and stimulated with L18 - MDP was assessed by ELISA . N = 3 - 8 experiments . Shown is average ± SEM . ns = P > 0 . 05 ; * = P ≤ 0 . 05 ; * * * = P ≤ 0 . 001 ; * * * * = P ≤ 0 . 0001 ; two - way ANOVA . E Ubiquitination of RIPK2 pocket mutants . RIPK2 - deficient THP - 1 cells were reconstituted with wild - type or mutant RIPK2 , left unstimulated or stimulated with L18 - MDP and subjected to UBA pulldown and immunoblotting .

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Figure 4C Activation of the NF-κB pathway by RIPK2 pocket mutants. RIPK2-deficient THP-1 cells were reconstituted with wild-type RIPK2 or RIPK2 mutants, stimulated with L18-MDP, harvested at indicated time points and analyzed by immunoblotting. D IL-8 production of RIPK2-deficient THP-1cells reconstituted with wild-type RIPK2 or RIPK2 mutants and stimulated with L18-MDP was assessed by ELISA. N = 3-8 experiments. Shown is average ± SEM. ns = P > 0.05; * = P ≤ 0.05; *** = P ≤ 0.001; **** = P ≤ 0.0001; two-way ANOVA.E Ubiquitination of RIPK2 pocket mutants. RIPK2-deficient THP-1 cells were reconstituted with wild-type or mutant RIPK2, left unstimulated or stimulated with L18-MDP and subjected to UBA pulldown and immunoblotting.


Figure 5A : Thermal stability assay using selected RIPK2 mutants . THP - 1 cells expressing RIPK2 were subjected to heat treatment and the non - denatured fraction was analysed by immunoblotting . B : Binding of RIPK2 to XIAP - BIR2 . Lysates from RIPK2 - deficient THP - 1 cells reconstituted with WT or mutant RIPK2 were subjected to pulldown experiments with recombinantly expressed XIAP - BIR2 and analysed by immunoblotting .

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Figure 5A: Thermal stability assay using selected RIPK2 mutants. THP-1 cells expressing RIPK2 were subjected to heat treatment and the non-denatured fraction was analysed by immunoblotting.B: Binding of RIPK2 to XIAP-BIR2. Lysates from RIPK2-deficient THP-1 cells reconstituted with WT or mutant RIPK2 were subjected to pulldown experiments with recombinantly expressed XIAP-BIR2 and analysed by immunoblotting.


Fig 1 ( B ) Gal4 DNA - binding domain fusions of MUPs were tested with Gal4 activation domain fusions of ubiquilins ( murine ) in Y2H . ( C ) HEK293 cells transfected with indicated MUP - V5 , myc - UBQLN1 ( human ) , or empty vector were immunoprecipitated ( IP ) with anti - myc or anti - UBQLN1 antibodies . Western blot ( WB ) of IP and lysate were probed as indicated . ( D - E ) WB of input lysate and IP from HEK293 cells expressing myc - UBQLN1 and Rv1926 - V5 ( D ) or Rv1566 - V5 ( E ) . IP was performed using antibodies directed against myc , V5 , or isotype control ( IgG ) .

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Fig 1(B) Gal4 DNA-binding domain fusions of MUPs were tested with Gal4 activation domain fusions of ubiquilins (murine) in Y2H.(C) HEK293 cells transfected with indicated MUP-V5, myc-UBQLN1 (human), or empty vector were immunoprecipitated (IP) with anti-myc or anti-UBQLN1 antibodies. Western blot (WB) of IP and lysate were probed as indicated.(D-E) WB of input lysate and IP from HEK293 cells expressing myc-UBQLN1 and Rv1926-V5 (D) or Rv1566-V5 (E). IP was performed using antibodies directed against myc, V5, or isotype control (IgG).


Fig 2 ( B ) Mtb were incubated with lysate from HEK293 cells transfected with vector control , myc - UBQLN1 , GFP , or V5 - parkin . WB analysis of the input ( I ) , 5th wash ( W5 ) , and elution ( E ) , probed with indicated antibodies . Actin is a loading control for the cell lysate and Ag85b for the bacterial pellet . ( C ) Mtb were incubated with lysate from HEK293 cells transfected with myc - UBQLN1 - ΔUBA , myc - UBQLN1 - ΔUBL , and myc - UBQLN1 - UBA . WB containing the input , 5th wash , and elution were incubated with anti - myc antibody . The predicted sizes of myc - UBQLN1 - ΔUBL , myc - UBQLN1 - ΔUBA , and myc - UBQLN1 - UBA are 55 kD , 50 kD , and 6 kD , respectively . ( D ) Mtb were incubated with recombinant GST - UBQLN1 , GST - UBQLN1 - ΔUBL , or GST - UBQLN1 - ΔUBA . WB containing the input and elution was probed with an anti - GST antibody .

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Fig 2(B) Mtb were incubated with lysate from HEK293 cells transfected with vector control, myc-UBQLN1, GFP, or V5-parkin. WB analysis of the input (I), 5th wash (W5), and elution (E), probed with indicated antibodies. Actin is a loading control for the cell lysate and Ag85b for the bacterial pellet.(C) Mtb were incubated with lysate from HEK293 cells transfected with myc-UBQLN1-ΔUBA, myc-UBQLN1-ΔUBL, and myc-UBQLN1-UBA. WB containing the input, 5th wash, and elution were incubated with anti-myc antibody. The predicted sizes of myc-UBQLN1-ΔUBL, myc-UBQLN1-ΔUBA, and myc-UBQLN1-UBA are 55 kD, 50 kD, and 6 kD, respectively.(D) Mtb were incubated with recombinant GST-UBQLN1, GST-UBQLN1-ΔUBL, or GST-UBQLN1-ΔUBA. WB containing the input and elution was probed with an anti-GST antibody.


Fig 3 ( A ) BMDMs were infected with GFP - expressing wt Mtb or ΔesxA for 24h and immunostained with UBQLN1 antibody . IFN - γ was added 1d prior to infection as indicated . ( B ) Quantification of results from A from 3 independent experiments with at least 100 bacteria analyzed per experiment ; * * * P < 0 . 001 , Fisher ' s exact test , comparing the proportion of UBQLN1 positive versus negative Mtb in indicated samples . ( C ) Cellular lysate from BMDMs that were treated with IFN - γ as indicated were analyzed by WB with an antibody that recognizes UBQLN1 and UBQLN2 . ( D ) BMDMs were transfected with siRNAs targeting UBQLN1 or a non - targeting control ( siCON ) and lysates were analyzed by WB using an antibody that recognizes UBQLN1 and UBQLN2 . ( C - D ) Actin served as a loading control . ( E ) BMDMs were infected with Mtb live / dead strain and treated with AnTc 24h prior to fixation . ( F ) BMDMs were transfected with siRNAs targeting UBQLN1 or siCON and infected with live / dead Mtb strain . IFN - γ was added 1d prior to infection if indicated . % Viable bacteria were quantified 24 and 48 hpi . Data are from 2 independent experiments . * P < 0 . 05 ; * * * P < 0 . 001 , Fisher ' s exact test , comparing the proportion of viable and non - viable bacteria in indicated samples . ( G ) BMDMs were treated with the indicated siRNAs three days prior to infection and then activated with IFN - γ one day prior to infection with H37Rv or ΔesxA . CFU were enumerated 4 and 96 hpi . * P < 0 . 05 , unpaired Student ' s t - test . Data are representative of three independent experiments . ( H ) BMDMs transfected with siRNA targeting UBQLN1 or siCON were infected with wt Mtb ( H37Rv ) and IFN - γ was measured by ELISA ( I ) UBQLN1 - silenced BMDMs and controls were infected with H37Rv or ΔesxA . ( H - I ) 24 hpi infected BMDMs were co - cultured with P25TCR - Tg CD4 + T cells , and IFN - γ was measured by ELISA . Data is representative of 3 independent experiments . * P < 0 . 05 , * * P < 0 . 005 , unpaired Student ' s t - test . ( B , F , Γ , H ) Results are mean + / - SEM ; ns , not significant .

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Fig 3(A) BMDMs were infected with GFP-expressing wt Mtb or ΔesxA for 24h and immunostained with UBQLN1 antibody. IFN-γ was added 1d prior to infection as indicated. (B) Quantification of results from A from 3 independent experiments with at least 100 bacteria analyzed per experiment; ***P< 0.001, Fisher's exact test, comparing the proportion of UBQLN1 positive versus negative Mtb in indicated samples.(C) Cellular lysate from BMDMs that were treated with IFN-γ as indicated were analyzed by WB with an antibody that recognizes UBQLN1 and UBQLN2.(D) BMDMs were transfected with siRNAs targeting UBQLN1 or a non-targeting control (siCON) and lysates were analyzed by WB using an antibody that recognizes UBQLN1 and UBQLN2. (C-D) Actin served as a loading control.(E) BMDMs were infected with Mtb live/dead strain and treated with AnTc 24h prior to fixation.(F) BMDMs were transfected with siRNAs targeting UBQLN1 or siCON and infected with live/dead Mtb strain. IFN-γ was added 1d prior to infection if indicated. %Viable bacteria were quantified 24 and 48 hpi. Data are from 2 independent experiments. *P<0.05; ***P<0.001, Fisher's exact test, comparing the proportion of viable and non-viable bacteria in indicated samples.(G) BMDMs were treated with the indicated siRNAs three days prior to infection and then activated with IFN-γ one day prior to infection with H37Rv or ΔesxA. CFU were enumerated 4 and 96 hpi. *P< 0.05, unpaired Student's t-test. Data are representative of three independent experiments.(H) BMDMs transfected with siRNA targeting UBQLN1 or siCON were infected with wt Mtb (H37Rv) and IFN-γ was measured by ELISA(I) UBQLN1-silenced BMDMs and controls were infected with H37Rv or ΔesxA. (H-I) 24 hpi infected BMDMs were co-cultured with P25TCR-Tg CD4+ T cells, and IFN-γ was measured by ELISA. Data is representative of 3 independent experiments. *P<0.05, **P<0.005, unpaired Student's t-test. (B, F, Γ, H) Results are mean +/- SEM; ns, not significant.


Fig 4 ( A ) BMDMs treated with IFN - γ and infected for 24h with GFP - Mtb were immunostained for UBQLN1 and either ubiquitin ( using the FK2 antibody ) or p62 . GFP - LC3 BMDMs infected with DsRed - Mtb were immunostained for UBQLN1 . ( B ) Quantification of those Mtb that co - localize with at least one of the cellular markers shown in A . More than 375 total bacteria were counted in two independent experiments , and the number co - localizing with at least one marker is indicated as " total " under each bar . ( C ) Wt macrophages ( Atg16L1flox / flox ; Cre - ) and autophagy - deficient macrophages ( Atg16L1flox / flox Lyz - cre ; Cre + ) were activated with IFN - γ or not , followed by infection with the live / dead Mtb reporter strain . % viable was quantified after addition of AnTc . * * P < 0 . 005 , * * * P < 0 . 0001 , Fisher ' s exact test ( in which the proportions of viable to non - viable Mtb was compared ) . ( D ) Atg16L1flox / flox Cre + and Cre - BMDMs treated with IFN - γ and infected for 24h with GFP - Mtb were immunostained for UBQLN1 and p62 . The number of Mtb co - localizating with the cellular markers is shown . ( C - D ) At least 100 bacteria were analyzed in three independent experiments . ( E ) Images from IFN - γ treated BMDMs of indicated genotype infected with GFP - Mtb and immunostained with UBQLN1 and p62 . Arrows indicates p62 + UBQLN1 + Mtb .

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Fig 4(A) BMDMs treated with IFN-γ and infected for 24h with GFP-Mtb were immunostained for UBQLN1 and either ubiquitin (using the FK2 antibody) or p62. GFP-LC3 BMDMs infected with DsRed-Mtb were immunostained for UBQLN1. (B) Quantification of those Mtb that co-localize with at least one of the cellular markers shown in A. More than 375 total bacteria were counted in two independent experiments, and the number co-localizing with at least one marker is indicated as "total" under each bar.(C) Wt macrophages (Atg16L1flox/flox; Cre-) and autophagy-deficient macrophages (Atg16L1flox/flox Lyz-cre; Cre+) were activated with IFN-γ or not, followed by infection with the live/dead Mtb reporter strain. % viable was quantified after addition of AnTc. **P<0.005, ***P<0.0001, Fisher's exact test (in which the proportions of viable to non-viable Mtb was compared).(D) Atg16L1flox/flox Cre+ and Cre- BMDMs treated with IFN-γ and infected for 24h with GFP-Mtb were immunostained for UBQLN1 and p62. The number of Mtb co-localizating with the cellular markers is shown. (C-D) At least 100 bacteria were analyzed in three independent experiments.(E) Images from IFN-γ treated BMDMs of indicated genotype infected with GFP-Mtb and immunostained with UBQLN1 and p62. Arrows indicates p62+ UBQLN1+ Mtb.


Fig 5 ( A - C ) BMDMs transfected with siRNA targeting UBQLN1 or control siRNA . Images show IFN - γ treated macrophages infected with GFP - Mtb ( A - B ) or DsRed - Mtb ( C ) for 24h . Cells were immunostained for ubiquitin ( Ub ) with the FK2 antibody ( A ) or p62 ( B ) , which are shown in red . ( C ) GFP - LC3 BMDMs were infected with DsRed - Mtb . LC3 is shown in green , whereas Mtb are red . ( A - C ) Quantification shows the percentage of Mtb that colocalized with the indicated cellular marker in macrophages that were treated with IFN - γ as indicated . Results are mean + / - SEM from two independent experiments , with at least 100 bacteria analyzed per experiment . * * * P < 0 . 001 , Fisher ' s exact test ( in which the proportion of Mtb associated with the cellular marker in a given condition was compared to the proportion found in the sample treated with siCON and IFN - γ ) .

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Fig 5(A-C) BMDMs transfected with siRNA targeting UBQLN1 or control siRNA. Images show IFN-γ treated macrophages infected with GFP-Mtb (A-B) or DsRed-Mtb (C) for 24h. Cells were immunostained for ubiquitin (Ub) with the FK2 antibody (A) or p62 (B), which are shown in red. (C) GFP-LC3 BMDMs were infected with DsRed-Mtb. LC3 is shown in green, whereas Mtb are red. (A-C) Quantification shows the percentage of Mtb that colocalized with the indicated cellular marker in macrophages that were treated with IFN-γ as indicated. Results are mean +/- SEM from two independent experiments, with at least 100 bacteria analyzed per experiment. ***P<0.001, Fisher's exact test (in which the proportion of Mtb associated with the cellular marker in a given condition was compared to the proportion found in the sample treated with siCON and IFN-γ).


Fig 6 ( A ) WT and Park2 - / - BMDMs were pretreated with IFN - γ , infected with GFP - Mtb for 24h , and immunostained with the FK2 antibody . Bacterial colocalization with FK2 immunoreactivity was quantified from 2 independent experiments . ( B ) Park2 - / - BMDMs pretreated or not treated with IFN - γ were infected with GFP - Mtb for 24h and immunostained with UBQLN1 specific antibody ; Arrows indicate Mtb co - localized with UBQLN1 . ns - not significant . ( C ) Park2 - / - BMDMs were transfected with siRNA targeting UBQLN1 or control siRNA 3d prior to infection , treated with IFN - γ , and infected with GFP - Mtb for 24h prior to immunostaining for ubiquitin ( FK2 antibody ) . Ubiquitin ( FK2 + ) bacteria were quantified from two independent experiments . ( A , C ) * * P < 0 . 01 , Fisher ' s exact test ( comparing the proportion of FK2 positive bacteria in indicated samples ) . Results are mean + / - SEM . A minimum of 100 Mtb were counted in each experiment .

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Fig 6(A) WT and Park2-/- BMDMs were pretreated with IFN-γ, infected with GFP-Mtb for 24h, and immunostained with the FK2 antibody. Bacterial colocalization with FK2 immunoreactivity was quantified from 2 independent experiments.(B) Park2-/- BMDMs pretreated or not treated with IFN-γ were infected with GFP-Mtb for 24h and immunostained with UBQLN1 specific antibody; Arrows indicate Mtb co-localized with UBQLN1. ns- not significant.(C) Park2-/- BMDMs were transfected with siRNA targeting UBQLN1 or control siRNA 3d prior to infection, treated with IFN-γ, and infected with GFP-Mtb for 24h prior to immunostaining for ubiquitin (FK2 antibody). Ubiquitin (FK2+) bacteria were quantified from two independent experiments. (A, C) **P<0.01, Fisher's exact test (comparing the proportion of FK2 positive bacteria in indicated samples). Results are mean +/- SEM. A minimum of 100 Mtb were counted in each experiment.


Figure 2C . Confocal images of U2OS cells transiently expressing lifeAct - GFP wild type and mutants of lifeAct residues interacting with F - actin , cells were additionally stained with SiR - actin to confirm the actin filaments . The line scan as indicated with yellow line on the cells shows the extent of lifeAct ( green ) and SiR - actin ( magenta ) co - staining of actin structures . Scale bar = 5µm . D . Binding affinities calculated from titration data of co - sedimentation assays of lifeAct - 14 ( 1 . 2µM ) and lifeAct - 17 ( 2 . 2µM ) as indicated . Data points for each concentration were averaged from 3 independent experiments , error bars represent S . D between independent experiments .

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Figure 2C. Confocal images of U2OS cells transiently expressing lifeAct-GFP wild type and mutants of lifeAct residues interacting with F-actin, cells were additionally stained with SiR-actin to confirm the actin filaments. The line scan as indicated with yellow line on the cells shows the extent of lifeAct (green) and SiR-actin (magenta) co-staining of actin structures. Scale bar = 5µm.D. Binding affinities calculated from titration data of co-sedimentation assays of lifeAct-14 (1.2µM) and lifeAct-17 (2.2µM) as indicated. Data points for each concentration were averaged from 3 independent experiments, error bars represent S.D between independent experiments.


Figure 3C . Representative TIRF images of lifeAct binding experiments as indicated . The remaining lifeAct concentration images are shown in Supplement Figure 4 . Scale bar = 5µm . D . Mean ratio of lifeAct fluorescent intensity bound to phalloidin and jasplakinolide F - actin ( mean and s . e . m ; n = 2 or 3 independent experiments with > 50 actin filament for each set ) .

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Figure 3C. Representative TIRF images of lifeAct binding experiments as indicated. The remaining lifeAct concentration images are shown in Supplement Figure 4. Scale bar = 5µm.D. Mean ratio of lifeAct fluorescent intensity bound to phalloidin and jasplakinolide F-actin (mean and s.e.m; n = 2 or 3 independent experiments with >50 actin filament for each set).


Figure 5A . Representative Coomassie stained SDS - PAGE gels of UTRN - ABD and UTRN - V87D co - sedimentation with F - actin . Pellet ( top ) and supernatant ( bottom ) fractions of individual co - sedimentation reactions of increasing utrophin concentrations , uncropped gel images of all the co - sedimentation reactions are presented in Supplement Figure 6 . B . Apparent Kd indicated was calculated from the titration data of co - sedimentation assays of utrophin wildtype ( 1 . 8µM ) and mutants ; H29A ( 2 . 8µM ) , I22D ( 38µM ) , V87D ( > 100µM ) and N109A ( 55µM ) as indicated . Data points for each concentration were averaged from 3 independent experiments , error bars represent S . D between independent experiments . C . Confocal images of U20S cells transfected with GFP tagged UTRN - mini and mCherry UTRN - ABD , stained with SIR - Actin shows F - actin structures , mainly stress fibers . Scale bar = 5µm .

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Figure 5A. Representative Coomassie stained SDS-PAGE gels of UTRN-ABD and UTRN-V87D co-sedimentation with F-actin. Pellet (top) and supernatant (bottom) fractions of individual co-sedimentation reactions of increasing utrophin concentrations, uncropped gel images of all the co-sedimentation reactions are presented in Supplement Figure 6.B. Apparent Kd indicated was calculated from the titration data of co-sedimentation assays of utrophin wildtype (1.8µM) and mutants; H29A (2.8µM), I22D (38µM), V87D (>100µM) and N109A (55µM) as indicated. Data points for each concentration were averaged from 3 independent experiments, error bars represent S.D between independent experiments.C. Confocal images of U20S cells transfected with GFP tagged UTRN-mini and mCherry UTRN-ABD, stained with SIR-Actin shows F-actin structures, mainly stress fibers. Scale bar = 5µm.


Figure 1A A neutral red - stained mouse pancreas is excised and digested with collagenase . Islets are then separated by hand picking and further digested into single islet cells . Scale bars : 10 mm ( left image ) ; 200 µm ( middle image ) ; 60 µm ( right image ) . B Epidydimal fat pads are isolated and processed mechanically and enzymatically . The obtained mixture , containing MVF and ECM , is filtrated for the purification of MVF . Scale bars : 20 mm ( left image ) ; 25 µm ( right image ) . C Western blots of CD31 , α - SMA and GAPDH in whole cell extracts of eEND , HDMEC , HUVEC and MVF . D Immunofluorescence stainings of CD31 / α - SMA and CD31 / PDGFR - β in MVF . Cell nuclei are stained with Hoechst 33342 ( blue ) . Scale bar : 20 µm . F PI and PI + MVF on days - 5 , - 3 and 0 . Scale bar : 200 µm . G Surface morphology of FI , CI , PI and PI + MVF on day 0 as visualized by scanning electron microscopy . Scale bar : 50 µm .

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Figure 1A A neutral red-stained mouse pancreas is excised and digested with collagenase. Islets are then separated by hand picking and further digested into single islet cells. Scale bars: 10 mm (left image); 200 µm (middle image); 60 µm (right image).B Epidydimal fat pads are isolated and processed mechanically and enzymatically. The obtained mixture, containing MVF and ECM, is filtrated for the purification of MVF. Scale bars: 20 mm (left image); 25 µm (right image).C Western blots of CD31, α-SMA and GAPDH in whole cell extracts of eEND, HDMEC, HUVEC and MVF.D Immunofluorescence stainings of CD31 / α-SMA and CD31 / PDGFR-β in MVF. Cell nuclei are stained with Hoechst 33342 (blue). Scale bar: 20 µm.F PI and PI+MVF on days -5, -3 and 0. Scale bar: 200 µm.G Surface morphology of FI, CI, PI and PI+MVF on day 0 as visualized by scanning electron microscopy. Scale bar: 50 µm.


Figure 2A Neutral red and trypan blue stainings of FI , CI , PI and PI + MVF . CI incubated for 24 h with 0 . 2 % H2O2 served as control . Scale bar : 100 µm . B Representative flow cytometric scatterplots of propidium iodide / annexin V - stained cells from PI + MVF and CI + H2O2 ( positive control ) . C Quantitative analysis of propidium iodide / annexin V - stained cells from FI , CI , PI , PI + MVF and CI + H2O2 subdivided in necrotic , necroptotic , apoptotic and vital cells in % of total cell number ( n = 3 each ) . D Schematic illustration of the mitochondrial ROS - sensor mito - roGFP2 - Orp1 . The fluorescence maximum of this sensor shifts from 488 nm to 405 nm with increasing mitochondrial H2O2 levels , as shown by representative fluorescence images of FI . Scale bar : 70 µm . E Quantitative analysis of the ROS sensor ratio ( 405 nm / 488 nm ) of FI , CI , PI and PI + MVF ( n = 25 each ) . Mean ± SD . One - way ANOVA and Tukey ' s multiple comparisons post hoc test were used for statistical analysis . * P < 0 . 05 vs . FI ; # P < 0 . 05 vs . CI ; + P < 0 . 05 vs . PI . F Representative Western blot analysis of β - actin and HO - 1 in whole cell extracts from FI , CI , PI and PI + MVF . G Quantitative analysis of HO - 1 expression in FI , CI , PI and PI + MVF ( n = 3 each ) . Mean ± SD . One - way ANOVA and Tukey ' s multiple comparisons post hoc test were used for statistical analysis . * P < 0 . 05 vs . FI ; # P < 0 . 05 vs . CI ; + P < 0 . 05 vs . PI . H Representative immunofluorescence stainings of HO - 1 / insulin and HO - 1 / CD31 in PI + MVF . Cell nuclei are stained with Hoechst 33342 ( blue ) . Scale bar : 50 µm . I Representative Western blot analysis of β - actin and HO - 1 in whole cell extracts from PI and MVF .

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Figure 2A Neutral red and trypan blue stainings of FI, CI, PI and PI+MVF. CI incubated for 24 h with 0.2% H2O2 served as control. Scale bar: 100 µm.B Representative flow cytometric scatterplots of propidium iodide / annexin V-stained cells from PI+MVF and CI+H2O2 (positive control). C Quantitative analysis of propidium iodide / annexin V-stained cells from FI, CI, PI, PI+MVF and CI+H2O2 subdivided in necrotic, necroptotic, apoptotic and vital cells in % of total cell number (n = 3 each). D Schematic illustration of the mitochondrial ROS-sensor mito-roGFP2-Orp1. The fluorescence maximum of this sensor shifts from 488 nm to 405 nm with increasing mitochondrial H2O2 levels, as shown by representative fluorescence images of FI. Scale bar: 70 µm.E Quantitative analysis of the ROS sensor ratio (405 nm / 488 nm) of FI, CI, PI and PI+MVF (n = 25 each). Mean ± SD. One-way ANOVA and Tukey's multiple comparisons post hoc test were used for statistical analysis. *P < 0.05 vs. FI; #P < 0.05 vs. CI; +P < 0.05 vs. PI.F Representative Western blot analysis of β-actin and HO-1 in whole cell extracts from FI, CI, PI and PI+MVF. G Quantitative analysis of HO-1 expression in FI, CI, PI and PI+MVF (n = 3 each). Mean ± SD. One-way ANOVA and Tukey's multiple comparisons post hoc test were used for statistical analysis. *P < 0.05 vs. FI; #P < 0.05 vs. CI; +P < 0.05 vs. PI. H Representative immunofluorescence stainings of HO-1 / insulin and HO-1 / CD31 in PI+MVF. Cell nuclei are stained with Hoechst 33342 (blue). Scale bar: 50 µm.I Representative Western blot analysis of β-actin and HO-1 in whole cell extracts from PI and MVF.


Figure 3A Representative immunofluorescence stainings of insulin / glucagon and insulin / somatostatin in FI , CI , PI and PI + MVF . Cell nuclei are stained with Hoechst 33342 ( blue ) . Scale bar : 60 µm . B - D Quantitative analysis of insulin - ( B ) , glucagon - ( C ) and somatostatin - positive cells ( D ) in FI , CI , PI and PI + MVF in % of all islet or organoid cells ( n = 20 each ) . Mean ± SD . One - way ANOVA and Tukey ' s multiple comparisons post hoc test were used for statistical analysis . * P < 0 . 05 vs . FI ; # P < 0 . 05 vs . CI ; + P < 0 . 05 vs . PI . E Western blot analysis of β - actin and insulin in whole cell extracts from FI , CI , PI and PI + MVF . Insulin secretion ( µU / mL ) ( normalized to DNA content ) of FI , CI , PI and PI + MVF exposed to 1 . 1 mM and 16 . 5 mM glucose ( n = 3 each ) . Mean ± SD . One - way ANOVA and Tukey ' s multiple comparisons post hoc test as well as an unpaired Student ' s t - test were used for statistical analysis . * P < 0 . 05 vs . insulin secretion at 1 . 1 mM glucose within each individual group ; + P < 0 . 05 vs . insulin secretion of PI at 16 . 5 mM glucose .

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Figure 3A Representative immunofluorescence stainings of insulin / glucagon and insulin / somatostatin in FI, CI, PI and PI+MVF. Cell nuclei are stained with Hoechst 33342 (blue). Scale bar: 60 µm. B-D Quantitative analysis of insulin- (B), glucagon- (C) and somatostatin-positive cells (D) in FI, CI, PI and PI+MVF in % of all islet or organoid cells (n = 20 each). Mean ± SD. One-way ANOVA and Tukey's multiple comparisons post hoc test were used for statistical analysis. *P < 0.05 vs. FI; #P < 0.05 vs. CI; +P < 0.05 vs. PI. E Western blot analysis of β-actin and insulin in whole cell extracts from FI, CI, PI and PI+MVF.Insulin secretion (µU/mL) (normalized to DNA content) of FI, CI, PI and PI+MVF exposed to 1.1 mM and 16.5 mM glucose (n = 3 each). Mean ± SD. One-way ANOVA and Tukey's multiple comparisons post hoc test as well as an unpaired Student's t-test were used for statistical analysis. *P < 0.05 vs. insulin secretion at 1.1 mM glucose within each individual group; +P < 0.05 vs. insulin secretion of PI at 16.5 mM glucose.


Figure 4A Representative immunofluorescence stainings of insulin and CD31 in CI , FI , PI and PI + MVF . Cell nuclei are stained with Hoechst 33342 ( blue ) . Scale bar : 100 µm . B Quantitative analysis of CD31 - positive cells in FI , CI , PI and PI + MVF in % of all islet or islet organoid cells ( n = 20 each ) . Mean ± SD . One - way ANOVA and Tukey ' s multiple comparisons post hoc test were used for statistical analysis . * P < 0 . 05 vs . FI ; # P < 0 . 05 vs . CI ; + P < 0 . 05 vs . PI . C Representative Western blot analysis of CD31 and β - actin in whole cell extracts from CI , FI , PI and PI + MVF . D Quantitative analysis of CD31 expression in FI , CI , PI and PI + MVF in % of FI ( n = 3 ) . Mean ± SD . One - way ANOVA and Tukey ' s multiple comparisons post hoc test were used for statistical analysis . * P < 0 . 05 vs . FI ; # P < 0 . 05 vs . CI ; + P < 0 . 05 vs . PI . E Confocal microscopic images of CD31 and cell nuclei ( blue ) in PI + MVF , demonstrating endothelial cells ( arrowheads ) . Scale bar : 100 µm . F Representative immunofluorescence staining of Ki67 and CD31 in PI + MVF . Cell nuclei are stained with Hoechst 33342 ( blue ) . Arrows mark endothelial cells expressing Ki67 . Scale bar : 100 µm . G Quantitative analysis of CD31 / Ki67 - positive cells in FI , CI , PI and PI + MVF in % of all endothelial cells ( n = 10 each ) . Mean ± SD . One - way ANOVA and Tukey ' s multiple comparisons post hoc test were used for statistical analysis . * P < 0 . 05 vs . FI ; # P < 0 . 05 vs . CI ; + P < 0 . 05 vs . PI . H Flow cytometric analysis of BrdU - positive cells from PI + MVF treated with DMSO ( vehicle ) or linsitinib during the entire fusion process ( n = 5 each ) . Mean ± SD . Unpaired Student ' s t - test was used for statistical analysis . * P < 0 . 05 vs . vehicle . I Representative Western blot analysis of pAkt , Akt , pERK1 / 2 and ERK1 / 2 from MVF cell extracts . MVF were treated with vehicle or insulin for 3 h . Quantitative analysis of pERK1 / 2 / ERK1 / 2 ( J ) ratio in PI + MVF treated with vehicle or insulin for 3 h . Data are expressed as relative intensities ( n = 3 each ) . Mean ± SD . Unpaired Student ' s t - test was used for statistical analysis . * P < 0 . 05 vs . vehicle . Quantitative analysis of pAkt / Akt ( K ) ratio in PI + MVF treated with vehicle or insulin for 3 h . Data are expressed as relative intensities ( n = 3 each ) . Mean ± SD . Unpaired Student ' s t - test was used for statistical analysis . * P < 0 . 05 vs . vehicle . L Representative bright field image of angiogenic sprouting from PI + MVF on 3 day of the sprouting assay . Scale bar : 150 µm . Insert : Immunofluorescence staining of insulin ( red ) and CD31 ( green ) of sprouted PI + MVF . Cell nuclei are stained with Hoechst 33342 ( blue ) . Scale bar : 30 µm . M Quantitative analysis of sprouting area of FI , CI , PI and PI + MVF in % of the initial size ( day 0 ) ( n = 10 each ) . Mean ± SD . One - way ANOVA and Tukey ' s multiple comparisons post hoc test were used for statistical analysis . * P < 0 . 05 vs . FI ; # P < 0 . 05 vs . CI ; + P < 0 . 05 vs . PI .

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Figure 4A Representative immunofluorescence stainings of insulin and CD31 in CI, FI, PI and PI+MVF. Cell nuclei are stained with Hoechst 33342 (blue). Scale bar: 100 µm. B Quantitative analysis of CD31-positive cells in FI, CI, PI and PI+MVF in % of all islet or islet organoid cells (n = 20 each). Mean ± SD. One-way ANOVA and Tukey's multiple comparisons post hoc test were used for statistical analysis. *P < 0.05 vs. FI; #P < 0.05 vs. CI; +P < 0.05 vs. PI. C Representative Western blot analysis of CD31 and β-actin in whole cell extracts from CI, FI, PI and PI+MVF.D Quantitative analysis of CD31 expression in FI, CI, PI and PI+MVF in % of FI (n = 3). Mean ± SD. One-way ANOVA and Tukey's multiple comparisons post hoc test were used for statistical analysis. *P < 0.05 vs. FI; #P < 0.05 vs. CI; +P < 0.05 vs. PI.E Confocal microscopic images of CD31 and cell nuclei (blue) in PI+MVF, demonstrating endothelial cells (arrowheads). Scale bar: 100 µm.F Representative immunofluorescence staining of Ki67 and CD31 in PI+MVF. Cell nuclei are stained with Hoechst 33342 (blue). Arrows mark endothelial cells expressing Ki67. Scale bar: 100 µm.G Quantitative analysis of CD31 / Ki67-positive cells in FI, CI, PI and PI+MVF in % of all endothelial cells (n = 10 each). Mean ± SD. One-way ANOVA and Tukey's multiple comparisons post hoc test were used for statistical analysis. *P < 0.05 vs. FI; #P < 0.05 vs. CI; +P < 0.05 vs. PI.H Flow cytometric analysis of BrdU-positive cells from PI+MVF treated with DMSO (vehicle) or linsitinib during the entire fusion process (n = 5 each). Mean ± SD. Unpaired Student's t-test was used for statistical analysis. *P < 0.05 vs. vehicle.I Representative Western blot analysis of pAkt, Akt, pERK1/2 and ERK1/2 from MVF cell extracts. MVF were treated with vehicle or insulin for 3 h. Quantitative analysis of pERK1/2 / ERK1/2 (J) ratio in PI+MVF treated with vehicle or insulin for 3 h. Data are expressed as relative intensities (n = 3 each). Mean ± SD. Unpaired Student's t-test was used for statistical analysis. *P < 0.05 vs. vehicle.Quantitative analysis of pAkt / Akt (K) ratio in PI+MVF treated with vehicle or insulin for 3 h. Data are expressed as relative intensities (n = 3 each). Mean ± SD. Unpaired Student's t-test was used for statistical analysis. *P < 0.05 vs. vehicle.L Representative bright field image of angiogenic sprouting from PI+MVF on 3 day of the sprouting assay. Scale bar: 150 µm. Insert: Immunofluorescence staining of insulin (red) and CD31 (green) of sprouted PI+MVF. Cell nuclei are stained with Hoechst 33342 (blue). Scale bar: 30 µm.M Quantitative analysis of sprouting area of FI, CI, PI and PI+MVF in % of the initial size (day 0) (n = 10 each). Mean ± SD. One-way ANOVA and Tukey's multiple comparisons post hoc test were used for statistical analysis. *P < 0.05 vs. FI; #P < 0.05 vs. CI; +P < 0.05 vs. PI.