( a ) Renilla luciferase expression controlled by an exogenous siRNA - siRNA * duplex targeting two partially complementary sites in HeLa cells depleted of ATG5 or ATG7 with siRNA ( black and grey bars correspond to two independent siRNAs ) . Results are normalized to firefly luciferase and Renilla luciferase expression in cells treated with a nonspecific siRNA - siRNA * duplex . n = 2 independent experiments .

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

(a) Renilla luciferase expression controlled by an exogenous siRNA-siRNA* duplex targeting two partially complementary sites in HeLa cells depleted of ATG5 or ATG7 with siRNA (black and grey bars correspond to two independent siRNAs). Results are normalized to firefly luciferase and Renilla luciferase expression in cells treated with a nonspecific siRNA-siRNA* duplex. n = 2 independent experiments.


( b ) Renilla luciferase expression controlled by let - 7 in HeLa cells depleted of NDP52 or ATG5 with siRNA ( black and grey bars are independent siRNAs ) . Results are normalized to firefly luciferase and a version of the Renilla luciferase construct with mutated let - 7 binding sites . n = 3 independent experiments . The asterisks denote mutations in the let - 7 target sites of the reporter .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Renilla luciferase expression controlled by let-7 in HeLa cells depleted of NDP52 or ATG5 with siRNA (black and grey bars are independent siRNAs). Results are normalized to firefly luciferase and a version of the Renilla luciferase construct with mutated let-7 binding sites. n = 3 independent experiments. The asterisks denote mutations in the let-7 target sites of the reporter.


( c ) Western blot analysis of endogenous miRNA - repressed proteins ( RAS , HMGA2 and CDK6 are repressed by let - 7 ; CDK6 is also repressed by miR - 16 ( bottom panel ) ) or control ( TUBA , α - tubulin ) in HeLa cells treated with control siRNA or either of two independent siRNAs targeting ATG7 . Cells were treated in parallel with nonspecific antagomir or let - 7 targeting antagomir .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Western blot analysis of endogenous miRNA-repressed proteins (RAS, HMGA2 and CDK6 are repressed by let-7; CDK6 is also repressed by miR-16 (bottom panel)) or control (TUBA, α-tubulin) in HeLa cells treated with control siRNA or either of two independent siRNAs targeting ATG7. Cells were treated in parallel with nonspecific antagomir or let-7 targeting antagomir.


( d ) Renilla luciferase expression controlled by the DICER 3 ′ UTR in HeLa cells treated with control antagomir or anti - let - 7 antagomir .

O O O O 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) Renilla luciferase expression controlled by the DICER 3′UTR in HeLa cells treated with control antagomir or anti-let-7 antagomir.


( e ) Western blot analysis of DICER in HeLa cells treated with control siRNA or either of two independent siRNAs targeting ATG7 . Cells were also treated with nonspecific antagomir or let - 7 targeting antagomir .

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

(e) Western blot analysis of DICER in HeLa cells treated with control siRNA or either of two independent siRNAs targeting ATG7. Cells were also treated with nonspecific antagomir or let-7 targeting antagomir.


( f ) Renilla luciferase expression controlled by the DICER 3 ′ UTR in HeLa cells treated with either of two independent siRNAs targeting ATG5 or a control siRNA . All error bars are s . e . m . Uncropped images of blots are shown in Supplementary Fig . S3 .

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

(f) Renilla luciferase expression controlled by the DICER 3′UTR in HeLa cells treated with either of two independent siRNAs targeting ATG5 or a control siRNA. All error bars are s.e.m. Uncropped images of blots are shown in Supplementary Fig. S3.


In vitro membrane pelleting assay comparing the TRAPPIII complex versus the core subunits . While both complexes exhibit a small amount of pelleting in the absence of synthetic liposomes , in their presence only the TRAPPIII complex is enriched in the membrane - bound fraction . ( S = supernatant , P = pellet ) Representative of n = 3 independent experiments . Quantification of D . Average % membrane binding between three assays is plotted , the error bars represent the 95 % confidence intervals . The value for each replicate is depicted . The negative values arise from subtraction of the average amount of protein that pellets in the absence of liposomes . Analyzed by unpaired , two - tailed Student ' s t - test with Welch ' s correction , * p = 0 . 0229 . n = 3 independent experiments .

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

In vitro membrane pelleting assay comparing the TRAPPIII complex versus the core subunits. While both complexes exhibit a small amount of pelleting in the absence of synthetic liposomes, in their presence only the TRAPPIII complex is enriched in the membrane-bound fraction. (S=supernatant, P=pellet) Representative of n=3 independent experiments.   Quantification of D. Average % membrane binding between three assays is plotted, the error bars represent the 95% confidence intervals. The value for each replicate is depicted. The negative values arise from subtraction of the average amount of protein that pellets in the absence   of liposomes. Analyzed by unpaired, two-tailed Student's t-test with Welch's correction, * p = 0.0229. n=3 independent experiments.


Representative Coomassie - stained gel of size exclusion chromatography fractions after purification of recombinant wild - type TRAPPIII complex . Representative of n = 3 independent experiments . As in A , using a Trs85 truncation containing only the final 198 amino acids . NOTE : Trs85 [ 501 - 698 ] is ~ 25kD in size and now migrates at the same size as some of the core subunits . The species migrating near 85kD are contaminants present due to the lower expression level of this mutant construct . Representative of n = 3 independent experiments . As in A / B , using a Trs85 mutant with a truncation of the loop from 575 - 603 . Trs85 [ ∆ 575 - 603 ] appears to dissociate from the core during chromatography , indicating a reduced affinity for the core subunits . Representative of n = 3 independent experiments .

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

Representative Coomassie-stained gel of size exclusion chromatography fractions after purification of recombinant wild-type TRAPPIII complex. Representative of n=3 independent experiments.   As in A, using a Trs85 truncation containing only the final 198 amino acids. NOTE: Trs85[501-698] is ~25kD in size and now migrates at the same size as some of the core subunits. The species migrating near 85kD are contaminants present due to the lower expression level of this mutant construct. Representative of n=3 independent experiments.   As in A/B, using a Trs85 mutant with a truncation of the loop from 575-603. Trs85[∆575-603] appears to dissociate from the core during chromatography, indicating a reduced affinity for the core subunits. Representative of n=3 independent experiments.


Coimmunoprecipitations of Trs23 - 6xHis - 3xFLAG with the same Trs85 mutants . Representative of n = 3 independent experiments . ( WCE = whole cell extract , WT = wild - type , EV = empty vector )

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

Coimmunoprecipitations of Trs23-6xHis-3xFLAG with the same Trs85 mutants. Representative of n=3 independent experiments. (WCE = whole cell extract, WT = wild-type, EV = empty vector)


Live cell fluorescence microscopy of Trs85 - mNeonGreen and mRFPmars - Ypt1 . Localization of Rab1 / Ypt1 to punctate structures is indicative of its activation by TRAPPIII . Scale bar is 2 microns . Representative of n = 3 independent experiments . ( WT = wild - type , EV = empty vector )

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

Live cell fluorescence microscopy of Trs85-mNeonGreen and mRFPmars-Ypt1. Localization of Rab1/Ypt1 to punctate structures is indicative of its activation by TRAPPIII. Scale bar is 2 microns. Representative of n=3 independent experiments. (WT = wild-type, EV = empty vector)


E . Quantification of the colocalization of TRAPPIII with mitochondrially - anchored Rab substrates . Average Mander ' s Overlap Coefficient ( MOC ) is plotted with 95 % confidence intervals . Each dot represents the MOC for a cropped image containing 2 - 9 cells . Samples are compared using an ordinary one - way ANOVA with Tukey ' s multiple comparisons test , * * * * p < 0 . 0001 . F . Quantification of the colocalization of TRAPPIII with mitochondrially - anchored Rab substrates . The sequence of wild - type Rab1 / Ypt1 HVD is in black , the substituted residues are colored to correspond with their location on the graph . The " CIM " motif is required for enzymatic prenylation of the Rab , but these chimeras are anchored to the plasma membrane independently of prenylation , via Fis1 - protein fusion . Average Mander ' s Overlap Coefficient ( MOC ) is plotted with 95 % confidence intervals . Each dot represents the MOC for a cropped image containing 2 - 9 cells . Samples are compared using an ordinary one - way ANOVA with Tukey ' s multiple comparisons test , but significance indicators are only present for each substitution mutant compared to " wild - type " Ypt31HVDYpt1 : vs . 181TTQ p = 0 . 9924 , vs . 184KKE p = 0 . 9997 , vs . 187DKGN * p = 0 . 0145 , vs . 191VNL , 194KGQS , 198LTNT * * * * p < 0 . 0001 , ns = not significant .

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

E. Quantification of the colocalization of TRAPPIII with mitochondrially-anchored Rab substrates. Average Mander's Overlap Coefficient (MOC) is plotted with 95% confidence intervals. Each dot represents the MOC for a cropped image containing 2-9 cells. Samples are compared using an ordinary one-way ANOVA with Tukey's multiple comparisons test, **** p < 0.0001. F. Quantification of the colocalization of TRAPPIII with mitochondrially-anchored Rab substrates. The sequence of wild-type Rab1/Ypt1 HVD is in black, the substituted residues are colored to correspond with their location on the graph. The "CIM" motif is required for enzymatic prenylation of the Rab, but these chimeras are anchored to the plasma membrane independently of prenylation, via Fis1-protein fusion. Average Mander's Overlap Coefficient (MOC) is plotted with 95% confidence intervals. Each dot represents the MOC for a cropped image containing 2-9 cells. Samples are compared using an ordinary one-way ANOVA with Tukey's multiple comparisons test, but significance indicators are only present for each substitution mutant compared to "wild-type" Ypt31HVDYpt1: vs. 181TTQ p = 0.9924, vs. 184KKE p = 0.9997, vs. 187DKGN * p = 0.0145, vs. 191VNL, 194KGQS, 198LTNT **** p < 0.0001, ns = not significant.


Coimmunoprecipitations of Trs23 - 6xHis - 3xFLAG with the same Trs85 substitution mutants . Representative of n = 3 independent experiments . ( WCE = whole cell extract , WT = wild - type , EV = empty vector )

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

Coimmunoprecipitations of Trs23-6xHis-3xFLAG with the same Trs85 substitution mutants. Representative of n=3 independent experiments. (WCE = whole cell extract, WT = wild-type, EV = empty vector)


Live cell fluorescence microscopy of Trs85 - mNeonGreen substitution mutants and mRFPmars - Ypt1 . Scale bar = 2 μm . Representative of n = 3 independent experiments . ( WT = wild - type , EV = empty vector )

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

Live cell fluorescence microscopy of Trs85-mNeonGreen substitution mutants and mRFPmars-Ypt1. Scale bar = 2 μm. Representative of n=3 independent experiments. (WT = wild-type, EV = empty vector)


GFP - Atg8 processing is monitored for either the TRAPP core binding mutations or the amphipathic helix ( membrane binding ) substitutions . With functional Trs85 , there is an accumulation of free GFP . Samples are from pre - rapamycin treatment ( - ) and 1hr after addition of rapamycin ( + ) . Representative of n = 3 independent experiments . ( WT = wild - type , EV = empty vector ) Quantification of A . The average of at least three replicates is plotted with error bars representing the 95 % confidence interval . The percent of free GFP for wild - type Trs85 is compared to each mutant using an unpaired , two - tailed Student ' s t - test with Welch ' s correction : vs . EV * * p = 0 . 0022 , vs . R618E , R619E , R620E , K621E * * * p = 0 . 0008 , vs . R618A , R619A , R620A , K621A * * * p = 0 . 0006 , vs . 501 - 698 * * * p = 0 . 0003 , vs . 4A p = 0 . 3295 , vs . 7A * * * p = 0 . 0008 , vs . 4E * * * p = 0 . 0005 , ns = not significant . ( WT = wild - type , EV = empty vector )

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

GFP-Atg8 processing is monitored for either the TRAPP core binding mutations or the amphipathic helix (membrane binding) substitutions. With functional Trs85, there is an accumulation of free GFP. Samples are from pre-rapamycin treatment (-) and 1hr after addition of rapamycin (+). Representative of n=3 independent experiments. (WT = wild-type, EV = empty vector)   Quantification of A. The average of at least three replicates is plotted with error bars representing the 95% confidence interval. The percent of free GFP for wild-type Trs85 is compared to each mutant using an unpaired, two-tailed Student's t-test with Welch's correction: vs. EV ** p =   0.0022, vs. R618E, R619E, R620E, K621E *** p = 0.0008, vs. R618A, R619A, R620A, K621A *** p = 0.0006, vs. 501-698 *** p = 0.0003, vs. 4A p = 0.3295, vs. 7A *** p = 0.0008, vs. 4E *** p = 0.0005, ns = not significant. (WT = wild-type, EV = empty vector)


( a ) Activation of the gB - specific CD8 + T cell hybridoma ( which expresses β - galactosidase as an indicator of T cell activation ) by macrophages infected for various times ( horizontal axis ) with HSV - 1 , then incubated for 12 h at 37 ° C with the hybridoma . A595 , absorbance at 595 nm .

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

(a) Activation of the gB-specific CD8+ T cell hybridoma (which expresses β-galactosidase as an indicator of T cell activation) by macrophages infected for various times (horizontal axis) with HSV-1, then incubated for 12 h at 37 °C with the hybridoma. A595, absorbance at 595 nm.


b ) Activation of the hybridoma as described in a , with the addition of dimethyl sulfoxide ( DMSO ; negative control ) , bafilomycin A ( Baf ) , brefeldin A ( BFA ) or MG - 132 at 2 h after macrophage infection .

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

b) Activation of the hybridoma as described in a, with the addition of dimethyl sulfoxide (DMSO; negative control), bafilomycin A (Baf), brefeldin A (BFA) or MG-132 at 2 h after macrophage infection.


( c ) Activation of the hybridoma as described in a , with the addition of bafilomycin A at 2 h after macrophage infection .

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

(c) Activation of the hybridoma as described in a, with the addition of bafilomycin A at 2 h after macrophage infection.


( d ) Immunofluorescence microscopy of gB ( blue ) and LAMP - 1 ( red ) in HSV - 1 - infected macrophages ; pink indicates colocalization . Original magnification , × 40 .

O O O O O O O O O O O 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) Immunofluorescence microscopy of gB (blue) and LAMP-1 (red) in HSV-1-infected macrophages; pink indicates colocalization. Original magnification, × 40.


( e ) CD8 + T cell - stimulatory capacity ( as described in a ) of uninfected macrophages ( Mock ) , of BMA ( BMA - HSV ) or J774 ( J774 - HSV ) macrophages infected for 8 h with HSV - 1 , and of cocultures of J774 macrophages ( H - 2d ) infected for 1 h with HSV - 1 , then mixed with uninfected BMA ( H - 2b ) macrophages at a ratio of 1 : 1 and cultured together for 8 h ( J774 - HSV + BMA ) .

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

(e) CD8+ T cell-stimulatory capacity (as described in a) of uninfected macrophages (Mock), of BMA (BMA-HSV) or J774 (J774-HSV) macrophages infected for 8 h with HSV-1, and of cocultures of J774 macrophages (H-2d) infected for 1 h with HSV-1, then mixed with uninfected BMA (H-2b) macrophages at a ratio of 1:1 and cultured together for 8 h (J774-HSV + BMA).


( a ) Immunofluorescence microscopy of LC3 expression in macrophages infected for 2 - 8 h ( left margin ) with HSV - 1 . Original magnification , × 20 .

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

(a) Immunofluorescence microscopy of LC3 expression in macrophages infected for 2-8 h (left margin) with HSV-1. Original magnification, × 20.


( b ) Activation of the gB - specific CD8 + T cell hybridoma ( described in Fig . 1a ) by macrophages infected for various times ( horizontal axis ) with HSV - 1 , with ( DMEM + 3 - MA ) or without ( DMEM ) the addition of 3 - methyladenine 2 h after infection , then incubated for 12 h at 37 ° C with the hybridoma .

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

(b) Activation of the gB-specific CD8+ T cell hybridoma (described in Fig. 1a) by macrophages infected for various times (horizontal axis) with HSV-1, with (DMEM + 3-MA) or without (DMEM) the addition of 3-methyladenine 2 h after infection, then incubated for 12 h at 37 °C with the hybridoma.


( c ) Activation of the hybridoma ( as described in b ) by macrophages transfected for 60 h with control ( Ctrl ) siRNA or Atg5 - specific siRNA , then infected for 8 h or 12 h with HSV - 1 .

O O O O O O O O O O O O O O O O O O O O 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) Activation of the hybridoma (as described in b) by macrophages transfected for 60 h with control (Ctrl) siRNA or Atg5-specific siRNA, then infected for 8 h or 12 h with HSV-1.


( d ) Immunoblot analysis of Atg - 5 in siRNA - treated macrophages .

(d) Immunoblot analysis of Atg-5 in siRNA-treated macrophages.


( e ) Activation of the hybridoma ( as described in b ) by macrophages infected with HSV - 1 and incubated at 37 ° C ( Basal ) , incubated for 12 h at 39 ° C before being infected with HSV - 1 ( heat shock ( HS ) ) , or treated with rapamycin during HSV - 1 infection ( Rapa ) , with ( + Baf ) or without the addition of bafilmycin A at 2 h after infection .

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

(e) Activation of the hybridoma (as described in b) by macrophages infected with HSV-1 and incubated at 37 °C (Basal), incubated for 12 h at 39 °C before being infected with HSV-1 (heat shock (HS)), or treated with rapamycin during HSV-1 infection (Rapa), with (+ Baf) or without the addition of bafilmycin A at 2 h after infection.


( f ) Activation of the hybridoma ( as described in b ) by macrophages transfected for 60 h with control siRNA or Atg5 - specific siRNA , then infected for 8 h with HSV - 1 , with ( + ) or without ( − ) the addition of rapamycin at 2 h after infection . Results in b , c , e , f are normalized to results obtained for CD8 + T cells stimulated with macrophages infected for 8 h at 37 ° C without further treatment ( b , e ) or infected macrophages treated with control siRNA ( c , f ) and are presented in arbitrary units . Data are representative of three independent experiments ( a , d ) or are from three independent experiments ( mean and s . e . m . of triplicate samples ; b , c , e , f ) .

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

(f) Activation of the hybridoma (as described in b) by macrophages transfected for 60 h with control siRNA or Atg5-specific siRNA, then infected for 8 h with HSV-1, with (+) or without (−) the addition of rapamycin at 2 h after infection. Results in b,c,e,f are normalized to results obtained for CD8+ T cells stimulated with macrophages infected for 8 h at 37 °C without further treatment (b,e) or infected macrophages treated with control siRNA (c,f) and are presented in arbitrary units. Data are representative of three independent experiments (a,d) or are from three independent experiments (mean and s.e.m. of triplicate samples; b,c,e,f).


( a - c ) Immunofluorescence microscopy of uninfected macrophages incubated at 37 ° C ( control ; a ) , subjected to mild heat shock ( b ) or treated with rapamycin ( c ) , then stained with anti - LC3 .

O O O O O O O O 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

(a-c) Immunofluorescence microscopy of uninfected macrophages incubated at 37 °C (control; a), subjected to mild heat shock (b) or treated with rapamycin (c), then stained with anti-LC3.


d ) Immunofluorescence microscopy of the expression of LC3 , gB and GFP ( VP26 ) by macrophages infected for various times ( left margin ) with HSV - 1 . White indicates colocalization .

d) Immunofluorescence microscopy of the expression of LC3, gB and GFP (VP26) by macrophages infected for various times (left margin) with HSV-1. White indicates colocalization.


( e ) Immunofluorescence microscopy of macrophages infected for 6 h or 8 h ( left margin ) with wild - type HSV - 1 ( WT ) or HSV - 1 lacking ICP34 . 5 ( Δ34 . 5 ) . Blue , staining of nuclei with DAPI ( 4 , 6 - diamidino - 2 - phenylindole ) . Original magnification , × 100 ( a - c ) or × 63 ( d , e ) . Results in a - e are representative of three independent experiments .

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

(e) Immunofluorescence microscopy of macrophages infected for 6 h or 8 h (left margin) with wild-type HSV-1 (WT) or HSV-1 lacking ICP34.5 (Δ34.5). Blue, staining of nuclei with DAPI (4,6-diamidino-2-phenylindole). Original magnification, × 100 (a-c) or × 63 (d,e). Results in a-e are representative of three independent experiments.


( f ) Activation of the gB - specific CD8 + T cell hybridoma ( as described in Fig . 1a ) by macrophages infected for various times ( horizontal axis ) with wild - type HSV - 1 strain 17 + ( WT 17 + ) or Δ34 . 5 HSV - 1 . Data are from three independent experiments ( mean and s . e . m . of triplicate samples ) .

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

(f) Activation of the gB-specific CD8+ T cell hybridoma (as described in Fig. 1a) by macrophages infected for various times (horizontal axis) with wild-type HSV-1 strain 17+ (WT 17+) or Δ34.5 HSV-1. Data are from three independent experiments (mean and s.e.m. of triplicate samples).


( g , h ) Immunoblot analysis ( IB ; g ) and flow cytometry ( h ) of the expression of HSV - 1 proteins ( g ) and gB ( h ) in macrophages infected for various times ( above lanes ( g ) or plots ( h ) ) with wild - type or Δ34 . 5 HSV - 1 . Ctrl , control ( uninfected BMA macrophages ; h ) . Data are representative of two ( g ) or three ( h ) independent experiments .

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

(g,h) Immunoblot analysis (IB; g) and flow cytometry (h) of the expression of HSV-1 proteins (g) and gB (h) in macrophages infected for various times (above lanes (g) or plots (h)) with wild-type or Δ34.5 HSV-1. Ctrl, control (uninfected BMA macrophages; h). Data are representative of two (g) or three (h) independent experiments.


( i ) Activation of the gB - specific CD8 + T cell hybridoma ( as described in Fig . 1a ) by macrophages infected for various times ( below graph ) with wild - type or Δ34 . 5 HSV - 1 , with ( + ) or without ( - ) the addition of bafilomycin A at 2 h after infection . Results in f , i are normalized to results obtained for CD8 + T cells stimulated with macrophages infected for 6 h with wild - type virus ( f ) or with infected macrophages incubated without bafilomycin ( i ) and are presented in arbitrary units . Data are from three independent experiments ( mean and s . e . m . of triplicate samples ) .

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

(i) Activation of the gB-specific CD8+ T cell hybridoma (as described in Fig. 1a) by macrophages infected for various times (below graph) with wild-type or Δ34.5 HSV-1, with (+) or without (-) the addition of bafilomycin A at 2 h after infection. Results in f,i are normalized to results obtained for CD8+ T cells stimulated with macrophages infected for 6 h with wild-type virus (f) or with infected macrophages incubated without bafilomycin (i) and are presented in arbitrary units. Data are from three independent experiments (mean and s.e.m. of triplicate samples).


Electron microscopy of macrophages 10 h after infection with HSV - 1 . ( a ) Arrows indicate membrane - coiled structures emerging from the nucleus of an infected cell . ( b - d ) Four - layered membrane structures formed by coiling of the nuclear membrane . ( e , f ) Glucose - 6 - phosphatase ( black deposits ) on autophagosome - like structures emerging from the nuclear envelope or free in the cytoplasm , and viral capsids in the cytoplasm engulfed in the lumen of an autophagosome - like compartment . N , nucleus ; VP , viral particles . Scale bars , 1 μm ( a ) , 0 . 25 μm ( b - d , f ) or 0 . 4 μm ( e ) . Images are representative of three independent experiments with at least 100 cell profiles in each .

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

Electron microscopy of macrophages 10 h after infection with HSV-1. (a) Arrows indicate membrane-coiled structures emerging from the nucleus of an infected cell. (b-d) Four-layered membrane structures formed by coiling of the nuclear membrane. (e,f) Glucose-6-phosphatase (black deposits) on autophagosome-like structures emerging from the nuclear envelope or free in the cytoplasm, and viral capsids in the cytoplasm engulfed in the lumen of an autophagosome-like compartment. N, nucleus; VP, viral particles. Scale bars, 1 μm (a), 0.25 μm (b-d,f) or 0.4 μm (e). Images are representative of three independent experiments with at least 100 cell profiles in each.


Immunoelectron microscopy of macrophages 10 h after infection with HSV - 1 . ( a - c ) Accumulation of LC3 ( a , b ) and gB ( c ) . ( d ) Fusion of four - layered membrane structures and lysosomes preloaded with bovine serum albumin - gold ( BSA - gold ; black dots ) . Original magnification , × 54 , 800 ( a , b ) , × 69 , 000 ( c ) or × 38 , 000 ( d ) . Images are representative of three ( a - c ) or two ( d ) independent experiments .

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

Immunoelectron microscopy of macrophages 10 h after infection with HSV-1. (a-c) Accumulation of LC3 (a,b) and gB (c). (d) Fusion of four-layered membrane structures and lysosomes preloaded with bovine serum albumin-gold (BSA-gold; black dots). Original magnification, × 54,800 (a,b), × 69,000 (c) or × 38,000 (d). Images are representative of three (a-c) or two (d) independent experiments.


( a ) Activation of the gB - specific CD8 + T cell hybridoma ( as described in Fig . 1a ) by macrophages exposed to DMSO ( negative control ) , mild heat shock , IL - 1β or IFN - γ .

(a) Activation of the gB-specific CD8+ T cell hybridoma (as described in Fig. 1a) by macrophages exposed to DMSO (negative control), mild heat shock, IL-1β or IFN-γ.


( b ) Dansylcadaverin staining of untreated macrophages ( Basal ) or macrophages exposed to mild heat shock , IFN - γ or IL - 1β and infected for 8 h with wild - type HSV - 1 , normalized to the signal obtained in basal conditions and presented in arbitrary units .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Dansylcadaverin staining of untreated macrophages (Basal) or macrophages exposed to mild heat shock, IFN-γ or IL-1β and infected for 8 h with wild-type HSV-1, normalized to the signal obtained in basal conditions and presented in arbitrary units.


( c - f ) Activation of the gB - specific CD8 + T cell hybridoma ( as described in Fig . 1a ) by macrophages incubated at 37 ° C ( c ) or exposed to mild heat shock ( d ) , IL - 1β ( e ) or IFN - γ ( f ) and infected for 8 h with wild - type HSV - 1 with the addition of DMSO ( negative control ) , 3 - methyladenine , bafilomycin , brefeldin A or MG - 132 at 2 h after infection . Results in a , c - f are normalized to results obtained for CD8 + T cells stimulated with macrophages incubated with DMSO in each condition and are presented in arbitrary units . Data are from three independent experiments ( mean and s . e . m . of triplicate samples ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-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 O O O O O O O O O O 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-f) Activation of the gB-specific CD8+ T cell hybridoma (as described in Fig. 1a) by macrophages incubated at 37 °C (c) or exposed to mild heat shock (d), IL-1β (e) or IFN-γ (f) and infected for 8 h with wild-type HSV-1 with the addition of DMSO (negative control), 3-methyladenine, bafilomycin, brefeldin A or MG-132 at 2 h after infection. Results in a,c-f are normalized to results obtained for CD8+ T cells stimulated with macrophages incubated with DMSO in each condition and are presented in arbitrary units. Data are from three independent experiments (mean and s.e.m. of triplicate samples).


Viability curve of cells following 24h of recombinant Pol II expression induction by removal of tetracycline ( Tet - Off system ) and treatment with α - amanitin ( black arrow - head ) . Untransfected Raji ( WT : black ) is shown for reference . For all experiments presented in this article , cells are collected after 24 hours of α - amanitin treatment for CTD - ∆ 5 and YFFF mutants expressing cells since CTD - ∆ 5 is unstable after 48h ( see panel C ) . Error bars representing top and bottom values are based on the average of 3 independent experiments .

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

Viability curve of cells following 24h of recombinant Pol II expression induction by removal of tetracycline (Tet-Off system) and treatment with α-amanitin (black arrow-head). Untransfected Raji (WT: black) is shown for reference. For all experiments presented in this article, cells are collected after 24 hours of α-amanitin treatment for CTD-∆5 and YFFF mutants expressing cells since CTD-∆5 is unstable after 48h (see panel C). Error bars representing top and bottom values are based on the average of 3 independent experiments.


Western blot analysis of the recombinant RPB1 expression ( rWT , YFFF , and ∆ 5 ) assessed by ( HA ) - tag expression after 24 or 48 hours of α - amanitin treatment following 24h of induction . Untransfected Raji extracts are shown for comparison ( WT ) . Total RPB1 levels are represented by the F12 western blot . Tubulin was used as a loading control .

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

Western blot analysis of the recombinant RPB1 expression (rWT, YFFF, and ∆5) assessed by (HA)-tag expression after 24 or 48 hours of α-amanitin treatment following 24h of induction. Untransfected Raji extracts are shown for comparison (WT). Total RPB1 levels are represented by the F12 western blot. Tubulin was used as a loading control.


Volcano plot of differentially expressed genes in chromatin associated RNA - seq ( ChrRNA ) data sets : protein coding genes UP ( purple ) and DOWN ( pink ) regulated in B relative to A after 24h hours α - amanitin treatment ( n = 22810 ) . Experiments were done in biological duplicates . Threshold : log2 fold change ≥ 3 , p - value < 0 . 05 .

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

Volcano plot of differentially expressed genes in chromatin associated RNA-seq (ChrRNA) data sets: protein coding genes UP (purple) and DOWN (pink) regulated in B relative to A after 24h hours α-amanitin treatment (n=22810). Experiments were done in biological duplicates. Threshold: log2 fold change ≥ 3, p-value < 0.05.


Example of ChrRNA read - through phenotype at the 5 ' ( anti - sense ) and 3 ' ( sense ) ends in CTD - ∆ 5 at the GBE1 locus .

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

Example of ChrRNA read-through phenotype at the 5' (anti-sense) and 3' (sense) ends in CTD-∆5 at the GBE1 locus.


Boxplots of ChrRNA read - through ( RT ) indexes based on signal over 20kb upstream ( anti - sense ) and 20kb downstream ( sense ) regions of coding genes in rWT ( dotted grey line ) and CTD - ∆ 5 mutant ( blue ) . Units are asinh transformed . Experiments were done in biological duplicates . Boxplots represent minimal and maximal values , first and third quartiles with median value as central band . P - values were calculated using two - sided Wilcoxon tests .

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

Boxplots of ChrRNA read-through (RT) indexes based on signal over 20kb upstream (anti-sense) and 20kb downstream (sense) regions of coding genes in rWT (dotted grey line) and CTD-∆5 mutant (blue). Units are asinh transformed. Experiments were done in biological duplicates. Boxplots represent minimal and maximal values, first and third quartiles with median value as central band. P-values were calculated using two-sided Wilcoxon tests.


Example of Pol - HA ChIP - seq showing global spreading of Pol II along the genome accompanied by a loss of Pol - II accumulation on coding genes in CTD - ∆ 5 ( orange ) compared to rWT ( black ) and their associated ChrRNA - signal .

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

Example of Pol-HA ChIP-seq showing global spreading of Pol II along the genome accompanied by a loss of Pol-II accumulation on coding genes in CTD-∆5 (orange) compared to rWT (black) and their associated ChrRNA-signal.


Average metagene profiles comparing Pol - II , H3K4me3 and H3K4me1 signals on active promoters ( top ) and active enhancers ( bottom ) in rWT and CTD - ∆ 5 . Experiments were done in biological duplicates however only the sample with the best signal to noise ratio was used for subsequent analysis . P - values were calculated using two - sided Wilcoxon tests .

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

Average metagene profiles comparing Pol-II, H3K4me3 and H3K4me1 signals on active promoters (top) and active enhancers (bottom) in rWT and CTD-∆5. Experiments were done in biological duplicates however only the sample with the best signal to noise ratio was used for subsequent analysis. P-values were calculated using two-sided Wilcoxon tests.


Example of Pol - HA , H3K4me3 and H3K4me1 Chip - seq signals in rWT and CTD - ∆ 5 illustrating the metagene profiles in figure 3B . Top : Light green rectangles highlight putative active enhancers in rWT .

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

Example of Pol-HA, H3K4me3 and H3K4me1 Chip-seq signals in rWT and CTD-∆5 illustrating the metagene profiles in figure 3B. Top: Light green rectangles highlight putative active enhancers in rWT.


Example showing ChrRNA - seq ( top ) and its associated PolyA RNA - seq signals ( bottom ) in rWT and CTD - ∆ 5 .

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

Example showing ChrRNA-seq (top) and its associated PolyA RNA-seq signals (bottom) in rWT and CTD-∆5.


Boxplots of PolyA read - through ( RT ) indexes based on signal over 20kb upstream ( anti - sense ) and 20kb downstream ( sense ) regions of coding genes in rWT ( dotted grey line ) and CTD - ∆ 5 mutant ( blue ) . Units are asinh transformed . Experiments were done in biological duplicates . Boxplots represent minimal and maximal values , first and third quartiles with median value as central band . P - values were calculated using two - sided Wilcoxon tests .

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

Boxplots of PolyA read-through (RT) indexes based on signal over 20kb upstream (anti-sense) and 20kb downstream (sense) regions of coding genes in rWT (dotted grey line) and CTD-∆5 mutant (blue). Units are asinh transformed. Experiments were done in biological duplicates. Boxplots represent minimal and maximal values, first and third quartiles with median value as central band. P-values were calculated using two-sided Wilcoxon tests.


Volcano plot comparing the Pol II interactome in rWT and CTD - ∆ 5 mutant . Left : Proteins lost in CTD - ∆ 5 , Right : Proteins enriched in CTD - ∆ 5 . Highlighted are the subunits of the Mediator ( purple ) and Integrator complexes ( orange ) CTD phosphatases scaffolds ( pink ) , Ubiquitin ligases ( light blue ) and SOSS complex subunits ( red ) . In black are highlighted some chosen interactors gained by the CTD - ∆ 5 Threshold : log2 fold change ≥ 4 ; p - value < 0 . 05 . Data based on three independent biological replicates . The table in the figure sums up the proteins that do not change significantly ; dotted lines connected to the table represent the region where these proteins are found in the

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

Volcano plot comparing the Pol II interactome in rWT and CTD-∆5 mutant. Left: Proteins lost in CTD-∆5, Right: Proteins enriched in CTD-∆5. Highlighted are the subunits of the Mediator (purple) and Integrator complexes (orange) CTD phosphatases scaffolds (pink), Ubiquitin ligases (light blue) and SOSS complex subunits (red). In black are highlighted some chosen interactors gained by the CTD-∆5 Threshold: log2 fold change ≥ 4; p-value < 0.05. Data based on three independent biological replicates. The table in the figure sums up the proteins that do not change significantly; dotted lines connected to the table represent the region where these proteins are found in the volcano


Example of CTD - ∆ 5 polymerases transcribing through TAD borders marked by CTCF sites ( highlighted in light purple ) . HiC map on top represents the rWT interactions shown in the zoom in panel A . Purple boxes represent TADs called in human B cells CTCF ChIP - seq in rWT and CTD - ∆ 5 are both shown in black and their associated ChrRNA - seq signal are also shown .

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

Example of CTD-∆5 polymerases transcribing through TAD borders marked by CTCF sites (highlighted in light purple). HiC map on top represents the rWT interactions shown in the zoom in panel A. Purple boxes represent TADs called in human B cells CTCF ChIP-seq in rWT and CTD-∆5 are both shown in black and their associated ChrRNA-seq signal are also shown.


Size exclusion chromatography ( SEC ) results for MICU2 _ WT and a set of mutants in the presence of Ca2 + monitored at 280 nm absorption wavelength . Size exclusion chromatography ( SEC ) results for MICU2 _ WT and a set of mutants in the presence of EGTA monitored at 280 nm absorption wavelength .

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

Size exclusion chromatography (SEC) results for MICU2_WT and a set of mutants in the presence of Ca2+ monitored at 280 nm absorption wavelength. Size exclusion chromatography (SEC) results for MICU2_WT and a set of mutants in the presence of EGTA monitored at 280 nm absorption wavelength.


MICU2 _ WT ( A ) , MICU2 _ K172A ( B ) were pulled - down by GST - MICU1 both in the absence and presence of 2 mM Ca2 + . MICU2 _ EF1mut was not pulled - down by GST - MICU1 in the presence of 2 mM Ca2 + ( C ) , while MICU2 _ EF2mut was pulled - down by GST - MICU1 both in the absence and presence of 2 mM Ca2 + ( D ) . MICU2 _ R352A was not pulled - down by GST - MICU1 in the absence of 2 mM Ca2 + ( E ) , while MICU2 _ E329A was pulled - down by GST - MICU1 both in the absence and presence of 2 mM Ca2 + ( F ) . MICU2 _ D185A was partially pulled - down by GST - MICU1 in the presence of Ca2 + , while they showed a strong interaction in the absence of 2 mM Ca2 + . MICU2 _ E196K was not pulled - down by GST - MICU1 in the presence of 2 mM Ca2 + , while a strong interaction was observed in the absence of 2 mM Ca2 + .

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

MICU2_WT (A), MICU2_K172A (B) were pulled-down by GST-MICU1 both in the absence and presence of 2 mM Ca2+. MICU2_EF1mut was not pulled-down by GST-MICU1 in the presence of 2 mM Ca2+ (C), while MICU2_EF2mut was pulled-down by GST-MICU1 both in the absence and presence of 2 mM Ca2+ (D). MICU2_R352A was not pulled-down by GST-MICU1 in the absence of 2 mM Ca2+ (E), while MICU2_E329A was pulled-down by GST-MICU1 both in the absence and presence of 2 mM Ca2+ (F). MICU2_D185A was partially pulled-down by GST-MICU1 in the presence of Ca2+, while they showed a strong interaction in the absence of 2 mM Ca2+. MICU2_E196K was not pulled-down by GST-MICU1 in the presence of 2 mM Ca2+, while a strong interaction was observed in the absence of 2 mM Ca2+.


GST - MICU1 _ EF1mut failed to pull down MICU2 _ WT in the absence of 2 mM Ca2 + , whereas they displayed a strong interaction in the presence of 2 mM Ca2 + . GST - MICU1 _ F383A & H385A failed to pull - down MICU2 _ WT in the presence of Ca2 + , whereas they displayed a strong interaction in the absence of 2 mM Ca2 + . C GST - MICU1 _ E242K failed to pull - down MICU2 _ WT in the absence of Ca2 + , whereas they displayed a strong interaction in the presence of 2 mM Ca2 + . GST - MICU1 _ D231A partly pulled - down MICU2 _ WT in the absence of Ca2 + , whereas they displayed a strong interaction in the presence of 2 mM Ca2 + . GST - MICU1 _ F383A failed to pull down MICU2 _ WT in the presence of Ca2 + , whereas they displayed a strong interaction in the absence of 2 mM Ca2 + . GST - MICU1 _ H385A exhibited a strong interaction with MICU2 both in the absence and presence of 2 mM Ca2 + .

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

GST-MICU1_EF1mut failed to pull down MICU2_WT in the absence of 2 mM Ca2+, whereas they displayed a strong interaction in the presence of 2 mM Ca2+. GST-MICU1_F383A & H385A failed to pull-down MICU2_WT in the presence of Ca2+, whereas they displayed a strong interaction in the absence of 2 mM Ca2+. C GST-MICU1_E242K failed to pull-down MICU2_WT in the absence of Ca2+, whereas they displayed a strong interaction in the presence of 2 mM Ca2+. GST-MICU1_D231A partly pulled-down MICU2_WT in the absence of Ca2+, whereas they displayed a strong interaction in the presence of 2 mM Ca2+. GST-MICU1_F383A failed to pull down MICU2_WT in the presence of Ca2+, whereas they displayed a strong interaction in the absence of 2 mM Ca2+. GST-MICU1_H385A exhibited a strong interaction with MICU2 both in the absence and presence of 2 mM Ca2+.


ITC analyses of MICU1 and MICU2 binding properties in the presence of 2 mM EGTA ( G ) and CaCl2 ( H ) .

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

ITC analyses of MICU1 and MICU2 binding properties in the presence of 2 mM EGTA (G) and CaCl2 (H).


( A ) Anti - NANP5 and anti - C - CSP serum IgG . Circles represent ELISA area under the curve ( AUC ) values for individual donors ( n = 12 ) . ( B ) Ten most frequent Ig heavy and light chain V gene pairs in PfCSP - reactive memory B cells ( MBCs ; n = 1172 ) .

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

(A) Anti-NANP5 and anti-C-CSP serum IgG. Circles represent ELISA area under the curve (AUC) values for individual donors (n=12). (B) Ten most frequent Ig heavy and light chain V gene pairs in PfCSP-reactive memory B cells (MBCs; n=1172).


somatic hypermutation ( SHM ) count ( E ) in IGHV3 - 21 and IGHV3 - 33 genes from

somatic hypermutation (SHM) count (E) in IGHV3-21 and IGHV3-33 genes from MBCs


( F ) NANP10 ( left ) and C - CSP ( right ) binding of FL - CSP - reactive mAbs ( n = 177 ) . Circles represent ELISA AUC values for each mAb .

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(F) NANP10 (left) and C-CSP (right) binding of FL-CSP-reactive mAbs (n=177). Circles represent ELISA AUC values for each mAb.


( C ) VH SHM load of VH3 - 21 and non - VH3 - 21 mAbs . mAbs with unmutated VH are highlighted in green .

O O O 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) VH SHM load of VH3-21 and non-VH3-21 mAbs. mAbs with unmutated VH are highlighted in green.


( D ) Amino acid ( aa ) VH replacement ( red bars ) and silent ( black bars ) SHM in VH3 - 21 mAbs ( n = 113 ) . FWR , framework region ; CDR , complementarity - determining region .

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(D) Amino acid (aa) VH replacement (red bars) and silent (black bars) SHM in VH3-21 mAbs (n=113). FWR, framework region; CDR, complementarity-determining region.


( D ) C - CSP reactivity of the α - TSR - specific mAb 1710 in a blocking ELISA with C - CSP - specific VH3 - 21 ( n = 21 ; upper panel ) or non - VH3 - 21 ( n = 17 ; lower panel ) mAbs with the indicated SPR affinities . mAb 1710 binding without blocking ( blue ) and after self - blocking ( gold ) is shown for comparison .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) C-CSP reactivity of the α-TSR-specific mAb 1710 in a blocking ELISA with C-CSP-specific VH3-21 (n=21; upper panel) or non-VH3-21 (n=17; lower panel) mAbs with the indicated SPR affinities. mAb 1710 binding without blocking (blue) and after self-blocking (gold) is shown for comparison.


( E ) C - CSP reactivity of mAb 1512 in a blocking ELISA with mAbs ( with the indicated SPR affinities ) that do not block C - CSP binding of mAb 1710 ( open circles ; D ) . mAb 1512 binding without blocking ( purple ) and after self - blocking ( magenta ) is shown for comparison .

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

(E) C-CSP reactivity of mAb 1512 in a blocking ELISA with mAbs (with the indicated SPR affinities) that do not block C-CSP binding of mAb 1710 (open circles; D). mAb 1512 binding without blocking (purple) and after self-blocking (magenta) is shown for comparison.


( F ) ELISA cross - reactivity with Pf - 7G8 FL - CSP of CSP - specific mAbs that block ( left ) or do not block ( right ) mAb 1710 binding to NF54 C - CSP .

(F) ELISA cross-reactivity with Pf-7G8 FL-CSP of CSP-specific mAbs that block (left) or do not block (right) mAb 1710 binding to NF54 C-CSP.


percentage 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 ) .

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

percentage 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 ) .

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

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 ) .

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

(E) 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

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

(F) 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


( 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 .

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

(A) 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.


C 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O O O O O 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 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 .

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.


C 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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.


A 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 .

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

A 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.


F 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 .

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

F 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.


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

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

A. 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 .

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

B. Representative 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 .

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

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 .

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


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

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

A. 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 .

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

B. Representative 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 .

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 .

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


A . The model of the Elp123 sub - complex .

A. 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 .

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


D . Crosslinks supporting the orientation of the Elp456 hexamer .

D. Crosslinks supporting the orientation of the Elp456 hexamer.


A . GST - pull down assays of purified GST - tagged Elp3 SAM domain ( aa72 - 389 ) 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 .

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

A. 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 .

O O O 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. 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 .

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 .

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


B . Same as A but tilted view to highlight 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 .

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


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

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


A . 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 .

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

A. 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.


A . 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 .

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

A. 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 .

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

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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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.


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 . 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 .

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

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. 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 .

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

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 .

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

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

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.


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 . A . FISH analysis to detect poly ( A ) + RNA and IF analysis against FUS protein . Images are a single 0 . 2 micron Z section .

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

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. 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 ) .

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

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 .

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

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

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.


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 .

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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.


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 .

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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 .

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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.


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 .

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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 .

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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.


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 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

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

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 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 acid


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 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

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

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 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 acid


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 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 -

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

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 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-kynurenine


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

A - 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.


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 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 .

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

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 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

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

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 acid


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

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 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.


A 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 ) .

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A 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 .

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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.