Representative Western Blot ( J ) relative bar plot ( K ) showing collagen expression in Fam134a knockout MEFs overexpressing wild type Fam134b . Representative Western Blot ( L ) relative bar plot ( M ) showing collagen expression in Fam134a knockout MEFs overexpressing wild type Fam134c .

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Representative Western Blot (J) and the relative bar plot (K) showing collagen expression in Fam134a knockout MEFs overexpressing wild type Fam134b. Representative Western Blot (L) and the relative bar plot (M) showing collagen expression in Fam134a knockout MEFs overexpressing wild type Fam134c.


C . Confocal of MDA - MB - 231 transfected with ZsGreen or ZsRASSF1C grown in 3D - collagen matrix . were imaged stained with Phalloidin - 568 ( red ) , pMLCII / Alexa ‑ 633 ( false ‑ coloured green ) or imaged for ZsGreen ( false - coloured ) . Scale 20 μm .

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C. Confocal images of MDA-MB-231 cells transfected with ZsGreen or ZsRASSF1C grown in 3D-collagen matrix. Cells were imaged and stained with Phalloidin-568 (red), pMLCII/Alexa‑633 (false‑coloured green) or imaged for ZsGreen (false-coloured white). Scale 20 μm.


D . Single of MDA - MB - 231 cultured in 3D - collagen analysed for 24 hours after transient knock - down using either a control ( NT , non - targeting ) or a targeting RASSF1 locus . RASSF1 knock - down marks a reduction in rounded . Data information : data are from n = 3 independent experiments . Data are analyzed by Student T - represented as mean ± . * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 .

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D. Single cell morphology assay of MDA-MB-231 cells cultured in 3D-collagen and analysed for morphology 24 hours after transient knock-down using either a control sequence (NT, non-targeting) or a sequence targeting the RASSF1 locus. RASSF1 knock-down marks a reduction in rounded cells. Data information: All data are from n=3 independent experiments. Data are analyzed by Student T-test and represented as mean ± SEM.* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.


C . Western blot of in MDA - MB - 231 transiently transfected with siRNA targeting a control ( NT ) or targeting RASSF1 . GAPDH was used as a loading control .

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C. Western blot analysis of proteins in MDA-MB-231 cells transiently transfected with siRNA targeting a control sequence (NT) or targeting RASSF1. GAPDH was used as a loading control.


D . Immunoprecipitation from MDA - MB - 231 transiently transfected with control ( pcDNA3 ) or HA ‑ RASSF1C plasmid . Pull ‑ down was performed using a SRC or same species IgG antibody blotted with indicated antibodies .

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D. Immunoprecipitation from MDA-MB-231 transiently transfected with control (pcDNA3) or HA‑RASSF1C plasmid. Pull‑down was performed using a SRC or same species IgG antibody and blotted with indicated antibodies.


H . of rounded versus elongated in single in 3D ‑ collagen indicating degree of mesenchymal - amoeboid transition of MDA - MB - 231 when RASSF1C or described mutants are expressed . I . Representative confocal show elongated or rounded per field of view of MDA ‑ MB - 231 transfected with DsRed , DsRASSF1C , DsRASSF1C - R197W or DsRASSF1C - R199F , grown in 3D ‑ collagen stained with Phalloidin - 568 ( red ) for F ‑ actin pMLCII / Alexa 633 . Scale bars represent 10 μm .

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H. Quantification of rounded versus elongated cells in single cell morphology assay in 3D‑collagen indicating the degree of mesenchymal-amoeboid transition of MDA-MB-231 cells when RASSF1C or the described mutants are expressed. I. Representative confocal images show elongated or rounded cells per field of view of MDA‑MB-231 cells transfected with DsRed, DsRASSF1C, DsRASSF1C-R197W or DsRASSF1C-R199F, grown in 3D‑collagen and stained with Phalloidin-568 (red) for F‑actin and pMLCII/Alexa 633. Scale bars represent 10 μm.


A . Left : Cartoon showing principle behind Cre - carrying EVs how recipient switch from DsRed + to eGFP + expression upon Cre - mediated excision of DsRed locus . Centre : representative , right : of T47DDsRed that converted to eGFP + in bottom wells of a transwell system in presence of MDA - MB - 231CFP ; Cre ; Control or MDA ‑ MB ‑ 231CFP ; Cre ; HA - RASSF1C seeded in upper wells . Scale bars represent 100 μm . Data information : data are from n = 3 independent experiments . Data are analyzed by Student T - represented as mean ± . * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * * p ≤ 0 . 0001 .

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A. Left: Cartoon showing the principle behind the Cre-carrying EVs and how recipient cells switch from DsRed+ to eGFP+ expression upon Cre-mediated excision of the DsRed locus. Centre: representative images, and right: quantification of T47DDsRed cells that converted to eGFP+ in the bottom wells of a transwell system in the presence of MDA-MB-231CFP;Cre;Control or MDA‑MB‑231CFP;Cre;HA-RASSF1C seeded in the upper wells. Scale bars represent 100 μm. Data information: All data are from n=3 independent experiments. Data are analyzed by Student T-test and represented as mean ± SEM.* p ≤ 0.05, ** p ≤ 0.01, **** p ≤ 0.0001.


C . Western blot of EVs isolated from MDA - MB - 231 transfected with Control ( pcDNA3 ) or FLAG - RASSF1C with indicated antibodies . Equal amounts of were loaded after normalization via microBCA .

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C. Western blot analysis of EVs isolated from MDA-MB-231 cells transfected with Control (pcDNA3) or FLAG-RASSF1C with indicated antibodies. Equal amounts of protein were loaded after normalization via microBCA assay.


F . Left : Representative of Boyden chamber invasion in 3D matrigel of MCF7 , treated with 20 ng / μl EVs derived from MDA - MB - 231 , transfected same constructs used in ( e ) . Right : of Boyden chamber invasion 24 hours after EV treatment . Scale bars represent 200µm . Data information : data are from n = 3 independent experiments . Data are analyzed by Student T - represented as mean ± . * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * * p ≤ 0 . 0001 .

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F. Left: Representative images of Boyden chamber invasion assay in 3D matrigel of MCF7 cells, treated with 20 ng/μl EVs derived from MDA-MB-231 cells, transfected the same constructs used in (e). Right: Quantification of Boyden chamber invasion 24 hours after EV treatment. Scale bars represent 200µm. Data information: All data are from n=3 independent experiments. Data are analyzed by Student T-test and represented as mean ± SEM.* p ≤ 0.05, ** p ≤ 0.01, **** p ≤ 0.0001.


D . Representative confocal ( upper row ) of of ALDH1 expression in MDA - MB - 231 expressing RASSF1C ( black bar in graph ) or after siRNA treatment against RASSF1 ( grey bar ) . Lower row shows merged with DAPI ( blue ) , ALDH1 ( green ) RASSF1C ( red ) . Scale bars represent 20 μm . Data Information : data are from n = 3 independent experiments . Data are analyzed by Student T - represented as mean ± . * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 .

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D. Representative confocal images (upper row) and quantification of fluorescent intensity of ALDH1 expression in MDA-MB-231 cells expressing RASSF1C (black bar in the graph) or after siRNA treatment against RASSF1 (grey bar). Lower row shows merged images with DAPI (blue), ALDH1 (green) and RASSF1C (red). Scale bars represent 20 μm. Data Information: All data are from n=3 independent experiments. Data are analyzed by Student T-test and represented as mean ± SEM.* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.


J . qRT ‑ PCR expression of NANOG , OCT4 , SOX2 in MCF7 24 h after exposure to 50 ng / μl EVs derived from MDA ‑ MB ‑ 231 ( treated as indicated ) . Data Information : data are from n = 3 independent experiments . Data are analyzed by Student T - represented as mean ± . * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 .

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J. qRT‑PCR expression of NANOG, OCT4, SOX2 in MCF7 cells 24 h after exposure to 50 ng/μl EVs derived from MDA‑MB‑231 cells (treated as indicated). Data Information: All data are from n=3 independent experiments. Data are analyzed by Student T-test and represented as mean ± SEM.* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.


B . Left : representative confocal of mammary formed by co - injection of T47DDsRed reporter with either MDA - MB - 231CFP ; Cre ; Control or MDA - MB - 231CFP ; Cre ; HA - RASSF1C . Scale 200 μm . Right : averaged values for mice for of eGFP + or CFP + as part of whole . Data information : data are from n = 3 independent experiments . Data are analyzed by Student T - represented as mean ± . * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001 .

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B. Left: representative confocal images of mammary tumor formed by the co-injection of T47DDsRed reporter cells with either MDA-MB-231CFP;Cre;Control or MDA-MB-231CFP;Cre;HA-RASSF1C. Scale 200 μm. Right: averaged values for all mice for the percent of the eGFP+ or CFP+ cells as part of the whole tumor. Data information: All data are from n=3 independent experiments. Data are analyzed by Student T-test and represented as mean ± SEM.* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001.


B . Kaplan - Meier plots of overall survival of 496 stage I II breast cancer with different of promoter / CpG island methylation of RASSF1A , previously reported to be associated with outcome in breast cancer . P values were obtained from a log - rank .

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B. Kaplan-Meier plots of overall survival of 496 stage I and II breast cancer patients with different levels of promoter/CpG island methylation of RASSF1A, previously reported to be associated with outcome in breast cancer patients. P values were obtained from a log-rank test.


C . Left : depicting methylation patterns across RASSF1 gene . In green , RASSF1 - 2γ CpG island is depicted ; in green , RASSF1 - 1α CpG island is depicted . Right : Kaplan ‑ Meyer survival curves depicting interaction of RASSF1A RASSF1C methylation to predict outcome of early stage breast cancer . P values were calculated from a log - rank . Color legend : yellow , low RASSF1A low RASSF1C ; green , low RASSF1A high RASSF1C ; red , high RASSF1A low RASSF1C ; grey , high RASSF1A high RASSF1C .

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C. Left: heatmap depicting methylation patterns across the RASSF1 gene. In light green, RASSF1-2γ CpG island is depicted; in dark green, RASSF1-1α CpG island is depicted. Right: Kaplan‑Meyer survival curves depicting interaction of RASSF1A and RASSF1C methylation to predict the outcome of early stage breast cancer patients. P values were calculated from a log-rank test. Color legend: yellow, low RASSF1A and low RASSF1C; green, low RASSF1A and high RASSF1C; red, high RASSF1A and low RASSF1C; grey, high RASSF1A and high RASSF1C.


Representative BLI traces showing decreased binding of Nxph1 to Nrxn1 LNS2SS2 - ( I401Q ) mutant compared to that of wild - type Nrxn1 LNS2SS2 - . Replicate numbers are shown in E .

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Representative BLI traces showing decreased binding of Nxph1 to the Nrxn1 LNS2SS2- (I401Q) mutant compared to that of wild-type Nrxn1 LNS2SS2-. Replicate numbers are shown in E.


Comparison of binding affinities for Nxph1 binding to mouse Nrxn1 - 3 LNS2SS2 - LNS2SS2 - ( I401Q ) , plotted on a logarithmic scale . Error bars represent replicate numbers are indicated in bars . Significance values were calculated using Welch ' s t - , * P < 0 . 05 , * * P < 0 . 01 .

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Comparison of binding affinities for Nxph1 binding to mouse Nrxn1-3 LNS2SS2- and LNS2SS2- (I401Q), plotted on a logarithmic scale. Error bars represent SEM and replicate numbers are indicated in bars. Significance values were calculated using Welch's t-test, *P < 0.05, **P < 0.01.


Representative BLI traces showing increased binding of Nxph1 to Nrxn1 LNS2SS2A + Nrxn1 LNS2SS2AB + compared to that of Nrxn1 LNS2SS2 - . Replicate numbers are shown in E .

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Representative BLI traces showing increased binding of Nxph1 to Nrxn1 LNS2SS2A+ and Nrxn1 LNS2SS2AB+ compared to that of Nrxn1 LNS2SS2-. Replicate numbers are shown in E.


Comparison of binding affinities for Nxph1 binding to mouse Nrxn1 LNS2 splice variants splice - insert containing Nrxn1 LNS2 with I401Q mutation ; error bars represent replicate numbers are indicated in or above bars . Significance values were calculated using Welch ' s t - , * P < 0 . 05 , * * P < 0 . 01 , * * * * P < 0 . 0001 .

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Comparison of binding affinities for Nxph1 binding to mouse Nrxn1 LNS2 splice variants and splice-insert containing Nrxn1 LNS2 with the I401Q mutation; error bars represent SEM and replicate numbers are indicated in or above bars. Significance values were calculated using Welch's t-test, *P < 0.05, **P < 0.01, ****P < 0.0001.


Representative polarization binding curves are shown for LNS2SS2 - LNS2SS2A + interacting with Nxph1 with or without Ca2 + added .

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Representative fluorescence polarization binding curves are shown for LNS2SS2- and LNS2SS2A+ interacting with Nxph1 with or without Ca2+ added.


Comparison of binding affinities of Nxph1 for Nrxn1 LNS2SS2 - Nrxn1 LNS2SS2A + with or without Ca2 + added ; replicate numbers are indicated in bars . Error bars represent , significance values were calculated using Welch ' s t - . ns , not significant .

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Comparison of binding affinities of Nxph1 for Nrxn1 LNS2SS2- and Nrxn1 LNS2SS2A+ with or without Ca2+ added; replicate numbers are indicated in bars. Error bars represent SEM, and significance values were calculated using Welch's t-test. ns, not significant.


Comparison of binding affinities of Nxph1 binding to different Nrxn1 - 3 LNS2 isoforms .

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Comparison of binding affinities of Nxph1 binding to different Nrxn1-3 LNS2 isoforms.


Comparison of binding affinities for Nxph3 binding to Nrxn1 - 3 LNS2SS2 - .

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Comparison of binding affinities for Nxph3 binding to Nrxn1-3 LNS2SS2-.


Sucrose gradient UV profile of 60S / 40S / IAPV - IRES reaction mixture after an overnight run . peaks corresponding to 80S 40S were used for extraction UREA - PAGE shown in inset .

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Sucrose gradient UV profile of 60S/40S/IAPV-IRES reaction mixture after an overnight run. The peaks corresponding to 80S and 40S were used for RNA extraction and UREA-PAGE shown in the inset.


Representative cryo - EM image where roughly half of particles correspond to 40S ( blue ) other half to 80S ( orange ) .

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Representative cryo-EM image where roughly half of the particles correspond to 40S (blue) and the other half to 80S (orange).


( H , I ) Induction of Ctcflos Ucp1 in vivo during cold - induced iWAT browning . Relative expression of ( H ) Ctcflos transcript 1 ( I ) Ucp1 in iWAT of C57BL / 6J mice held at 5 ° C for one week compared to matched mice held at 30 ° C or 23 ° C , assessed by quantitative PCR ( qPCR ) . Mean values , n = 7 ( biological replicates ) , one - way ANOVA ( Šídák - ) , * * p < 0 . 01 , * * * * p < 0 . 0001 .

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(H, I) Induction of Ctcflos and Ucp1 in vivo during cold-induced iWAT browning. Relative expression levels of (H) Ctcflos transcript 1 and (I) Ucp1 in iWAT of C57BL/6J mice held at 5°C for one week compared to age and weight matched mice held at 30°C or 23°C, assessed by quantitative PCR (qPCR). Mean and individual values, n=7 (biological replicates), one-way ANOVA (Šídák-test), **p< 0.01, ****p<0.0001.


( D ) Relative transcript of Ctcflos Ucp1 in various tissues of 129S6 mice , assessed by qPCR . Mean values ± SD , n = 1 ( biological replicate ) for Ucp1n = 3 ( biological replicates ) for Ctcflos .

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(D) Relative transcript levels of Ctcflos and Ucp1 in various tissues of 129S6 mice, assessed by qPCR. Mean values ± SD, n=1 (biological replicate) for Ucp1n=3 (biological replicates) for Ctcflos .


( E ) Ctcflos , 45SrRNA 12SrRNA transcript abundance in , mitochondrial subfractions of brite adipocytes of 129S6 mice , assessed by qPCR . Mean values ± SD , n = 6 ( biological replicates ) , two - way ANOVA ( Tukey - ) , * * * * p < 0 . 0001 .

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(E) Ctcflos, 45SrRNA and 12SrRNA transcript abundance in nuclear, cytosolic and mitochondrial subfractions of primary brite adipocytes of 129S6 mice, assessed by qPCR. Mean values ± SD, n=6 (biological replicates), two-way ANOVA (Tukey-test), ****p< 0.0001.


( E ) Relative Ucp1 transcript in response to Ctcflos tr1 , 3 4 KD by ASO 1 , 3 , 4 2 , 5 6 , respectively , compared to nontargeting controls , assessed by qPCR . Mean values ± SD , n = 3 - 4 ( biological replicates ) , one - way ANOVA ( Šídák - ) , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

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(E) Relative Ucp1 transcript levels in response to Ctcflos tr1, 3 and 4 KD by ASO 1, 3, 4 and 2, 5 6, respectively, compared to nontargeting controls, assessed by qPCR. Mean values ± SD, n=3-4 (biological replicates), one-way ANOVA (Šídák-test), ***p<0.001, ****p<0.0001.


( F ) Relative expression of - inducing fragmentation factor alpha - like effector A ( Cidea ) cytochrome c oxidase subunit 7a1 ( Cox7a1 ) in Ctcflos tr1 , 3 4 KD compared to control samples , assessed by qPCR . Mean values ± SD , n = 3 ( biological replicates ) - 4 , unpaired t or in grey parenthesis two - way ANOVA ( Šídák - ) , n . s . p > 0 . 05 , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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(F) Relative expression levels of cell death-inducing DNA fragmentation factor alpha-like effector A (Cidea) and cytochrome c oxidase subunit 7a1 (Cox7a1) in Ctcflos tr1, 3 and 4 KD compared to control samples, assessed by qPCR. Mean values ± SD, n=3 (biological replicates)-4, unpaired t tests or in grey parenthesis two-way ANOVA (Šídák-test), n.s. p>0.05, *p<0.05, **p<0.01, ***p<0.001.


( H ) Immunocytochemistry of UCP1 in response to Ctcflos tr1 KD by ASO 1 ( UCP1 ( red ) ; by DAPI ( green ) ; phase contrast ( PC ) ( gray ) ) .

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(H) Immunocytochemistry of UCP1 protein in response to Ctcflos tr1 KD by ASO 1 (UCP1 (red); DNA staining by DAPI (green); phase contrast (PC) (gray)).


Impact of Ctcflos KD on mitochondrial biogenesis . ( R ) Microscopic of Mito Tracker stained brite adipocytes ( Mito tracker ( blue ) ; by DAPI ( green ) ; phase contrast ( PC ) ( gray ) )

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Impact of Ctcflos KD on mitochondrial biogenesis. (R) Microscopic images of Mito Tracker stained brite adipocytes (Mito tracker (blue); DNA staining by DAPI (green); phase contrast (PC) (gray))


( D ) Relative expression of brite adipocyte marker genes uncoupling 1 ( Ucp1 ) , - inducing fragmentation factor alpha - like effector A ( Cidea ) , cytochrome c oxidase subunit 7a1 ( Cox7a1 ) , peroxisome proliferator activated receptor γ coactivator 1α ( Pgc1a ) elongation of very long chain fatty - like 3 ( Elovl3 ) , comparing Ctcflos tr1 ( ASO 1 ) tr 3 4 ( ASO 2 ) KD with their respective controls . Mean values ± SD , n = 6 ( biological replicates ) , unpaired t or in grey parenthesis two - way ANOVA ( Šídák - ) n . s . p > 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

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

(D) Relative expression levels of brite adipocyte marker genes uncoupling protein 1 (Ucp1), cell death-inducing DNA fragmentation factor alpha-like effector A (Cidea), cytochrome c oxidase subunit 7a1 (Cox7a1), peroxisome proliferator activated receptor γ coactivator 1α (Pgc1a) and elongation of very long chain fatty acids-like 3 (Elovl3), comparing Ctcflos tr1 (ASO 1) and tr 3 and 4 (ASO 2) KD with their respective controls. Mean values ± SD, n=6 (biological replicates), unpaired t tests or in grey parenthesis two-way ANOVA (Šídák-test) n.s. p>0.05, **p<0.01, ***p<0.001, ****p<0.0001.


( K - M ) Rescue of Ctcflos KD impact on Ucp1 gene transcription by Prdm16 overexpression . iWAT infected with Prdm16 - or turbo green ( GFP ) - expressing viral particles at second day of proliferation , followed by reverse transfection at first day of differentiation using nontargeting control ( NC ) or Ctcflos - targeting ASO1 . Gene expression analyzed 72 hours later by qPCR ( K ) Transcript of Ctcflos tr1 relative to Fabp4 ( L ) Transcript of Prdm16 relative to Fabp4 ( M ) Transcript of Ucp1 relative to Fabp4 . Mean values . Each graph presents pooled data from two experiments slightly varying titers . RM One - way ANOVA ( Tukey - ) , n . s . p > 0 . 05 , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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

(K-M) Rescue of Ctcflos KD impact on Ucp1 gene transcription by Prdm16 overexpression. Primary iWAT cells infected with Prdm16- or turbo green fluorescent protein (GFP)-expressing viral particles at the second day of proliferation, followed by reverse transfection at the first day of differentiation using nontargeting control (NC) or Ctcflos-targeting ASO1. Gene expression analyzed 72 hours later by qPCR (K) Transcript levels of Ctcflos tr1 relative to Fabp4 (L) Transcript levels of Prdm16 relative to Fabp4 (M) Transcript levels of Ucp1 relative to Fabp4. Mean and individual values. Each graph presents pooled data from two experiments slightly varying virus titers. RM One-way ANOVA (Tukey-test), n.s. p>0.05, *p<0.05, **p<0.01, ***p<0.001.


( B - D ) Impact of splicing inhibition by different concentrations of general splicing inhibitor isoginkgetin on brite adipogenesis , ( B ) expressed as Ucp1 relative to Fabp4 mRNA , ( C ) Ucp1 relative to Gtf2b ( D ) Fabp4 relative to Gtf2b . Mean values ± SD , n = 3 ( biological replicates ) , one - way ANOVA ( Šídák - ) , n . s . p > 0 . 05 , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

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(B-D) Impact of splicing inhibition by different concentrations of general splicing inhibitor isoginkgetin on brite adipogenesis, (B) expressed as Ucp1 relative to Fabp4 mRNA levels, (C) Ucp1 relative to Gtf2b and (D) Fabp4 relative to Gtf2b. Mean values ± SD, n=3 (biological replicates), one-way ANOVA (Šídák-test), n.s. p>0.05, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.


Effect of Ctcflos KD on Serine / Arginine rich Splicing factor 2 ( SC35 / SRSF2 ) speckle abundance in Ctcflos tr1 ( ASO 1 ) KD control ( KD day 1 of differentiation , after 48h ) . ( E ) Immunocytochemistry of SC35 . SC35 signal ( blue ) alone ( middle ) SC35 ( blue ) , by DAPI ( green ) phase contrast ( PC ) ( gray ) combined ( outer ) .

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Effect of Ctcflos KD on Serine/Arginine rich Splicing factor 2 (SC35/SRSF2) and nuclear speckle abundance in Ctcflos tr1 (ASO 1) KD and control cells (KD day 1 of differentiation, analysis after 48h). (E) Immunocytochemistry of SC35. SC35 signal (blue) alone (middle images) and SC35 (blue), DNA staining by DAPI (green) and phase contrast (PC) (gray) combined (outer images).


Influence of Ctcflos KD on Prdm16 alternative splicing . ( O ) Relative Prdm16 total , ( P ) Relative Prdm16 long transcript 24 hours after Ctcflos KD , comparing Ctcflos tr1 ( ASO1 ) tr3 , 4 ( ASO2 ) KD control samples , assessed by qPCR , Mean values ± SD , n = 3 - 4 ( biological replicates ) , unpaired t , n . s . p > 0 . 05 , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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

Influence of Ctcflos KD on Prdm16 alternative splicing. (O) Relative Prdm16 total, (P) Relative Prdm16 long transcript levels 24 hours after Ctcflos KD, comparing Ctcflos tr1 (ASO1) and tr3, 4 (ASO2) KD and control samples, assessed by qPCR, Mean values ± SD, n=3-4 (biological replicates), unpaired t tests, n.s. p>0.05, * p<0.05, **p<0.01, ***p<0.001.


Influence of Ctcflos KD on Prdm16 alternative splicing . ( R , S ) Fold change of Prdm16 short Prdm16 long ( R ) ASO1 to NC ( S ) ASO2 NC 24 hours after Ctcflos KD , n = 3 - 4 ( biological replicates ) , unpaired t , n . s . p > 0 . 05 , * * p < 0 . 01 .

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

Influence of Ctcflos KD on Prdm16 alternative splicing. (R, S) Fold change of Prdm16 short and Prdm16 long (R) ASO1 to NC and (S) ASO2 and NC 24 hours after Ctcflos KD, n=3-4 (biological replicates), unpaired t tests, n.s. p>0.05, ** p<0.01.


B accessibility as measured by number of ( TSS distal ) ATAC - Seq peaks ( read ≥ 10 ) detected in DN2 ( n = 5 ) , DN3 ( n = 5 ) , ISP ( n = 5 ) , DPCD3 - ( n = 6 ) , DPCD3 + ( n = 6 ) , SPCD4 + ( n = 5 ) , SPCD8 + ( n = 5 ) populations . Horizontal lines of box plot indicate median , lower upper limits of each box correspond to first third quartiles ( 25th 75th percentiles ) lower upper whiskers extend from min to max ; pval = 0 . 035 ( Kruskal - Wallis ) .

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B Chromatin accessibility as measured by the number of all (TSS and distal) ATAC-Seq peaks (read count ≥10) detected in DN2 (n=5), DN3 (n=5), ISP (n=5), DPCD3- (n=6), DPCD3+ (n=6), SPCD4+ (n=5), SPCD8+ (n=5) populations. Horizontal lines of the box plot indicate the median, lower and upper limits of each box correspond to the first and third quartiles (the 25th and 75th percentiles) and the lower and upper whiskers extend from min to max; pval=0.035 (Kruskal-Wallis Test).


C Fraction of OCRs having increasing , decreasing , steady fluctuating accessibility pattern during T - development ( x - axis ) . Average number of normalized reads in OCRs ( y - axis ) .

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C Fraction of OCRs having increasing, decreasing, steady and fluctuating accessibility pattern during T-cell development (x-axis). Average number of normalized reads in OCRs (y-axis).


A Unsupervised hierarchical clustering on variance stabilizing transformation ( VST ) normalized read ( each row is also normalized by row mean ) of 2 , 021 signature open regions of 7 stages

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A Unsupervised hierarchical clustering based on the variance stabilizing transformation (VST) normalized read counts (each row is also normalized by the row mean) of the 2,021 signature open chromatin regions of 7 stages


A Unsupervised hierarchical clustering on variance stabilizing transformation ( VST ) normalized read ( each row is also normalized by row mean ) of 2 , 021 signature open regions of 7

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B ATAC - Seq genome tracks showing three signature peaks located near genes SAE1 , RAG1 , CD8A . Framed regions indicate - type - specific signature ATAC - Seq peaks . Blue triangles indicate known promoter or enhancer regions , which are annotated in GeneHancer ( GH ) Regulatory Elements database

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B ATAC-Seq genome tracks showing three signature peaks located near the genes SAE1, RAG1, and CD8A. Framed regions indicate cell-type-specific signature ATAC-Seq peaks. Blue triangles indicate known promoter or enhancer regions, which are annotated in the GeneHancer (GH) Regulatory Elements database


B ATAC - Seq genome tracks showing three signature peaks located near genes SAE1 , RAG1 , CD8A . Framed regions indicate - type - specific signature ATAC - Seq peaks . Blue triangles indicate known promoter or enhancer regions , which are annotated in GeneHancer ( GH ) Regulatory Elements

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A rationale of footprinting as illustrated by actual ATAC - Seq peaks , HINT - ATAC footprint hits , example TF motif instances near RAG1 gene in DN2 population of thymus donor 6 .

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A The rationale of footprinting analysis as illustrated by actual ATAC-Seq peaks, HINT-ATAC footprint hits, and example TF motif instances near RAG1 gene in DN2 population of thymus donor 6.


B of ln ( odds ) of 50 TFs showing most differential abundance of binding motifs in open regions . TFs with an SD of motif < 10 are excluded .

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B Heatmap of ln(odds ratio) of the 50 TFs showing the most differential abundance of binding motifs in the open chromatin regions. TFs with an SD of the motif counts <10 are excluded.


D accessibility as assessed by number of ATAC peaks identified in sorted populations of normal thymic T - precursors in T - ALLs , respectively ( n = 10 / 19 have two biological replicates , n = 9 / 19 have one replicate ) . Horizontal lines of box plot indicate median , lower upper limits of each box correspond to first third quartiles ( 25th 75th percentiles ) lower upper whiskers extend from min to max ; pval = 4 . 4e - 08 ( Kruskal - Wallis ) .

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D Chromatin accessibility as assessed by the number of ATAC peaks identified in the sorted populations of normal thymic T-cell precursors and in T-ALLs, respectively (n=10/19 patients have two biological replicates, n=9/19 patients have one replicate). Horizontal lines of the box plot indicate the median, lower and upper limits of each box correspond to the first and third quartiles (the 25th and 75th percentiles) and the lower and upper whiskers extend from min to max; pval=4.4e-08 (Kruskal-Wallis Test).


E representing enrichment depletion of binding motifs of top 50 TFs in OCRs of T - ALLs in comparison to healthy T - precursors . on x - axis ; same order as in panel ( C ) . Gray represent TFs having non - significant enrichment or depletion .

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E Heatmap representing the enrichment and depletion of binding motifs of top 50 TFs in OCRs of T-ALLs in comparison to healthy T-cell precursors. Patients on the x-axis; same order as in panel (C). Gray cells represent TFs having non-significant enrichment or depletion.


C log2 fold changes of 292 genes which are differentially expressed ( x - axis , - Seq : non - sorted bulk thymi vs . T - ALLs ) differentially accessible ( y - axis , ATAC - Seq : sorted T - populations vs . T - ALLs ) in T - ALLs in comparison to healthy T - precursors . Dot color indicate frequency ( number of having dysregulation of gene ) . Genes with ≥ 5 / 19 frequency are shown on plot . coding genes with ≥ 12 / 19 frequency are labelled . For plotting average LFC values of ATAC are used per gene per quartile . Q1 - 4 stands for quadrant 1 - 4 .

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C log2 fold changes of 292 genes which are differentially expressed (x-axis, RNA-Seq: non-sorted bulk thymi vs. T-ALLs) and differentially accessible (y-axis, ATAC-Seq: sorted T-cell populations vs. T-ALLs) in T-ALLs in comparison to healthy T-cell precursors. Dot size and color indicate the frequency (number of patients having dysregulation of the gene). Genes with ≥5/19 frequency are shown on the plot. Protein coding genes with ≥12/19 frequency are labelled. For plotting the average LFC values of ATAC and RNA are used per gene per quartile. Q1 - 4 stands for the quadrant 1 - 4.


D of VST normalized read ( each row is also normalized by row mean ) of differentially expressed genes ( pval < 0 . 1 ; DESeq2 ) in three in comparison to remaining 16 . Orange triangles indicate three with no DAB1 but high SPI1 expression motif .

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D Heatmap of VST normalized read counts (each row is also normalized by the row mean) of differentially expressed genes (pval<0.1; DESeq2) in three patients in comparison to remaining 16. Orange triangles indicate three patients with no DAB1 but high SPI1 expression and motif counts.


( B ) Bar graph representing residual thrombin activity ( ΔOD / min ) in presence of sdAbs without heparin .

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(B) Bar graph representing residual thrombin activity (ΔOD/min) in presence of the sdAbs without heparin.


( C ) Bar graph representing residual thrombin activity ( ΔOD / min ) in presence of sdAbs heparin .

(C) Bar graph representing residual thrombin activity (ΔOD/min) in presence of the sdAbs and heparin.


( D ) Bar graph representing residual activated factor X ( FXa ) activity ( ΔOD / min ) in presence of sdAbs heparin .

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

(D) Bar graph representing residual activated factor X (FXa) activity (ΔOD/min) in presence of the sdAbs and heparin.


( B - C ) Graphs reporting recovery of thrombin - FXa - mediated substrate conversion in presence of increasing doses of KB - AT - 23 , in presence of antithrombin heparin .

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(B-C) Graphs reporting the recovery of thrombin- and FXa-mediated substrate conversion in the presence of increasing doses of KB-AT-23, in presence of antithrombin and heparin.


( D - E ) Thrombin generation profiles of FVIII - deficient plasma spiked with different concentrations of recombinant FVIII or 2 doses of KB - AT - 23 .

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(D-E) Thrombin generation profiles of FVIII-deficient plasma spiked with different concentrations of recombinant FVIII or 2 doses of KB-AT-23.


( B ) Purified KB - AT - 23 - fus KB - hFX - 11 - fus were given intravenously to wild - type C57B6 mice ( 10 mg / kg ) . At indicated time - points , blood was collected residual was measured . Plotted is residual versus time after injection . Green circles : KB - AT - 23 - fus ; Red circles : KB - hFX - 11 - fus .

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(B) Purified KB-AT-23-fus and KB-hFX-11-fus were given intravenously to wild-type C57B6 mice (10 mg/kg). At indicated time-points, blood was collected and residual protein was measured. Plotted is residual protein versus time after injection. Green circles: KB-AT-23-fus; Red circles: KB-hFX-11-fus.


( D ) Graph reporting volume of blood loss ( µL ) in treated mice ( n = 4 - 9 per group ) observed during 30 minutes post TVT . Dosing was 10 mg / kg for KB - AT - 23 1 mg / kg for FVIIa . grey represents range of blood loss in FVIII - treated mice ( 33 - 245 μL ) , which is similar to that of wild - type C57BL / 6 mice ( 49 - 308 μL ; Johansen et al . 2016 ) .

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(D) Graph reporting the volume of blood loss (µL) in treated mice (n=4-9 per group) observed during 30 minutes post TVT. Dosing was 10 mg/kg for KB-AT-23 and 1 mg/kg for FVIIa. The grey area represents the range of blood loss in FVIII-treated mice (33-245 μL), which is similar to that of wild-type C57BL/6 mice (49-308 μL; Johansen et al. 2016).


( B ) Western blot of KB - AT - 23 secretion in conditioned media 72 hours post - transfection of HuH7 . In lower panel western blot via densitometry is depicted . Data are presented as mean ± SD , were analysed via 1 - way ANOVA with Dunnett ' s correction for multiple comparisons . * * * * p < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Western blot analysis of KB-AT-23 protein secretion in conditioned media 72 hours post-transfection of HuH7 cells. In the lower panel the western blot quantification via densitometry analysis is depicted. Data are presented as mean ±SD, and were analysed via 1-way ANOVA with Dunnett's correction for multiple comparisons. ****p<0.001.


( C ) HuH7 - celles were collected 72 hours post - transfection . Presented is Western blot of lysed for presence of KB - AT - 23 . Cntr - refers to non - transfected , while Cntr + refers to purified recombinant KB - AT - 23 .

(C) HuH7-celles were collected 72 hours post-transfection. Presented is the Western blot analysis of lysed cells for the presence of KB-AT-23. Cntr- refers to non-transfected cells, while Cntr+ refers to purified recombinant KB-AT-23.


( B ) Representative western blot analyses of KB - AT - 23 antithrombin expression on plasma samples ( n = 4 ) collected from 27 post - AAV injection . kDa , molecular marker .

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(B) Representative western blot analyses of KB-AT-23 and antithrombin expression on plasma samples (n=4) collected from animals 27 weeks post-AAV injection. kDa, molecular weight marker.


( C ) Measurement of D - dimers ( D2D ) plasma 27 post vector administration . Data are presented as mean ± SD ( n = 6 - 9 / group ) .

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(C) Measurement of D-dimers (D2D) plasma concentration 27 weeks post vector administration. Data are presented as mean ±SD (n=6-9/group).


( D ) Measurement of anti - KB - AT - 23 mouse IgG in plasma samples collected from mice ( n = 6 - 9 / group ) 8 post - AAV administration . Data represent mean ± SD .

O O O O O O O O O O O O O O O O O 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) Measurement of anti-KB-AT-23 mouse IgG in plasma samples collected from mice (n=6-9/group) 8 weeks post-AAV administration. Data represent mean±SD.


( E ) TVT on ( n = 4 - 5 per group ) performed 29 post vector administration . volume of blood loss ( µL ) during 30 minutes observation time is reported . grey represents range of blood loss in FVIII - treated mice ( 33 - 245 μL ) , which is similar to that of wild - type C57BL / 6 mice ( 49 - 308 μL ; Johansen et al . 2016 ) . Boxes represent 25 - 75 % quartiles ; central indicates median . Whiskers span min to max . Data were analysed via 1 - way ANOVA with Dunnett ' s correction for multiple comparisons .

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(E) TVT test on males and females (n=4-5 per group) performed 29 weeks post vector administration. The volume of blood loss (µL) during 30 minutes observation time is reported. The grey area represents the range of blood loss in FVIII-treated mice (33-245 μL), which is similar to that of wild-type C57BL/6 mice (49-308 μL; Johansen et al. 2016). Boxes represent 25-75% quartiles; the central line indicates the median. Whiskers span min to max. Data were analysed via 1-way ANOVA with Dunnett's correction for multiple comparisons.


( F ) Vector genome copy number ( VGCN ) per in liver samples collected at sacrifice ( n = 8 / group ) . Data represent mean ± SD .

(F) Vector genome copy number (VGCN) per cell in liver samples collected at sacrifice (n=8/group). Data represent mean±SD.


( C ) Measurement of anti - hFIX mouse IgG in plasma samples collected from mice injected with FIX or PBS ( n = 5 / group ) at different time points . Data represent mean ± .

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(C) Measurement of anti-hFIX mouse IgG in plasma samples collected from mice injected with FIX or PBS (n=5/group) at different time points. Data represent mean±SEM.


( D ) Representative western blot analyses on plasma samples ( n = 4 ) collected from 8 post - AAV injection .

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(D) Representative western blot analyses on plasma samples (n=4) collected from animals 8 weeks post-AAV injection.


( E ) Antithrombin activity in plasma samples collected from mice at different time points . Data are presented as mean ± SD ( n = 3 - 4 per datapoint ) . Data were analyzed in a 2 - way ANOVA with Tukey ' s correction for multiple comparisons . * * * p < 0 . 01 * * * * p < 0 . 001 ( 2 - way ANOVA with Tukey ' s multiple comparison )

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(E) Antithrombin activity in plasma samples collected from mice at different time points. Data are presented as mean±SD (n=3-4 per datapoint). Data were analyzed in a 2-way ANOVA with Tukey's correction for multiple comparisons. ***p<0.01 ****p<0.001 (2-way ANOVA with Tukey's multiple comparison test)


( F ) Graph reporting volume of blood loss ( µL ) over 30 minutes following tail clip . WT : wild - type of F9 - / - mice . grey represents range of blood loss of F9 - / - mice that received AAV - vectors expressing human FIX . Data are presented as mean ± SD ( n = 3 - 4 / group ) , were analyzed in a 1 - way ANOVA with Tukey ' s correction for multiple comparisons . * * p < 0 . 01 Boxes represent 25 % - 75 % quartiles , with indicating median . Whiskers span min to max .

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(F) Graph reporting the volume of blood loss (µL) over 30 minutes following the tail clip. WT: wild-type littermates of F9-/- mice. The grey area represents the range of blood loss of F9-/- mice that received AAV-vectors expressing human FIX. Data are presented as mean±SD (n=3-4/group), and were analyzed in a 1-way ANOVA with Tukey's correction for multiple comparisons. **p<0.01 Boxes represent 25%-75% quartiles, with the line indicating the median. Whiskers span min to max.


G Measurement of anti - KB - AT - 23 mouse IgG in plasma samples collected from mice ( n = 5 / group ) 8 post - AAV administration . Data represent mean ± SD .

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G Measurement of anti-KB-AT-23 mouse IgG in plasma samples collected from mice (n=5/group) 8 weeks post-AAV administration. Data represent mean±SD.


( C ) Representative western blot analyses on plasma samples ( n = 4 ) collected from 4 post - AAV injection . Cntrl + : positive controls represented by loading of purified KB - AT - 23 or KB - AT - 113 .

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(C) Representative western blot analyses on plasma samples (n=4) collected from animals 4 weeks post-AAV injection. Cntrl +: positive controls represented by the loading of the purified KB-AT-23 or KB-AT-113 proteins.


( D - H ) Measurement of anti - sdAbs mouse IgG in plasma samples collected at days 28 57 post - AAV injection . ELISA plate was coated with purified KB - AT - 01 ( D )

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(D-H) Measurement of anti-sdAbs mouse IgG in plasma samples collected at days 28 and 57 post-AAV injection. The ELISA plate was coated with the purified KB-AT-01 (D)


Measurement of anti - sdAbs mouse IgG in plasma samples collected at days 28 57 post - AAV injection . ELISA plate was coated with purified KB - AT - 01 , - 02 ( E ) , - 03 ( F ) , - 23 ( G ) or - 113 ( H ) sdAbs .

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Measurement of anti-sdAbs mouse IgG in plasma samples collected at days 28 and 57 post-AAV injection. The ELISA plate was coated with the purified KB-AT-01 , -02 (E), -03 (F), -23 (G) or -113 (H) sdAbs.


( D ) RipAC overexpression inhibits flg22 - triggered MAPK activation in Arabidopsis . 100 nM flg22Pto was used to treat Arabidopsis seedlings samples were collected at indicated time points . Immunoblots were analysed using anti - pMAPK anti - RipAC antibodies . Coomassie brilliant blue ( CBB ) a non - specific band were used as loading control . Molecular ( kDa ) marker bands are indicated for reference .

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(D) RipAC overexpression inhibits flg22-triggered MAPK activation in Arabidopsis. 100 nM flg22Pto was used to treat Arabidopsis seedlings and the samples were collected at indicated time points. Immunoblots were analysed using anti-pMAPK and anti-RipAC antibodies. Coomassie brilliant blue (CBB) staining and a non-specific band were used as loading control. Molecular weight (kDa) marker bands are indicated for reference.


( A - B ) Interaction between RipAC SlPUB4 / AtPUB4 was confirmed by Split - LUC qualitatively ( A ) quantitatively ( B ) in Nicotiana benthamiana . Values are means ± SE ( n = 8 biological replicates ) . Asterisks indicate significant differences compared to RipAC - nLUC / cLUC - AtPIP2A negative control ( Student ' s t , * * * * p < 0 . 0001 ) . RLU : relative luminescence units .

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(A-B) Interaction between RipAC and SlPUB4/AtPUB4 was confirmed by Split-LUC assay qualitatively (A) and quantitatively (B) in Nicotiana benthamiana. Values are means ± SE (n=8 biological replicates). Asterisks indicate significant differences compared to RipAC-nLUC/cLUC-AtPIP2A negative control (Student's t test, **** p<0.0001). RLU: relative luminescence units.


( C ) Co - immunoprecipitation of SlPUB4 / AtPUB4 - GFP RipAC - nLUC after co - expression in N . benthamiana . Two days after Agrobacterium infiltration , tissues were collected then subjected to anti - GFP immunoprecipitation . Immunoblots were analyzed using anti - LUC anti - GFP antibodies . Molecular ( kDa ) marker bands are indicated for reference .

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(C) Co-immunoprecipitation of SlPUB4/AtPUB4-GFP and RipAC-nLUC after co-expression in N. benthamiana. Two days after Agrobacterium infiltration, the plant tissues were collected and then subjected to anti-GFP immunoprecipitation. Immunoblots were analyzed using anti-LUC and anti-GFP antibodies. Molecular weight (kDa) marker bands are indicated for reference.


( E ) flg22 - triggered MAPK activation in pub4 T - mutants . 100 nM flg22Pto was used to treat Arabidopsis seedlings samples were collected at indicated time points for western blots . Immunoblots were analyzed using anti - pMAPK anti - FLS2 antibodies . Coomassie brilliant blue ( CBB ) was used as loading control . Molecular ( kDa ) marker bands are indicated for reference .

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(E) flg22-triggered MAPK activation in pub4 T-DNA mutants. 100 nM flg22Pto was used to treat Arabidopsis seedlings and the samples were collected at indicated time points for western blots. Immunoblots were analyzed using anti-pMAPK and anti-FLS2 antibodies. Coomassie brilliant blue (CBB) staining was used as loading control. Molecular weight (kDa) marker bands are indicated for reference.


( B ) Co - immunoprecipitation of PUB4 BIK1 in N . benthamiana . PUB4 - GFP or GFP - LTI6b were transiently co - expressed with BIK1 - HA in leaves of N . benthamiana . After treatment with mock or 1 μM flg22Paer for 10 min , total ( input ) were extracted subjected for immunoprecipitation with anti - GFP beads .

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(B) Co-immunoprecipitation of PUB4 and BIK1 in N. benthamiana. PUB4-GFP or GFP-LTI6b were transiently co-expressed with BIK1-HA in the leaves of N. benthamiana. After treatment with mock or 1 μM flg22Paer for 10 min, total proteins (input) were extracted and subjected for immunoprecipitation with anti-GFP beads.


( C ) Co - immunoprecipitation of PUB4 with PRR members in Arabidopsis . PUB4 co - immunoprecipitated with FLS2 BAK1 specifically after 10 min of 1 μM flg22Paer treatment , constantly with BIK1 . Total extracts ( input ) from Col - 0 PUB4 - FLAG were subjected to immunoprecipitation with anti - FLAG beads .

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(C) Co-immunoprecipitation of PUB4 with PRR complex members in Arabidopsis. PUB4 co-immunoprecipitated with FLS2 and BAK1 specifically after 10 min of 1 μM flg22Paer treatment, and constantly with BIK1. Total protein extracts (input) from Col-0 and PUB4-FLAG plants were subjected to immunoprecipitation with anti-FLAG beads.


( D ) PUB4 directly interacts with BIK1 in vitro . Immunoblots were analyzed using indicated antibodies . Molecular ( kDa ) marker bands are indicated for reference .

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(D) PUB4 directly interacts with BIK1 in vitro. Immunoblots were analyzed using the indicated antibodies. Molecular weight (kDa) marker bands are indicated for reference.


RipAC does not affect PUB4 association with PRR members . PUB4 - FLAG , PUB4 - FLAG RipAC - GFP Col - 0 were treated for 10 min with water ( as mock ) or 1 μM flg22Paer elf18Ecol treatment . Total extracts ( input ) were subjected for immunoprecipitation with anti - FLAG beads . Immunoblots were analyzed using indicated antibodies . Molecular ( kDa ) marker bands are indicated for reference . Data information : experiment was performed 3 times with similar results .

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RipAC does not affect PUB4 association with PRR complex members. PUB4-FLAG, PUB4-FLAG RipAC-GFP and Col-0 plants were treated for 10 min with water (as mock) or 1 μM flg22Paer and elf18Ecol treatment. Total protein extracts (input) were subjected for immunoprecipitation with anti-FLAG beads. Immunoblots were analyzed using the indicated antibodies. Molecular weight (kDa) marker bands are indicated for reference. Data information: The experiment was performed 3 times with similar results.


( A ) PUB4 has auto - ubiquitination activity in vitro . PUB4 autoubiquitination using His - Ubiquitin , UBA1 ( E1 ) , HA - UBC8 ( E2 ) MBP - PUB4 or MBP - PUB4 Cys239Ala inactive mutant reconstituted in . Ubiquitinated products were purified under denaturing conditions by immobilized metal ion chromatography ( IMAC ) . Input precipitated were resolved in 10 % 8 % acrylamide gels , respectively , analyzed by immunoblot with indicated antibodies . indicates autoubiquitinated PUB4 .

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(A) PUB4 has auto-ubiquitination activity in vitro. PUB4 autoubiquitination assay using His-Ubiquitin, UBA1 (E1), HA-UBC8 (E2) and MBP-PUB4 or MBP-PUB4 Cys239Ala inactive mutant reconstituted in bacteria. Ubiquitinated products were purified under denaturing conditions by immobilized metal ion chromatography (IMAC). Input and precipitated proteins were resolved in 10% and 8% acrylamide gels, respectively, and analyzed by immunoblot with indicated antibodies. The line indicates autoubiquitinated PUB4.


( B ) PUB4 ubiquitinates non - active BIK1 , does not ubiquitinate active BIK1 . Operon containing His - Ubiquitin , UBA1 ( E1 ) , HA - UBC8 ( E2 ) , MBP - PUB4 were co - transformed with GST , GST - BIK1 , or GST - BIK1 kinase dead ( BIK1 - KD ) in presence of PUB4 . Ubiquitinated products were purified under denaturing conditions by IMAC , modified further enriched by GSH chromatography . Input precipitated were resolved in 10 % 8 % acrylamide gels , respectively , analyzed by immunoblot with indicated antibodies . indicates ubiquitination products .

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(B) PUB4 ubiquitinates non-active BIK1, and does not ubiquitinate active BIK1. Operon containing His-Ubiquitin, UBA1 (E1), HA-UBC8 (E2), and MBP-PUB4 were co-transformed with GST, GST-BIK1, or GST-BIK1 kinase dead (BIK1-KD) in the presence of PUB4. Ubiquitinated products were purified under denaturing conditions by IMAC, and modified proteins further enriched by GSH chromatography. Input and precipitated proteins were resolved in 10% and 8% acrylamide gels, respectively, and analyzed by immunoblot with indicated antibodies. The line indicates the ubiquitination products.


( A ) of BIK1 - HA accumulation in BIK1 - HA + / - , pub4 - / - BIK1 - HA + / - , PUB4 - FLAG + / - BIK1 - HA + / - in 4 - to 5 - week - old . Prior to extraction leave disks were treated for 6 h with 100 mM MG132 , 50 mM CHX , for 10 min with water ( as mock ) or 1 mM flg22Paer . were extracted in SDS buffer analysed by western blot . BAK1 was used as a loading control . Numbers correspond to quantitation of BIK1 - HA band , normalized to quantitation of BAK1 control band in same sample .

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(A) Analysis of BIK1-HA protein accumulation in BIK1-HA+/-, pub4-/- BIK1-HA+/-, and PUB4-FLAG+/- BIK1-HA+/- in 4- to 5-week-old plants. Prior to protein extraction leave disks were treated for 6 h with 100 mM MG132, 50 mM CHX, and for 10 min with water (as mock) or 1 mM flg22Paer. Proteins were extracted in SDS buffer and analysed by western blot. BAK1 was used as a loading control. Numbers correspond to the quantitation of the BIK1-HA band, normalized to the quantitation of the BAK1 control band in the same sample.


( B ) BIK1 - HA accumulation is reduced in PUB4 - FLAG + / - BIK1 - HA + / - compared to BIK1 - HA + / - in absence of PAMP treatment . Total extracts were analysed by western blot . Tubulin was used as a loading control .

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(B) BIK1-HA accumulation is reduced in PUB4-FLAG+/- BIK1-HA+/- line compared to BIK1-HA+/- line in the absence of PAMP treatment. Total protein extracts were analysed by western blot. Tubulin was used as a loading control.


( E ) BIK1 - HA accumulation is reduced in RipAC - GFP / BIK1 - HA compared to Col - 0 / BIK1 - HA . Total extracts were analysed by western blot . Actin was used as a loading control .

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(E) BIK1-HA accumulation is reduced in RipAC-GFP/BIK1-HA line compared to Col-0/BIK1-HA line. Total protein extracts were analysed by western blot. Actin was used as a loading control.


Transfection of poly ( I : C ) induces ACE2 expression . HEK293 were cultured in six - well plates transfected with poly ( I : C ) as indicated above using Lipofectamine 2000 . HEK293 were then collected for isolation RT - qPCR at 9 hrs post - transfection . Results in each panel are representative of three independent experiments .

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Transfection of poly (I:C) induces ACE2 expression.HEK293 cells were cultured in six-well plates and transfected with poly (I:C) as indicated above using Lipofectamine 2000. HEK293 cells were then collected for RNA isolation and RT-qPCR analysis at 9 hrs post-transfection. Results in each panel are representative of three independent experiments.


VSV - eGFP infection induces ACE2 expression . HEK293 were cultured in six - well plates infected with VSV - eGFP as indicated above . 6 hrs later , HEK293 were collected for isolation RT - qPCR . Results in each panel are representative of three independent experiments .

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VSV-eGFP infection induces ACE2 expression.HEK293 cells were cultured in six-well plates and infected with VSV-eGFP as indicated above. 6 hrs later, HEK293 cells were collected for RNA isolation and RT-qPCR analysis. Results in each panel are representative of three independent experiments.


Overexpression of TBK1 or TRIF induces ACE2 expression . TBK1 is an essential activator of - sensing pathway . HEK293 were cultured in 24 - well plates . 24 hrs later , plasmids as shown above were transfected into HEK293 . 36 hrs after transfection , total was isolation for subsequent RT - qPCR . Results in each panel are representative of three independent experiments .

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Overexpression of TBK1 or TRIF induces ACE2 expression.TBK1 is an essential activator of RNA-sensing pathway. HEK293 cells were cultured in 24-well plates. 24 hrs later, plasmids as shown above were transfected into HEK293 cells. 36 hrs after transfection, total RNA was isolation for subsequent RT-qPCR analysis. Results in each panel are representative of three independent experiments.


ACE2 expression is upregulated in lungs from SARS - CoV MA15 infected C57Bl / 6 mouse . Intranasal instillation of 102 , 103 PFU of SARS - CoV MA15 in 50 µl of PBS or mock - infected with PBS alone , lungs were harvested 24 hrs later total was isolated subjected to microarray . ACE2 expression data was retrieved from Gene Expression Omnibus ( GEO ) microarray database ( NCBI Accession : PRJNA149057 ; GEO : GSE33266 ) .

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ACE2 expression is upregulated in the lungs from SARS-CoV MA15 infected C57Bl/6 mouse.Intranasal instillation of 102, 103 PFU of SARS-CoV MA15 in 50 µl of PBS or mock-infected with PBS alone, lungs were harvested 24 hrs later and total RNA was isolated and subjected to microarray analysis. ACE2 expression data was retrieved from Gene Expression Omnibus (GEO) microarray database (NCBI Accession: PRJNA149057; GEO: GSE33266).


ACE2 expression is upregulated in human airway epithelial in response to wild type MERS - CoV ( icMERS - CoV EMC2012 ) . Human airway epithelial were infected with a multiplicity of infection of 5 PFU per . Infected samples were collected as indicated above . ACE2 expression data was retrieved from GEO microarray database ( NCBI Accession : PRJNA391962 ; GEO : GSE100504 ) .

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ACE2 expression is upregulated in primary human airway epithelial cells in response to wild type MERS-CoV (icMERS-CoV EMC2012).Human airway epithelial cells were infected with a multiplicity of infection of 5 PFU per cell. Infected samples were collected as indicated above. ACE2 expression data was retrieved from GEO microarray database (NCBI Accession: PRJNA391962; GEO: GSE100504).


ACE2 expression is upregulated in human airway epithelial in response to rhinovirus infection . epithelial from 4 donors were exposed to rhinovirus or medium only ( used as control ) . 24 hrs post - infection , gene expression was assessed using Affymetrix U133 plus 2 . 0 human GeneChips . ACE2 expression data was retrieved from GEO microarray database ( NCBI Accession : PRJNA137767 ; GEO : GSE27973 ) .

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ACE2 expression is upregulated in human airway epithelial cells in response to rhinovirus infection.Primary epithelial cells from 4 donors were exposed to rhinovirus or medium only (used as control). 24 hrs post-infection, gene expression was assessed using Affymetrix U133 plus 2.0 human GeneChips. ACE2 expression data was retrieved from GEO microarray database (NCBI Accession: PRJNA137767; GEO: GSE27973).


ACE2 expression is upregulated in human bronchial epithelial ( HBECs ) after IFNβ H1N1 influenza stimulation . HBECs were treated with IFNβ ( 1000U / ml ) or wild type H1N1 influenza ( A / PR / 8 / 34 ) . Control samples were incubated with fresh medium under same conditions . HBECs were harvested at indicated time points ( 0 . 25 , 0 . 5 , 1 , 1 . 5 , 2 , 4 , 6 , 8 , 12 , 18 hours post - treatment ) . Total was extracted analyzed using Affymetrix High Throughput Array . ACE2 expression data was retrieved from GEO microarray database ( NCBI Accession : PRJNA121751 ; GEO : GSE19392 ) .

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ACE2 expression is upregulated in human bronchial epithelial cells (HBECs) after IFNβ and H1N1 influenza stimulation.HBECs were treated with IFNβ (1000U/ml) or wild type H1N1 influenza (A/PR/8/34). Control samples were incubated with fresh medium under the same conditions. HBECs were harvested at indicated time points (0.25, 0.5, 1, 1.5, 2, 4, 6, 8, 12, and 18 hours post-treatment). Total RNA was extracted and analyzed using the Affymetrix High Throughput Array. ACE2 expression data was retrieved from GEO microarray database (NCBI Accession: PRJNA121751; GEO: GSE19392).


ACE2 expression is upregulated in human keratinocytes exposed to IFNα IFNγ . keratinocytes from 3 donors were either untreated ( control ) or exposed to cytokines IFNα , IFNγ . 24 hrs after treatment , total was extracted for subsequent microarray . ACE2 expression data was retrieved from GEO microarray database ( NCBI Accession : PRJNA153023 ; GEO : GSE36287 ) .

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ACE2 expression is upregulated in human primary keratinocytes exposed to IFNα and IFNγ.Primary keratinocytes from 3 donors were either untreated (control) or exposed to cytokines IFNα, IFNγ. 24 hrs after treatment, total RNA was extracted for subsequent microarray analysis. ACE2 expression data was retrieved from GEO microarray database (NCBI Accession: PRJNA153023; GEO: GSE36287).


( A ) NIH - 3T3 were transduced with retroviral vectors expressing M45 or GFP . Two days after transduction were stimulated with TLR4 agonist LPS ( 10 µg / ml ) , TLR2 agonist LTA - SA ( 10 µg / ml ) , or IL - 1β ( 20 ng / ml ) . IκBα were determined by immunoblotting .

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(A) NIH-3T3 cells were transduced with retroviral vectors expressing M45 or GFP. Two days after transduction cells were stimulated with the TLR4 agonist LPS (10 µg/ml), the TLR2 agonist LTA-SA (10 µg/ml), or IL-1β (20 ng/ml). IκBα levels were determined by immunoblotting.


( B ) NIH - 3T3 were transduced with an M45 - expressing or an empty retroviral vector . 30 min after stimulation with IL - 1β ( 10 ng / ml ) were fixed , subcellular localization of NF - κB p65 subunit was analyzed by immunofluorescence .

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(B) NIH-3T3 cells were transduced with an M45-expressing or an empty retroviral vector. 30 min after stimulation with IL-1β (10 ng/ml) cells were fixed, and subcellular localization of the NF-κB p65 subunit was analyzed by immunofluorescence.


( C ) NIH - 3T3 expressing an NF - κB - dependent secreted alkaline phosphatase ( SEAP ) reporter were transduced with retroviral vectors expressing M45 or GFP stimulated 78 h later with TLR2 agonist Pam3CSK4 ( Pam . , 1 µg / ml ) , TLR4 agonist LPS ( 0 . 1 µg / ml ) , or IL - 1β ( 5 ng / ml ) . SEAP activity in supernatant was quantified 15 h after stimulation is shown as fold induction of SEAP activity of stimulated compared to non - stimulated ( mean ± SD )

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(C) NIH-3T3 cells expressing an NF-κB-dependent secreted alkaline phosphatase (SEAP) reporter were transduced with retroviral vectors expressing M45 or GFP and stimulated 78 h later with the TLR2 agonist Pam3CSK4 (Pam.,1 µg/ml), the TLR4 agonist LPS (0.1 µg/ml), or IL-1β (5 ng/ml). SEAP activity in the supernatant was quantified 15 h after stimulation and is shown as fold induction of SEAP activity of stimulated cells compared to non-stimulated cells (mean ± SD)


( D ) NIH - 3T3 transduced with retroviral vectors expressing M45 or GFP were stimulated with IL - 1β ( 20 ng / ml , 15 min ) or TNFα ( 10 ng / ml , 5 min ) . Phosphorylated total p38 in lysates was detected by immunoblotting .

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(D) NIH-3T3 cells transduced with retroviral vectors expressing M45 or GFP were stimulated with IL-1β (20 ng/ml, 15 min) or TNFα (10 ng/ml, 5 min). Phosphorylated and total p38 in cell lysates was detected by immunoblotting.


( A ) NIH - 3T3 were infected with wt MCMV - GFP , an M45 deletion mutant ( ΔM45 ) or a revertant ( RM45 ) at an MOI of 10 treated 5 h postinfection with TLR2 agonist Pam3CSK4 ( Pam . , 0 . 1 µg / ml ) , TLR4 agonist LPS ( 10 µg / ml ) , or IL - 1β ( 20 ng / ml ) for indicated times . of indicated were analyzed by immunoblotting .

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(A) NIH-3T3 cells were infected with wt MCMV-GFP, an M45 deletion mutant (ΔM45) or a revertant virus (RM45) at an MOI of 10 and treated 5 h postinfection with the TLR2 agonist Pam3CSK4 (Pam., 0.1 µg/ml), the TLR4 agonist LPS (10 µg/ml), or IL-1β (20 ng/ml) for the indicated times. Levels of the indicated proteins were analyzed by immunoblotting.


( B ) BMDMs were infected with wt MCMV - GFP , ΔM45 , or RM45 at an MOI of 3 stimulated 8 h postinfection for 16 h with TLR7 agonist R848 ( 0 . 1 µM ) . TNFα IL - 6 in supernatant were determined by ELISA ( mean ± SD ) .

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(B) BMDMs were infected with wt MCMV-GFP, ΔM45, or RM45 at an MOI of 3 and stimulated 8 h postinfection for 16 h with the TLR7 agonist R848 (0.1 µM). TNFα and IL-6 levels in the supernatant were determined by ELISA (mean ± SD).


( C ) RAW264 . 7 macrophages were infected with wt MCMV - GFP or ΔM45 at an MOI of 0 . 1 , stimulated 24 h postinfection for 4 hours with TLR agonists Pam3CSK4 ( Pam . ) or Malp - 2 ( TLR2 ) , LPS ( TLR4 ) , R837 ( TLR7 ) , or CpG ( TLR9 ) , in presence of brefeldin A . were fixed , permeabilized , stained with a TNFα - specific antibody . percentages of TNFα - positive within infected ( GFP - positive ) populations were determined by FACS ( mean ± SD ) .

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(C) RAW264.7 macrophages were infected with wt MCMV-GFP or ΔM45 at an MOI of 0.1, stimulated 24 h postinfection for 4 hours with TLR agonists Pam3CSK4 (Pam.) or Malp-2 (TLR2), LPS (TLR4), R837 (TLR7), or CpG (TLR9), in the presence of brefeldin A. Cells were fixed, permeabilized, and stained with a TNFα-specific antibody. The percentages of TNFα-positive cells within infected (GFP-positive) cell populations were determined by FACS analysis (mean ± SD).


( A ) 293A were transfected with plasmids expressing HA - tagged M45 or an unrelated MCMV control ( m143 ) Flag - tagged IKK subunits . Lysates were subjected to immunoprecipitation ( IP ) using an anti - HA antibody . Immunoprecipitates were analyzed by immunoblotting ( IB ) using anti - Flag antibody . Whole lysates ( WCL ) were immunoblotted using Flag - , HA - , actin - specific antibodies , respectively .

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(A) 293A cells were transfected with plasmids expressing HA-tagged M45 or an unrelated MCMV control protein (m143) and Flag-tagged IKK subunits. Lysates were subjected to immunoprecipitation (IP) using an anti-HA antibody. Immunoprecipitates were analyzed by immunoblotting (IB) using anti-Flag antibody. Whole cell lysates (WCL) were immunoblotted using Flag-, HA-, and actin-specific antibodies, respectively.


( B ) Nemo − / − MEFs were transfected with HA - tagged IKK subunits HA - tagged M45 or an unrelated MCMV control ( m142 ) , respectively . IP IB were performed using anti - HA anti - Flag antibodies , respectively . WCL were immunoblotted with anti - NEMO antibody as described for panel A .

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(B) Nemo−/− MEFs were transfected with HA-tagged IKK subunits and HA-tagged M45 or an unrelated MCMV control protein ( m142), respectively. IP and IB were performed using anti-HA and anti-Flag antibodies, respectively. WCL were immunoblotted with anti-NEMO antibody and as described for panel A.


( C ) rip1 − / − MEFs were transfected with HA - tagged M45 or an unrelated MCMV control ( m142 ) Flag - tagged NEMO . IP was done with an anti - HA antibody . Immunoprecipitates WCL were analyzed by immunoblotting using indicated antibodies .

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(C) rip1−/− MEFs were transfected with HA-tagged M45 or an unrelated MCMV control protein (m142) and Flag-tagged NEMO. IP was done with an anti-HA antibody. Immunoprecipitates and WCL were analyzed by immunoblotting using the indicated antibodies.


( D ) 293A were transfected with plasmids expressing HA - tagged M45 or m143 ( as a control ) . Lysates were subjected to anti - HA immunoprecipitation . Immunoblotting of precipitates WCL was done with antibodies as indicated .

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(D) 293A cells were transfected with plasmids expressing HA-tagged M45 or m143 (as a control). Lysates were subjected to anti-HA immunoprecipitation. Immunoblotting of precipitates and the WCL was done with antibodies as indicated.


( E ) NIH - 3T3 were infected for 4 hours with RM45 ( MCMV expressing HA - tagged M45 ) , ΔM45 , or Rm142 ( MCMV expressing HA - tagged m142 ) at an MOI of 5 . lysates were subjected to IP IB with antibodies as indicated .

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(E) NIH-3T3 cells were infected for 4 hours with RM45 (MCMV expressing HA-tagged M45), ΔM45, or Rm142 (MCMV expressing HA-tagged m142) at an MOI of 5. Cell lysates were subjected to IP and IB with antibodies as indicated.


( B ) NIH - 3T3 were transduced with retroviruses expressing full M45 , truncated M45 , or GFP . After stimulation with IL - 1β ( 20 ng / ml , 15 min ) , IκBα were determined by immunoblotting .

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(B) NIH-3T3 cells were transduced with retroviruses expressing full length M45, truncated M45 proteins, or GFP. After stimulation with IL-1β (20 ng/ml, 15 min), IκBα levels were determined by immunoblotting.


( C ) 293A were transfected with plasmids encoding Flag - tagged NEMO HA - tagged full - M45 , truncated M45 , or an unrelated MCMV control ( m142 ) , respectively . Lysates were subjected to immunoprecipitation ( IP ) with an anti - HA antibody . Immunoprecipitates whole lysates ( WCL ) were analyzed by immunoblotting ( IB ) with indicated antibodies .

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(C) 293A cells were transfected with plasmids encoding Flag-tagged NEMO and HA-tagged full-length M45, truncated M45, or an unrelated MCMV control protein (m142), respectively. Lysates were subjected to immunoprecipitation (IP) with an anti-HA antibody. Immunoprecipitates and the whole cell lysates (WCL) were analyzed by immunoblotting (IB) with the indicated antibodies.


( D ) BMDMs were mock infected or infected with GFP - expressing wt MCMV ( wt ) , ΔM45 mutant ( ΔM45 ) , M45 revertant ( RM45 ) , or MCMVs expressing Ct or Nt3 ( RCt RNt3 ) at an MOI of 1 . 17 h postinfection were stimulated for 4 hours with TLR9 agonist CpG ( 0 . 5 µM ) or TLR7 agonist R838 ( 0 . 1 µM ) in presence of brefeldin A . were fixed , permeabilized , stained with a TNFα - specific antibody . percentages of TNFα - positive within infected ( GFP - positive ) populations were determined by FACS ( mean ± ) .

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

(D) Primary BMDMs were mock infected or infected with GFP-expressing wt MCMV (wt), ΔM45 mutant (ΔM45), M45 revertant (RM45), or MCMVs expressing Ct or Nt3 (RCt and RNt3) at an MOI of 1. 17 h postinfection cells were stimulated for 4 hours with TLR9 agonist CpG (0.5 µM) or TLR7 agonist R838 (0.1 µM) in the presence of brefeldin A. Cells were fixed, permeabilized, and stained with a TNFα-specific antibody. The percentages of TNFα-positive cells within infected (GFP-positive) cell populations were determined by FACS analysis (mean ± SEM).


( A ) NIH - 3T3 were infected with MCMV ΔM45 or revertant , RM45 , at an MOI of 3 . were harvested at indicated time points postinfection , of IKK subunits , M45 , IE1 , actin in lysates were analyzed by immunoblotting .

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(A) NIH-3T3 cells were infected with MCMV ΔM45 or the revertant virus, RM45, at an MOI of 3. Cells were harvested at indicated time points postinfection, and the levels of IKK subunits, M45, IE1, and actin in lysates were analyzed by immunoblotting.


( B ) Murine fibroblasts ( NIH - 3T3 , 10 . 1 ) or human embryonic kidney 293A were transduced with retroviral vectors expressing M45 or GFP , harvested 72 hours later , analyzed by immunoblotting .

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(B) Murine fibroblasts (NIH-3T3, 10.1) or human embryonic kidney 293A cells were transduced with retroviral vectors expressing M45 or GFP, harvested 72 hours later, and analyzed by immunoblotting.


( C ) ts20 harboring a immunoblotting - sensitive E1 enzyme were incubated for 14 h at a permissive ( 35 ° C ) or a restrictive ( 40 . 5 ° C ) . were then mock - infected or infected with RM45 , analyzed 10 h postinfection by

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(C) ts20 cells harboring a immunoblotting-sensitive E1 enzyme were incubated for 14 h at a permissive (35°C) or a restrictive (40.5°C) temperature. Cells were then mock-infected or infected with RM45, and analyzed 10 h postinfection by immunoblotting


( D ) NIH - 3T3 were mock infected or infected with RM45 ( MOI of 5 ) treated 2 hpi with 10 mM ammonium chloride ( NH4Cl ) or a mix of lysosomal protease inhibitors ( PI - mix ) . 9 hpi , of IKK subunits , M45 , IE1 , actin were determined by immunoblotting . (

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(D) NIH-3T3 cells were mock infected or infected with RM45 (MOI of 5) and treated 2 hpi with 10 mM ammonium chloride (NH4Cl) or a mix of lysosomal protease inhibitors (PI-mix). 9 hpi, levels of IKK subunits, M45, IE1, and actin were determined by immunoblotting. (


( E ) NIH - 3T3 were stably transduced with retroviral vectors expressing M45 or GFP , treated for indicated times with PI - mix , 10 mM NH4Cl , or 5 µM lactacystin , respectively . of indicated were analyzed by immunoblotting .

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(E) NIH-3T3 cells were stably transduced with retroviral vectors expressing M45 or GFP, and treated for the indicated times with PI-mix, 10 mM NH4Cl, or 5 µM lactacystin, respectively. Levels of the indicated proteins were analyzed by immunoblotting.


( A ) NIH - 3T3 stably expressing Flag - tagged NEMO were mock infected or infected with wt MCMV - GFP or MCMV - GFP - ΔM45 at an MOI of 6 . were fixed 6 hpi , NEMO distribution was analyzed by immunofluorescence . arrow indicates region which is shown in higher magnification in right corner of picture .

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(A) NIH-3T3 cells stably expressing Flag-tagged NEMO were mock infected or infected with wt MCMV-GFP or MCMV-GFP-ΔM45 at an MOI of 6. Cells were fixed 6 hpi, and NEMO distribution was analyzed by immunofluorescence. The arrow indicates the region which is shown in higher magnification in the right corner of the picture.


( B ) NIH - 3T3 were transfected with Flag - NEMO M45 - HA expressing plasmids as indicated . 24 h later , were fixed double immunofluorescence was performed using antibodies against HA Flag tags . Regions shown in higher magnification in left corner of pictures are indicated by arrows .

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(B) NIH-3T3 cells were transfected with Flag-NEMO and M45-HA expressing plasmids as indicated. 24 h later, cells were fixed and double immunofluorescence staining was performed using antibodies against the HA and Flag tags. Regions shown in higher magnification in the left corner of the pictures are indicated by arrows.


( C ) NIH - 3T3 stably expressing Flag - NEMO were mock infected or infected with MCMV - M45 - HA or MCMV - ΔM45 at an MOI of 7 . Seven hpi , were fixed analyzed as described for panel B .

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(C) NIH-3T3 cells stably expressing Flag-NEMO were mock infected or infected with MCMV-M45-HA or MCMV-ΔM45 at an MOI of 7. Seven hpi, cells were fixed and analyzed as described for panel B.


( D ) NIH - 3T3 were transfected with expression plasmids for Flag - NEMO M45 - HA or empty vector as indicated . Plasmids encoding GFP - tagged LC3 , Rab5 , or Rab7 were cotransfected . 24 hours posttransfection were fixed used for anti - Flag immunofluorescence .

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(D) NIH-3T3 cells were transfected with expression plasmids for Flag-NEMO and M45-HA or empty vector as indicated. Plasmids encoding GFP-tagged LC3, Rab5, or Rab7 were cotransfected. 24 hours posttransfection cells were fixed and used for anti-Flag immunofluorescence staining.


( A ) atg5 − / − atg5 + / + MEFs were mock infected or infected with MCMVΔM45 or revertant , RM45 , at an MOI of 10 . Eight hpi were harvested of indicated were analyzed by immunoblotting .

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(A) atg5−/− and atg5+/+ MEFs were mock infected or infected with MCMVΔM45 or the revertant virus, RM45, at an MOI of 10. Eight hpi cells were harvested and levels of the indicated proteins were analyzed by immunoblotting.


( B ) 10 . 1 fibroblasts were mock infected or infected with wt MCMV or ΔM45 at an MOI of 5 . were left untreated or treated 30 min after infection with 10 mM NH4Cl to block lysosomal degradation of LC3 - II . were harvested at indicated time points , LC3 - II in lysates were analyzed by immunoblotting using an LC3 - II - specific antibody . LC3 - II were quantified by densitometric normalized using corresponding actin . Fold increases are shown relative to mock - infected .

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(B) 10.1 fibroblasts were mock infected or infected with wt MCMV or ΔM45 at an MOI of 5. Cells were left untreated or treated 30 min after infection with 10 mM NH4Cl to block lysosomal degradation of LC3-II. Cells were harvested at the indicated time points, and LC3-II levels in cell lysates were analyzed by immunoblotting using an LC3-II-specific antibody. LC3-II levels were quantified by densitometric analysis and normalized using the corresponding actin levels. Fold increases are shown relative to mock-infected cells.


( A ) NIH - 3T3 stably expressing GFP - LC3 were transduced with retroviral vectors expressing M45 or Ct3 . Three days later , were analyzed by confocal scanning microscopy . GFP - LC3 dots were counted in 50 per sample . Results are shown as box whisker plots . bottom top of box represent first third quartile , respectively . horizontal within box represents median . minima maxima within 1 . 5 - fold interquartile range are shown as whiskers . Dots represent outliers . Significance was determined using ANOVA ( * * * , p < 0 . 001 ) . M45 Ct3 were detected by

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

(A) NIH-3T3 cells stably expressing GFP-LC3 were transduced with retroviral vectors expressing M45 or Ct3. Three days later, cells were analyzed by confocal laser scanning microscopy. GFP-LC3 dots were counted in 50 cells per sample. Results are shown as box and whisker plots. The bottom and top of the box represent the first and third quartile, respectively. The horizontal line within the box represents the median. The minima and maxima within 1.5-fold interquartile range are shown as whiskers. Dots represent outliers. Significance was determined using ANOVA (***, p<0.001). M45 and Ct3 were detected by immunoblot


( B ) of described above . Large GFP - LC3 are indicated by arrow heads .

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(B) Fluorescence images of cells described above. Large GFP-LC3 aggregates are indicated by arrow heads.


( C ) Electron micrograph of a double - autophagosome in an M45 - expressing .

(C) Electron micrograph of a double-membrane autophagosome in an M45-expressing cell.


( D ) Autophagosomes were counted in 10 randomly selected M45 - Ct3 - expressing ( 2 - 3 sections per ) . Results are shown in box whisker plots as described for panel A . Significance was determined using student ' s t - ( * * * , p < 0 . 001 ) .

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(D) Autophagosomes were counted in 10 randomly selected M45- and Ct3-expressing cells (2-3 TEM sections per cell). Results are shown in box and whisker plots as described for panel A. Significance was determined using the student's t-test (***, p<0.001).


( E ) Epifluorescence image of an M45 - expressing with large GFP - LC3

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(E) Epifluorescence image of an M45-expressing cell with large GFP-LC3 structures


( F ) image of same reveals large marked with ' A ' . ( G , H ) Successive enlargement of region marked in panel F shows an example of an aggregate - associated autophagosome .

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(F) TEM image of the same cell reveals large aggregates marked with 'A'. (G, H) Successive enlargement of the region marked in panel F shows an example of an aggregate-associated autophagosome.


( A ) Clone P15 induces HA - LC3 lipidation . 293T were transfected with P15 plus plasmids expressing HA - LC3A / or apoptotic inhibitor p35 ( as shown ) , lysed for western blotting against indicated molecules ( anti ‐ HA for HA - LC3A ) . figure shows that expression of P15 induces HA - LC3A conversion to a lower molecular form indicative of lipidation . This activity remains unchanged by p35 .

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(A) Clone P15 induces HA-LC3 lipidation. 293T cells were transfected with P15 plus plasmids expressing HA-LC3A and/or the apoptotic inhibitor p35 (as shown), and lysed for western blotting against the indicated molecules (anti‐HA for HA-LC3A). The figure shows that expression of P15 induces HA-LC3A conversion to a lower molecular weight form indicative of protein lipidation. This activity remains unchanged by p35.


( B ) P15 induces GFP - LC3 translocation to a vesiculated pattern . 293T were transfected with indicated plasmids mixed with vectors expressing GFP - LC3A p35 . known autophagic inducer bNIP3L constituted a positive control . Representative confocal microscopy pictures are shown .

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(B) P15 induces GFP-LC3 translocation to a vesiculated pattern. 293T cells were transfected with the indicated plasmids mixed with vectors expressing GFP-LC3A and p35. The known autophagic inducer bNIP3L constituted a positive control. Representative confocal microscopy pictures are shown.


( C ) TMEM59 induces HA - LC3 lipidation in different lines lacking SV40 large T − antigen plasmid amplification system . were transfected with shown plasmids , vectors expressing HA - LC3A ( left ) or HA - LC3B ( right ) GST ( as transfection control ) , lysed for western blotting against indicated molecules .

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(C) TMEM59 induces HA-LC3 lipidation in different cell lines lacking the SV40 large T−antigen plasmid amplification system. Cells were transfected with the shown plasmids, vectors expressing HA-LC3A (left) or HA-LC3B (right) and GST (as transfection control), and lysed for western blotting against the indicated molecules.


( D ) TMEM59 induces GFP - LC3 activation . 293 were transfected with indicated plasmids GFP - LC3A ( top ) or GFP - LC3B ( bottom ) . Representative confocal microscopy pictures are shown .

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(D) TMEM59 induces GFP-LC3 activation. 293 cells were transfected with the indicated plasmids and GFP-LC3A (top) or GFP-LC3B (bottom). Representative confocal microscopy pictures are shown.


( E ) of phenotype in D . number of GFP - LC3 ‐ positive vesicles per transfected was scored for at least fifty . Data are expressed as means ± s . d . of one representative experiment of three repetitions . Source data for this figure is available on online supplementary information page .

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(E) Quantification of the phenotype in D. The number of GFP-LC3‐positive vesicles per transfected cell was scored for at least fifty cells. Data are expressed as means ±s.d. of one representative experiment of three repetitions.Source data for this figure is available on the online supplementary information page.


( A ) TMEM59 ID is required for HA - LC3 lipidation . 293 were transfected with full ‐ TMEM59 ( FL ) or a deleted version lacking ID ( ΔID , Δ263 - 323 ) , HA - LC3A ( left ) or HA - LC3B ( right ) GST . were lysed for western blotting against shown molecules .

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(A) TMEM59 ID is required for HA-LC3 lipidation. 293 cells were transfected with full‐length TMEM59 (FL) or a deleted version lacking the ID (ΔID, Δ263-323), HA-LC3A (left) or HA-LC3B (right) and GST. Cells were lysed for western blotting against the shown molecules.


( B ) ID is necessary for GFP - LC3 activation . 293 were transfected with indicated TMEM59 constructs GFP - LC3A or GFP - LC3B ( as indicated ) . Representative confocal pictures are shown .

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(B) The ID is necessary for GFP-LC3 activation. 293 cells were transfected with the indicated TMEM59 constructs and GFP-LC3A or GFP-LC3B (as indicated). Representative confocal pictures are shown.


( C ) of phenotype in B . Scoring data expression were as in Figure 1E .

(C) Quantification of the phenotype in B. Scoring and data expression were as in Figure 1E.