C . Representative of semithin section of WT Pmp22 - / - sciatic nerves at P30 . Tomacula are abundant in Pmp22 - / - nerves at this , where both with thicker myelin ( in range of < 3 - 4 μm in diameter ) thinner myelin ( with diameters greater than 4 μm , particularly in motor fasicles ) can be observed . C ' . Niaspan does not ameliorate phenotype in Pmp22 - / - mice , as indicated by number of tomacula ( of of tomacula in entire sciatic nerve section , results are mean ± , p = 0 . 7400 , Mann Whitney U , n = 15 per condition ) .

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C. Representative images of semithin section analysis of WT and Pmp22-/- sciatic nerves at P30. Tomacula are abundant in Pmp22-/- nerves at this age, where both fibers with thicker myelin (in the range of <3-4 μm in diameter) and thinner myelin (with diameters greater than 4 μm, particularly in motor fasicles) can be observed.C'. Niaspan does not ameliorate the phenotype in Pmp22-/- mice, as indicated by the number of tomacula (quantification of the percentage of tomacula in the entire sciatic nerve section, results are mean ± SEM, p=0.7400, Mann Whitney U test, n= 15 animals per condition).


D . Growth curve of Pmp22 - / - mice treated with Niaspan administered by i . p . injection daily at 160mg / Kg starting at P15 for 15 days . A significant time by group effect is noted between WT saline Pmp22 - / - saline ( group effect p = 0 . 9907 time effect p = 0 . 0057 ) , indicating that two groups start with similar values but then WT saline group ( n = 6 ) grows more in time . When Pmp22 - / - saline ( n = 5 ) Pmp22 - / - Niaspan - treated ( n = 6 ) are compared , group effect is statistically significant ( p = 0 . 0076 ) while time effect is not ( p = 0 . 6677 ) , suggesting that these two groups have a similar growth trend but Pmp22 - / - Niaspan - treated group starts with a higher baseline .

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

D. Growth curve of Pmp22-/- mice treated with Niaspan administered by i.p. injection daily at 160mg/Kg and starting at P15 for 15 days. A significant time by group effect is noted between WT saline and Pmp22-/- saline (group effect p=0.9907 and time effect p=0.0057), indicating that the two groups start with similar weight values but then the WT saline group (n=6 animals) grows more in time. When Pmp22-/- saline (n=5) and Pmp22-/- Niaspan-treated (n=6) are compared, the group effect is statistically significant (p=0.0076) while the time effect is not (p=0.6677), suggesting that these two groups have a similar growth trend but Pmp22-/- Niaspan-treated group starts with a higher baseline weight.


E . Niaspan does not ameliorate myelin thickness as indicated by g - , with axonal diameter distribution : WT 0 . 68 ± 0 . 003 , 2945 , n = 5 ; Pmp22 - / - 0 . 64 ± 0 . 003 , 1557 , n = 6 ; Pmp22 - / - Niaspan 0 . 63 ± 0 . 003 , 1445 , n = 6 , results are mean ± . WT saline as compared to Pmp22 - / - saline - treated p < 0 . 0001 ; WT saline as compared to Pmp22 - / - Niaspan - treated p < 0 . 0001 , repeated measures ANOVA . Note loss of myelinated in Pmp22 - / - nerves for caliber axons .

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E. Niaspan does not ameliorate myelin thickness as indicated by g-ratio analysis, with axonal diameter distribution: WT 0.68 ± 0.003, 2945 fibers, n=5 animals; Pmp22-/- 0.64 ± 0.003, 1557 fibers, n= 6 animals; Pmp22-/- Niaspan 0.63 ± 0.003, 1445 fibers, n=6 animals, results are mean ± SEM. WT saline as compared to Pmp22-/- saline-treated p<0.0001; WT saline as compared to Pmp22-/- Niaspan-treated p<0.0001, repeated measures ANOVA. Note the loss of myelinated fibers in Pmp22-/- nerves for all caliber axons.


Dynamics of histone modifications after an acclimatizing HS ( ACC ) or no HS ( NHS ) in Col - 0 wild type at HSP22 . 0 ( A ) HSP70 ( B ) . Seedlings were subjected to ACC or a control treatment ( NHS ) 4 d after germination . At indicated time points after treatments , ChIP - qPCR was performed with antibodies against H3K9ac , H3K4me3 , H3K4me2 H3 . Schematics show positions of regions analyzed . Amplicon positions relative to TSS are HSP22 . 0 : 1 , - 2570 bp ; 2 , + 235 bp . HSP70 : 1 , 4192 bp downstream of 3 ' UTR ; 2 , + 47 bp . Data shown are averages over three biological replicates . Amplification values were normalized to input , H3 4 h NHS region 2 . bottom panel shows H3 signal normalized to input 4 h NHS region 2 . Squares triangles within bars mark significant differences ( p < 0 . 01 p < 0 . 05 , respectively , Student ´ s t - ) between ACC NHS samples of same time point . Error bars indicate SE .

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Dynamics of histone modifications after an acclimatizing HS (ACC) or no HS (NHS) in Col-0 wild type at HSP22.0 (A) and HSP70 (B). Seedlings were subjected to ACC or a control treatment (NHS) 4 d after germination. At the indicated time points after the treatments, ChIP-qPCR was performed with antibodies against H3K9ac, H3K4me3, H3K4me2 and H3. Schematics show positions of regions analyzed. Amplicon positions relative to TSS are HSP22.0: 1, -2570 bp; 2, +235 bp. HSP70: 1, 4192 bp downstream of the 3'UTR; 2, +47 bp. Data shown are averages over three biological replicates. Amplification values were normalized to input, H3 and 4 h NHS region 2. The bottom panel shows the H3 signal normalized to input and 4 h NHS region 2. Squares and triangles within bars mark significant differences (p<0.01 and p<0.05, respectively, Student´s t-test) between ACC and NHS samples of the same time point. Error bars indicate SE.


A . Transcript of indicated HS - inducible genes after ACC in Col - 0 ( blue bars ) hsfa2 ( orange bars ) as determined by quantitative Reverse Transcription - PCR ( qRT - PCR ) . Transcript were normalized to TUB6 respective NHS harvested at same time point ( [ GENE OF INTERESTACC x h / TUB6ACC x h ] / [ GENE OF INTERESTNHS x h / TUB6NHS x h ] ) . Note that y - axis is on a log10 scale . Error bars show SE of at least three biological replicates . Asterisks show significant differences between genotypes at same time point on Student ´ s t - ( * : p < 0 . 05 ; * * : p < 0 . 01 ) .

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

A. Transcript levels of the indicated HS-inducible genes after ACC in Col-0 (blue bars) and hsfa2 (orange bars) as determined by quantitative Reverse Transcription-PCR (qRT-PCR). Transcript levels were normalized to TUB6 and the respective NHS harvested at the same time point ([GENE OF INTERESTACC x h/ TUB6ACC x h]/ [GENE OF INTERESTNHS x h/ TUB6NHS x h]). Note that the y-axis is on a log10 scale. Error bars show SE of at least three biological replicates. Asterisks show significant differences between the genotypes at the same time point based on Student´s t-test (*: p<0.05; **: p<0.01).


B . Immunoblotting of HSP21 HSP101 in Col - 0 ( C ) hsfa2 ( h ) at indicated time points after ACC or NHS . TUBULIN was used as a loading control . M , marker .

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B. Immunoblotting of HSP21 and HSP101 in Col-0 (C) and hsfa2 (h) at the indicated time points after ACC or NHS. TUBULIN was used as a loading control. M, marker.


H3K4me3 ( A ) or H3K4me2 ( B ) after an acclimatizing HS ( ACC ) in Col - 0 hsfa2 at HSP18 . 2 , HSP22 . 0 , HSP70 APX2 as detected by ChIP - qPCR . Col - 0 ( blue bars ) hsfa2 ( orange bars ) seedlings were subjected to ACC or no treatment ( NHS ) 4 d after germination . At indicated time points after treatment , ChIP - qPCR was performed with antibodies against H3K4me3 ( A ) , H3K4me2 ( B ) H3 ( for normalization ) . Schematics show positions of regions analyzed ( grey bars , UTR ; black bar , exons ) . Intergenic control region 1 is 3123 bp ( APX2 ) or 2570 bp ( HSP22 . 0 ) upstream of TSS , or 5311 bp ( HSP18 . 2 ) or 6725 bp ( HSP70 ) downstream of TSS , respectively . Data are averages over four biological replicates . Amplification values were normalized to input H3 Col - 0 4h NHS region 2 ( HSP18 . 2 , HSP22 . 0 HSP70 ) or region 3 ( for APX2 ) . * , p < 0 . 05 ; * * , p < 0 . 01 for differences between genotypes at same time point treatment ; squares triangles within bars mark significant differences ( p < 0 . 01 p < 0 . 05 , respectively ) between ACC NHS samples of same time point genotype , Student ´ s t - . Error bars indicate SE .

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

H3K4me3 (A) or H3K4me2 (B) levels after an acclimatizing HS (ACC) in Col-0 and hsfa2 at HSP18.2, HSP22.0, HSP70 and APX2 as detected by ChIP-qPCR. Col-0 (blue bars) and hsfa2 (orange bars) seedlings were subjected to ACC or no treatment (NHS) 4 d after germination. At the indicated time points after the treatment, ChIP-qPCR was performed with antibodies against H3K4me3 (A), H3K4me2 (B) and H3 (for normalization). Schematics show positions of regions analyzed (grey bars, UTR; black bar, exons). Intergenic control region 1 is 3123 bp (APX2) or 2570 bp (HSP22.0) upstream of the TSS, or 5311 bp (HSP18.2) or 6725 bp (HSP70) downstream of the TSS, respectively. Data are averages over four biological replicates. Amplification values were normalized to input and H3 and the Col-0 4h NHS region 2 (HSP18.2, HSP22.0 and HSP70) or region 3 (for APX2). *, p<0.05; **, p<0.01 for differences between genotypes at the same time point and treatment; squares and triangles within bars mark significant differences (p<0.01 and p<0.05, respectively) between ACC and NHS samples of the same time point and genotype, Student´s t-test. Error bars indicate SE.


Figure 4 : Histone H3K9ac profiles after ACC in Col - 0 hsfa2 at HSP18 . 2 , HSP22 . 0 , HSP70 APX2 as detected by ChIP - qPCR . Col - 0 ( blue bars ) hsfa2 ( orange bars ) seedlings were subjected to ACC or control treatment ( NHS ) 4 d after germination . At indicated time points after treatments , ChIP - qPCR was performed with antibodies against H3K9ac or H3 . Schematics ( grey bars , UTR ; black bar , exons ) show positions of regions analyzed . Intergenic control region 1 is 3123 bp ( APX2 ) or 2570 bp ( HSP22 . 0 ) upstream of TSS , or 5311 bp ( HSP18 . 2 ) or 6725 bp ( HSP70 ) downstream of TSS , respectively . Data are averages over four biological replicates . Amplification values were normalized to input H3 Col - 0 4h NHS region 2 ( HSP18 . 2 , HSP22 . 0and HSP70 ) or 3 ( for APX2 ) . * , p < 0 . 05 ; * * , p < 0 . 01 for differences between genotypes at same time point treatment ; squares triangles within bars mark significant differences ( p < 0 . 01 p < 0 . 05 , respectively ) between ACC NHS samples of same time point genotype , Student ´ s t - . Error bars indicate SE .

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Figure 4: Histone H3K9ac profiles after ACC in Col-0 and hsfa2 at HSP18.2, HSP22.0, HSP70 and APX2 as detected by ChIP-qPCR.Col-0 (blue bars) and hsfa2 (orange bars) seedlings were subjected to ACC or control treatment (NHS) 4 d after germination. At the indicated time points after the treatments, ChIP-qPCR was performed with antibodies against H3K9ac or H3. Schematics (grey bars, UTR; black bar, exons) show positions of regions analyzed. Intergenic control region 1 is 3123 bp (APX2) or 2570 bp (HSP22.0) upstream of the TSS, or 5311 bp (HSP18.2) or 6725 bp (HSP70) downstream of the TSS, respectively. Data are averages over four biological replicates. Amplification values were normalized to input and H3 and the Col-0 4h NHS region 2 (HSP18.2, HSP22.0and HSP70) or 3 (for APX2). *, p<0.05; **, p<0.01 for differences between genotypes at the same time point and treatment; squares and triangles within bars mark significant differences (p<0.01 and p<0.05, respectively) between ACC and NHS samples of the same time point and genotype, Student´s t-test. Error bars indicate SE.


Figure 5 : HSFA2 associates with HS memory - related genes transiently after an acclimatizing HS . Binding of HSFA2 - YFP to promoter regions of HSP18 . 2 ( A ) , HSP22 . 0 ( B ) , APX2 ( C ) , HSP70 ( D ) ACTIN7 ( E ) was determined by ChIP - qPCR . Quantifications represent enrichment relative to input in % . Red - orange - yellow bars represent time points ( 0 . 5 h - 4 h - 28 h ) after a ACC grey bars represent respective NHS control samples . Schematics show promoter region of respective gene with regions analyzed ( arrow , TSS ; grey bar , 5 ' UTR ; black bar , exon ; black boxes , heat shock elements ( 5 ' - nnGnAnnTnCtn - 3 ' ) . Regions are drawn to scale . Intergenic control region 1 is 4610 bp ( HSP18 . 2 ) 4192 bp ( HSP70 ) downstream of 3 ' UTR 2570 bp ( HSP22 . 0 ) or 3123 bp ( APX2 ) upstream of TSS , respectively . One representative experiment out of at least four biological replicates is shown . Error bars are SD of three replicates .

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Figure 5: HSFA2 associates with HS memory-related genes transiently after an acclimatizing HS.Binding of HSFA2-YFP to the promoter regions of HSP18.2 (A), HSP22.0 (B), APX2 (C), HSP70 (D) and ACTIN7 (E) was determined by ChIP-qPCR. Quantifications represent the enrichment relative to input in %. Red-orange-yellow bars represent time points (0.5 h-4 h-28 h) after a ACC and grey bars represent the respective NHS control samples. Schematics show the promoter region of the respective gene with regions analyzed (arrow, TSS; grey bar, 5'UTR; black bar, exon; black boxes, heat shock elements (5'-nnGnAnnTnCtn-3'). Regions are drawn to scale. Intergenic control region 1 is 4610 bp (HSP18.2) and 4192 bp (HSP70) downstream of the 3'UTR and 2570 bp (HSP22.0) or 3123 bp (APX2) upstream of the TSS, respectively. One representative experiment out of at least four biological replicates is shown. Error bars are SD of three replicates.


Figure 6 : Expression profiles of HS memory - related genes after a recurring HS show HSFA2 - dependent transcriptional memoryResponse of selected HS - inducible loci upon recurring HS as determined by qRT - PCR . Transcript of APX2 ( A ) , HSP18 . 2 ( B ) , HSP22 . 0 ( C ) , HSP70 ( D ) in Col - 0 ( blue bars ) hsfa2 ( orange bars ) at indicated time points after a single HS or two HS separated by 2 d . were subjected to 37 ° C for 60 min on either d 4 , d 6 or on d 4 + d 6 after germination . NHS samples were harvested at same time points as HS d 4 samples . Transcript of four biological replicates normalized to TUB6 are shown . Small brackets compare two genotypes at same condition , large brackets compare same genotype across two treatments ( at 1 h after treatment ) with Col - 0 indicated first ( Col - 0 / hsfa2 ) . Error bars show SE over four biological replicates . * , p < 0 . 05 ; * * , p < 0 . 01 ; Student ´ s t - .

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Figure 6: Expression profiles of HS memory-related genes after a recurring HS show HSFA2-dependent transcriptional memoryResponse of selected HS-inducible loci upon recurring HS as determined by qRT-PCR. Transcript levels of APX2 (A), HSP18.2 (B), HSP22.0 (C), and HSP70 (D) in Col-0 (blue bars) and hsfa2 (orange bars) at the indicated time points after a single HS or two HS separated by 2 d. Plants were subjected to 37°C for 60 min on either d 4, d 6 or on d 4 + d 6 after germination. NHS samples were harvested at the same time points as HS d 4 samples. Transcript levels of four biological replicates normalized to TUB6 are shown. Small brackets compare the two genotypes at the same condition, large brackets compare the same genotype across two treatments (at 1 h after treatment) with Col-0 indicated first (Col-0/ hsfa2). Error bars show SE over four biological replicates. *, p<0.05; **, p<0.01; Student´s t-test.


b ) Histograms of GFP expression ( x - axis ) are presented from A549 transduced with different viral vectors .

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b) Histograms of GFP expression (x-axis) are presented from A549 cells transduced with the different viral vectors.


c ) of GFP positive from experiment described in ( b ) .

c) Percentage of GFP positive cells from the experiment described in (b).


. d ) A PCR along with primers , as illustrated in Appendix Figure S2a , was used to detect formation of LTR junction thereby circularization of vector . expected of PCR product is 552 bp from a 1 - LTR circle 1029 bp from a 2 - LTR circle .

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. d) A PCR assay along with primers, as illustrated in Appendix Figure S2a, was used to detect formation of LTR junction and thereby circularization of the vector. The expected size of PCR product is 552 bp from a 1-LTR circle and 1029 bp from a 2-LTR circle.


. e ) Southern Blot to detect integration events in LV - NILV - S / MAR - transduced single clones with probed against viral genome . A 6 . 8 kb positive control from a linearized plasmid ( lane 3 ) was included for indication of of circles formed by non - integrated NILV - S / MAR vector .

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. e) Southern Blot analysis to detect integration events in LV- and NILV-S/MAR-transduced single cell clones with DNA probed against the viral genome. A 6.8 kb positive control from a linearized plasmid (lane 3) was included for indication of the size of DNA circles formed by non-integrated NILV-S/MAR vector.


f - h ) A padlock probe , as illustrated in Appendix Figure S4 , was used to demonstrate persistence of proviral either in a circular or integrated form . Representative show persistence in ( f ) LV - , ( g ) NILV - or ( h ) NILV - S / MAR - transduced . Each red dot ( Cy3 ) represents a single molecule nuclei were stained with Hoechst 33342 .

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f-h) A padlock probe assay, as illustrated in Appendix Figure S4, was used to demonstrate persistence of proviral DNA either in a circular or integrated form. Representative images show DNA persistence in (f) LV-, (g) NILV- or (h) NILV-S/MAR-transduced cells. Each red dot (Cy3) represents a single DNA molecule and cell nuclei were stained with Hoechst 33342.


c ) Flow cytometry histograms of RFP expression ( x - axis ) of A549 / RFP + Luc + at day 20 27 after NILV - S / MAR ( GFP , shNT ) or NILV - S / MAR ( GFP , shRFP ) transduction .

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c) Flow cytometry histograms of RFP expression (x-axis) of A549/RFP+Luc+ cells at day 20 and 27 after NILV-S/MAR(GFP, shNT) or NILV-S/MAR(GFP, shRFP) transduction.


d ) Luciferase activity of A549 / RFP + Luc + at day 20 day 44 after NILV - S / MAR ( GFP , shNT ) - or NILV - S / MAR ( GFP , shRFP ) - transduction .

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d) Luciferase activity of the A549/RFP+Luc+ cells at day 20 and day 44 after NILV-S/MAR(GFP, shNT)- or NILV-S/MAR(GFP, shRFP)-transduction.


e - h ) Representative microscopy show expression of RFP from either NILV - S / MAR - transduced or un - transduced A549 / RFP + Luc + with nuclei stained with Hoechst 33342 ( blue ) . RFP signal ( orange ) were collected with a fixed exposure time ( 300 ms ) for equal comparison .

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e-h) Representative fluorescence microscopy images show the expression of RFP from either NILV-S/MAR-transduced or un-transduced A549/RFP+Luc+ cells with cell nuclei stained with Hoechst 33342 (blue). RFP signal (orange) were collected with a fixed exposure time (300 ms) for equal comparison.


c ) of CD19CAR expression on CD3 + T from each independent donor . d ) Histograms of CD19 CAR expression on T form one representative donor before after expansion . e ) CD107a expression on transduced T ( CD3 + ) after exposure to CD19 + target ( Daudi ) for 24 h ( n = 6 ) . f ) IFN - γ production in supernatant was analyzed by ELISA after 24 h of exposure of transduced T to target ( n = 6 ) . g ) Relative viability of target ( CD19 + Daudi ) , as an indication of T killing efficacy , were measured at different effector T to target ( E : T ) normalized to signal from target alone ( n = 6 ) . h ) Transduced T were labeled with a dye co - cultured with CD19 + Daudi for 4 days . Dye dilution upon T division was monitored by flow cytometry representative histograms are shown .

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

c) Percentage of CD19CAR expression on CD3+ T cells from each independent donor. d) Histograms of CD19 CAR expression on T cells form one representative donor before and after expansion. e) CD107a expression on transduced T cells (CD3+) after exposure to CD19+ target cells (Daudi) for 24 h (n=6). f) IFN-γ production in the supernatant was analyzed by ELISA after 24 h of exposure of transduced T cells to target cells (n=6). g) Relative viability of target cells (CD19+ Daudi), as an indication of T cell killing efficacy, were measured at different effector T cell to target cell ratio (E:T ratio) and normalized to the signal from target cells alone (n=6). h) Transduced T cells were labeled with a fluorescent cell membrane dye and co-cultured with CD19+ Daudi cells for 4 days. Dye dilution upon T cell division was monitored by flow cytometry and representative histograms are shown.


i - m ) Mice were treated by intravenous injection of NILV - S / MAR ( Mock ) , LV ( CD19CAR ) NILV - S / MAR ( CD19CAR ) - transduced T ( 8x106 / injection ) at days 18 , 21 , 24 after implantation . growth was monitored by caliper measurements . i - k ) volume of mice in each treatment groups are presented . l ) average volume of each treatment group is presented values on day 37 were statistically compared ( n = 12 in LV group , n = 9 in NILV - S / MAR group n = 11 in Mock control group ) . m ) survival of mice is shown as Kaplan - Meier curves .

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 O O

i-m) Mice were treated by intravenous injection of NILV-S/MAR(Mock), LV(CD19CAR) and NILV-S/MAR(CD19CAR)-transduced T cells (8x106 cells/injection) at days 18, 21, 24 after tumor implantation. Tumor growth was monitored by caliper measurements. i-k) Tumor volume of individual mice in each treatment groups are presented. l) The average tumor volume of each treatment group is presented and the values on day 37 were statistically compared (n=12 in LV group, n=9 in NILV-S/MAR group and n=11 in Mock control group). m) The survival of mice is shown as Kaplan-Meier curves.


n - p ) Representative showing infiltration of adoptively transferred human T into tumors . T were visualized by for human CD3 ( red ) nucleus ( blue ) . q ) of CD3 + T from each treatment group is presented ( n = 5 ) .

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

n-p) Representative images showing infiltration of adoptively transferred human T cells into tumors. T cells were visualized by staining for human CD3 (red) and nucleus (blue). q) Quantification of CD3+ T cells from each treatment group is presented (n=5).


of Prdx4 WT KO mice over 72 h course of LPS ( 4 . 5 mg / kg BW ) or PBS injection ( i . p . ) . Each circle represents a mean of n = 7 mice , vertical lines indicate

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

Percent body weight of male Prdx4 WT and KO mice over the 72 h course of LPS (4.5 mg/kg BW) or PBS injection (i.p.). Each circle represents a mean of n=7 mice, vertical lines indicate SEM


Cytokine in Prdx4 WT KO mice in response to LPS injection . ( B ) Cxcl1 in serum ( left ) or peritoneal lavage ( right ) at indicated time points after LPS or PBS injection . ( C ) TNF - α in serum ( left ) or peritoneal lavage ( right ) at indicated time points after LPS or PBS injection . ( D ) IL - 1β in serum ( left ) or peritoneal lavage ( right ) at indicated time points after LPS or PBS injection . Each dot ( B - D ) represents an mouse . Horizontal lines indicate mean .

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

Cytokine concentration in Prdx4 WT and KO mice in response to LPS injection. (B) Cxcl1 levels in serum (left) or peritoneal lavage (right) at indicated time points after LPS or PBS injection. (C) TNF-α levels in serum (left) or peritoneal lavage (right) at indicated time points after LPS or PBS injection. (D) IL-1β levels in serum (left) or peritoneal lavage (right) at indicated time points after LPS or PBS injection. Each dot (B-D) represents an individual mouse. Horizontal lines indicate mean.


of Prdx4 WT KO mice over 48 h course of LPS ( 4 . 5 mg / kg BW ) injection ( i . p . ) treatment with IL - 1 receptor antagonist ( IL - 1RA ) Anakinra ( 200 µg / mouse ) or control . Arrows indicate time point of Anakinra injection . Each circle represents a mean of n = 5 mice , vertical lines indicate .

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

Percent body weight of male Prdx4 WT and KO mice over the 48 h course of LPS (4.5 mg/kg BW) injection (i.p.) and treatment with IL-1 receptor antagonist (IL-1RA) Anakinra (200 µg/mouse) or control. Arrows indicate time point of Anakinra injection. Each circle represents a mean of n=5 mice, vertical lines indicate SEM.


Serum of Cxcl1 , TNF - α IL - 1β in Prdx4 WT KO mice injected with LPS , LPS IL - 1RA or control . Horizontal lines indicate mean .

Serum concentration of Cxcl1, TNF-α and IL-1β in Prdx4 WT and KO mice injected with LPS, LPS and IL-1RA or control. Horizontal lines indicate mean.


of Prdx4 - flox Prdx4 - ∆ LysMCre mice over 48 h course of 4 . 5 mg / kg BW LPS ( i . p . ) . Each circle represents a mean of n = 7 mice , vertical lines indicate .

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

Percent body weight of male Prdx4-flox and Prdx4-∆LysMCre mice over the 48 h course of 4.5 mg/kg BW LPS ( i.p.). Each circle represents a mean of n=7 mice, vertical lines indicate SEM.


Serum of Cxcl1 , TNF - α IL - 1β in Prdx4 - flox Prdx4 - ∆ LysMCre mice injected with LPS . Each dot ( B , D ) represents an mouse . Horizontal lines indicate mean .

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

Serum concentration of Cxcl1, TNF-α and IL-1β in Prdx4-flox and Prdx4-∆LysMCre mice injected with LPS. Each dot (B, D) represents an individual mouse. Horizontal lines indicate mean.


of Cxcl1 , TNF - α IL - 1β in supernatants of Prdx4 WT KO BMDMs in response to a time course of LPS stimulation ( 100 ng / ml LPS , time points indicated ) .

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

Concentration of Cxcl1, TNF-α and IL-1β in the supernatants of Prdx4 WT and KO BMDMs in response to a time course of LPS stimulation (100 ng/ml LPS, time points indicated).


IL - 1β release of Prdx4 WT KO BMDMs , untreated or primed for 6 h with LPS ( 100 ng / ml ) then pulsed for indicated time points with ATP ( 5 mM ) .

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

IL-1β release of Prdx4 WT and KO BMDMs, untreated or primed for 6 h with LPS (100 ng/ml) and then pulsed for indicated time points with ATP (5 mM).


Western blot of IL - 1β in lysates supernatants of Prdx4 WT KO BMDMs , primed with LPS ( 100 ng / ml ) pulsed with ATP ( 5 mM ) for 4 h or left untreated . Dashed indicates vertical slice .

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

Western blot analysis of IL-1β in cell lysates and supernatants of Prdx4 WT and KO BMDMs, primed with LPS (100 ng/ml) and pulsed with ATP (5 mM) for 4 h or left untreated. Dashed line indicates vertical slice.


Immunofluorescence microscopy of ASC speck formation in Prdx4 WT KO BMDMs in response to Nigericin ( 10 µg / ml ) stimulation for 45 min of LPS - primed . were stained with an antibody to ASC nuclei were counterstained using DAPI . Scale bar indicates 20 µm . ASC speck - positive were counted expressed as of total . Bars represent a mean of n = 4 mice , vertical lines indicate SD . n . s . not significant ( two - t - ) .

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

Immunofluorescence microscopy of ASC speck formation in Prdx4 WT and KO BMDMs in response to Nigericin (10 µg/ml) stimulation for 45 min of LPS-primed cells. Cells were stained with an antibody to ASC and nuclei were counterstained using DAPI. Scale bar indicates 20 µm. ASC speck-positive cells were counted and expressed as percentage of total cells. Bars represent a mean of n=4 mice, vertical lines indicate SD. n.s. not significant (two-tailed t-test).


Western blot of caspase - 1 cleavage in supernatant of Prdx4 WT KO BMDMs in response to Nigericin ( 10 µg / ml ) stimulation for 1 h after priming with LPS ( 100 ng / ml ) , LPS - priming alone or without stimulation . Whole lysates were analyzed for pro - caspase - 1 Gapdh .

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

Western blot analysis of caspase-1 cleavage in the supernatant of Prdx4 WT and KO BMDMs in response to Nigericin (10 µg/ml) stimulation for 1 h after priming with LPS (100 ng/ml), LPS-priming alone or without stimulation. Whole cell lysates were analyzed for pro-caspase-1 and Gapdh levels.


IL - 1β release in Prdx4 WT KO BMDMs , untreated or primed for 6 h with LPS ( 100 ng / ml ) then pulsed for 3 h with ATP ( 5 mM ) or Nigericin ( 10 µg / ml ) or transfected for 3 h with poly ( dA : dT ) or Flagellin ( 1 µg / ml each ) or treated with transfection agent only .

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

IL-1β release in Prdx4 WT and KO BMDMs, untreated or primed for 6 h with LPS (100 ng/ml) and then pulsed for 3 h with ATP (5 mM) or Nigericin (10 µg/ml) or transfected for 3 h with poly (dA:dT) or Flagellin (1 µg/ml each) or treated with transfection agent only.


of by LDH - release in Prdx4 WT KO BMDMs , untreated or primed for 6 h with LPS ( 100 ng / ml ) then pulsed for 3 h with ATP ( 5 mM ) or Nigericin ( 10 µg / ml ) or transfected for 3 h with poly ( dA : dT ) or flagellin ( 1 µg / ml each ) or treated with transfection agent only .

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

Quantification of cell death by LDH-release in Prdx4 WT and KO BMDMs, untreated or primed for 6 h with LPS (100 ng/ml) and then pulsed for 3 h with ATP (5 mM) or Nigericin (10 µg/ml) or transfected for 3 h with poly (dA:dT) or flagellin (1 µg/ml each) or treated with transfection agent only.


Western blot of rPRDX4 , rCASP - 1 co - incubated rPRDX4 rCASP - 1 after non - reducing SDS - PAGE . Black arrows at 250 kDa indicate corresponding molecular weights of rCASP - 1 rPRDX4 oligomers decrease of Prdx4 band upon co - incubation with rCASP - 1 . Black arrow at approx . 50 kDa indicates appearance of an additional rPRDX4 band which corresponds to molecular of a Prdx4 dimer .

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

Western blot analysis of rPRDX4, rCASP-1 and co-incubated rPRDX4 and rCASP-1 after non-reducing SDS-PAGE. Black arrows at 250 kDa indicate the corresponding molecular weights of rCASP-1 and rPRDX4 oligomers and decrease of the Prdx4 band intensity upon co-incubation with rCASP-1. Black arrow at approx. 50 kDa indicates the appearance of an additional rPRDX4 band which corresponds to the molecular weight of a Prdx4 dimer.


Western blot of rPRDX4 ( = P ) rCASP - 1 ( = C ) after alkylation ( = A ) non - reducing SDS - PAGE . Black arrows indicate termination of integration of rCASP - 1 into rPRDX4 decamer / HMWC upon alkylation .

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

Western blot analysis of rPRDX4 (=P) and rCASP-1 (=C) after alkylation (=A) and non-reducing SDS-PAGE. Black arrows indicate the termination of the integration of rCASP-1 into the rPRDX4 decamer/HMWC upon alkylation.


Rate of caspase - 1 activity in presence of non - reduced decameric rPrdx4 , reduced dimeric monomeric rPRDX4 , YVAD or control .

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

Rate of caspase-1 activity in the presence of non-reduced decameric rPrdx4, reduced dimeric and monomeric rPRDX4, YVAD or control.


Foldchange in IL - 1β in supernatants of HEK293 transfected with plasmids for NLRP3 , ASC , IL - 1β caspase - 1 WT or Cys - to - Ser mutants C362S or C397S co - transfected with Prdx4 or control . were stimulated with 2 . 5 mM ATP for 30 min before .

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

Foldchange in IL-1β concentration in supernatants of HEK293 cells transfected with plasmids for NLRP3, ASC, IL-1β and caspase-1 WT or Cys-to-Ser mutants C362S or C397S and co-transfected with Prdx4 or control. Cells were stimulated with 2.5 mM ATP for 30 min before analysis.


Western blot of non - reducing SDS - PAGE of lysates from HEK293 transfected with caspase - 1 WT , C362S , C397S , or C362S plus C397S mutants co - transfected with Prdx4 - GFP or GFP as control .

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 B-CONTROLLED_VAR O O O O O O O

Western blot analysis of non-reducing SDS-PAGE of cell lysates from HEK293 cells transfected with caspase-1 WT, C362S, C397S, or C362S plus C397S mutants and co-transfected with Prdx4-GFP or GFP as control.


Western blot of co - immunoprecipitation using HA - magnetic beads from lysates of HEK293 transfected with HA - tagged caspase - 1 WT , C362S , C397S , or C362S plus C397S mutants co - transfected with Prdx4 - GFP or GFP as control .

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 B-CONTROLLED_VAR O O O O O O O

Western blot analysis of co-immunoprecipitation using HA-magnetic beads from cell lysates of HEK293 cells transfected with HA-tagged caspase-1 WT, C362S, C397S, or C362S plus C397S mutants and co-transfected with Prdx4-GFP or GFP as control.


IL - 1β in supernatants of HEK293 transfected with plasmids for NLRP3 , ASC , IL - 1β caspase - 1 co - transfected with Prdx4 WT or Cys - to - Ala mutants C51A , C124A , C245A or DM C124A / C245A or control . were stimulated with 2 . 5 mM ATP for 30 min before .

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

IL-1β concentration in supernatants of HEK293 cells transfected with plasmids for NLRP3, ASC, IL-1β and caspase-1 and co-transfected with Prdx4 WT or Cys-to-Ala mutants C51A, C124A, C245A or DM C124A/C245A or control. Cells were stimulated with 2.5 mM ATP for 30 min before analysis.


Western blot of co - immunoprecipitation using HA - magnetic beads from lysates of HEK293 transfected with HA - tagged caspase - 1 WT or control co - transfected with Prdx4 WT or Cys - to - Ala mutants C51A , C124A , C245A or DM C124A / C245A .

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

Western blot analysis of co-immunoprecipitation using HA-magnetic beads from cell lysates of HEK293 cells transfected with HA-tagged caspase-1 WT or control and co-transfected with Prdx4 WT or Cys-to-Ala mutants C51A, C124A, C245A or DM C124A/C245A.


Western blot of Prdx4 , pro - caspase ‑ 1 , Gapdh E - Cadherin from insoluble fraction of LPS / or ATP - stimulated BMDMs or untreated controls .

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

Western blot analysis of Prdx4, pro-caspase‑1, Gapdh and E-Cadherin from the cytosolic and insoluble cell fraction of LPS and/or ATP-stimulated BMDMs or untreated controls.


Prdx4 in supernatants of Prdx4 WT or KO BMDMs , primed for 6 h with LPS pulsed for indicated time points with 5 mM ATP . Each circle represents a mean of n = 3 mice , vertical lines indicate SD . * * , p < 0 . 01 ; n . s . not significant ( two - t - ) .

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

Prdx4 concentration in supernatants of Prdx4 WT or KO BMDMs, primed for 6 h with LPS and pulsed for indicated time points with 5 mM ATP. Each circle represents a mean of n=3 mice, vertical lines indicate SD. **, p < 0.01; n.s. not significant (two-tailed t-test).


of Prdx4 in serum of WT mice , injected with LPS ( 4 . 5 mg / kg BW ) for time points indicated . Each dot represents an mouse . Horizontal lines indicate mean . * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; n . s . not significant ( two - t - ) .

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

Concentration of Prdx4 in the serum of WT mice, injected with LPS (4.5 mg/kg BW) for the time points indicated. Each dot represents an individual mouse. Horizontal lines indicate mean. *, p < 0.05; **, p < 0.01; ***, p < 0.001; n.s. not significant (two-tailed t-test).


of Prdx4 in supernatants of Prdx4 WT KO BMDMs . were primed with LPS ( 100 ng / ml ) for 6 h , followed by pretreatment with 20 µM YVAD or DMSO as control for 30 min stimulated with 5 mM ATP for 4 h or no further stimulation . Each bar represents a mean of n = 3 mice , vertical lines indicate SD . * * , p < 0 . 01 ; n . s . not significant ( two - t - ) .

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

Concentration of Prdx4 in supernatants of Prdx4 WT and KO BMDMs. Cells were primed with LPS (100 ng/ml) for 6 h, followed by pretreatment with 20 µM YVAD or DMSO as control for 30 min and stimulated with 5 mM ATP for 4 h or no further stimulation. Each bar represents a mean of n=3 mice, vertical lines indicate SD. **, p < 0.01; n.s. not significant (two-tailed t-test).


Relative of Prdx4 secretion in response to LPS + ATP stimulation pretreatment with either Glycine , NSA or GW4869 relative of caspase - 1 secretion in response to LPS + ATP stimulation pretreatment with in response to LPS + ATP stimulation pretreatment with GW4869 . Each bar represents a mean of n = 3 biological with 2 technical replicates , vertical lines indicate SD . * , p < 0 . 05 ; * * , p < 0 . 01 ; n . s . not significant ( two - t - ) .

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

Relative levels of Prdx4 secretion in response to LPS+ATP stimulation and pretreatment with either Glycine, NSA or GW4869 and relative levels of caspase-1 secretion in response to LPS+ATP stimulation and pretreatment with in response to LPS+ATP stimulation and pretreatment with GW4869. Each bar represents a mean of n=3 biological with 2 technical replicates, vertical lines indicate SD. *, p < 0.05; **, p < 0.01; n.s. not significant (two-tailed t-test).


Western blot of subcellular organelle fractions . OptiPrep density gradient ultracentrifugation was used to fractionate subcellular organelles from Prdx4 WT BMDMs that were primed with LPS ( 100 ng / ml ) for 12 h stimulated with 5 mM ATP for 4 h .

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

Western blot analysis of subcellular organelle fractions. OptiPrep density gradient ultracentrifugation was used to fractionate subcellular organelles from Prdx4 WT BMDMs that were primed with LPS (100 ng/ml) for 12 h and stimulated with 5 mM ATP for 4 h.


BCA of EV in EVs isolates . Each bar represents a mean of n = 3 technical replicates , vertical lines indicate SD . * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; n . s . not significant ( two - t - ) .

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BCA analysis of EV protein concentration in EVs isolates. Each bar represents a mean of n=3 technical replicates, vertical lines indicate SD. *, p < 0.05; **, p < 0.01; ***, p < 0.001; n.s. not significant (two-tailed t-test).


Exocet of EVs particle numbers . Each bar represents a mean of n = 3 technical replicates , vertical lines indicate SD . * , p < 0 . 05 ; * * * , p < 0 . 001 ; n . s . not significant ( two - t - ) .

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Exocet quantification assay of EVs particle numbers. Each bar represents a mean of n=3 technical replicates, vertical lines indicate SD. *, p < 0.05; ***, p < 0.001; n.s. not significant (two-tailed t-test).


Transmission electron microscopy ( ) of EV isolates . Three representative pictures are displayed . Scale bar indicates 200 nm .

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Transmission electron microscopy (TEM) of EV isolates. Three representative pictures are displayed. Scale bar indicates 200 nm.


of distribution of EVs . Each dot indicates diameter in nm of an vesicle . SD . * * * , p < 0 . 001 ; n . s . not significant ( one - way ANOVA , followed by a Tukey multiple comparison ) .

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Analysis of size distribution of EVs. Each dot indicates the diameter in nm of an individual vesicle. SD. ***, p < 0.001; n.s. not significant (one-way ANOVA, followed by a Tukey multiple comparison test).


Prdx4 in EV lysates . Each bar represents a mean of n = 2 technical replicates , vertical lines indicate SD . * * , p < 0 . 01 ; * * * , p < 0 . 001 ; n . s . not significant ( two - t - ) .

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Prdx4 concentration in EV lysates. Each bar represents a mean of n= 2 technical replicates, vertical lines indicate SD. **, p < 0.01; ***, p < 0.001; n.s. not significant (two-tailed t-test).


Caspase - 1 IL - 1β in EV lysates . Each bar represents a mean of n = 2 technical replicates , vertical lines indicate SD . * * , p < 0 . 01 ; * * * , p < 0 . 001 ; n . s . not significant ( two - t - ) .

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Caspase-1 and IL-1β concentration in EV lysates. Each bar represents a mean of n= 2 technical replicates, vertical lines indicate SD. **, p < 0.01; ***, p < 0.001; n.s. not significant (two-tailed t-test).


Western blot of Prdx4 , NLRP3 , ASC , caspase ‑ 1 , IL - 1β CD63 from EV lysates ( upper panel ) or caspase - 1 after immunoprecipitation against caspase - 1 ( lower panel ) .

Western blot analysis of Prdx4, NLRP3, ASC, caspase‑1, IL-1β and CD63 from EV lysates (upper panel) or caspase-1 after immunoprecipitation against caspase-1 (lower panel).


Cxcl1 in supernatants of caspase - 1 - deficient BMDMs stimulated with EVs from LPS , LPS ATP or control - treated Prdx4 WT or KO BMDMs , as well as caspase - 1 - deficient BMDMs pre - treated with Anakinra stimulated with EVs from LPS ATP - treated Prdx4 WT or KO BMDMs . Each bar represents a mean of n = 3 biological with 2 technical replicates , vertical lines indicate SD .

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Cxcl1 concentration in supernatants of caspase-1-deficient BMDMs stimulated with EVs from LPS, LPS and ATP or control-treated Prdx4 WT or KO BMDMs, as well as caspase-1-deficient BMDMs pre-treated with Anakinra and stimulated with EVs from LPS and ATP-treated Prdx4 WT or KO BMDMs. Each bar represents a mean of n=3 biological with 2 technical replicates, vertical lines indicate SD.


Serum Cxcl1 in C57Bl6 / N mice injected with either PBS or EVs from LPS ATP or control - treated Prdx4 WT or KO or ASC KO BMDMs . Each dot represents an mouse . Horizontal lines indicate mean .

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Serum Cxcl1 in C57Bl6/N mice injected with either PBS or EVs from LPS and ATP or control-treated Prdx4 WT or KO or ASC KO BMDMs. Each dot represents an individual mouse. Horizontal lines indicate mean.


( B ) Western blot of SARS - CoV - 2 Spike 1 - 1273 , 1 - 674 , 319 - 591 expression in HEK293T . black arrows indicate location of induced target .

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(B) Western blot analysis of SARS-CoV-2 Spike 1-1273, 1-674, and 319-591 expression in HEK293T cells. The black arrows indicate the location of the induced target proteins.


( C ) Detection of decoy antibody ACE2 - Fc Spike S1 - Fc chimeric homodimer formation in nonreducing Coomassie Brilliant Blue .

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(C) Detection of decoy antibody ACE2-Fc and Spike S1-Fc chimeric homodimer formation in nonreducing Coomassie Brilliant Blue staining.


( D ) Recognition of ACE2 - Fc Spike S1 - Fc by anti - human IgG Fc antibody in nonreducing sodium dodecyl sulfate - polyacrylamide gel electrophoresis ( SDS - PAGE ) .

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(D) Recognition of the ACE2-Fc and Spike S1-Fc by anti-human IgG Fc antibody in nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).


( E , F ) Deglycosylation of ACE2 - Fc Spike 1 - 674 - Fc by PNGase F . PNGase F digested ACE2 - Fc ( 500 ng ) Spike 1 - 674 - Fc ( 500 ng ) were subjected to Coomassie Brilliant Blue ( E ) Western blot by anti - human IgG Fc antibody ( F ) .

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(E, F) Deglycosylation of ACE2-Fc and Spike 1-674-Fc by PNGase F. PNGase F digested ACE2-Fc (500 ng) and Spike 1-674-Fc (500 ng) were subjected to Coomassie Brilliant Blue staining (E) and Western blot analysis by anti-human IgG Fc antibody (F).


( A ) Competitive ELISA . ACE2 - Fc can compete with ACE2 - Fc - Biotin for Spike 1 - 674 binding in a dose - dependent manner . competition effects of ACE2 - Fc on ACE2 - Spike binding was normalized to PBS control . Error bars represent standard deviation ( SD ) , n = 2 . Statistical was performed by unpaired two tail t - . * * P < 0 . 01 , * * * P < 0 . 001 .

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(A) Competitive ELISA. ACE2-Fc can compete with ACE2-Fc-Biotin for Spike 1-674 binding in a dose-dependent manner. The competition effects of ACE2-Fc on ACE2-Spike protein binding was normalized to PBS control. Error bars represent the standard deviation (SD), n=2. Statistical analysis was performed by unpaired two tail t-test. **P < 0.01, ***P < 0.001.


( B ) Recognition of full - Spike on H1975 ( left panel as negative control ) or H1975 - Spike ( right panel ) by ACE2 - Fc using flow cytometry . Isotype control : 40 μg / mL mouse IgG - FITC .

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(B) Recognition of full-length Spike on H1975 (left panel as negative control) or H1975-Spike (right panel) cells by ACE2-Fc using flow cytometry analysis. Isotype control: 40 μg/mL mouse IgG-FITC.


( C ) Confocal microscopy of H1975 - Spike - overexpressing . Spikes on H1975 were stained with anti - Spike antibody Alexa Fluor ® 594 - conjugated secondary antibody on ice for 1 hr . After that , ACE2 - Fc - FITC was incubated for another 1 hr . Scale bars equal to 20 µm . DAPI was used as a counterstain .

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(C) Confocal microscopy of H1975-Spike-overexpressing cells. Spikes on H1975 cells were stained with anti-Spike antibody and Alexa Fluor® 594-conjugated secondary antibody on ice for 1 hr. After that, ACE2-Fc-FITC was incubated for another 1 hr. Scale bars equal to 20 µm. DAPI was used as a nuclear counterstain.


( D ) Preservation of ACE2 - Fc peptidase activity . peptidase activity of ACE2 - Fc was measured by cleavage of peptide substrates .

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(D) Preservation of ACE2-Fc peptidase activity. The peptidase activity of ACE2-Fc was measured by cleavage of the fluorescent peptide substrates.


( E ) Inhibition of angiotensin II ( Ang II ) - induced TNF - α production by ACE2 - Fc . Ang II was preincubated with indicated amounts of ACE2 - Fc or IgG for 30 minutes . After that , mixtures were added to RAW264 . 7 for 12 hr . of TNF - α in culture medium was determined by ELISA . Error bars represent standard deviation ( SD ) , n = 3 . Statistical was performed by unpaired two tail t - . * P < 0 . 05 . dotted represents mean value of TNF - α in control group .

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(E) Inhibition of angiotensin II (Ang II)-induced TNF-α production by ACE2-Fc. Ang II was preincubated with indicated amounts of ACE2-Fc or IgG for 30 minutes. After that, the mixtures were added to RAW264.7 cells for 12 hr. The concentration of TNF-α in the culture medium was determined by ELISA. Error bars represent the standard deviation (SD), n=3. Statistical analysis was performed by unpaired two tail t-test. *P < 0.05. The dotted line represents the mean value of TNF-α in control group.


( F ) Inhibition of Ang II - induced ADAM17 ( a disintegrin metalloprotease 17 ) phosphorylation by ACE2 - Fc . extracts from ( E ) were immunoblotted with indicated antibodies ( left panel ) . β - actin served as loading control . signal was normalized to only ( right panel ) . Data are representative of three independent experiments , values are expressed as mean ± SD ( right panel ) . Error bars represent standard deviation ( SD ) , n = 2 . Statistical was performed by unpaired two tail t - . * P < 0 . 05 . IgG represents human normal IgG control .

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(F) Inhibition of Ang II-induced ADAM17 (a disintegrin and metalloprotease 17) phosphorylation by ACE2-Fc. The protein extracts from (E) were immunoblotted with the indicated antibodies (left panel). β-actin served as the loading control. The signal intensity was normalized to cells only (right panel). Data are representative of three independent experiments, and the values are expressed as the mean ± SD (right panel). Error bars represent the standard deviation (SD), n=2. Statistical analysis was performed by unpaired two tail t-test. *P < 0.05. IgG represents the human normal IgG control.


( B ) HEK293 H1975 were transduced with full - ACE2 by lentivirus . extracts were immunoblotted with indicated antibodies . O / E represents overexpression .

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(B) HEK293 and H1975 cells were transduced with full-length ACE2 by lentivirus. Protein extracts were immunoblotted with the indicated antibodies. O/E represents overexpression.


( C ) GFP full - Spike co - transfected HEK293T were used as effector ( 293 - S ) . HEK293 / ACE2 ( HEK293T - overexpressing ACE2 ) ( left panel ) or H1975 / ACE2 ( H1975 - overexpressing ACE2 ) ( right panel ) were used as target . 293 - S were preincubated with normal human IgG or ACE2 - Fc at 37 ° C for 1 hr . After that , mixtures were added to target for 4 hr ( - fusion ) or 24 hr ( syncytia formation ) . areas were measured by inverted microscopy Metamorph software ( Metamorphosis ) . Scale bars equal to 50 µm . arrows indicate - fusion or syncytia formation .

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(C) GFP and full-length Spike co-transfected HEK293T cells were used as effector cells (293-S). HEK293/ACE2 (HEK293T-overexpressing ACE2) (left panel) or H1975/ACE2 (H1975-overexpressing ACE2) (right panel) cells were used as target cells. The 293-S cells were preincubated with normal human IgG or ACE2-Fc at 37°C for 1 hr. After that, the mixtures were added to target cells for 4 hr (cell-cell fusion) or 24 hr (syncytia formation). The fluorescent areas were measured by inverted fluorescence microscopy and Metamorph software (Metamorphosis). Scale bars equal to 50 µm. The white arrows indicate the cell-cell fusion or syncytia formation.


( D ) inhibition of - fusion syncytia formation by ACE2 - Fc in HEK293 / ACE2 H1975 / ACE2 was determined with formula described in Methods section . Error bars represent standard deviation ( SD ) , n = 6 . Statistical was performed by unpaired two tail t - . * * P < 0 . 01 , * * * P < 0 . 001 .

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(D) The inhibition of cell-cell fusion and syncytia formation by ACE2-Fc in HEK293/ACE2 and H1975/ACE2 was determined with the formula described in the Methods section. Error bars represent the standard deviation (SD), n=6. Statistical analysis was performed by unpaired two tail t-test. ** P < 0.01, *** P < 0.001.


( A ) ACE2 - Fc blocked entry of Spike - expressing pseudotyped lentivirus into HEK293T - ACE2 H1975 - ACE2 . relative luciferase activities , normalized to only group , represent efficiency of entry . MOI : Multiplicity of infection .

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(A) ACE2-Fc blocked the entry of Spike-expressing pseudotyped lentivirus into HEK293T-ACE2 and H1975-ACE2 cells. The relative luciferase activities, normalized to the only virus group, represent the efficiency of virus entry. MOI: Multiplicity of infection.


( B ) Confocal microscopy of airway organoids . green represents specific of indicated monoclonal antibodies Alexa Fluor 488 - conjugated secondary antibodies . DAPI was used as a counterstain . Hematoxylin eosin ( H & E ) stain : hematoxylin stained nuclei in blue color ; eosin stained cytoplasm in pink color . Scale bars equal to 20 µm .

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(B) Confocal microscopy images of the airway organoids. The green fluorescence represents the specific staining of indicated monoclonal antibodies and Alexa Fluor 488-conjugated secondary antibodies. DAPI was used as a nuclear counterstain. Hematoxylin and eosin (H&E) stain: hematoxylin stained the nuclei in blue color; eosin stained the cytoplasm in pink color. Scale bars equal to 20 µm.


( C ) Lung cancer A549 , human normal bronchial epithelial ( HBEpc ) , HBEpc - differentiated ( airway organoids ) were harvested for immunoblotting with indicated antibodies .

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(C) Lung cancer A549 cells, human normal bronchial epithelial cells (HBEpc), and HBEpc-differentiated cells (airway organoids) were harvested for immunoblotting with the indicated antibodies.


( D ) Blockage of pseudovirus entry into airway organoids by ACE2 - Fc . Mixtures of pseudotyped lentivirus with or without ACE2 - Fc were cocultured with airway organoids for 72 hr . entry was determined by quantifying luciferase activity in lysates .

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(D) Blockage of pseudovirus entry into airway organoids by ACE2-Fc. Mixtures of pseudotyped lentivirus with or without ACE2-Fc were cocultured with airway organoids for 72 hr. The virus entry was determined by quantifying the luciferase activity in the cell lysates.


( A ) Inhibition of SARS - CoV - 2 infection by ACE2 - Fc in plaque . Mixtures of ACE2 - Fc SARS - CoV - 2 were incubated for one hour before adding to Vero E6 for another 1 hr at 37 ° C . ACE2 - Fc SARS - CoV - 2 premixtures were removed , were washed once with PBS overlaid with methylcellulose with 2 % FBS for 5 - 7 days before being stained with crystal violet . Those results showed increase of plaque formation was regarded as no inhibition of plaque formation . TPCK - treated trypsin : N - tosyl - L - phenylalanine chloromethyl ketone - treated trypsin .

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(A) Inhibition of SARS-CoV-2 infection by ACE2-Fc in the plaque assay. Mixtures of ACE2-Fc and SARS-CoV-2 were incubated for one hour before adding to Vero E6 cells for another 1 hr at 37°C. The ACE2-Fc and SARS-CoV-2 premixtures were removed, and the cells were washed once with PBS and overlaid with methylcellulose with 2% FBS for 5-7 days before being stained with crystal violet. Those results showed increase of plaque formation was regarded as no inhibition of plaque formation. TPCK-treated trypsin: N-tosyl-L-phenylalanine chloromethyl ketone-treated trypsin.


Yield reduction was performed to determine inhibitory effects of ACE2 - Fc on titers culture medium extracts were harvested 24 hours post - infection for real - time PCR ( B )

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Yield reduction assay was performed to determine the inhibitory effects of ACE2-Fc on virus titers The culture medium and cell extracts were harvested 24 hours post-infection for real-time PCR (B)


Yield reduction was performed to determine inhibitory effects of ACE2 - Fc on expression . culture medium extracts were harvested 24 hours post - infection for western blot ( C ) . NP / PCNA represents relative NP expression as compared to that of PCNA , which served as a loading control . NP / PCNA numbers below panel are ratios of NP / PCNA normalized to that of human IgG control group . PCNA : Proliferating antigen .

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Yield reduction assay was performed to determine the inhibitory effects of ACE2-Fc on protein expression. The culture medium and cell extracts were harvested 24 hours post-infection for western blot (C). The NP/PCNA represents the relative NP expression as compared to that of PCNA, which served as a loading control. The NP/PCNA numbers below the panel are the ratios of NP/PCNA normalized to that of the human IgG control group. PCNA: Proliferating cell nuclear antigen.


( E ) lysates from pretreatment full - time experiments were harvested immunoblotted with indicated antibodies .

(E) The cell lysates from the pretreatment and full-time experiments were harvested and immunoblotted with the indicated antibodies.


( F ) SARS - CoV - 2 titer in pretreatment full - time experiments was analyzed by real - time PCR .

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(F) The SARS-CoV-2 titer in the pretreatment and full-time experiments was analyzed by real-time PCR.


( A ) ( A ) NP in lysates were determined by Western blot .

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(A) The (A) NP proteins in the cell lysates were determined by Western blot analysis.


( B , C ) in ( B ) culture medium ( C ) lysates was quantified by real - time RT - PCR . genetic information reference numbers for 5 SARS - CoV - 2 strains were summarized in Table EV1 . Error bars represent standard deviation ( SD ) , n = 3 . Statistical was performed by unpaired two tail t - . * * P < 0 . 01 , * * * P < 0 . 001 . Experiments were performed at least three times with similar results .

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(B, C) The virus RNA in the (B) culture medium and (C) cell lysates was quantified by real-time RT-PCR. The genetic information and sequence reference numbers for the 5 SARS-CoV-2 strains were summarized in Table EV1. Error bars represent the standard deviation (SD), n=3. Statistical analysis was performed by unpaired two tail t-test. ** P < 0.01, *** P < 0.001. Experiments were performed at least three times with similar results.


( A ) H1975 were transduced with full - Spike by Lentiviral vector . extracts were immunoblotted with indicated antibodies . O / E represents overexpress .

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(A) H1975 cells were transduced with full-length Spike by Lentiviral vector. Protein extracts were immunoblotted with the indicated antibodies. O/E represents overexpress.


( B ) effect of ACE2 - Fc activation on degranulative capacity of NK . experiments were performed with human NK derived from three independent donors . Error bars represent standard deviation ( SD ) , n = 3 . Statistical was performed by unpaired two tail t - . * P < 0 . 05 ( each of ACE2 - Fc vs control ) .

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(B) The effect of ACE2-Fc activation on degranulative capacity of NK cells. The experiments were performed with the primary human NK cells derived from three independent donors. Error bars represent the standard deviation (SD), n= 3. Statistical analysis was performed by unpaired two tail t-test. * P < 0.05 (each concentration of ACE2-Fc vs control).


( C ) CD107a , IFN - γ , TNF - α expression were determined by flow cytometry after NK H1975 - Spike coincubation in presence or absence of ACE2 - Fc or ACE2 control . Error bars represent standard deviation ( SD ) , n = 3 . Statistical was performed by unpaired two tail t - . * P < 0 . 05 . of positive in both groups were normalized to NK / H1975 - Spike only group .

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(C) The CD107a, IFN-γ, and TNF-α expression levels were determined by flow cytometry after the NK cells and H1975-Spike cells coincubation in the presence or absence of ACE2-Fc or ACE2 control. Error bars represent the standard deviation (SD), n=3. Statistical analysis was performed by unpaired two tail t-test. * P < 0.05. The percentage of positive cells in both groups were normalized to the NK/H1975-Spike only group.


( D ) differential expression of degranulation markers after treatment of ACE2 - Fc or ACE2 by flow cytometry . numbers in each plot indicate percentages of positive .

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(D) The differential expression of degranulation markers after the treatment of ACE2-Fc or ACE2 by the flow cytometry. The numbers in each plot indicate the percentages of positive cells.


Immunoblot of biotinylated isolated as in ( A ) . Parental HeLa - T - REx or expressing H3 . 1 - BirA * - HA treated with biotin for indicated times .

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Immunoblot analysis of proteins biotinylated and isolated as in (A). Parental HeLa-T-REx cells or cells expressing H3.1-BirA*-HA and treated with biotin for indicated times.


Immunoblot of TAP - H3 . 1 or - CENP - A pulldown of UBR7 - LAP ( schematic , upper ) . 293T were co - transfected with UBR7 - LAP TAP - tagged constructs , which were purified on IgG - conjugated sepharose beads .

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Immunoblot analysis of TAP-H3.1 or -CENP-A pulldown of UBR7-LAP (schematic, upper). 293T cells were co-transfected with UBR7-LAP and TAP-tagged constructs, which were purified on IgG-conjugated sepharose beads.


Immunoblot of anti - FLAG IP of 3x - FLAG tagged GFP , or FLAG - HA tagged H3 . 1 or H2B , from non - nucleosomal fraction of 293T .

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Immunoblot of anti-FLAG IP of 3x-FLAG tagged GFP, or FLAG-HA tagged H3.1 or H2B, from the non-nucleosomal fraction of 293T cells.


Representative of HeLa transfected with UBR7 - FL . is visualized via DAPI shown in magenta immunofluorescence for 3xFLAG is shown in green . within yellow dotted box in ( E ) is magnified 4x in ( E ' ) . Scale bar = 5 μm . of anti - FLAG immunofluorescence in ( E ) . Error bars represent mean + / - standard deviation . One way ANOVA was used to calculate p - values ( italics ) associated with differences between treatments . Experiment in ( E - F ) was performed twice .

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Representative images of HeLa cells transfected with UBR7-FL. DNA is visualized via DAPI staining shown in magenta and immunofluorescence for 3xFLAG is shown in green. The area within yellow dotted box in (E) is magnified 4x in (E'). Scale bar = 5 μm.   Quantification of the nuclear anti-FLAG immunofluorescence intensity in (E). Error bars represent mean +/- standard deviation. One way ANOVA was used to calculate p-values (italics) associated with differences between treatments. Experiment in (E-F) was performed twice.


Immunoblot of anti - FLAG pulldown of full UBR7 - FLAG ( residues 1 - 425 ) mutant UBR7 bearing deletion of UBR7 C - terminus ( ∆ C - term , residues 1 - 215 ) , UBR box ( ∆ UBR , residues 117 - 425 ) , UBR box PHD ( ∆ UBR ∆ PHD , residues 216 - 425 ) , or PHD C - terminus ( ∆ PHD ∆ C - term , residues 1 - 116 ) . 293T were co - transfected with H3 . 1 - HA UBR7 - FLAG constructs ( schematic , upper ) . Asterisk represents non - specific band . Numbers below blot indicate FLAG - normalized anti - HA immunoblot signal relative to full UBR7 - FLAG .

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Immunoblot analysis of anti-FLAG pulldown of full length UBR7-FLAG (residues 1-425) mutant UBR7 bearing deletion of UBR7 C-terminus (∆C-term, residues 1-215), UBR box (∆UBR, residues 117-425), UBR box and PHD (∆UBR∆PHD, residues 216-425), or PHD and C-terminus (∆PHD∆C-term, residues 1-116). 293T cells were co-transfected with H3.1-HA and UBR7-FLAG constructs (schematic, upper). Asterisk represents non-specific band. Numbers below blot indicate FLAG-normalized anti-HA immunoblot signal relative to full length UBR7-FLAG.


Immunoblot of anti - H3 . 1 - HA pulldown of full - UBR7 - LAP UBR7PHD fragment ( residues 117 - 215 ) ( schematic , upper ) . 293T were transfected as in ( A ) . Values below pulldown represent H3 . 1 - HA - normalized GFP - UBR7 immunoblot signal relative to UBR7 - FL . Data were acquired from a continuous , image is cropped to omit irrelevant samples . Numbers below blot indicate HA - normalized anti - GFP immunoblot signal relative to full UBR7 - LAP .

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Immunoblot analysis of anti-H3.1-HA pulldown of full-length UBR7-LAP and UBR7PHD fragment (residues 117-215) (schematic, upper). 293T cells were transfected as in (A). Values below pulldown represent H3.1-HA-normalized GFP-UBR7 immunoblot signal relative to UBR7-FL. Data were acquired from a continuous membrane, and the image is cropped to omit irrelevant samples. Numbers below blot indicate HA-normalized anti-GFP immunoblot signal relative to full length UBR7-LAP.


Anti - GFP immunoblot of streptavidin pulldown of indicated biotin - conjugated H3 peptides following incubation with 293T - GFP - UBR7 lysate after mutation of residues highlighted in ( C ) .

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Anti-GFP immunoblot analysis of streptavidin pulldown of the indicated biotin-conjugated H3 peptides following incubation with 293T-GFP-UBR7 cell lysate after mutation of residues highlighted in (C).


Immunoblot for total modified histones in anti - FLAG pulldown of constructs from soluble fractions demonstrates that UBR7 UBR box PHD are necessary for binding to post - nucleosomal H3 / H4 .

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Immunoblot analysis for total and modified histones in anti-FLAG pulldown of constructs from soluble cellular fractions demonstrates that UBR7 UBR box and PHD are necessary for binding to post-nucleosomal H3/H4.


Immunoblot of streptavidin pulldown from proximity biotinylation described in ( G ) . 4 . 5 h represent newly synthesized histones 48 h represent pre - existing histones . Wedge represents loading titration : 1x > 0 . 1x > 0 . 01x > 0 . 001x . other lanes are loaded at 1x . Immunoblot signals were normalized to anti - FLAG signal . of anti - FLAG ( H3 . 1 - APEX2 - 3xFLAG ) anti - UBR7 immunoblot represented in ( G - H ) . Lanes 8 11 were quantified . Error bar represents mean + / - standard deviation . A two - one - sample t - was used to calculate p - value ( italics ) associated with group mean difference from a hypothetical value of 1 . 0 ( represented by dashed ) .

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Immunoblot analysis of streptavidin pulldown from proximity biotinylation described in (G). 4.5 h represent newly synthesized histones and 48 h represent pre-existing histones. Wedge represents loading titration: 1x > 0.1x > 0.01x > 0.001x. All other lanes are loaded at 1x concentration. Immunoblot signals were normalized to anti-FLAG signal.   Quantification of anti-FLAG (H3.1-APEX2-3xFLAG) and anti-UBR7 immunoblot represented in (G-H). Lanes 8 and 11 were quantified. Error bar represents mean +/- standard deviation. A two-tailed one-sample t-test was used to calculate p-value (italics) associated with group mean difference from a hypothetical value of 1.0 (represented by dashed line).


Immunoblot for total modified H3 H4 in anti - FLAG IP . Experiment was performed as in ( D ) . Dot plot error bars represent mean + / - standard deviation . Immunoblot signals were normalized to anti - H4 signal . A two - one sample t - was used to calculate p - value associated with group mean difference from a hypothetical value of 1 . 0 . Experiment was performed twice .

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Immunoblot analysis for total and modified H3 and H4 in anti-FLAG IP. Experiment was performed as in (D). Dot plot error bars represent mean +/- standard deviation. Immunoblot signals were normalized to anti-H4 signal. A two-tailed one sample t-test was used to calculate p-value associated with group mean difference from a hypothetical value of 1.0. Experiment was performed twice.


Representative of HeLa transfected with sNASP - FL . is visualized via DAPI shown in magenta immunofluorescence for 3xFLAG is shown in green . Scale bar = 5 μm . of anti - FLAG immunofluorescence in ( G ) demonstrating that sNASP - FL is highly susceptible to pre - extraction with Triton X - 100 . One way ANOVA was used to determine p - values ( italics ) associated with differences between treatments . Error bars represent mean + / - standard deviation . Experiment in ( G - H ) was performed twice .

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Representative images of HeLa cells transfected with sNASP-FL. DNA is visualized via DAPI staining shown in magenta and immunofluorescence for 3xFLAG is shown in green. Scale bar = 5 μm.   Quantification of the nuclear anti-FLAG immunofluorescence intensity in (G) demonstrating that sNASP-FL is highly susceptible to pre-extraction with Triton X-100. One way ANOVA was used to determine p-values (italics) associated with differences between treatments. Error bars represent mean +/- standard deviation. Experiment in (G-H) was performed twice.


A , C . Immunoblot for modified histone in anti - FLAG pulldown of UBR7 - FL ( A ) or sNASP - FL ( C ) from non - nucleosomal fractions of untreated or treated with replication inhibitors ( hydroxyurea , aphidicolin ) or transcription inhibitors ( α - amanitin , triptolide ) . B , D . Plots represent either normalized immunoblot signals or prior to fractionation . Immunoblot signals were normalized to anti - FLAG signal . Error bars represent mean + / - standard deviation , with grey dotted marking mean of No Treat condition . A two - one sample t - was used to calculate p - values ( italics ) associated with group means difference from a hypothetical value of 1 . 0 . Experiments were performed three times . numbers were counted in two of three experiments .

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A,C. Immunoblot analysis for modified histone in anti-FLAG pulldown of UBR7-FL (A) or sNASP-FL (C) from non-nucleosomal fractions of untreated cells or cells treated with DNA replication inhibitors (hydroxyurea, aphidicolin) or transcription inhibitors (α-amanitin, triptolide). B,D. Plots represent either normalized immunoblot signals or cell counts prior to fractionation. Immunoblot signals were normalized to anti-FLAG signal. Error bars represent mean +/- standard deviation, with grey dotted line marking mean of No Treat condition. A two-tailed one sample t-test was used to calculate p-values (italics) associated with group means difference from a hypothetical value of 1.0. Experiments were performed three times. Cells numbers were counted in two of the three experiments.


Immunoblot of H3 , UBR7 , GAPDH in non - nucleosomal fraction of parental 293T - UBR7KO . of anti - H3 immunoblot represented in ( A ) . Immunoblot signals were normalized to anti - GAPDH signal . Error bars represent mean + / - standard deviation . A two - one - sample t - was used to calculate p - value ( italics ) associated with group mean difference from a hypothetical value of 1 . 0 ( represented by dashed ) . Experiment in ( A - B ) was performed in three times .

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Immunoblot analysis of H3, UBR7, and GAPDH in the non-nucleosomal fraction of parental and 293T-UBR7KO cells.   Quantification of anti-H3 immunoblot represented in (A). Immunoblot signals were normalized to anti-GAPDH signal. Error bars represent mean +/- standard deviation. A two-tailed one-sample t-test was used to calculate p-value (italics) associated with group mean difference from a hypothetical value of 1.0 (represented by dashed line). Experiment in (A-B) was performed in three times.


Immunoblot of anti - FLAG pulldown of sNASP - FL in parental or 293T - UBR7KO . 3xFLAG tagged sNASP - FL was immunoprecipitated from non - nucleosomal fraction eluted with FLAG peptide . Experiment was performed three times . of anti - FLAG ( sNASP - FL ) , anti - histone immunoblots represented in ( C ) . Immunoblot signals were normalized to anti - FLAG signal . Error bars represent mean + / - standard deviation . A two - one sample t - was used to calculate p - values ( italics ) associated with group means difference from a hypothetical value of 1 . 0 ( represented by dashed ) . Experiments were performed two ( sNASP , H4 ) to three ( other targets ) times .

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Immunoblot analysis of anti-FLAG pulldown of sNASP-FL in parental or 293T-UBR7KO cells. 3xFLAG tagged sNASP-FL was immunoprecipitated from non-nucleosomal fraction and eluted with FLAG peptide. Experiment was performed three times.   Quantification of anti-FLAG (sNASP-FL), and anti-histone immunoblots represented in (C). Immunoblot signals were normalized to anti-FLAG signal. Error bars represent mean +/- standard deviation. A two-tailed one sample t-test was used to calculate p-values (italics) associated with group means difference from a hypothetical value of 1.0 (represented by dashed line). Experiments were performed two (sNASP, H4) to three (all other targets) times.


Immunoblot for LAMP2A in parental 293T or expressing UBR7 - FL ( left ) or sNASP - FL ( right ) following treatment with siCTRL , siLAMP2A , or MG - 132 .

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Immunoblot for LAMP2A in parental 293T cells or cells expressing UBR7-FL (left) or sNASP-FL (right) following treatment with siCTRL, siLAMP2A, or MG-132.


of data represented in ( C , D ) . Error bars represent mean + / - standard deviation . A two - one sample t - was used to calculate p - values ( italics ) associated with group means difference from a hypothetical value of 1 . 0 ( represented by dashed ) . Experiments in ( C - E ) were performed three times .

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Quantification of data represented in (C, D). Error bars represent mean +/- standard deviation. A two-tailed one sample t-test was used to calculate p-values (italics) associated with group means difference from a hypothetical value of 1.0 (represented by dashed line). Experiments in (C-E) were performed three times.


Heatmaps of ATAC - seq , anti - GFP ( GFP - UBR7 ) ChIP - seq , anti - H3K4me3 CUT & RUN in parental UBR7KO . Plots are centered on TSS that show changes in ATAC - seq signal in UBR7KO versus parental . Sites are clustered by unsupervised k - means . Cluster color key is defined by cluster labels .

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Heatmaps of ATAC-seq, anti-GFP (GFP-UBR7) ChIP-seq, and anti-H3K4me3 CUT&RUN in parental and UBR7KO cells. Plots are centered on TSS that show changes in ATAC-seq signal in UBR7KO versus parental cells. Sites are clustered by unsupervised k-means analysis. Cluster color key is defined by cluster labels.


Low magnification views of a CD1 Otof - / - organ of Corti ( P23 ) transduced with otoferlin dual ‑ AAV ‑ TS vectors . IHCs : inner hair , OHCs : outer hair maximum projections of confocal optical

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Low magnification views of a CD1 Otof-/- organ of Corti (P23) transduced with otoferlin dual‑AAV‑TS vectors. IHCs: inner hair cells, OHCs: outer hair cells maximum intensity projections of confocal optical sections


High magnification views of CD1B6F1 Otof - / - IHCs transduced with otoferlin dual ‑ AAV ‑ TS ( P26 ) ( B ) dual ‑ AAV ‑ Hyb ( P26 ) ( C ) vectors . eGFP otoferlin immunostainings are depicted as color lookup tables in ( A - C ) with warmer colors representing higher pixel intensities ) , maximum projections of confocal optical

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High magnification views of CD1B6F1 Otof-/- IHCs transduced with otoferlin dual‑AAV‑TS (P26) (B) and dual‑AAV‑Hyb (P26) (C) vectors. Individual eGFP and otoferlin immunostainings are depicted as color lookup tables in (A-C) with warmer colors representing higher pixel intensities ), maximum intensity projections of confocal optical sections


of N ‑ C ‑ terminal otoferlin labelled IHCs in dual ‑ AAV ‑ TS ( n = 10 mice ) dual ‑ AAV ‑ Hyb ( n = 9 mice ) injected CD1B6F1 Otof - / - mice ( P18 ‑ 30 )

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Percentage of N‑ and C‑terminal otoferlin labelled IHCs in dual‑AAV‑TS (n=10 mice) and dual‑AAV‑Hyb (n=9 mice) injected CD1B6F1 Otof-/- mice (P18‑30)


Average N - terminal C ‑ terminal otoferlin immunofluorescence in dual ‑ AAV transduced Otof - / - wild - type IHCs ( P23 - 30 ) . Otoferlin were normalized to immunofluorescence in non - transduced B6 wild - type IHCs for each antibody

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Average N-terminal and C‑terminal otoferlin immunofluorescence levels in dual‑AAV transduced Otof-/- and wild-type IHCs (P23-30). Otoferlin levels were normalized to immunofluorescence levels in non-transduced B6 wild-type IHCs for each antibody separately


High magnification views of IHCs immunolabeled for otoferlin synaptic ribbons ( CtBP2 ) from wild ‑ type ( B6 : P27 , CD1B6F1 : P29 ) , dual - AAV injected CD1B6F1 Otof - / - ( dual - AAV - TS : P26 , dual - AAV - Hyb : P28 ) , their non - injected ears . ( * ) Transduced . Maximum projections of optical confocal sections . Scale bars : 5

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High magnification views of IHCs immunolabeled for otoferlin and synaptic ribbons (CtBP2) from wild‑type (B6: P27, CD1B6F1: P29), dual-AAV injected CD1B6F1 Otof-/- (dual-AAV-TS: P26, dual-AAV-Hyb: P28), and their contralateral non-injected ears. (*) Transduced cells. Maximum intensity projections of optical confocal sections. Scale bars: 5 µm


Synaptic ribbon numbers quantified from IHCs in apical cochlear turns of wild ‑ type ( B6 : n = 48 IHCs , CD1B6F1 : n = 108 IHCs ) , transduced Otof - / - ( dual ‑ AAV ‑ TS : n = 59 IHCs , dual - AAV - Hyb : n = 37 IHCs ) , non - transduced Otof - / - IHCs from injected ( - AAV injected ear , n = 65 IHCs ) non - injected ( - AAV non - injected ear , n = 46 IHCs ) ears ( P25 - 29 )

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Synaptic ribbon numbers quantified from IHCs in apical cochlear turns of wild‑type (B6: n=48 IHCs, CD1B6F1: n=108 IHCs), transduced Otof-/- (dual‑AAV‑TS: n=59 IHCs, dual-AAV-Hyb: n=37 IHCs), and non-transduced Otof-/- IHCs from injected (-AAV injected ear, n=65 IHCs) and contralateral non-injected (-AAV non-injected ear, n=46 IHCs) ears (P25-29)


synapses labelled with CtBP2 postsynaptic marker Shank1a in B6 wild - type Otof - / - P6 P14 organs of Corti . Maximum projections of optical confocal sections . Scale bars : 5 µm . D Synapse numbers quantified from IHCs in apical cochlear turns ( C ) of B6 wild - type ( P6 : n = 53 IHCs ; P14 : n = 73 IHCs ) B6 Otof - / - ( P6 : n = 62 IHCs ; P14 : n = 65 IHCs ) mice at two different developmental stages ( P6 P14 )

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IHC synapses labelled with CtBP2 and the postsynaptic marker Shank1a in B6 wild-type and Otof-/- P6 and P14 organs of Corti. Maximum intensity projections of optical confocal sections. Scale bars: 5 µm. D Synapse numbers quantified from IHCs in apical cochlear turns (C) of B6 wild-type (P6: n= 53 IHCs; P14: n=73 IHCs) and B6 Otof-/- (P6: n=62 IHCs; P14: n=65 IHCs) mice at two different developmental stages (P6 and P14)


Ca2 + ‑ current - voltage relationship of control CD1B6F1 wild ‑ type ( n = 6 IHCs ) , dual - AAV - TS transduced ( n = 8 IHCs ) , non ‑ transduced CD1B6F1 Otof - / - ( n = 10 IHCs ) IHCs ( P14 ‑ 18 )

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Ca2+‑current-voltage relationship of control CD1B6F1 wild‑type (n=6 IHCs), dual-AAV-TS transduced (n=8 IHCs), and non‑transduced CD1B6F1 Otof-/- (n=10 IHCs) IHCs (P14‑18)


Representative Ca2 + ‑ currents ( Ica ) plasma capacitance increments ( ΔCm ) of a wild ‑ type control , transduced , non ‑ transduced Otof - / - in response to a 20 ms depolarization at maximum Ca2 + ‑ current potentials ( typically - 14 mV )

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Representative Ca2+‑currents (Ica) and IHC plasma membrane capacitance increments (ΔCm) of a wild‑type control, transduced, and non‑transduced Otof-/- IHC in response to a 20 ms depolarization pulse at maximum Ca2+‑current potentials (typically -14 mV)


Average exocytosis level measured as ΔCm ( G ) corresponding Ca2 + ‑ current integrals ( QCa2 + ) ( H ) in wild ‑ type ( CD1B6F1 : n = 6 IHCs ; B6 : n = 11 IHCs ( B6 data replotted from Strenzke et al , 2016 ) ) , dual - AAV - TS transduced Otof - / - ( n = 8 IHCs ) , non ‑ transduced ( n = 11 IHCs ) Otof - / - IHCs . Transduced IHCs that showed eGFP expression , but had almost no exocytosis are depicted as dashed lines ( not included into average )

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

Average exocytosis level measured as ΔCm (G) and corresponding Ca2+‑current integrals (QCa2+) (H) in wild‑type (CD1B6F1: n=6 IHCs; B6: n=11 IHCs (B6 data replotted from Strenzke et al, 2016)), dual-AAV-TS transduced Otof-/- (n=8 IHCs), and non‑transduced (n=11 IHCs) Otof-/- IHCs. Transduced IHCs that showed eGFP expression, but had almost no exocytosis are depicted as dashed lines (not included into the average)


Representative ABR wave traces in response to broadband click sound stimuli from otoferlin dual ‑ AAV ‑ TS dual ‑ AAV ‑ Hyb injected CD1B6F1 Otof - / - . AAV2 / 6 . eGFP ( + AAV . eGFP ) dual ‑ AAV ‑ TS injected CD1B6F1 wild ‑ type , non ‑ injected control Otof - / - littermate ( - AAV ) mice served as controls . SP : summating potential ; ABR waves are indicated from I -

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 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 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

Representative ABR wave traces in response to broadband click sound stimuli from otoferlin dual‑AAV‑TS and dual‑AAV‑Hyb injected CD1B6F1 Otof-/- animals. AAV2/6.eGFP (+AAV.eGFP) and dual‑AAV‑TS injected CD1B6F1 wild‑type, and non‑injected control Otof-/- littermate (-AAV) mice served as controls. SP: summating potential; ABR waves are indicated from I-V


Average ABRs evoked by 80 dB SPL click sound stimuli for 20 clicks /

Average ABRs evoked by 80 dB SPL click sound stimuli for 20 clicks/s