( C ) U2OS ( EGFP - STGC - 1731 ) cell lines carrying Dox - inducible Cas9 / sgRNA - 1731 ( Dox - Cas9 ) or Cas9n / sgRNA ( Dox - Cas9n ) were incubated with or without Dox ( 5 µg / ml ) , and the percentage of EGFP positive cells was quantified by FACS analysis 2 days later ( top ) . U2OS ( EGFP - STGC - 1731 , Dox - Cas9 or Dox - Cas9n ) cells expressing shRNAs for RAD51 , POLD3 and PIF1 or shRNA control ( Ctrl ) were incubated with Dox ( 5 µg / ml ) , and the percentage of EGFP positive cells was quantified by FACS analysis after 2 days ( bottom ) . Data information : Error bars represent the standard deviation ( SD ) of at least three independent experiments . Signiﬁcance of the differences was assayed by Two - tailed non - paired parameters were applied in Student ' s t - test . The P value is indicated as * * P < 0 . 01 and n . s . ( not significant ) P > 0 . 05 .

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

(C) U2OS (EGFP-STGC-1731) cell lines carrying Dox-inducible Cas9/sgRNA-1731 (Dox-Cas9) or Cas9n/sgRNA (Dox-Cas9n) were incubated with or without Dox (5 µg/ml), and the percentage of EGFP positive cells was quantified by FACS analysis 2 days later (top). U2OS (EGFP-STGC-1731, Dox-Cas9 or Dox-Cas9n) cells expressing shRNAs for RAD51, POLD3 and PIF1 or shRNA control (Ctrl) were incubated with Dox (5 µg/ml), and the percentage of EGFP positive cells was quantified by FACS analysis after 2 days (bottom). Data information: Error bars represent the standard deviation (SD) of at least three independent experiments. Signiﬁcance of the differences was assayed by Two-tailed non-paired parameters were applied in Student's t-test. The P value is indicated as **P < 0.01 and n.s. (not significant) P>0.05.


( E ) U2OS ( EGFP - Flex1 - STGC - 1541 ) cells were treated with or without 2 mM HU for 24 hr , and the percentage of EGFP positive cells by HU induction was quantified by FACS analysis 4 days after removal of HU ( left ) . U2OS ( EGFP - Flex1 - STGC - 1541 ) cells expressing shRNAs for RAD51 , POLD3 and PIF1 or shRNA vector ( Ctrl ) were treated with 2 mM HU for 24 hr , and the percentage of EGFP positive cells was quantified by FACS analysis 4 days later ( right ) . Data information : Error bars represent the standard deviation ( SD ) of at least three independent experiments . Signiﬁcance of the differences was assayed by Two - tailed non - paired parameters were applied in Student ' s t - test . The P value is indicated as * * P < 0 . 01 and n . s . ( not significant ) P > 0 . 05 .

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 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 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

(E) U2OS (EGFP-Flex1-STGC-1541) cells were treated with or without 2 mM HU for 24 hr, and the percentage of EGFP positive cells by HU induction was quantified by FACS analysis 4 days after removal of HU (left). U2OS (EGFP-Flex1-STGC-1541) cells expressing shRNAs for RAD51, POLD3 and PIF1 or shRNA vector (Ctrl) were treated with 2 mM HU for 24 hr, and the percentage of EGFP positive cells was quantified by FACS analysis 4 days later (right). Data information: Error bars represent the standard deviation (SD) of at least three independent experiments. Signiﬁcance of the differences was assayed by Two-tailed non-paired parameters were applied in Student's t-test. The P value is indicated as **P < 0.01 and n.s. (not significant) P>0.05.


( F ) U2OS ( EGFP - Flex1 - STGC - 1541 ) cells were infected by retroviruses expressing RAS or empty vector , and the percentage of EGFP positive cells was assayed by FACS 5 days following infection ( left ) . U2OS ( EGFP - Flex1 - STGC - 1541 ) cells expressing shRNAs for RAD51 , POLD3 and PIF1 shRNAs or shRNA vector were infected by retroviruses expressing RAS , and the percentage of EGFP positive cells was assayed by FACS 5 days after infection ( middle ) . Expression level of RAD51 and POLD3 is shown by Western blot analysis ( right ) . Data information : Error bars represent the standard deviation ( SD ) of at least three independent experiments . Signiﬁcance of the differences was assayed by Two - tailed non - paired parameters were applied in Student ' s t - test . The P value is indicated as * * P < 0 . 01 and n . s . ( not significant ) P > 0 . 05 .

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

(F) U2OS (EGFP-Flex1-STGC-1541) cells were infected by retroviruses expressing RAS or empty vector, and the percentage of EGFP positive cells was assayed by FACS 5 days following infection (left). U2OS (EGFP-Flex1-STGC-1541) cells expressing shRNAs for RAD51, POLD3 and PIF1 shRNAs or shRNA vector were infected by retroviruses expressing RAS, and the percentage of EGFP positive cells was assayed by FACS 5 days after infection (middle). Expression level of RAD51 and POLD3 is shown by Western blot analysis (right). Data information: Error bars represent the standard deviation (SD) of at least three independent experiments. Signiﬁcance of the differences was assayed by Two-tailed non-paired parameters were applied in Student's t-test. The P value is indicated as **P < 0.01 and n.s. (not significant) P>0.05.


( D ) U2OS ( EGFP / STGC - mCherry / LTGC - 5034 , Dox - Cas9 ( left ) or Dox - Cas9n ( right ) ) cells expressing shRNAs for POLD3 and PIF1 or shRNA vector ( Ctrl ) were incubated with Dox ( 5 µg / ml ) . The percentage of EGFP or mCherry positive cells after induction was quantified by FACS analysis 2 days later to determine the percentage of EGFP or mCherry positive cells . Data information : Error bars represent the standard deviation ( SD ) of at least three independent experiments . Signiﬁcance of the differences was assayed by Two - tailed non - paired parameters were applied in Student ' s t - test . The P value is indicated as * * P < 0 . 01 and n . s . ( not significant ) P > 0 . 05 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) U2OS (EGFP/STGC-mCherry/LTGC-5034, Dox-Cas9 (left) or Dox-Cas9n (right)) cells expressing shRNAs for POLD3 and PIF1 or shRNA vector (Ctrl) were incubated with Dox (5 µg/ml). The percentage of EGFP or mCherry positive cells after induction was quantified by FACS analysis 2 days later to determine the percentage of EGFP or mCherry positive cells. Data information: Error bars represent the standard deviation (SD) of at least three independent experiments. Signiﬁcance of the differences was assayed by Two-tailed non-paired parameters were applied in Student's t-test. The P value is indicated as **P < 0.01 and n.s. (not significant) P>0.05.


( E ) U2OS ( EGFP - Flex1 - STGC - 1541 ) cells expressing shRNAs for RFC1 and PCNA or vector ( Ctrl ) were treated with 2 mM HU for 24 hr . The percentage of EGFP positive cells after HU treatment was quantified by FACS analysis 3 days after HU removal ( left ) . U2OS ( EGFP - STGC - 1731 ) cells expressing shRNAs for RFC1 and PCNA or vector ( Ctrl ) were infected by lentivirus expressing I - SceI . The percentage of EGFP positive cells by I - SceI induction was quantified by FACS analysis 4 days later ( right ) . Data information : Error bars represent the standard deviation ( SD ) of at least three independent experiments . Signiﬁcance of the differences was assayed by Two - tailed non - paired parameters were applied in Student ' s t - test . The P value is indicated as * * P < 0 . 01 and n . s . ( not significant ) P > 0 . 05 .

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

(E) U2OS (EGFP-Flex1-STGC-1541) cells expressing shRNAs for RFC1 and PCNA or vector (Ctrl) were treated with 2 mM HU for 24 hr. The percentage of EGFP positive cells after HU treatment was quantified by FACS analysis 3 days after HU removal (left). U2OS (EGFP-STGC-1731) cells expressing shRNAs for RFC1 and PCNA or vector (Ctrl) were infected by lentivirus expressing I-SceI. The percentage of EGFP positive cells by I-SceI induction was quantified by FACS analysis 4 days later (right). Data information: Error bars represent the standard deviation (SD) of at least three independent experiments. Signiﬁcance of the differences was assayed by Two-tailed non-paired parameters were applied in Student's t-test. The P value is indicated as **P < 0.01 and n.s. (not significant) P>0.05.


( F ) U2OS ( EGFP / STGC - mCherry / LTGC - 5034 , Dox - Cas9 ( left ) or Dox - Cas9n ( right ) ) cells expressing shRNAs for RFC1 and PCNA or vector ( Ctrl ) were incubated with Dox ( 5 µg / ml ) . The percentage of EGFP or mCherry positive cells after induction was quantified by FACS analysis 2 days later . Data information : Error bars represent the standard deviation ( SD ) of at least three independent experiments . Signiﬁcance of the differences was assayed by Two - tailed non - paired parameters were applied in Student ' s t - test . The P value is indicated as * * P < 0 . 01 and n . s . ( not significant ) P > 0 . 05 .

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

(F) U2OS (EGFP/STGC-mCherry/LTGC-5034, Dox-Cas9 (left) or Dox-Cas9n (right)) cells expressing shRNAs for RFC1 and PCNA or vector (Ctrl) were incubated with Dox (5 µg/ml). The percentage of EGFP or mCherry positive cells after induction was quantified by FACS analysis 2 days later. Data information: Error bars represent the standard deviation (SD) of at least three independent experiments. Signiﬁcance of the differences was assayed by Two-tailed non-paired parameters were applied in Student's t-test. The P value is indicated as **P < 0.01 and n.s. (not significant) P>0.05.


( G ) FLAG - PIF1 was stably expressed in U2OS ( EGFP - Flex1 - STGC - 1541 ) cells by lentiviral infection . Enrichment of FLAG - PIF1 at Flex1 site or GAPDH site with or without HU ( 2 mM , 10 hr ) treatment was calculated by anti - FLAG ChIP ( left ) . When PCNA was depleted by shRNA using vector as a control ( Ctrl ) , enrichment of FLAG - PIF1 at Flex1 site was calculated by anti - FLAG ChIP ( right ) . ChIP value in cells without HU treatment is set up as 1 for normalization . Data information : Error bars represent the standard deviation ( SD ) of at least three independent experiments . Signiﬁcance of the differences was assayed by Two - tailed non - paired parameters were applied in Student ' s t - test . The P value is indicated as * * P < 0 . 01 and n . s . ( not significant ) P > 0 . 05 .

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

(G) FLAG-PIF1 was stably expressed in U2OS (EGFP-Flex1-STGC-1541) cells by lentiviral infection. Enrichment of FLAG-PIF1 at Flex1 site or GAPDH site with or without HU (2 mM, 10 hr) treatment was calculated by anti-FLAG ChIP (left). When PCNA was depleted by shRNA using vector as a control (Ctrl), enrichment of FLAG-PIF1 at Flex1 site was calculated by anti-FLAG ChIP (right). ChIP value in cells without HU treatment is set up as 1 for normalization. Data information: Error bars represent the standard deviation (SD) of at least three independent experiments. Signiﬁcance of the differences was assayed by Two-tailed non-paired parameters were applied in Student's t-test. The P value is indicated as **P < 0.01 and n.s. (not significant) P>0.05.


( D ) Growth curves of WT or FANCM - KO HCT116 cells were plotted after expressing POLD3 ( left ) or PIF1 ( right ) shRNA or shRNA vector ( Ctrl ) . Cell number was normalized to that on day one . Data information : Error bars represent the standard deviation ( SD ) of at least three independent experiments . Signiﬁcance of the differences was assayed by Two - tailed non - paired parameters were applied in Student ' s t - test . The P value is indicated as * * P < 0 . 01 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Growth curves of WT or FANCM-KO HCT116 cells were plotted after expressing POLD3 (left) or PIF1 (right) shRNA or shRNA vector (Ctrl). Cell number was normalized to that on day one. Data information: Error bars represent the standard deviation (SD) of at least three independent experiments. Signiﬁcance of the differences was assayed by Two-tailed non-paired parameters were applied in Student's t-test. The P value is indicated as **P < 0.01.


( C ) Anti - γH2AX ChIP analysis at FRA3B , FRA16D and GAPDH genomic loci was performed in U2OS WT and PIF1 - KO cells before and after APH treatment ( 0 . 4 μM , 24 hr ) . Enrichment of γH2AX at FRA3B , FRA16D and GAPDH loci in WT and PIF1 KO cells was calculated using ChIP value in WT cells as 1 for normalization . Data information : Error bars represent the standard deviation ( SD ) of at least three independent experiments . Signiﬁcance of the differences was assayed by Two - tailed non - paired parameters were applied in Student ' s t - test . The P value is indicated as * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and n . s . ( not significant ) P > 0 . 05 .

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

(C) Anti-γH2AX ChIP analysis at FRA3B, FRA16D and GAPDH genomic loci was performed in U2OS WT and PIF1-KO cells before and after APH treatment (0.4 μM, 24 hr). Enrichment of γH2AX at FRA3B, FRA16D and GAPDH loci in WT and PIF1 KO cells was calculated using ChIP value in WT cells as 1 for normalization. Data information: Error bars represent the standard deviation (SD) of at least three independent experiments. Signiﬁcance of the differences was assayed by Two-tailed non-paired parameters were applied in Student's t-test. The P value is indicated as *P < 0.05, **P < 0.01, ***P < 0.001 and n.s. (not significant) P>0.05.


( E ) U2OS ( EGFP - BIR - 5085 ) cells expressing PIF1 WT or L319P mutant with endogenous PIF1 silenced by shRNA were infected by lentiviruses to express I - SceI . The percentage of EGFP positive cells was quantified by FACS analysis 5 days after I - SceI infection . Data information : Error bars represent the standard deviation ( SD ) of at least three independent experiments . Signiﬁcance of the differences was assayed by Two - tailed non - paired parameters were applied in Student ' s t - test . The P value is indicated as * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and n . s . ( not significant ) P > 0 . 05 .

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

(E) U2OS (EGFP-BIR-5085) cells expressing PIF1 WT or L319P mutant with endogenous PIF1 silenced by shRNA were infected by lentiviruses to express I-SceI. The percentage of EGFP positive cells was quantified by FACS analysis 5 days after I-SceI infection. Data information: Error bars represent the standard deviation (SD) of at least three independent experiments. Signiﬁcance of the differences was assayed by Two-tailed non-paired parameters were applied in Student's t-test. The P value is indicated as *P < 0.05, **P < 0.01, ***P < 0.001 and n.s. (not significant) P>0.05.


( I ) Anti - γH2AX ChIP analysis at FRA3B , FRA16D and GAPDH genomic loci was performed in U2OS WT and PIF1 - KO cells , or PIF1 - KO cells expressing PIF1 - WT , and E307Q and L319P mutants before and after APH treatment ( 0 . 4 μM , 24 hr ) . Enrichment of γH2AX at FRA3B , FRA16D and GAPDH loci was calculated using ChIP value in WT cells as 1 for normalization . Data information : Error bars represent the standard deviation ( SD ) of at least three independent experiments . Signiﬁcance of the differences was assayed by Two - tailed non - paired parameters were applied in Student ' s t - test . The P value is indicated as * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and n . s . ( not significant ) P > 0 . 05 .

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

(I) Anti-γH2AX ChIP analysis at FRA3B, FRA16D and GAPDH genomic loci was performed in U2OS WT and PIF1-KO cells, or PIF1-KO cells expressing PIF1-WT, and E307Q and L319P mutants before and after APH treatment (0.4 μM, 24 hr). Enrichment of γH2AX at FRA3B, FRA16D and GAPDH loci was calculated using ChIP value in WT cells as 1 for normalization. Data information: Error bars represent the standard deviation (SD) of at least three independent experiments. Signiﬁcance of the differences was assayed by Two-tailed non-paired parameters were applied in Student's t-test. The P value is indicated as *P < 0.05, **P < 0.01, ***P < 0.001 and n.s. (not significant) P>0.05.


Vector mediated eGFP expression after intravitreal injection of novel capsid variants compared to controls in 2 - month - old mice . ( A ) In vivo cSLO examinations of the mouse retina at 1 - and 2 - weeks post injection ( WPI ) of 2E9 total vg ( in 1 µl ) of sc - CMV - eGFP packaged with parental serotype AAV2 , state - of - art AAV2 . 7m8 , novel AAV2 . GL and AAV2 . NN capsid variants .

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

Vector mediated eGFP expression after intravitreal injection of novel capsid variants compared to controls in 2-month-old mice. (A) In vivo cSLO examinations of the mouse retina at 1- and 2-weeks post injection (WPI) of 2E9 total vg (in 1 µl) of sc-CMV-eGFP packaged with parental serotype AAV2 , state-of-art AAV2.7m8, novel AAV2.GL and AAV2.NN capsid variants.


( B ) Confocal scans of retinal cross sections immunolabelled for eGFP and DAPI . Acquisition settings were kept constant for all samples . Scale bar : 100 μm . ONL , outer nuclear layer ; INL , inner nuclear layer .

O O O O O O O O O O O O O O O O O O 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) Confocal scans of retinal cross sections immunolabelled for eGFP and DAPI. Acquisition settings were kept constant for all samples. Scale bar: 100 μm. ONL, outer nuclear layer; INL, inner nuclear layer.


( C ) Confocal tile - scans of retinal cross sections immunolabelled for eGFP and Hoechst . Scale bar : 500

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

(C) Confocal tile-scans of retinal cross sections immunolabelled for eGFP and Hoechst. Scale bar: 500 μm


( D - E ) Immunolabelled cross sections of mouse retina infected with AAV2 . GL and AAV2 . NN respectively , with focus on the ONL and INL , stained for eGFP , recoverin and DAPI . Scale bar : 50 μm ONL , outer nuclear layer ; INL , inner nuclear layer .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-E) Immunolabelled cross sections of mouse retina infected with AAV2.GL and AAV2.NN respectively, with focus on the ONL and INL, stained for eGFP, recoverin and DAPI. Scale bar: 50 μm ONL, outer nuclear layer; INL, inner nuclear layer.


( F ) qRT - PCR data comparing fold change in eGFP transcript levels from retinal samples infected with capsid variants AAV2 . 7m8 , AAV2 . GL and AAV2 . NN compared to AAV2 .

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

(F) qRT-PCR data comparing fold change in eGFP transcript levels from retinal samples infected with capsid variants AAV2.7m8, AAV2.GL and AAV2.NN compared to AAV2.


Vector mediated eGFP expression after intravitreal injection of novel capsid variants compared to controls in 2 - month - old mice . ( A ) In vivo cSLO examinations of the mouse retina at 1 - , 2 - and 3 - weeks post injection ( WPI ) of 8E9 total vg of ss - hRho - eGFP packaged with parental serotype AAV2 , state - of - art AAV2 . 7m8 , novel AAV2 . GL and AAV2 . NN capsid variants .

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

Vector mediated eGFP expression after intravitreal injection of novel capsid variants compared to controls in 2-month-old mice. (A) In vivo cSLO examinations of the mouse retina at 1-, 2- and 3- weeks post injection (WPI) of 8E9 total vg of ss-hRho-eGFP packaged with parental serotype AAV2, state-of-art AAV2.7m8, novel AAV2.GL and AAV2.NN capsid variants.


( B ) qRT - PCR data comparing fold change in eGFP transcript levels relative to ITR2 amplicons from retinal samples infected with all four capsid variants .

O O O O 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) qRT-PCR data comparing fold change in eGFP transcript levels relative to ITR2 amplicons from retinal samples infected with all four capsid variants.


( C ) Confocal scans of retinal cross sections , with focus on the ONL , immunolabelled for DAPI , eGFP and recoverin . The upper panel shows only the DAPI and eGFP signal in the ONL and the lower panel shows the same images ( noted with asterisk * ) with merged DAPI , eGFP and recoverin signal . Acquisition settings were kept constant for all samples . Scale bar : 50 μm . ONL , outer nuclear layer .

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

(C) Confocal scans of retinal cross sections, with focus on the ONL, immunolabelled for DAPI, eGFP and recoverin. The upper panel shows only the DAPI and eGFP signal in the ONL and the lower panel shows the same images (noted with asterisk *) with merged DAPI, eGFP and recoverin signal. Acquisition settings were kept constant for all samples. Scale bar: 50 μm. ONL, outer nuclear layer.


Vector mediated eGFP expression after intravitreal injection in 10 - month - old dog eyes . ( A ) In vivo cSLO examinations of the dog retina at four , five and six weeks post intravitreal injection of 2E11 total vg ( in 200 µl ) of sc - CMV - eGFP packaged with wildtype AAV2 ( right eye ) , AAV2 . GL ( right eye ) and AAV2 . NN ( left eye ) capsid variants .

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

Vector mediated eGFP expression after intravitreal injection in 10-month-old dog eyes. (A) In vivo cSLO examinations of the dog retina at four, five and six weeks post intravitreal injection of 2E11 total vg (in 200 µl) of sc-CMV-eGFP packaged with wildtype AAV2 (right eye), AAV2.GL (right eye) and AAV2.NN (left eye) capsid variants.


( B - C ) Confocal scans of immunolabelled retinal cross sections ( 14 μm ) stained for GFP and DAPI at 6 WPI time point . : DAPI was used a nuclear marker . GFP = eGFP ; RPE , retinal pigmented epithelium ; OS , outer segments ; ONL , outer nuclear layer ; INL , inner nuclear layer ; GCL , ganglion cell layer .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-C) Confocal scans of immunolabelled retinal cross sections (14 μm) stained for GFP and DAPI at 6 WPI time point. : DAPI was used a nuclear marker. GFP=eGFP; RPE, retinal pigmented epithelium; OS, outer segments; ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer.


( D - E ) Confocal scans of immunolabelled retinal cross sections ( 14 μm ) at 6 WPI time point , focusing on the photoreceptors , using antibodies against eGFP , Rhodopsin ( Rho ) and M - opsin ( MO ) . OS , outer segments ONL , outer nuclear

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(D-E) Confocal scans of immunolabelled retinal cross sections (14 μm) at 6 WPI time point, focusing on the photoreceptors, using antibodies against eGFP, Rhodopsin (Rho) and M-opsin (MO). OS, outer segments ONL, outer nuclear layer


Vector mediated expression after intravitreal injection in ~ 3 - year old cynomolgus macaque eyes . ( A - B ) In vivo cSLO examination images collaged to provide NHP fundus overview at 8 WPI of E12 total vg ( in 38 . 9 - 100 µl ) of sc - CMV - eGFP packaged with AAV2 . GL and AAV2 . NN capsid variants , respectively .

Vector mediated expression after intravitreal injection in ~3-year old cynomolgus macaque eyes. (A-B) In vivo cSLO examination images collaged to provide NHP fundus overview at 8 WPI of E12 total vg (in 38.9-100 µl) of sc-CMV-eGFP packaged with AAV2.GL and AAV2.NN capsid variants, respectively.


( C - D ) Confocal scans of immunolabelled retinal cross sections ( 14 μm ) of the fovea centralis from AAV2 . GL and AAV2 . NN treated animals , respectively .

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(C-D) Confocal scans of immunolabelled retinal cross sections (14 μm) of the fovea centralis from AAV2.GL and AAV2.NN treated animals, respectively.


( E ) Percentage quantification of eGFP + photoreceptors in the ONL of foveal cross sections . AAV2 . GL technical repeats n = 4 , AAV2 . NN technical repeats n = 3 .

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(E) Percentage quantification of eGFP+ photoreceptors in the ONL of foveal cross sections. AAV2.GL technical repeats n=4, AAV2.NN technical repeats n=3.


( F - G ) Confocal scans of immunolabelled retinal cross sections ( 14 μm ) of the periphery from AAV2 . GL ( F ) and AAV2 . NN ( G ) treated retinae . ( Η - Ι ) Higher magnification confocal scans of immunolabelled peripheral retina . ONL , outer nuclear layer ; INL , inner nuclear

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(F-G) Confocal scans of immunolabelled retinal cross sections (14 μm) of the periphery from AAV2.GL (F) and AAV2.NN (G) treated retinae. (Η-Ι) Higher magnification confocal scans of immunolabelled peripheral retina. ONL, outer nuclear layer; INL, inner nuclear layer


Vector mediated eGFP expression in human retinal explants . ( B ) EVOS epifluorescence images at 2x magnification of the human retinal explants at three , six and nine days in vitro ( DIV ) after transduction with 1E11 total vg of sc - CMV - eGFP packaged with AAV2 . GL and AAV2 . NN capsid variants . White signal indicates eGFP .

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Vector mediated eGFP expression in human retinal explants. (B) EVOS epifluorescence images at 2x magnification of the human retinal explants at three, six and nine days in vitro (DIV) after transduction with 1E11 total vg of sc-CMV-eGFP packaged with AAV2.GL and AAV2.NN capsid variants. White signal indicates eGFP.


( C ) Confocal scans of day 10 tissue sections immunolabelled for eGFP and Hoechst . Scale bar : 50 μm .

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(C) Confocal scans of day 10 tissue sections immunolabelled for eGFP and Hoechst. Scale bar: 50 μm.


( A ) Representative photopic ERG flicker series at 1 , 5 and 10 Hz with 3 cd . s / m2 illumination at 10 WPI . Arrowheads indicate light stimulus .

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(A) Representative photopic ERG flicker series at 1, 5 and 10 Hz with 3 cd.s/m2 illumination at 10 WPI. Arrowheads indicate light stimulus.


( B ) Representative photopic single flash ERG signal with 1 , 3 and 10 cd . s / m2 illumination . The vertical dotted line indicates light stimulus . ( C ) Quantification of photopic B - wave amplitude from single flash responses to 1 , 3 and 10 cd . s / m2 illumination of wildtype C57BL6 / J x 129 / sv eyes ( WT , n = 3 ) compared to treated eyes ( IVT / GL , n ≥ 3 ) and untreated eyes ( UN , n ≥ 3 ) at 10 weeks post injection .

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(B) Representative photopic single flash ERG signal with 1, 3 and 10 cd.s/m2 illumination. The vertical dotted line indicates light stimulus. (C) Quantification of photopic B-wave amplitude from single flash responses to 1, 3 and 10 cd.s/m2 illumination of wildtype C57BL6/J x 129/sv eyes (WT, n = 3) compared to treated eyes (IVT/GL, n ≥ 3) and untreated eyes (UN, n ≥ 3) at 10 weeks post injection.


( E - F ) Confocal images of sections proximal to the injection site in Q2 ( E ) and distal to the injection site in Q4 ( F ) , showing Cnga3 expression in PNA - positive cone outer segments . OS , outer segment layer ; ONL , outer nuclear layer ; INL , inner nuclear layer .

(E-F) Confocal images of sections proximal to the injection site in Q2 (E) and distal to the injection site in Q4 (F), showing Cnga3 expression in PNA-positive cone outer segments. OS, outer segment layer; ONL, outer nuclear layer; INL, inner nuclear layer.


( G - I ) Confocal images of Cnga3 - / - treated ( G ) , wildtype ( H ) and Cnga3 - / - untreated ( I ) retinal sections stained for Cnga3 . Note some non - specific signal detected in INL and adjacent synaptic layer . Scale bar : 50 μm . OS , outer segment layer ; ONL , outer nuclear layer ; INL , inner nuclear layer .

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(G-I) Confocal images of Cnga3-/- treated (G), wildtype (H) and Cnga3-/- untreated (I) retinal sections stained for Cnga3. Note some non-specific signal detected in INL and adjacent synaptic layer. Scale bar: 50 μm. OS, outer segment layer; ONL, outer nuclear layer; INL, inner nuclear layer.


The refined models of four crystal structures of the ribosome bound to aminoacyl - or peptidyl - tRNA analogs are displayed in their respective unbiased electron density Fo - Fc maps ( contoured at 2 . 5σ ) . The maps were calculated using phases produced by rigid body refinement of the ligand - free test structure put into the Fo data set . A , B . A - site bound substrates ACC - Puromycin ( A ) and ACCA - Pro ( B ) .

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

The refined models of four crystal structures of the ribosome bound to aminoacyl- or peptidyl-tRNA analogs are displayed in their respective unbiased electron density Fo-Fc maps (contoured at 2.5σ). The maps were calculated using phases produced by rigid body refinement of the ligand-free test structure put into the Fo data set.A, B. A-site bound substrates ACC-Puromycin (A) and ACCA-Pro (B).


The refined models of four crystal structures of the ribosome bound to aminoacyl - or peptidyl - tRNA analogs are displayed in their respective unbiased electron density Fo - Fc maps ( contoured at 2 . 5σ ) . The maps were calculated using phases produced by rigid body refinement of the ligand - free test structure put into the Fo data set . C , D . P - site bound substrates ACCA - Leu - Phe ( C ) and ACCA - Pro - Pro ( D ) .

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

The refined models of four crystal structures of the ribosome bound to aminoacyl- or peptidyl-tRNA analogs are displayed in their respective unbiased electron density Fo-Fc maps (contoured at 2.5σ). The maps were calculated using phases produced by rigid body refinement of the ligand-free test structure put into the Fo data set.C, D. P-site bound substrates ACCA-Leu-Phe (C) and ACCA-Pro-Pro (D).


This figure compares conformation of aminoacyl residues of the two A - site substrates : A . ACC - PuromycinB . ACCA - Pro . To show position of the A - site substrates relative to the P - site , the model P - site substrate tRNA - Phe is shown as in the pre - attack complex of the ribosome ( pdb id 1vy4 ) . In both illustrations , the A site is viewed as through the peptide exit tunnel . In addition to S . cerevisiae residues numbering , E . coli numbering is shown in parenthesis . Comparison of the two structures shows that the side chain of the methyl - tyrosine is bound to the conserved hydrophobic A - site cleft , whereas the side chain of the proline residue penetrates the actual site of peptide bond formation where it may interfere with alignment of the reacting groups and proper positioning of the attacking electron pair of the α - amine .

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

This figure compares conformation of aminoacyl residues of the two A-site substrates:A. ACC-PuromycinB. ACCA-Pro.To show position of the A-site substrates relative to the P-site, the model P-site substrate tRNA-Phe is shown as in the pre-attack complex of the ribosome (pdb id 1vy4). In both illustrations, the A site is viewed as through the peptide exit tunnel. In addition to S. cerevisiae residues numbering, E. coli numbering is shown in parenthesis. Comparison of the two structures shows that the side chain of the methyl-tyrosine is bound to the conserved hydrophobic A-site cleft, whereas the side chain of the proline residue penetrates the actual site of peptide bond formation where it may interfere with alignment of the reacting groups and proper positioning of the attacking electron pair of the α-amine.


This figure compares conformation of two dipeptides in the ribosome nascent peptide tunnel : A . Phe - Leu peptideB . Pro - Pro peptide .

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This figure compares conformation of two dipeptides in the ribosome nascent peptide tunnel:A. Phe-Leu peptideB. Pro-Pro peptide.


Mapping of open chromatin as well as active and repressive histone modifications at upstream regulatory regions of the Prdm1 locus in pro - B cells and plasmablasts . Open chromatin was determined by ATAC - seq ( Buenrostro et al . , 2013 ) , and active ( H3K4me2 , H3K4me3 , H3K9ac ) and repressive ( H3K27me3 ) histone marks were mapped by ChIP - seq analysis in ex vivo sorted pro - B cells ( ATAC - seq and H3K27me3 , this study ) , short - term in vitro cultured pro - B cells ( H3K4me2 , H3K4me3 , H3K9ac ) ( Revilla - i - Domingo et al . , 2012 ) and in vitro LPS - induced plasmablasts ( Minnich et al . , 2016 ) ( Table EV4 ) . The indicated upstream regions were previously shown by 3C - analysis to interact with the Prdm1 promoter ( Wöhner et al . , 2016 ) . The mm9 genomic coordinates of mouse chromosome 10 and the respective positions of two human SNPs ( rs658431 and rs548234 ) are indicated . RPM , reads per million mapped sequence

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Mapping of open chromatin as well as active and repressive histone modifications at upstream regulatory regions of the Prdm1 locus in pro-B cells and plasmablasts. Open chromatin was determined by ATAC-seq (Buenrostro et al., 2013), and active (H3K4me2, H3K4me3, H3K9ac) and repressive (H3K27me3) histone marks were mapped by ChIP-seq analysis in ex vivo sorted pro-B cells (ATAC-seq and H3K27me3, this study), short-term in vitro cultured pro-B cells (H3K4me2, H3K4me3, H3K9ac) (Revilla-i-Domingo et al., 2012) and in vitro LPS-induced plasmablasts (Minnich et al., 2016) (Table EV4). The indicated upstream regions were previously shown by 3C-analysis to interact with the Prdm1 promoter (Wöhner et al., 2016). The mm9 genomic coordinates of mouse chromosome 10 and the respective positions of two human SNPs (rs658431 and rs548234) are indicated. RPM, reads per million mapped sequence reads


Mapping of nascent transcripts or mature mRNA at the Prdm1 and Atg5 loci by GRO - seq or RNA - seq analyses of ex vivo sorted pro - B and FO B cells , respectively ( Table EV4 ) . Strand - specific GRO - seq reads are indicated in blue and red , respectively Quantification of nascent transcript and mRNA levels . The nascent transcription or mRNA expression of the Atg5 and Prdm1 genes is shown as mean expression value ( TPM ) with SEM based on two different GRO - seq or RNA - seq experiments for each B cell type , respectively . TPM , transcripts per million ( Wagner et al . , 2012 )

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Mapping of nascent transcripts or mature mRNA at the Prdm1 and Atg5 loci by GRO-seq or RNA-seq analyses of ex vivo sorted pro-B and FO B cells, respectively (Table EV4). Strand-specific GRO-seq reads are indicated in blue and red, respectively Quantification of nascent transcript and mRNA levels. The nascent transcription or mRNA expression of the Atg5 and Prdm1 genes is shown as mean expression value (TPM) with SEM based on two different GRO-seq or RNA-seq experiments for each B cell type, respectively. TPM, transcripts per million (Wagner et al., 2012)


Expression of Prdm1 and Atg5 mRNA in ex vivo sorted pro - B and pre - B cells from the bone marrow of Prdm1ihCd2 / + or wild - type ( WT ) mice . mRNA expression is shown as mean expression value ( TPM ) with SEM based on two independent RNA - seq experiments for each cell type and

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Expression of Prdm1 and Atg5 mRNA in ex vivo sorted pro-B and pre-B cells from the bone marrow of Prdm1ihCd2/+ or wild-type (WT) mice. mRNA expression is shown as mean expression value (TPM) with SEM based on two independent RNA-seq experiments for each cell type and genotype


RT - qPCR analysis of nascent Prdm1 and Atg5 transcripts in ex vivo sorted pre - B cells and in in vitro differentiated plasmablasts ( PB ) of the indicated genotypes . Plasmablasts were generated by stimulation of splenic FO B cells for 4 days with LPS . The data were normalized to those of the ubiquitously expressed control gene Tbp and are presented relative to those of the wild - type pre - B cells ( set as 1 ) . Nascent transcripts were PCR - amplified with primers located in the indicated introns ( Table EV3 ) . Statistical data are shown as mean value with SEM and were analyzed by the Student " s t - test ; * P < 0 . 05 , * * P < 0 . 01 . Each dot corresponds to

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RT-qPCR analysis of nascent Prdm1 and Atg5 transcripts in ex vivo sorted pre-B cells and in in vitro differentiated plasmablasts (PB) of the indicated genotypes. Plasmablasts were generated by stimulation of splenic FO B cells for 4 days with LPS. The data were normalized to those of the ubiquitously expressed control gene Tbp and are presented relative to those of the wild-type pre-B cells (set as 1). Nascent transcripts were PCR-amplified with primers located in the indicated introns (Table EV3). Statistical data are shown as mean value with SEM and were analyzed by the Student"s t-test; *P < 0.05, **P < 0.01. Each dot corresponds to one


Flow cytometric analysis of hCD2 cell surface expression and intracellular Blimp1 expression of the indicated cell types . Bone marrow of Prdm1ihCd2 / + ( red ) or wild - type ( WT , gray ) mice was used to analyz ALPs , BLPs , pro - B , pre - B , immature B cells whereas the spleen was used for flow cytometric analysis o FO B , MZ B and plasma cell of both genotypes The histograms show hCD2 ( top row ) and Blimp1 ( bottom row ) expression for the different cell types Wild - type FO B cells ( dashed line ) were used as negative control for the Blimp1 staining in plasma cells . The difference in mean fluorescence intensity ( ΔMFI ) between the two genotypes is shown for each cell

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Flow cytometric analysis of hCD2 cell surface expression and intracellular Blimp1 expression of the indicated cell types. Bone marrow of Prdm1ihCd2/+ (red) or wild-type (WT, gray) mice was used to analyz ALPs, BLPs, pro-B, pre-B, immature B cells whereas the spleen was used for flow cytometric analysis o FO B, MZ B and plasma cell of both genotypes The histograms show hCD2 (top row) and Blimp1 (bottom row) expression for the different cell types Wild-type FO B cells (dashed line) were used as negative control for the Blimp1 staining in plasma cells. The difference in mean fluorescence intensity (ΔMFI) between the two genotypes is shown for each cell type


Flow cytometric analysis of hCD2 cell surface expression and intracellular Blimp1 expression of the indicated cell types . Bone marrow of Prdm1ihCd2 / + ( red ) or wild - type ( WT , gray ) mice was used to analyz NK cells and granulocytes , whereas the spleen was used for flow cytometric analysis o naïve CD4 T and naïve CD8 T cells of both genotypes The histograms show hCD2 ( top row ) and Blimp1 ( bottom row ) expression for the different cell types The difference in mean fluorescence intensity ( ΔMFI ) between the two genotypes is shown for each cell

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

Flow cytometric analysis of hCD2 cell surface expression and intracellular Blimp1 expression of the indicated cell types. Bone marrow of Prdm1ihCd2/+ (red) or wild-type (WT, gray) mice was used to analyz NK cells and granulocytes, whereas the spleen was used for flow cytometric analysis o naïve CD4 T and naïve CD8 T cells of both genotypes The histograms show hCD2 (top row) and Blimp1 (bottom row) expression for the different cell types The difference in mean fluorescence intensity (ΔMFI) between the two genotypes is shown for each cell type


Immunoblot analysis of Blimp1 and the TATA - binding protein ( TBP ) in nuclear extracts prepared from short - term cultured wild - type or Prdm1ihCd2 / + pro - B cells . Marker proteins of the indicated size ( in kilodaltons ) are shown to the

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Immunoblot analysis of Blimp1 and the TATA-binding protein (TBP) in nuclear extracts prepared from short-term cultured wild-type or Prdm1ihCd2/+ pro-B cells. Marker proteins of the indicated size (in kilodaltons) are shown to the right


Flow cytometric analysis of total B cells ( A ) as well as pro - B , pre - B , immature ( imm ) B and recirculating ( recirc ) B cells ( B ) from the bone marrow of wild - type ( WT , gray ) , Prdm1ihCd2 / + ( red ) and Prdm1ihCd2 / ihCd2 ( white ) mice at the age of 6 - 8 weeks Flow cytometric analysis of total B , FO B and MZ B cells from the spleens of wild - type , Prdm1ihCd2 / + and Prdm1ihCd2 / ihCd2

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Flow cytometric analysis of total B cells (A) as well as pro-B, pre-B, immature (imm) B and recirculating (recirc) B cells (B) from the bone marrow of wild-type (WT, gray), Prdm1ihCd2/+ (red) and Prdm1ihCd2/ihCd2 (white) mice at the age of 6-8 weeks Flow cytometric analysis of total B, FO B and MZ B cells from the spleens of wild-type, Prdm1ihCd2/+ and Prdm1ihCd2/ihCd2 mice


Determination of cell death of ex vivo pro - B and pre - B cells from the bone marrow of Prdm1ihCd2 / + ( red ) and wild - type ( gray ) mice by flow cytometric analysis of the loss of plasma membrane asymmetry ( F2N12S ratiometric dye staining ) and the loss of cell membrane integrity ( SYTOX staining ) . Representative flow cytometry plots ( left ) and the quantification of dead and apoptotic ( apop ) cells ( right ) are

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Determination of cell death of ex vivo pro-B and pre-B cells from the bone marrow of Prdm1ihCd2/+ (red) and wild-type (gray) mice by flow cytometric analysis of the loss of plasma membrane asymmetry (F2N12S ratiometric dye staining) and the loss of cell membrane integrity (SYTOX staining). Representative flow cytometry plots (left) and the quantification of dead and apoptotic (apop) cells (right) are shown


BrdU labeling of immature ( imm ) and FO B cells in the spleen of 3 - month - old Prdm1ihCd2 / + mice ( red dots ) and control wild - type littermates ( gray dots ) . The percentage of BrdU + B cells was determined for each B cell type by flow cytometric analysis after 10 days of continuous BrdU labeling ( day 10 , white bar ) or after a subsequent 15 - day chase period ( day 25 ; hatched bar ) without BrdU in the drinking

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BrdU labeling of immature (imm) and FO B cells in the spleen of 3-month-old Prdm1ihCd2/+ mice (red dots) and control wild-type littermates (gray dots). The percentage of BrdU+ B cells was determined for each B cell type by flow cytometric analysis after 10 days of continuous BrdU labeling (day 10, white bar) or after a subsequent 15-day chase period (day 25; hatched bar) without BrdU in the drinking wate


BrdU labeling of immature ( imm ) and FO B cells in the spleen of 3 - month - old Prdm1ihCd2 / + mice ( red dots ) and control wild - type littermates ( gray dots ) A diagram ( below ) indicates the design of the BrdU labeling and chase experiments . Flow cytometric data obtained with FO B cells from one mouse of each genotype are shown in ( F ) The percentage of BrdU + FO B cells is shown above the indicated gates ( F )

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

BrdU labeling of immature (imm) and FO B cells in the spleen of 3-month-old Prdm1ihCd2/+ mice (red dots) and control wild-type littermates (gray dots) A diagram (below) indicates the design of the BrdU labeling and chase experiments. Flow cytometric data obtained with FO B cells from one mouse of each genotype are shown in (F) The percentage of BrdU+ FO B cells is shown above the indicated gates (F)


Scatter plot of gene expression differences in ex vivo sorted Prdm1ihCd2 / + and wild - type ( WT ) pro - B ( A ) and pre - B ( B ) cells , based on two RNA - seq experiments for each cell type and genotype . The expression data of individual genes ( indicated by dots ) were plotted as normalized ( norm ) rlog values . Genes with an expression difference of > 3 - fold , an adjusted P value of < 0 . 05 and a TPM value of > 5 ( in one of the two pro - B or pre - B cell types , respectively ) are colored in blue or red , corresponding to activation or repression by Blimp1 . ( C ) Expression of commonly activated ( blue ) and commonly repressed ( red ) genes in pro - B and pre - B cells . The log2 - fold expression change observed between Prdm1ihCd2 / + and wild - type pro - B cells ( horizontal axis ) as well as between Prdm1ihCd2 / + and wild - type pre - B cells ( vertical axis ) is plotted for each gene . Lines indicated 2 - fold ( 2x ) and 3 - fold ( 3x ) expression

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Scatter plot of gene expression differences in ex vivo sorted Prdm1ihCd2/+ and wild-type (WT) pro-B (A) and pre-B (B) cells, based on two RNA-seq experiments for each cell type and genotype. The expression data of individual genes (indicated by dots) were plotted as normalized (norm) rlog values. Genes with an expression difference of > 3-fold, an adjusted P value of < 0.05 and a TPM value of > 5 (in one of the two pro-B or pre-B cell types, respectively) are colored in blue or red, corresponding to activation or repression by Blimp1. (C) Expression of commonly activated (blue) and commonly repressed (red) genes in pro-B and pre-B cells. The log2-fold expression change observed between Prdm1ihCd2/+ and wild-type pro-B cells (horizontal axis) as well as between Prdm1ihCd2/+ and wild-type pre-B cells (vertical axis) is plotted for each gene. Lines indicated 2-fold (2x) and 3-fold (3x) expression differences


Identification of 762 Blimp1 peaks in short - term cultured Prdm1ihCd2 / + pro - B cells , as detected by ChIP - seq with an anti - V5 antibody and MACS peak calling with a P value of < 10 - 10 . The 9320 Blimp1 peaks identified in plasmablasts ( PB ; Minnich et al . , 2016 ) are shown for comparison . Common ( black ) and unique ( white ) peaks are shown for both cell types Densities of Blimp1 binding . Average read density profiles aligned at the center of the Blimp1 peak are shown for the common Blimp peaks of both cell

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

Identification of 762 Blimp1 peaks in short-term cultured Prdm1ihCd2/+ pro-B cells, as detected by ChIP-seq with an anti-V5 antibody and MACS peak calling with a P value of < 10-10. The 9320 Blimp1 peaks identified in plasmablasts (PB; Minnich et al., 2016) are shown for comparison. Common (black) and unique (white) peaks are shown for both cell types Densities of Blimp1 binding. Average read density profiles aligned at the center of the Blimp1 peak are shown for the common Blimp peaks of both cell types


Blimp1 binding at activated and repressed genes in Prdm1ihCd2 / + pro - B and pre - B cells . Regulated genes , which are bound by Blimp1 in Prdm1ihCd2 / + pro - B cells , are shown in

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Blimp1 binding at activated and repressed genes in Prdm1ihCd2/+ pro-B and pre-B cells. Regulated genes, which are bound by Blimp1 in Prdm1ihCd2/+ pro-B cells, are shown in black


Blimp1 binding and regulation of the commonly repressed target genes Sell ( CD62L ) and B3gnt5 . The ChIP - seq data ( left ) were obtained with Prdm1ihCd2 / + pro - B cells ( this study ) and Prdm1Bio / Bio Rosa26BirA / BirA plasmablasts ( PB ; Minnich et al . , 2016 ) . The RNA - seq data ( right ) were determined in pro - B and pre - B cells of the Prdm1ihCd2 / + ( red ) and wild - type ( gray ) genotype and in Blimp1 - deficient ( Prdm1Gfp / ∆ , blue ) and wild - type ( grey ) pre - plasmablasts ( Pre - PB ; Minnich et al . , 2016 ) . Cell types expressing Blimp1 ( + ) are

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Blimp1 binding and regulation of the commonly repressed target genes Sell (CD62L) and B3gnt5. The ChIP-seq data (left) were obtained with Prdm1ihCd2/+ pro-B cells (this study) and Prdm1Bio/Bio Rosa26BirA/BirA plasmablasts (PB; Minnich et al., 2016). The RNA-seq data (right) were determined in pro-B and pre-B cells of the Prdm1ihCd2/+ (red) and wild-type (gray) genotype and in Blimp1-deficient (Prdm1Gfp/∆, blue) and wild-type (grey) pre-plasmablasts (Pre-PB; Minnich et al., 2016). Cell types expressing Blimp1 (+) are indicated


Expression of selected Blimp1 - activated and Blimp1 - repressed genes coding for transcriptional regulators ( H ) and intracellular signal transducer in Prdm1ihCd2 / + ( red ) and wild - type ( gray ) pre - B cells . Blimp1 - bound genes are underlined . The mRNA expression of the indicated genes is shown as mean expression value ( TPM ) with SEM , based on two different RNA - seq experiments for pre - B cells of each

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Expression of selected Blimp1-activated and Blimp1-repressed genes coding for transcriptional regulators (H) and intracellular signal transducer in Prdm1ihCd2/+ (red) and wild-type (gray) pre-B cells. Blimp1-bound genes are underlined. The mRNA expression of the indicated genes is shown as mean expression value (TPM) with SEM, based on two different RNA-seq experiments for pre-B cells of each genotype


Flow cytometric analysis of plasma cells from the spleen ( A ) and bone marrow ( B ) of Prdm1ihCd2 / + and wild - type ( WT ) mice under steady - state conditions . Representative flow cytometric data are shown for 12 - month - old mice ( left ) , while bar graphs present absolute numbers of plasma cells for Prdm1ihCd2 / + ( red ) and wild - type ( gray ) mice at the age of 2 , 4 and 12 months ( right )

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Flow cytometric analysis of plasma cells from the spleen (A) and bone marrow (B) of Prdm1ihCd2/+ and wild-type (WT) mice under steady-state conditions. Representative flow cytometric data are shown for 12-month-old mice (left), while bar graphs present absolute numbers of plasma cells for Prdm1ihCd2/+ (red) and wild-type (gray) mice at the age of 2, 4 and 12 months (right)


T cell - dependent immune responses . Prdm1ihCd2 / + ( red dots ) and wild - type ( gray dots ) mice at the age of 2 months were immunized with NP - KLH ( in alum ) and analyzed at day 14 after immunization by ELISPOT assa The number of anti - NP - IgM antibody - secreting cells ( ASCs ) in the spleen was determined by ELISPOT assay ( C ) using NP24 - BSA - coated plates for detecting cells secreting total anti - NP - IgM antibodies . ELISPOT results are shown as representative pictures ( left ) or absolute ASC numbers ( right ) ( C ) Dot plots display the serum immunoglobulin titers of Prdm1ihCd2 / + ( red dots ) and wild - type ( gray dots ) mice NP - specific IgG1 concentrations ( μg / ml ) were determined relative to a standard NP - binding IgG1

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T cell-dependent immune responses. Prdm1ihCd2/+ (red dots) and wild-type (gray dots) mice at the age of 2 months were immunized with NP-KLH (in alum) and analyzed at day 14 after immunization by ELISPOT assa The number of anti-NP-IgM antibody-secreting cells (ASCs) in the spleen was determined by ELISPOT assay (C) using NP24-BSA-coated plates for detecting cells secreting total anti-NP-IgM antibodies. ELISPOT results are shown as representative pictures (left) or absolute ASC numbers (right) (C) Dot plots display the serum immunoglobulin titers of Prdm1ihCd2/+ (red dots) and wild-type (gray dots) mice NP-specific IgG1 concentrations (μg/ml) were determined relative to a standard NP-binding IgG1 antibody


T cell - dependent immune responses . Prdm1ihCd2 / + ( red dots ) and wild - type ( gray dots ) mice at the age of 2 months were immunized with NP - KLH ( in alum ) and analyzed at day 14 after immunization b ELISA The serum titers of anti - NP - specific IgM , IgG1 and IgG2b antibodies were analyzed by ELISA using NP7 - BSA - or NP24 - BSA - coated plates for detecting high - affinity IgG1 or total IgM , IgG1 and IgG2b antibodies , respectivel Dot plots display the serum immunoglobulin titers of Prdm1ihCd2 / + ( red dots ) and wild - type ( gray dots ) mice . NP - specific IgG1 concentrations ( μg / ml ) were determined relative to a standard NP - binding IgG1 antibody , whereas IgM and IgG2b amounts are indicated as relative units ( RU ) by setting the WT data to

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T cell-dependent immune responses. Prdm1ihCd2/+ (red dots) and wild-type (gray dots) mice at the age of 2 months were immunized with NP-KLH (in alum) and analyzed at day 14 after immunization b ELISA The serum titers of anti-NP-specific IgM, IgG1 and IgG2b antibodies were analyzed by ELISA using NP7-BSA- or NP24-BSA-coated plates for detecting high-affinity IgG1 or total IgM, IgG1 and IgG2b antibodies, respectivel Dot plots display the serum immunoglobulin titers of Prdm1ihCd2/+ (red dots) and wild-type (gray dots) mice. NP-specific IgG1 concentrations (μg/ml) were determined relative to a standard NP-binding IgG1 antibody, whereas IgM and IgG2b amounts are indicated as relative units (RU) by setting the WT data to 1


Flow cytometric analysis of splenic GC B cells in Prdm1ihCd2 / + ( red ) and wild - type ( gray ) mice ( at the age of 2 months ) at day 14 after NP - KLH immunization . Bar graphs display absolute numbers of GC B cells detected in the spleen of both genotype The absence of Bcl6 expression in non - GC B cells ( black ) is shown as

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Flow cytometric analysis of splenic GC B cells in Prdm1ihCd2/+ (red) and wild-type (gray) mice (at the age of 2 months) at day 14 after NP-KLH immunization. Bar graphs display absolute numbers of GC B cells detected in the spleen of both genotype The absence of Bcl6 expression in non-GC B cells (black) is shown as reference


Flow cytometric analysis of splenic GC B cells in Prdm1ihCd2 / + ( red ) and wild - type ( gray ) mice ( at the age of 2 months ) at day 14 after NP - KLH immunization Cell surface expression of hCD2 and intracellular staining of Bcl6 are shown for Prdm1ihCd2 / + ( red ) and wild - type ( gray ) GC B cell The absence of Bcl6 expression in non - GC B cells ( black ) is shown as

Flow cytometric analysis of splenic GC B cells in Prdm1ihCd2/+ (red) and wild-type (gray) mice (at the age of 2 months) at day 14 after NP-KLH immunization Cell surface expression of hCD2 and intracellular staining of Bcl6 are shown for Prdm1ihCd2/+ (red) and wild-type (gray) GC B cell The absence of Bcl6 expression in non-GC B cells (black) is shown as reference


Flow cytometric analysi in Prdm1ihCd2 / + ( red ) and wild - type ( gray ) mice ( at the age of 2 months ) at day 14 after NP - KLH immunization Flow cytometric analysis of splenic TFH cell Bar graphs indicate absolute numbers of TFH cell in hCD2lo ( black ) and hCD2hi ( blue ) Prdm1ihCd2 / + as well as wild - type ( gray ) TFH

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

Flow cytometric analysi in Prdm1ihCd2/+ (red) and wild-type (gray) mice (at the age of 2 months) at day 14 after NP-KLH immunization Flow cytometric analysis of splenic TFH cell Bar graphs indicate absolute numbers of TFH cell in hCD2lo (black) and hCD2hi (blue) Prdm1ihCd2/+ as well as wild-type (gray) TFH cells


Flow cytometric analysi in Prdm1ihCd2 / + ( red ) and wild - type ( gray ) mice ( at the age of 2 months ) at day 14 after NP - KLH immunization Flow cytometric analysis of splenic TFH cell histograms display cell surface expression of hCD2 and intracellular staining of Bcl6 ( H ) in hCD2lo ( black ) and hCD2hi ( blue ) Prdm1ihCd2 / + as well as wild - type ( gray ) TFH

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Flow cytometric analysi in Prdm1ihCd2/+ (red) and wild-type (gray) mice (at the age of 2 months) at day 14 after NP-KLH immunization Flow cytometric analysis of splenic TFH cell histograms display cell surface expression of hCD2 and intracellular staining of Bcl6 (H) in hCD2lo (black) and hCD2hi (blue) Prdm1ihCd2/+ as well as wild-type (gray) TFH cells


Efficient in vitro plasmablast differentiation of immature B cells . Immature B cells sorted from the bone marrow of Prdm1ihCd2 / + ( red ) and wild - type ( gray ) mice were stimulated with CpG oligodeoxynucleotides or LPS for 60 hours followed by flow cytometric analysis of CD138 + CD22lo plasmablasts ( PB ) . Bar graphs ( right ) summarize the plasmablast data of the CpG and LPS stimulation

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Efficient in vitro plasmablast differentiation of immature B cells. Immature B cells sorted from the bone marrow of Prdm1ihCd2/+ (red) and wild-type (gray) mice were stimulated with CpG oligodeoxynucleotides or LPS for 60 hours followed by flow cytometric analysis of CD138+CD22lo plasmablasts (PB). Bar graphs (right) summarize the plasmablast data of the CpG and LPS stimulation experiments


ELISA detection of Igκ - containing antibodies in the supernatants of immature B cells at 60 h of CpG or LPS

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ELISA detection of Igκ-containing antibodies in the supernatants of immature B cells at 60 h of CpG or LPS stimulation


Flow cytometric analysis of total B cells and different B cell subsets in the bone marrow of non - immunized Eβ - / - Prdm1ihCd2 / + ( red ) and Eβ - / - Prdm1 + / + ( gray ) chimeric mice . Bar graphs indicate absolute numbers of the different B cell types in these chimeric

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Flow cytometric analysis of total B cells and different B cell subsets in the bone marrow of non-immunized Eβ-/- Prdm1ihCd2/+ (red) and Eβ-/- Prdm1+/+ (gray) chimeric mice. Bar graphs indicate absolute numbers of the different B cell types in these chimeric mice


Flow cytometric analysis of total B cells and different B cell subsets in th splee of non - immunized Eβ - / - Prdm1ihCd2 / + ( red ) and Eβ - / - Prdm1 + / + ( gray ) chimeric mice . Bar graphs indicate absolute numbers of the different B cell types in these chimeric mice Histograms display the expression of hCD2 ( Blimp1 ) in splenic FO B , GC B and naïve CD4 T cells from the chimeric mice of the indicated

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

Flow cytometric analysis of total B cells and different B cell subsets in th splee of non-immunized Eβ-/- Prdm1ihCd2/+ (red) and Eβ-/- Prdm1+/+ (gray) chimeric mice. Bar graphs indicate absolute numbers of the different B cell types in these chimeric mice Histograms display the expression of hCD2 (Blimp1) in splenic FO B, GC B and naïve CD4 T cells from the chimeric mice of the indicated genotypes


Presence of IgG antibodies detecting dsDNA , cardiolipin , SSA ( Ro - 52 ) and SSB ( La ) in the serum of Prdm1ihCd2 / + ( red dots ) and wild - type ( gray dots ) mice at the age of 2 , 4 and 12 months . The titers of the different IgG antibodies were determined in the serum of male and female mice by ELISA and are displayed as arbitrary units ( AU ) . The serum of 6 - month - old Faslgld / gld mice was used as positive

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Presence of IgG antibodies detecting dsDNA, cardiolipin, SSA (Ro-52) and SSB (La) in the serum of Prdm1ihCd2/+ (red dots) and wild-type (gray dots) mice at the age of 2, 4 and 12 months. The titers of the different IgG antibodies were determined in the serum of male and female mice by ELISA and are displayed as arbitrary units (AU). The serum of 6-month-old Faslgld/gld mice was used as positive control


Representative images of anti - nuclear antibody ( ANA ) staining obtained with serum from 12 - month - old Prdm1ihCd2 / + or wild - type mice ( right ) , as detected by indirect immunofluorescence assay using HEp - 2 cells and an Alexa488 - conjugated anti - mouse IgG detection antibody . The serum of Faslgld / gld mice was used as positive control . The presence of ANA - IgG antibodies was analyzed for 22 Prdm1ihCd2 / + and 12 wild - type mice , as summarized in the pie - chart ( right )

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Representative images of anti-nuclear antibody (ANA) staining obtained with serum from 12-month-old Prdm1ihCd2/+ or wild-type mice (right), as detected by indirect immunofluorescence assay using HEp-2 cells and an Alexa488-conjugated anti-mouse IgG detection antibody. The serum of Faslgld/gld mice was used as positive control. The presence of ANA-IgG antibodies was analyzed for 22 Prdm1ihCd2/+ and 12 wild-type mice, as summarized in the pie-chart (right)


Periodic acid - Schiff ( PAS ) staining of paraffin - embedded kidney sections of Prdm1ihCd2 / + and wild - type mice at 12 months of age . The mean score of kidney pathology was determined for each mouse by evaluating 40 individual glomeruli to assess the severity of glomerulonephritis as shown in Table EV2 and described in the Appendix Supplementary Methods . Representative pictures of glomeruli of Prdm1ihCd2 / + kidneys with different pathology scores are shown to the left , and dot plots with average scores for male and female mice ( Table EV2 ) are shown to the right . Arrows denote intracapillary hyaline " thrombi " , asterisks indicate mesangial sclerosis with obscured capillary loops , and the arrowhead points to an obsolescent glomerulus with collapse of the glomerular tuft architecture . Statistical data ( A , C ) are shown as mean value with SEM and were analyzed by the Mann - Whitney test ( A ) or the Student " s t - test ( C ) ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Each dot

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Periodic acid-Schiff (PAS) staining of paraffin-embedded kidney sections of Prdm1ihCd2/+ and wild-type mice at 12 months of age. The mean score of kidney pathology was determined for each mouse by evaluating 40 individual glomeruli to assess the severity of glomerulonephritis as shown in Table EV2 and described in the Appendix Supplementary Methods. Representative pictures of glomeruli of Prdm1ihCd2/+ kidneys with different pathology scores are shown to the left, and dot plots with average scores for male and female mice (Table EV2) are shown to the right. Arrows denote intracapillary hyaline "thrombi", asterisks indicate mesangial sclerosis with obscured capillary loops, and the arrowhead points to an obsolescent glomerulus with collapse of the glomerular tuft architecture. Statistical data (A,C) are shown as mean value with SEM and were analyzed by the Mann-Whitney test (A) or the Student"s t-test (C); *P < 0.05, **P < 0.01, ***P < 0.001. Each dot correspon


Hydrolysis of fluorogenic substrates ( Suc - LLVY - AMC ) for the chymotrypsin - like activity of human ( B ) or mouse ( C ) 20S constitutive proteasome ( CP ) or 20S immunoproteasome ( IP ) at various PRN1126 concentrations . Data are presented as the means ± s . d . from quadruplicate assays . The highest fluorescence value was set to 100 % . The experiments were repeated twice with similar results .

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Hydrolysis of fluorogenic substrates (Suc-LLVY-AMC) for the chymotrypsin-like activity of human (B) or mouse (C) 20S constitutive proteasome (CP) or 20S immunoproteasome (IP) at various PRN1126 concentrations. Data are presented as the means ± s.d. from quadruplicate assays. The highest fluorescence value was set to 100%. The experiments were repeated twice with similar results.


Presentation of UTY246 - 254 on splenocytes after exposure to indicated concentrations of PRN1126 . Data are presented as the mean absorbance ± s . d . of three replicate cultures . The experiment has been performed twice , yielding similar results .

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Presentation of UTY246-254 on splenocytes after exposure to indicated concentrations of PRN1126. Data are presented as the mean absorbance ± s.d. of three replicate cultures. The experiment has been performed twice, yielding similar results.


Flow cytometry analysis of H - 2Kb surface expression on splenocytes derived from C57BL / 6 mice treated with the indicated concentrations of PRN1126 overnight . Pooled data from three independent experiments ( n = 9 ) are shown as the means of median fluorescent intensity ± SEM .

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Flow cytometry analysis of H-2Kb surface expression on splenocytes derived from C57BL/6 mice treated with the indicated concentrations of PRN1126 overnight. Pooled data from three independent experiments (n=9) are shown as the means of median fluorescent intensity ± SEM.


Splenocytes from C57BL / 6 mice ( C ) were exposed ( continuous treatment ) to 300 nM PRN1126 or vehicle ( DMSO ) or 300 nM ONX 0914 and stimulated with LPS overnight . IL - 6 concentrations in the supernatant were analyzed by ELISA . ( C ) IL - 6 concentrations are presented as mean and individual data points from triplicate wells . The experiment has been performed twice , yielding similar results .

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Splenocytes from C57BL/6 mice (C) were exposed (continuous treatment) to 300 nM PRN1126 or vehicle (DMSO) or 300 nM ONX 0914 and stimulated with LPS overnight. IL-6 concentrations in the supernatant were analyzed by ELISA. (C) IL-6 concentrations are presented as mean and individual data points from triplicate wells. The experiment has been performed twice, yielding similar results.


Splenocytes from human PBMCs ( D ) were exposed ( continuous treatment ) to 300 nM PRN1126 or vehicle ( DMSO ) or 300 nM ONX 0914 and stimulated with LPS overnight . IL - 6 concentrations in the supernatant were analyzed by ELISA . Data are presented as single dots from three independent donors . The highest cytokine concentration was set to 100 % . * P < 0 . 05 . Unpaired student ´ s t - test .

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Splenocytes from human PBMCs (D) were exposed (continuous treatment) to 300 nM PRN1126 or vehicle (DMSO) or 300 nM ONX 0914 and stimulated with LPS overnight. IL-6 concentrations in the supernatant were analyzed by ELISA. Data are presented as single dots from three independent donors. The highest cytokine concentration was set to 100%. *P < 0.05. Unpaired student´s t-test.


Colitis was induced by oral administration of 3 % DSS for 5 d . Mice were treated daily ( s . c . ) with either PRN1126 ( 40 mg / kg ) , or ONX 0914 ( 10 mg / kg ) , or vehicle . Data points represent mean ± SEM of 15 mice pooled from three independent experiments .

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Colitis was induced by oral administration of 3 % DSS for 5 d. Mice were treated daily (s.c.) with either PRN1126 (40 mg/kg), or ONX 0914 (10 mg/kg), or vehicle. Data points represent mean ± SEM of 15 mice pooled from three independent experiments.


Mice were immunized with MOG35 - 55 peptide and were monitored daily for clinical symptoms of EAE . Mice were treated three times a week ( s . c . ) with either PRN1126 ( 40 mg / kg ) , or ONX 0914 ( 10 mg / kg ) , or vehicle . All data were statistically compared to the vehicle treated group . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Two - way ANOVA . Shown are the means of the clinical scores ± SEM ( n = 6 per group ) .

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

Mice were immunized with MOG35-55 peptide and were monitored daily for clinical symptoms of EAE. Mice were treated three times a week (s.c.) with either PRN1126 (40 mg/kg), or ONX 0914 (10 mg/kg), or vehicle. All data were statistically compared to the vehicle treated group. *P < 0.05, **P < 0.01, ***P < 0.001. Two-way ANOVA. Shown are the means of the clinical scores ± SEM (n = 6 per group).


Hydrolysis of fluorogenic substrates ( Ac - PAL - AMC ) for LMP2 - activity of human ( upper panel ) or mouse ( lower panel ) 20S immunoproteasome at various concentrations of PRN1126 or ONX 0914 . Data are presented as the means of fluorescence ± s . d . from quadruplicate assays . The experiments were repeated three times with similar results .

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Hydrolysis of fluorogenic substrates (Ac-PAL-AMC) for LMP2-activity of human (upper panel) or mouse (lower panel) 20S immunoproteasome at various concentrations of PRN1126 or ONX 0914. Data are presented as the means of fluorescence ± s.d. from quadruplicate assays. The experiments were repeated three times with similar results.


Altered electrophoretic mobility of IP subunits by covalent modification with ONX 0914 . Ficoll enriched lymphocytes from C57BL / 6 mice were treated with DMSO or 300 nM ONX 0914 for 2 h in vitro . SDS - PAGE and immunoblotting against indicated proteins was performed . Shown are representative western blots out of three independent experiments with similar outcome .

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Altered electrophoretic mobility of IP subunits by covalent modification with ONX 0914. Ficoll enriched lymphocytes from C57BL/6 mice were treated with DMSO or 300 nM ONX 0914 for 2 h in vitro. SDS-PAGE and immunoblotting against indicated proteins was performed. Shown are representative western blots out of three independent experiments with similar outcome.


Flow cytometry analysis of H - 2Kb surface expression on splenocytes derived from C57BL / 6 wild type ( left side ) or LMP7 - / - ( right side ) mice treated with vehicle ( DMSO ) , ONX 0914 ( 300 nM ) , PRN1126 ( 300 nM ) , ML604440 ( 300 nM ) , LU - 001i ( 300 nM ) , PRN1126 + ML604440 ( 300 nM each ) , or PRN1126 + LU - 001i ( 300 nM each ) overnight . Unstained splenocytes were used as negative control . Pooled data from three independent experiments ( n = 9 ) are shown as the means of median fluorescent intensity ± SEM . All data were statistically compared to the DMSO treated group . * * P < 0 . 01 , * * * P < 0 . 001 . One - way ANOVA .

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

Flow cytometry analysis of H-2Kb surface expression on splenocytes derived from C57BL/6 wild type (left side) or LMP7-/- (right side) mice treated with vehicle (DMSO), ONX 0914 (300 nM), PRN1126 (300 nM), ML604440 (300 nM), LU-001i (300 nM), PRN1126+ML604440 (300 nM each), or PRN1126+LU-001i (300 nM each) overnight. Unstained splenocytes were used as negative control. Pooled data from three independent experiments (n=9) are shown as the means of median fluorescent intensity ± SEM. All data were statistically compared to the DMSO treated group. **P < 0.01, ***P < 0.001. One-way ANOVA.


Splenocytes from C57BL / 6 mice ( A ) or human PBMCs ( B ) were incubated ( continuous treatment ) with DMSO , ONX 0914 ( 300 nM ) , PRN1126 ( 300 nM ) , ML604440 ( 300 nM ) , LU - 001i ( 300 nM ) , PRN1126 + ML604440 ( 300 nM each ) , or PRN1126 + LU - 001i ( 300 nM each ) and stimulated with LPS overnight . The IL - 6 concentration in the supernatant was analyzed by ELISA . The highest IL - 6 concentration from each experiment was set to 100 % . ( A ) Data are presented as the mean ± SEM of 4 independent experiments each measured in triplicates . ( B ) Data are presented as the mean ± SEM of 6 different healthy donors . All data were statistically compared to the DMSO treated group .

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

Splenocytes from C57BL/6 mice (A) or human PBMCs (B) were incubated (continuous treatment) with DMSO, ONX 0914 (300 nM), PRN1126 (300 nM), ML604440 (300 nM), LU-001i (300 nM), PRN1126+ML604440 (300 nM each), or PRN1126+LU-001i (300 nM each) and stimulated with LPS overnight. The IL-6 concentration in the supernatant was analyzed by ELISA. The highest IL-6 concentration from each experiment was set to 100%. (A) Data are presented as the mean ± SEM of 4 independent experiments each measured in triplicates. (B) Data are presented as the mean ± SEM of 6 different healthy donors. All data were statistically compared to the DMSO treated group.


The differentiation of CD4 + T cells isolated from the spleens of C57BL / 6 mice that were exposed ( continuous treatment ) to DMSO , ONX 0914 ( 300 nM ) , PRN1126 ( 300 nM ) , ML604440 ( 300 nM ) , LU - 001i ( 300 nM ) , PRN1126 + ML604440 ( 300 nM each ) , or PRN1126 + LU - 001i ( 300 nM each ) and that were stimulated with plate bound antibodies to CD3 / CD28 in the presence of TGF - β and IL - 6 and scavenging antibodies neutralising IL - 4 and IFN - γ was measured in 3 days cultures . IL - 17 expression was detected by intracellular cytokine staining after a short restimulation with PMA / Ionomycin . IL - 17 expression in activated T cells cultured in the absence of Th17 - polarizing conditions is shown as a comparison ( no cocktail ) . Values reflect the percentage of CD4 + cells that are IL - 17A + . Data are presented as the means ± SEM of 3 independent experiments each measured in triplicates . All data were statistically compared to the DMSO treated group .

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

The differentiation of CD4+ T cells isolated from the spleens of C57BL/6 mice that were exposed (continuous treatment) to DMSO, ONX 0914 (300 nM), PRN1126 (300 nM), ML604440 (300 nM), LU-001i (300 nM), PRN1126+ML604440 (300 nM each), or PRN1126+ LU-001i (300 nM each) and that were stimulated with plate bound antibodies to CD3/CD28 in the presence of TGF-β and IL-6 and scavenging antibodies neutralising IL-4 and IFN-γ was measured in 3 days cultures. IL-17 expression was detected by intracellular cytokine staining after a short restimulation with PMA/Ionomycin. IL-17 expression in activated T cells cultured in the absence of Th17-polarizing conditions is shown as a comparison (no cocktail). Values reflect the percentage of CD4+ cells that are IL-17A+. Data are presented as the means ± SEM of 3 independent experiments each measured in triplicates. All data were statistically compared to the DMSO treated group.


Colitis was induced by oral administration of 3 % DSS . Mice were treated daily ( s . c . ) with LU - 001i ( 15 mg / kg ) , PRN1126 ( 40 mg / kg ) , PRN1126 + LU - 001i ( 40 mg / kg + 15 mg / kg ) , or vehicle starting from the begin of the experiment . Data points represent means ± SEM of 15 mice pooled from three independent experiments . ( A ) The body weight of individual mice was monitored daily and the percent weight loss ( y - axis ) was plotted versus time ( x - axis ) . All data were statistically compared to the vehicle treated group . *

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

Colitis was induced by oral administration of 3% DSS. Mice were treated daily (s.c.) with LU-001i (15 mg/kg), PRN1126 (40 mg/kg), PRN1126+ LU-001i (40 mg/kg+15 mg/kg), or vehicle starting from the begin of the experiment. Data points represent means ± SEM of 15 mice pooled from three independent experiments. (A) The body weight of individual mice was monitored daily and the percent weight loss (y-axis) was plotted versus time (x-axis). All data were statistically compared to the vehicle treated group. *


Colitis was induced by oral administration of 3 % DSS . Mice were treated daily ( s . c . ) with LU - 001i ( 15 mg / kg ) , PRN1126 ( 40 mg / kg ) , PRN1126 + LU - 001i ( 40 mg / kg + 15 mg / kg ) , or vehicle starting from the begin of the experiment . Data points represent means ± SEM of 15 mice pooled from three independent experiments . On day 9 after initiation of DSS treatment , colon lengths were measured ( n = 15 ) . Naïve mice ( n = 5 ) were used as healthy controls . All data were statistically compared to the vehicle treated group .

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

Colitis was induced by oral administration of 3% DSS. Mice were treated daily (s.c.) with LU-001i (15 mg/kg), PRN1126 (40 mg/kg), PRN1126+ LU-001i (40 mg/kg+15 mg/kg), or vehicle starting from the begin of the experiment. Data points represent means ± SEM of 15 mice pooled from three independent experiments. On day 9 after initiation of DSS treatment, colon lengths were measured (n=15). Naïve mice (n=5) were used as healthy controls. All data were statistically compared to the vehicle treated group.


Mice were immunized with MOG35 - 55 peptide and were monitored daily for clinical symptoms of EAE . Mice were treated intermittently with three times a week ( s . c . ) schedule with LU - 001i ( 15 mg / kg ) , PRN1126 ( 40 mg / kg ) , PRN1126 + LU - 001i ( 40 mg / kg + 15 mg / kg ) , or vehicle starting from the start of the experiment . Data points represent the means of the clinical scores ± SEM of 12 mice pooled from two independent experiments . All data were statistically compared to the vehicle treated group .

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

Mice were immunized with MOG35-55 peptide and were monitored daily for clinical symptoms of EAE. Mice were treated intermittently with three times a week (s.c.) schedule with LU-001i (15 mg/kg), PRN1126 (40 mg/kg), PRN1126+ LU-001i (40 mg/kg+15 mg/kg), or vehicle starting from the start of the experiment. Data points represent the means of the clinical scores ± SEM of 12 mice pooled from two independent experiments. All data were statistically compared to the vehicle treated group.


( C ) miR - 574 - 5p and miR - 574 - 3p were highly expressed in the explanted failing hearts from chronic HF patients ( n = 17 ) versus donor non - failing hearts ( n = 6 ) .

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(C) miR-574-5p and miR-574-3p were highly expressed in the explanted failing hearts from chronic HF patients (n=17) versus donor non-failing hearts (n=6).


( D ) miR - 574 - 5p and miR - 574 - 3p were induced in the hearts of mice with isoproterenol ( ISO ) infusion ( 4 weeks ; n = 10 - 12 ) .

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(D) miR-574-5p and miR-574-3p were induced in the hearts of mice with isoproterenol (ISO) infusion (4 weeks; n=10-12).


( E ) miR - 574 - 5p and miR - 574 - 3p were induced in the hearts of mice under transverse aortic constriction ( TAC ) surgery ( 4 weeks ; n = 8 - 10 ) .

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(E) miR-574-5p and miR-574-3p were induced in the hearts of mice under transverse aortic constriction (TAC) surgery (4 weeks; n=8-10).


( A ) H & E of hearts from WT and miR - 574 - / - mice with or without isoproterenol ( ISO ) treatment for 4 weeks . Mice were between 8 - 10 weeks old females .

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(A) H&E of hearts from WT and miR-574-/- mice with or without isoproterenol (ISO) treatment for 4 weeks. Mice were between 8-10 weeks old females.


( B ) Ratio of HW / TL ( heart weight / tibia length ) in WT and miR - 574 - / - mice ( n = 8 / 7 / 8 / 6 for 4 groups of WT , Veh . ; WT , ISO ; KO , Veh . ; KO , ISO ) .

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(B) Ratio of HW/TL (heart weight/tibia length) in WT and miR-574-/- mice (n=8/7/8/6 for 4 groups of WT, Veh.; WT, ISO; KO, Veh.; KO, ISO).


( C ) WGA ( wheat germ agglutinin ) staining in WT and miR - 574 - / - mice . Cross - sectional area ( CSA ) of CMs was measured and quantified ( n ≥ 500 cells ) . Scale bar : 50 μm . In the violin plot , black line shows median value for the group and peach dashed lines represent two quartile lines in each group .

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(C) WGA (wheat germ agglutinin) staining in WT and miR-574-/- mice. Cross-sectional area (CSA) of CMs was measured and quantified (n≥500 cells). Scale bar: 50 μm. In the violin plot, black line shows median value for the group and peach dashed lines represent two quartile lines in each group.


( D ) Picrosirius red staining in WT and miR - 574 - / - mice ( n = 6 per group ) . Scale bar : 1 mm .

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(D) Picrosirius red staining in WT and miR-574-/- mice (n=6 per group). Scale bar: 1 mm.


( E ) RT - qPCR of fetal cardiac genes in WT and miR - 574 - / - mice with ISO treatment ( n = 3 per group ) . ANF , atrial natriuretic factor ; BNP , B - type natriuretic peptide ; Myh6 / Myh7 , myosin heavy polypeptide 6 / 7 ; Col1a2 / Col3a1 , procollagen , type I , α2 ; type III , α1 .

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(E) RT-qPCR of fetal cardiac genes in WT and miR-574-/- mice with ISO treatment (n=3 per group). ANF, atrial natriuretic factor; BNP, B-type natriuretic peptide; Myh6/Myh7, myosin heavy polypeptide 6/7; Col1a2/Col3a1, procollagen, type I, α2; type III, α1.


( F ) TUNEL assay for heart tissue sections from WT and miR - 574 - / - mice under ISO versus vehicle treatment ( n = 6 per group ) . Scale bar : 10 μm .

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(F) TUNEL assay for heart tissue sections from WT and miR-574-/- mice under ISO versus vehicle treatment (n=6 per group). Scale bar: 10 μm.


( A ) H & E staining of hearts from WT and miR - 574 - / - mice 4 weeks after transverse aortic constriction ( TAC ) surgery .

(A) H&E staining of hearts from WT and miR-574-/- mice 4 weeks after transverse aortic constriction (TAC) surgery.


( B ) Ratio of HW / TL in WT and miR - 574 - / - mice ( n = 8 per group ) .

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(B) Ratio of HW/TL in WT and miR-574-/- mice (n=8 per group).


( C ) WGA staining of hearts from WT and miR - 574 - / - mice 4 weeks after TAC surgery ( n ≥ 500 cells ) . Scale bar : 20 μm . In the violin plot , black line shows median value for the group and peach dashed lines represent two quartile lines in each group .

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(C) WGA staining of hearts from WT and miR-574-/- mice 4 weeks after TAC surgery (n≥500 cells). Scale bar: 20 μm. In the violin plot, black line shows median value for the group and peach dashed lines represent two quartile lines in each group.


( D ) Picrosirius red staining of hearts from WT and miR - 574 - / - mice under TAC surgery ( n = 8 per group ) . Scale bar : 1 mm .

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(D) Picrosirius red staining of hearts from WT and miR-574-/- mice under TAC surgery (n=8 per group). Scale bar: 1 mm.


( E ) RT - qPCR of fetal cardiac genes in WT and miR - 574 - / - mice with TAC surgery ( n = 4 per group ) .

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(E) RT-qPCR of fetal cardiac genes in WT and miR-574-/- mice with TAC surgery (n=4 per group).


( F ) Echocardiography measurement of cardiac functions of hearts from WT and miR - 574 - / - mice 4 weeks post TAC ( n = 8 per group ) . FS , fractional shortening .

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(F) Echocardiography measurement of cardiac functions of hearts from WT and miR-574-/- mice 4 weeks post TAC (n=8 per group). FS, fractional shortening.


The schematic of therapeutic treatment of the TAC model using miRNA mimics followed by phenotypic characterizations . HW / TL ratio is measured ( n = 4 - 10 per group ) . Ctrl - miR : negative control miRNA mimics .

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The schematic of therapeutic treatment of the TAC model using miRNA mimics followed by phenotypic characterizations. HW/TL ratio is measured (n=4-10 per group). Ctrl-miR: negative control miRNA mimics.


( B ) H & E and WGA staining of murine hearts in the therapeutic models . Scale bar : 1 mm for H & E and 50 μm for WGA . Cardiomyocyte surface area was quantified from WGA staining in the right panel . > 120 CMs are quantified in each group . The black line shows medium value for the group and the dotted lines represent two quartile lines in each group .

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(B) H&E and WGA staining of murine hearts in the therapeutic models. Scale bar: 1 mm for H&E and 50 μm for WGA. Cardiomyocyte surface area was quantified from WGA staining in the right panel. >120 CMs are quantified in each group. The black line shows medium value for the group and the dotted lines represent two quartile lines in each group.


( C ) Picrosirius red staining of murine hearts in the therapeutic models . Scale bar : 1 mm . The fibrotic area was quantified in the right panel ( n = 4 - 10 per group ) .

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(C) Picrosirius red staining of murine hearts in the therapeutic models. Scale bar: 1 mm. The fibrotic area was quantified in the right panel (n=4-10 per group).


( D ) Echocardiography analysis of the cardiac function of mice in the therapeutic models at the end - point of 6 weeks post - surgery ( n = 4 - 10 per group ) .

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(D) Echocardiography analysis of the cardiac function of mice in the therapeutic models at the end-point of 6 weeks post-surgery (n=4-10 per group).


( A ) Volcano curve analysis of dysregulated genes in miR - 574 - / - versus WT heart . The Padj is the P - values adjusted for multiple testing with correction using the Benjamini - Hochberg procedure . The dots above the dashed line show Padj < 0 . 05 .

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(A) Volcano curve analysis of dysregulated genes in miR-574-/- versus WT heart. The Padj is the P-values adjusted for multiple testing with correction using the Benjamini-Hochberg procedure. The dots above the dashed line show Padj<0.05.


( B ) Heatmap of significantly upregulated genes in miR - 574 - / - mice at baseline analyzed by RNA - Seq . P60 male mice , n = 3 per group , Padj < 0 . 05 . The Padj is the P - values adjusted for multiple testing with correction using the Benjamini - Hochberg procedure .

(B) Heatmap of significantly upregulated genes in miR-574-/- mice at baseline analyzed by RNA-Seq. P60 male mice, n=3 per group, Padj<0.05. The Padj is the P-values adjusted for multiple testing with correction using the Benjamini-Hochberg procedure.