Figure 8 ( A ) Convertase - labeled bacteria were exposed to a concentration range of either preassembled C5b6 ( C5b6MAC ) or a mixture of C5 and C6 ( Conv - MAC ) , in the presence of 100 nM C7 . After washing , 20 nM C8 and 100 nM C9 - Cy3 was added . Controls at 0 nM C5b6 or C5 - C6 confirm that the detected C9 - Cy3 deposition is specifically related to MAC formation ( solid lines ) . Proper insertion of pores was assessed by a previously described shaving method with trypsin ( Moskovich & Fishelson , 2007 ) . Bacteria were first incubated with MAC components for 30 minutes and subsequently treated with 20 µg / ml trypsin for 15 min at 37 ° C ( dotted lines ) . Data information : ( A ) Data represent mean ± SD of 3 independent experiments . ( B ) Convertase labeled perimCherry / cytoGFP bacteria ( Green ) exposed to C5b6MAC or Conv - MAC . Conditions were similar to those in ( A ) , however C9 - Cy5 was used to detect MAC pores ( Red ) . 100 nM of C5 and C6 or C5b6 were used in combination with 100 nM C7 , 20 nM C8 and 100 nM C9 - Cy5 . Conv + C5b6MAC and conv - MAC images were taken in separate experiments in which laser settings were adjusted to the staining intensity of C9 - Cy5 to properly visualize pore distribution . Scale bars = 3 µm . ( C Atomic force microscopy analysis of E . coli BL21 and MG1655 immobilized using the Poly - L - Lysine protocol . ( C ) Entire bacteria and high - resolution comparisons of untreated and convertase - labeled E . coli BL21 exposed to C5 - C9 ( Conv - MAC ) for 10 minutes . Scale bars : 800 nm ( left ) and 30 nm ( right ) . Height Scales : 1 . 01 µm ( left ) , 8 nm ( top right ) , 22 nm ( bottom right ) . Width of magnification boxes ( left ) : 42 nm , height scales : 8 nm ( top left ) and 13 nm ( bottom left ) . Arrows highlight E . coli porin structures , an asterisk highlights the C5b - 7 stalk . Height profiles ( bottom ) are shown for the white dashed lines in the images . ( D ) Atomic force microscopy ( height and phase images ) of convertase - labeled E . coli MG1655 exposed to a mixture of C5 and C6 ( Conv - MAC ) or preassembled C5b6 ( Conv + C5b6MAC ) , in the presence of C7 , C8 , C9 , FB and FD . Images were generated in the same experiment . Scale bars : 50 nm . Height scales : 15 nm .

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

Figure 8(A) Convertase-labeled bacteria were exposed to a concentration range of either preassembled C5b6 (C5b6MAC) or a mixture of C5 and C6 (Conv-MAC), in the presence of 100 nM C7. After washing, 20 nM C8 and 100 nM C9-Cy3 was added. Controls at 0 nM C5b6 or C5-C6 confirm that the detected C9-Cy3 deposition is specifically related to MAC formation (solid lines). Proper insertion of pores was assessed by a previously described shaving method with trypsin (Moskovich & Fishelson, 2007). Bacteria were first incubated with MAC components for 30 minutes and subsequently treated with 20 µg/ml trypsin for 15 min at 37°C (dotted lines). Data information: (A) Data represent mean ± SD of 3 independent experiments.(B) Convertase labeled perimCherry/cytoGFP bacteria (Green) exposed to C5b6MAC or Conv-MAC. Conditions were similar to those in (A), however C9-Cy5 was used to detect MAC pores (Red). 100 nM of C5 and C6 or C5b6 were used in combination with 100 nM C7, 20 nM C8 and 100 nM C9-Cy5. Conv + C5b6MAC and conv-MAC images were taken in separate experiments in which laser settings were adjusted to the staining intensity of C9-Cy5 to properly visualize pore distribution. Scale bars = 3 µm.(C Atomic force microscopy analysis of E. coli BL21 and MG1655 immobilized using the Poly-L-Lysine protocol. (C) Entire bacteria and high-resolution comparisons of untreated and convertase-labeled E. coli BL21 exposed to C5-C9 (Conv-MAC) for 10 minutes. Scale bars: 800 nm (left) and 30 nm (right). Height Scales: 1.01 µm (left), 8 nm (top right), 22 nm (bottom right). Width of magnification boxes (left): 42 nm, height scales: 8 nm (top left) and 13 nm (bottom left). Arrows highlight E. coli porin structures, an asterisk highlights the C5b-7 stalk. Height profiles (bottom) are shown for the white dashed lines in the images.(D) Atomic force microscopy (height and phase images) of convertase-labeled E. coli MG1655 exposed to a mixture of C5 and C6 (Conv-MAC) or preassembled C5b6 (Conv + C5b6MAC), in the presence of C7, C8, C9, FB and FD. Images were generated in the same experiment. Scale bars: 50 nm. Height scales: 15 nm.


Figure 1Immunoblots and qRT - PCRs showing that expression levels of miR - 203 , miR - 200 , and E - cadherin are increased after ZEB1 knockdown in Panc1 , MDA - MB - 231 . Vice versa , the drug - resistant clones of BxPC3 , H358 , and DU - 145 show increased expression of ZEB1 and decreased expression of the miRNAs and E - cadherin . n = 3 , mean ± SEM , except for H358 ( data from microarray ) . Unpaired Student ' s t - test . Lentiviral overexpression of miR - 200c and miR - 203 in Panc1 and hPaca1 induces sensitivity to gemcitabine treatment as measured by MTT assay . For the changes in EC50 values , see Table1 . n = 3 , mean ± SEM , Dunnett ' s multiple comparisons test ( P - values in the graphs are * P = 0 . 01 - 0 . 05 , * * P = 0 . 001 - 0 . 01 , * * * P < 0 . 001 , and * * * * P < 0 . 0001 ; for exact P - values , see Supplementary Table S4 ) . Overexpression of miR - 203 decreases expression of the anti - apoptotic factor survivin and sensitizes to gemcitabine - triggered apoptosis as evaluated by cleaved caspase - 3 in Western blot and immunofluorescence . Panc1 and hPaca1 were treated with 50 and 5 nM gemcitabine , respectively , for 48 h . Scale bar 20 μm . MTT assay showing increase in gemcitabine resistance after inhibition of endogenous miRNAs in hPaca2 by specific antagomirs against miR - 203 or all miR - 200 members . For the changes in EC80 values , see Table1 . n = 3 , mean ± SEM , Dunnett ' s multiple comparisons test ( P - values in the graphs are * P = 0 . 01 - 0 . 05 , * * P = 0 . 001 - 0 . 01 , * * * P < 0 . 001 , and * * * * P < 0 . 0001 ; for exact P - values , see Supplementary Table S4 ) . Overexpression of miR - 203 shows reduced numbers of the CD24 / CD44 double - positive cancer stem cell population as determined by FACS analysis . The arrow indicates the reduction in the CD24 high subpopulation and reduction in CD133 by miR - 203 overexpression in hPaca1 cells . Cancer stem cell sphere assay showing reduced sphere - forming capacity of Panc1 and hPaca1 in miR - 203 overexpression cells . Colonies with a diameter greater than 75 μM for Panc1 and greater 30 μM for hPaca1 cells were counted as spheres . n = 3 , mean ± SEM , Mann - Whitney U - test .

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

Figure 1Immunoblots and qRT-PCRs showing that expression levels of miR-203, miR-200, and E-cadherin are increased after ZEB1 knockdown in Panc1, MDA-MB-231. Vice versa, the drug-resistant clones of BxPC3, H358, and DU-145 show increased expression of ZEB1 and decreased expression of the miRNAs and E-cadherin. n = 3, mean ± SEM, except for H358 (data from microarray). Unpaired Student's t-test.Lentiviral overexpression of miR-200c and miR-203 in Panc1 and hPaca1 induces sensitivity to gemcitabine treatment as measured by MTT assay. For the changes in EC50 values, see Table1. n = 3, mean ± SEM, Dunnett's multiple comparisons test (P-values in the graphs are *P = 0.01-0.05, **P = 0.001-0.01, ***P < 0.001, and ****P < 0.0001; for exact P-values, see Supplementary Table S4).Overexpression of miR-203 decreases expression of the anti-apoptotic factor survivin and sensitizes to gemcitabine-triggered apoptosis as evaluated by cleaved caspase-3 in Western blot and immunofluorescence. Panc1 and hPaca1 were treated with 50 and 5 nM gemcitabine, respectively, for 48 h. Scale bar 20 μm.MTT assay showing increase in gemcitabine resistance after inhibition of endogenous miRNAs in hPaca2 by specific antagomirs against miR-203 or all miR-200 members. For the changes in EC80 values, see Table1. n = 3, mean ± SEM, Dunnett's multiple comparisons test (P-values in the graphs are *P = 0.01-0.05, **P = 0.001-0.01, ***P < 0.001, and ****P < 0.0001; for exact P-values, see Supplementary Table S4).Overexpression of miR-203 shows reduced numbers of the CD24/CD44 double-positive cancer stem cell population as determined by FACS analysis. The arrow indicates the reduction in the CD24 high subpopulation and reduction in CD133 by miR-203 overexpression in hPaca1 cells.Cancer stem cell sphere assay showing reduced sphere-forming capacity of Panc1 and hPaca1 in miR-203 overexpression cells. Colonies with a diameter greater than 75 μM for Panc1 and greater 30 μM for hPaca1 cells were counted as spheres. n = 3, mean ± SEM, Mann-Whitney U-test.


Figure 2B , C Histone marks were analyzed using ChIP coupled to qRT - PCR for Panc1 control versus shZEB , MDA - MB - 231 control versus shZEB ( B ) , and BxPC3 control versus gemcitabine resistant ( gr ) ( C ) . In MDA - MB - 231 and Panc1 , the active histone marks H3K4me3 , H3ac , H4ac , and H3K9ac were enriched . Vice versa , in the drug - resistant clones of BxPC3 , the active marks were reduced in the CpG islands . The repressive histone mark H3K27me3 was not detectable in the miR - 200 loci , but in the loci of miR - 203 and E - cadherin in Panc1 and MDA - MB - 231 . DNA methylation status was determined by bisulfite sequencing . Depletion of ZEB1 in MDA - MB - 231 resulted in almost complete demethylation , whereas the selection of drug - resistant , ZEB1 - expressing clones in BxPC3 induced complete methylation . n = 2 ( Panc1 ) or 3 ( MDA - MB - 231 and BxPC3 ) , mean ± SEM ; unpaired Student ' s t - test .

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Figure 2B, C Histone marks were analyzed using ChIP coupled to qRT-PCR for Panc1 control versus shZEB, MDA-MB-231 control versus shZEB (B), and BxPC3 control versus gemcitabine resistant (gr) (C). In MDA-MB-231 and Panc1, the active histone marks H3K4me3, H3ac, H4ac, and H3K9ac were enriched. Vice versa, in the drug-resistant clones of BxPC3, the active marks were reduced in the CpG islands. The repressive histone mark H3K27me3 was not detectable in the miR-200 loci, but in the loci of miR-203 and E-cadherin in Panc1 and MDA-MB-231. DNA methylation status was determined by bisulfite sequencing. Depletion of ZEB1 in MDA-MB-231 resulted in almost complete demethylation, whereas the selection of drug-resistant, ZEB1-expressing clones in BxPC3 induced complete methylation. n = 2 (Panc1) or 3 (MDA-MB-231 and BxPC3), mean ± SEM; unpaired Student's t-test.


Figure 3Heat map showing the relative expression levels after drug treatment for 48 h in Panc1 . Values measured by qRT - PCR were depicted with the software GENE - E . Only mocetinostat upregulated the miRNAs and downregulated ZEB1 . Relative expression of indicated genes in Panc1 measured by qRT - PCR after treatment with different HDAC inhibitors . Note the downregulation of ZEB1 and upregulation of miR - 203 , miR - 200 , and E - cadherin by mocetinostat . n = 3 , mean ± SEM ; unpaired Student ' s t - test . For significance , see Supplementary Table S1 . Immunoblot and immunofluorescence showing that mocetinostat treatment ( 1 μM , 48 h ) reduced ZEB1 expression and induced E - cadherin in Panc1 . Expression of histone deacetylases was not altered by mocetinostat , but histone acetylation was induced . Scale bar 10 μm . Chromatin immunoprecipitation analysis validated mocetinostat - induced ( 1 μM , 48 h ) enrichment of the active histone marks H3ac , H4ac , H3K9ac , and H3K4me3 at ZEB1 target gene loci in Panc1 . n = 3 , mean ± SEM ; unpaired Student ' s t - test . Mocetinostat treatment reduced expression of the anti - apoptotic miR - 203 target survivin and sphere - forming capacity in Panc1 when pre - treated with mocetinostat for 48 h . n = 3 , mean ± SEM ; Mann - Whitney U - test .

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

Figure 3Heat map showing the relative expression levels after drug treatment for 48 h in Panc1. Values measured by qRT-PCR were depicted with the software GENE-E. Only mocetinostat upregulated the miRNAs and downregulated ZEB1.Relative expression of indicated genes in Panc1 measured by qRT-PCR after treatment with different HDAC inhibitors. Note the downregulation of ZEB1 and upregulation of miR-203, miR-200, and E-cadherin by mocetinostat. n = 3, mean ± SEM; unpaired Student's t-test. For significance, see Supplementary Table S1.Immunoblot and immunofluorescence showing that mocetinostat treatment (1 μM, 48 h) reduced ZEB1 expression and induced E-cadherin in Panc1. Expression of histone deacetylases was not altered by mocetinostat, but histone acetylation was induced. Scale bar 10 μm.Chromatin immunoprecipitation analysis validated mocetinostat-induced (1 μM, 48 h) enrichment of the active histone marks H3ac, H4ac, H3K9ac, and H3K4me3 at ZEB1 target gene loci in Panc1. n = 3, mean ± SEM; unpaired Student's t-test.Mocetinostat treatment reduced expression of the anti-apoptotic miR-203 target survivin and sphere-forming capacity in Panc1 when pre-treated with mocetinostat for 48 h. n = 3, mean ± SEM; Mann-Whitney U-test.


Figure 4Immunoblot and immunofluorescence for cleaved caspase - 3 showing strong increase in apoptosis in gemcitabine - resistant Panc1 after combined treatment with mocetinostat ( 1 μM ) and gemcitabine ( 50 nM ) . Scale bar 20 μm . MTT assay for Panc1 treated with the indicated concentrations of mocetinostat ( left ) or SAHA ( middle ) and gemcitabine ( 72 h ) . Combined treatment of mocetinostat and gemcitabine significantly reduced cell viability . In contrast , a combination with SAHA had no effect . For calculation of the CI and synergy between the drugs , see Table2 . Comparison of mocetinostat and SAHA alone ( right ) . n = 3 , mean ± SEM , Dunnett ' s multiple comparisons test ( P - values in the graphs are * * * P < 0 . 001 and * * * * P < 0 . 0001 ; for exact P - values , see Supplementary Table S4 ) . Effects of mocetinostat on cleaved caspase expression and susceptibility to gemcitabine in the patient - derived pancreatic cancer cells . Note that hPaca1 behaves similar to Panc1 , but mocetinostat had no significant effect in hPaca2 . Scale bar 20 μm . n = 3 , mean ± SEM , Dunnett ' s multiple comparisons test ( P - values in the graphs are * P = 0 . 01 - 0 . 05 , * * P = 0 . 001 - 0 . 01 , * * * P < 0 . 001 , and * * * * P < 0 . 0001 ; for exact P - values , see Supplementary Table S4 ) . MTT assays comparing the effects of docetaxel and mocetinostat in the prostate cancer cell line DU - 145 and the docetaxel - resistant subclone DU - 145 DR ( left ) . Mocetinostat treatment of DU - 145 DR downregulates ZEB1 , upregulates E - cadherin , miR - 200 , and miR - 203 expression . For relative miRNA expression , the expression levels in original DU - 145 were set to 1 ( middle panels , the immunoblot panel derives from the same experiment shown in Fig1A ) . Mocetinostat sensitizes DU - 145 DR to docetaxel ( right ) . For calculation of the CI and synergy between the drugs , see Table2 . n = 3 , mean ± SEM , Dunnett ' s multiple comparisons test ( P - values in the graphs are * P = 0 . 01 - 0 . 05 ; for exact P - values , see Supplementary Table S4 ) .

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

Figure 4Immunoblot and immunofluorescence for cleaved caspase-3 showing strong increase in apoptosis in gemcitabine-resistant Panc1 after combined treatment with mocetinostat (1 μM) and gemcitabine (50 nM). Scale bar 20 μm.MTT assay for Panc1 treated with the indicated concentrations of mocetinostat (left) or SAHA (middle) and gemcitabine (72 h). Combined treatment of mocetinostat and gemcitabine significantly reduced cell viability. In contrast, a combination with SAHA had no effect. For calculation of the CI and synergy between the drugs, see Table2. Comparison of mocetinostat and SAHA alone (right). n = 3, mean ± SEM, Dunnett's multiple comparisons test (P-values in the graphs are ***P < 0.001 and ****P < 0.0001; for exact P-values, see Supplementary Table S4).Effects of mocetinostat on cleaved caspase expression and susceptibility to gemcitabine in the patient-derived pancreatic cancer cells. Note that hPaca1 behaves similar to Panc1, but mocetinostat had no significant effect in hPaca2. Scale bar 20 μm. n = 3, mean ± SEM, Dunnett's multiple comparisons test (P-values in the graphs are *P = 0.01-0.05, **P = 0.001-0.01, ***P < 0.001, and ****P < 0.0001; for exact P-values, see Supplementary Table S4).MTT assays comparing the effects of docetaxel and mocetinostat in the prostate cancer cell line DU-145 and the docetaxel-resistant subclone DU-145 DR (left). Mocetinostat treatment of DU-145 DR downregulates ZEB1, upregulates E-cadherin, miR-200, and miR-203 expression. For relative miRNA expression, the expression levels in original DU-145 were set to 1 (middle panels, the immunoblot panel derives from the same experiment shown in Fig1A). Mocetinostat sensitizes DU-145 DR to docetaxel (right). For calculation of the CI and synergy between the drugs, see Table2. n = 3, mean ± SEM, Dunnett's multiple comparisons test (P-values in the graphs are *P = 0.01-0.05; for exact P-values, see Supplementary Table S4).


Figure 5Relative tumor volume ( RTV ) of Panc1 - derived tumors in NMRI nu / nu mice . Eleven days after implantation , mice were randomized according to tumor volume . Treatment with mocetinostat ( 60 or 90 mg / kg / day ) and gemcitabine ( 25 mg / kg / day ) was implemented ( day 0 ) as depicted in the scheme . Shown are the group medians of the RTVs over time ( left ) and the individual RTVs on day 32 ( right ) . n = 5 for each treatment group ; nonparametric Mann - Whitney U - test . Representative immunohistochemical stainings of serial sections showing reduced ZEB1 and increased E - cadherin in tumor tissues of mice treated with mocetinostat . Scale bar 40 μm , inserts for higher magnifications 10 μm . Squares indicate magnified regions . Representative pictures of in situ hybridization for miR - 203 and control probe showing gain of miR - 203 and associated loss of ZEB1 detected by immunohistochemistry in serial sections of mocetinostat - treated xenograft tumors . Scale bar 40 μm , inserts for higher magnifications 5 μm . Squares indicate magnified regions . Schematic outline and results for xenografts of ex vivo treated Panc1 in Foxn1 nude mice . Panc1 cells were pre - treated with mocetinostat ( 1 μM ) and / or gemcitabine ( 50 nM ) for 48 h , followed by a 7 - day recovery period before being injected subcutaneously ( left ) . Equal numbers of viable cells were injected in 75 μl volume . At day 9 after injection , tumor growth was detectable in all groups ( lower right ) . To better visualize and compare tumor growth , the tumor volume at day 9 was set to 1 and the increasing slope of the tumor volume to day 37 is depicted ( upper right ) . The individual absolute tumor volumes on day 37 ( lower left ) and the group medians of the absolute tumor volumes over time ( lower right ) are shown . For cells pre - treated with the combination of mocetinostat and gemcitabine , tumor growth was arrested . n = 4 for each treatment group ; nonparametric Mann - Whitney U - test .

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

Figure 5Relative tumor volume (RTV) of Panc1-derived tumors in NMRI nu/nu mice. Eleven days after implantation, mice were randomized according to tumor volume. Treatment with mocetinostat (60 or 90 mg/kg/day) and gemcitabine (25 mg/kg/day) was implemented (day 0) as depicted in the scheme. Shown are the group medians of the RTVs over time (left) and the individual RTVs on day 32 (right). n = 5 for each treatment group; nonparametric Mann-Whitney U-test.Representative immunohistochemical stainings of serial sections showing reduced ZEB1 and increased E-cadherin in tumor tissues of mice treated with mocetinostat. Scale bar 40 μm, inserts for higher magnifications 10 μm. Squares indicate magnified regions.Representative pictures of in situ hybridization for miR-203 and control probe showing gain of miR-203 and associated loss of ZEB1 detected by immunohistochemistry in serial sections of mocetinostat-treated xenograft tumors. Scale bar 40 μm, inserts for higher magnifications 5 μm. Squares indicate magnified regions.Schematic outline and results for xenografts of ex vivo treated Panc1 in Foxn1 nude mice. Panc1 cells were pre-treated with mocetinostat (1 μM) and/or gemcitabine (50 nM) for 48 h, followed by a 7-day recovery period before being injected subcutaneously (left). Equal numbers of viable cells were injected in 75 μl volume. At day 9 after injection, tumor growth was detectable in all groups (lower right). To better visualize and compare tumor growth, the tumor volume at day 9 was set to 1 and the increasing slope of the tumor volume to day 37 is depicted (upper right). The individual absolute tumor volumes on day 37 (lower left) and the group medians of the absolute tumor volumes over time (lower right) are shown. For cells pre-treated with the combination of mocetinostat and gemcitabine, tumor growth was arrested. n = 4 for each treatment group; nonparametric Mann-Whitney U-test.


Figure 6A , B Relative expression levels of miR - 203 and miR - 200c in pancreatic adenocarcinomas . ( A ) In normal versus tumor tissue of the same case ( n = 6 cases ) , ( B ) in the non - recurrence ( n = 10 cases ) versus the recurrence group ( n = 11 cases ) . The mean value of the lower group in each figure was set to 1 . Nonparametric Mann - Whitney U - test .

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Figure 6A, B Relative expression levels of miR-203 and miR-200c in pancreatic adenocarcinomas. (A) In normal versus tumor tissue of the same case (n = 6 cases), (B) in the non-recurrence (n = 10 cases) versus the recurrence group (n = 11 cases). The mean value of the lower group in each figure was set to 1. Nonparametric Mann-Whitney U-test.


Figure 2Normalized RPF count profile averaged for all E . coli transcripts . Profiles generated for cells grown in the absence and presence of IPTG ( 1 mM ) . Start and stop codons are shaded . Bar chart of all measured RBS initiation rates ranked by their strength . Strong RBSs with initiation rates > 1 ribosome / s are highlighted in red . ( D ) Bar chart of all measured translation termination efficiencies at stop codons ranked by their strength . Stop codons with translation termination efficiency > 0 . 99 are highlighted in red . ( E ) Distribution of initiation rates for cells grown in the absence and presence of IPTG ( 1 mM ) . ( F ) Distribution of translation termination efficiencies for cells grown in the absence and presence of IPTG ( 1 mM ) .

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Figure 2Normalized RPF count profile averaged for all E. coli transcripts. Profiles generated for cells grown in the absence and presence of IPTG (1 mM). Start and stop codons are shaded.Bar chart of all measured RBS initiation rates ranked by their strength. Strong RBSs with initiation rates >1 ribosome/s are highlighted in red. (D) Bar chart of all measured translation termination efficiencies at stop codons ranked by their strength. Stop codons with translation termination efficiency >0.99 are highlighted in red. (E) Distribution of initiation rates for cells grown in the absence and presence of IPTG (1 mM). (F) Distribution of translation termination efficiencies for cells grown in the absence and presence of IPTG (1 mM).


Figure 3Comparison of protein synthesis rate of endogenous E . coli genes measured using Ribo - seq from this study ( in molecules / s units ) and from that by Li et al . ( Li et al , 2014 ) ( in molecules / generation units ) . Each point corresponds to a single gene and color denotes the ratio of transcription initiation rate to translation initiation rate ( giving RNAP / ribosome ) capturing whether transcription ( light yellow ) or translation ( dark blue ) is more dominant . Transcription ( bottom ) and translation ( top ) profiles for yggN , rpoH and greA , computed from the RNA - seq and Ribo - seq data without induction . Positions of the genetic parts and gene are shown below the profiles . ( C ) Promoter strengths in RNAP / s units and RBS initiation rates in ribosome / s units . Transcription ( bottom ) and translation ( top ) profiles for lacZ . Profiles are shown for cells in the absence and presence of IPTG ( 1 mM ) . Position of genetic parts and gene is shown below the profiles . RBS is omitted from the genetic design due to its size . ( E ) Measured promoter strength in RNAP / s units , RBS initiation rate in ribosomes / s units , and the transcriptional terminator and translation termination efficiency for lacZ . Data shown for cells in the absence and presence of IPTG ( 1 mM ) .

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

Figure 3Comparison of protein synthesis rate of endogenous E. coli genes measured using Ribo-seq from this study (in molecules/s units) and from that by Li et al. (Li et al, 2014) (in molecules/generation units). Each point corresponds to a single gene and color denotes the ratio of transcription initiation rate to translation initiation rate (giving RNAP/ribosome) capturing whether transcription (light yellow) or translation (dark blue) is more dominant.Transcription (bottom) and translation (top) profiles for yggN, rpoH and greA, computed from the RNA-seq and Ribo-seq data without induction. Positions of the genetic parts and gene are shown below the profiles. (C) Promoter strengths in RNAP/s units and RBS initiation rates in ribosome/s units.Transcription (bottom) and translation (top) profiles for lacZ. Profiles are shown for cells in the absence and presence of IPTG (1 mM). Position of genetic parts and gene is shown below the profiles. RBS is omitted from the genetic design due to its size. (E) Measured promoter strength in RNAP/s units, RBS initiation rate in ribosomes/s units, and the transcriptional terminator and translation termination efficiency for lacZ. Data shown for cells in the absence and presence of IPTG (1 mM).


Figure 4Translation profiles for the PK - LacZ construct in cells cultured in the absence ( bottom ) and presence ( top ) of IPTG ( 1 mM ) . The gene10 , middle , and lacZ regions are labelled above the profiles . Shaded region denotes the PK , and dashed lines denote the start codon and stop codons of gene10 and LacZ . Fraction of the total RPFs and mRNA reads in each reading frame for the gene10 , PK or middle , and lacZ regions schematically shown below and are of the PK - LacZ construct . Data shown separately for cells cultured in the absence and presence of IPTG ( 1 mM ) . Violin plots of the distributions of fractions of total RPFs and mRNA reads in each reading frame for all E . coli transcripts .

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

Figure 4Translation profiles for the PK-LacZ construct in cells cultured in the absence (bottom) and presence (top) of IPTG (1 mM). The gene10, middle, and lacZ regions are labelled above the profiles. Shaded region denotes the PK, and dashed lines denote the start codon and stop codons of gene10 and LacZ.Fraction of the total RPFs and mRNA reads in each reading frame for the gene10, PK or middle, and lacZ regions schematically shown below and are of the PK-LacZ construct. Data shown separately for cells cultured in the absence and presence of IPTG (1 mM).Violin plots of the distributions of fractions of total RPFs and mRNA reads in each reading frame for all E. coli transcripts.


Figure 5Change in expression of chromosomal genes in E . coli cells following induction of PK - lacZ expression ( 1 mM IPTG ) . Each point represents a transcript . Differentially expressed genes ( mRNA count : P < 0 . 001 and absolute log2 fold - change > 1 . 37 ; translation efficiency : P < 0 . 01 ) are highlighted in color and by an alternative point shape ( transcriptional regulation : purple cross ; translational regulation : orange open circle ) . Translation initiation rates for all E . coli RBSs in cells harboring the LacZ and PK - LacZ constructs in the absence and presence of IPTG ( 1 mM ) . Solid line shows the same initiation rate for both conditions . Dotted lines denote linear regressions for the data with no offset . Fractions of mRNA reads and RPFs mapping to each synthetic expression construct ( LacZ and PK - LacZ ) and E . coli transcripts , which are divided into three major categories : ribosomal , metabolic , and other functions . Data shown for cells cultured in the absence and presence of IPTG ( 1 mM ) .

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

Figure 5Change in expression of chromosomal genes in E. coli cells following induction of PK-lacZ expression (1 mM IPTG). Each point represents a transcript. Differentially expressed genes (mRNA count: P < 0.001 and absolute log2 fold-change > 1.37; translation efficiency: P < 0.01) are highlighted in color and by an alternative point shape (transcriptional regulation: purple cross; translational regulation: orange open circle).Translation initiation rates for all E. coli RBSs in cells harboring the LacZ and PK-LacZ constructs in the absence and presence of IPTG (1 mM). Solid line shows the same initiation rate for both conditions. Dotted lines denote linear regressions for the data with no offset.Fractions of mRNA reads and RPFs mapping to each synthetic expression construct (LacZ and PK-LacZ) and E. coli transcripts, which are divided into three major categories: ribosomal, metabolic, and other functions. Data shown for cells cultured in the absence and presence of IPTG (1 mM).


Figure 1Growth phenotypes of the wild - type ( WT , Col - 0 ) , bri1 - null mutant , and two independent ubp12i / ubp13 double mutant lines grown on 1 / 2MS medium supplemented with 10 μM DEX ( DEX medium ) for 10 days and transferred to DEX medium containing 1 μM BL or mock solution ( 0 . 1 % [ v / v ] EtOH ) for additionally 8 days . Scale bars : 10 mm . Phenotype of the WT and ubp12i / ubp13 double mutant grown on DEX medium in the presence of increasing concentrations of BL in the light for 6 days . Scale bars : 10 mm . Hypocotyl length relative to the mock control of seedlings ( B ) . Experiments were done in triplicate ( n > 15 seedlings for each line ) . Western blot ( WB ) analysis of BES1 dephosphorylation after BL treatment with the α - BES1 antibody . Total proteins were isolated from 6 - day - old seedlings grown on DEX medium in the light , either in the presence of 100 nM BL or mock . PEPC was used as a loading control . Percentage of dephosphorylated BES1 relative to the total BES1 from ( D ) ( n = 6 biological replicates ) .

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

Figure 1Growth phenotypes of the wild-type (WT, Col-0), bri1-null mutant, and two independent ubp12i/ubp13 double mutant lines grown on 1/2MS medium supplemented with 10 μM DEX (DEX medium) for 10 days and transferred to DEX medium containing 1 μM BL or mock solution (0.1% [v/v] EtOH) for additionally 8 days. Scale bars: 10 mm.Phenotype of the WT and ubp12i/ubp13 double mutant grown on DEX medium in the presence of increasing concentrations of BL in the light for 6 days. Scale bars: 10 mm.   Hypocotyl length relative to the mock control of seedlings (B). Experiments were done in triplicate (n >15 seedlings for each line).  Western blot (WB) analysis of BES1 dephosphorylation after BL treatment with the α-BES1 antibody. Total proteins were isolated from 6-day-old seedlings grown on DEX medium in the light, either in the presence of 100 nM BL or mock. PEPC was used as a loading control.   Percentage of dephosphorylated BES1 relative to the total BES1 from (D) (n = 6 biological replicates).


Figure 2Western blot analysis of BRI1 protein amount in ubp12i / ubp13 plants with the α - BRI1 antibody . Total proteins were isolated from 18 - day - old plants grown on 1 / 2MS medium supplemented with 10 μM DEX ( + ) or not ( - ) . Western blot analysis of BRI1 protein level in concanamycin A ( ConcA ) - treated plants . Eighteen - day - old plants were grown on DEX medium and exposed to 1 μM ConcA for 4 h before total protein isolation .

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Figure 2Western blot analysis of BRI1 protein amount in ubp12i/ubp13 plants with the α-BRI1 antibody. Total proteins were isolated from 18-day-old plants grown on 1/2MS medium supplemented with 10 μM DEX (+) or not (-).   Western blot analysis of BRI1 protein level in concanamycin A (ConcA)-treated plants. Eighteen-day-old plants were grown on DEX medium and exposed to 1 μM ConcA for 4 h before total protein isolation.


Figure 3Deubiquitinating activity of UBP13 against K48 - and K63 - linked ubiquitination . Diubiquitins linked through K48 or K63 ( K48 Di - Ub or K63 Di - Ub ) were incubated alone or with GST - UBP13 or GST - UBP13C207S for 18 h . Deubiquitination of the diubiquitins was analyzed by western blot with the α - ubiquitin ( α - Ub , P4D1 ) antibody . The presence of GST - UBP13 / UBP13C207S recombinant proteins was confirmed by the α - GST antibody . UBP13 deubiquitination of K63 - ubiquitinated BRI1 in vitro . Polyubiquitinated MBP - BRI1CD was generated by incubation with GST - PUB13 ( Ub reaction ) and then with GST - UBP13 or GST - UBP13C207S ( DUB reaction - MBP pull - down , input ) . After in vitro deubiquitination , MBP - BRI1CD was purified by an MBP pull - down to analyze the BRI1CD ubiquitination ( DUB reaction - MBP pull - down , SE ) . The ubiquitinated proteins were detected by western blot with an α - ubiquitin antibody ( FK2 ) . Ubiquitin chain specificity was detected with an α - K63Ub antibody ( Apu3 ) . The presence of recombinant proteins was confirmed by the α - GST and α - MBP antibodies .

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

Figure 3Deubiquitinating activity of UBP13 against K48- and K63-linked ubiquitination. Diubiquitins linked through K48 or K63 (K48 Di-Ub or K63 Di-Ub) were incubated alone or with GST-UBP13 or GST-UBP13C207S for 18 h. Deubiquitination of the diubiquitins was analyzed by western blot with the α-ubiquitin (α-Ub, P4D1) antibody. The presence of GST-UBP13/UBP13C207S recombinant proteins was confirmed by the α-GST antibody.UBP13 deubiquitination of K63-ubiquitinated BRI1 in vitro. Polyubiquitinated MBP-BRI1CD was generated by incubation with GST-PUB13 (Ub reaction) and then with GST-UBP13 or GST-UBP13C207S (DUB reaction-MBP pull-down, input). After in vitro deubiquitination, MBP-BRI1CD was purified by an MBP pull-down to analyze the BRI1CD ubiquitination (DUB reaction-MBP pull-down, SE). The ubiquitinated proteins were detected by western blot with an α-ubiquitin antibody (FK2). Ubiquitin chain specificity was detected with an α-K63Ub antibody (Apu3). The presence of recombinant proteins was confirmed by the α-GST and α-MBP antibodies.


Figure 4Growth phenotype comparison of BRI1 - mCit / bri1 / ubp12i / ubp13 and BRI125KR - mCit / bri1 / ubp12i / ubp13 . The indicated transgenic lines were grown on DEX medium for 18 days . Scale bars : 10 mm . Western blot analysis monitoring the BRI1 protein accumulation in BRI125KR - mCit / bri1 / ubp12i / ubp13 plants with the α - BRI1 antibody . Total proteins were isolated from the roots of the lines indicated ( A ) . PEPC was used as a loading control .

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Figure 4Growth phenotype comparison of BRI1-mCit/bri1/ubp12i/ubp13 and BRI125KR-mCit/bri1/ubp12i/ubp13. The indicated transgenic lines were grown on DEX medium for 18 days. Scale bars: 10 mm.Western blot analysis monitoring the BRI1 protein accumulation in BRI125KR-mCit/bri1/ubp12i/ubp13 plants with the α-BRI1 antibody. Total proteins were isolated from the roots of the lines indicated (A). PEPC was used as a loading control.


Figure 5A - D Analysis of the BRI1 vacuolar targeting on Arabidopsis seedlings expressing BRI1 ( A ) or BRI125KR ( C ) tagged with mCitrine ( mCit ) in either bri1 / UBP12 / UBP13 or bri1 / ubp12i / ubp13 background . Six - day - old seedlings were grown on DEX medium , and kept in the dark for 2 h in the presence of 50 μM CHX before imaging . Confocal images of epidermal cells from root meristem are shown . Scale bars : 10 μm . Measurements of the relative PM / intracellular BRI1 - mCit fluorescence intensity . For each line , at least 55 and 25 cells from over 25 and 15 seedlings were measured in ( B ) and ( D ) , respectively .

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Figure 5A-D Analysis of the BRI1 vacuolar targeting on Arabidopsis seedlings expressing BRI1 (A) or BRI125KR (C) tagged with mCitrine (mCit) in either bri1/UBP12/UBP13 or bri1/ubp12i/ubp13 background. Six-day-old seedlings were grown on DEX medium, and kept in the dark for 2 h in the presence of 50 μM CHX before imaging. Confocal images of epidermal cells from root meristem are shown. Scale bars: 10 μm. Measurements of the relative PM/intracellular BRI1-mCit fluorescence intensity. For each line, at least 55 and 25 cells from over 25 and 15 seedlings were measured in (B) and (D), respectively.


Figure 1A . SDS - PAGE analysis of the D391N DIS3L2 - RNA complexes . RNAs were radioactively labeled at the 5 ' end and DIS3L2 - bound RNAs were detected by autoradiography . The negative control was performed with HEK293T - Rex cells ( Control ) B . DIS3L2 CLIP reads possess oligo ( U ) 3 ' termini . Histograms of reads with 3 ' - terminal stretches of identical nucleotides . Only oligo ( U ) tails longer than 4 nucleotides are observed . C . The untemplated uridylation is mostly found in the proximity of mature 3 ' RNA ends . The heat map shows the coverage of various RNA types by uridylated reads . RNA class and number of uridylated genes / transcripts for each category are indicated on the left . The length of each mature RNA was scaled to 100 % . D . List of DIS3L2 - bound uridylated miRNAs with the number of uridylated reads identified in all three replicate CLIPs . Members of the let - 7 family are shown in bold . F . Secondary structure of pre - hsa - let - 7i , with both 5p and 3p mature products highlighted in bold . The intramolecular ligation position is indicated in dash line between nt 28 and 61 . It corresponds to cleavage positions of RNaseT1 , which leaves 3 ' - terminal guanosine residues . G . D391N DIS3L2 overexpression does not cause abundance changes of CLIPed uridylated RNAs . Scatter plot of transcript abundances estimated from RNA - seq from the cell line overexpressing D391N DIS3L2 and control HEK293T - Rex cells . CLIPed transcripts are marked in white .

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Figure 1A. SDS-PAGE analysis of the D391N DIS3L2-RNA complexes. RNAs were radioactively labeled at the 5' end and DIS3L2-bound RNAs were detected by autoradiography. The negative control was performed with HEK293T-Rex cells (Control)B. DIS3L2 CLIP reads possess oligo(U) 3' termini. Histograms of reads with 3'-terminal stretches of identical nucleotides. Only oligo(U) tails longer than 4 nucleotides are observed.C. The untemplated uridylation is mostly found in the proximity of mature 3' RNA ends. The heat map shows the coverage of various RNA types by uridylated reads. RNA class and number of uridylated genes/transcripts for each category are indicated on the left. The length of each mature RNA was scaled to 100%.D. List of DIS3L2-bound uridylated miRNAs with the number of uridylated reads identified in all three replicate CLIPs. Members of the let-7 family are shown in bold.F. Secondary structure of pre-hsa-let-7i, with both 5p and 3p mature products highlighted in bold. The intramolecular ligation position is indicated in dash line between nt 28 and 61. It corresponds to cleavage positions of RNaseT1, which leaves 3'-terminal guanosine residues.G. D391N DIS3L2 overexpression does not cause abundance changes of CLIPed uridylated RNAs. Scatter plot of transcript abundances estimated from RNA-seq from the cell line overexpressing D391N DIS3L2 and control HEK293T-Rex cells. CLIPed transcripts are marked in white.


Figure 2A . Types of uridylated ncRNAs identified by DIS3L2 CLIP - seq . B . U + read coverage in downstream regions of mature U12 snRNA , Vault1 and 7SL ncRNAs . The full dark box is representing mature ncRNA , the thin line indicates the region downstream of the mature 3 ' end ( extension ) . C . Quantification of DIS3L2 D391N RIP analysis of U12 and U5D snRNAs showing number of snRNA clones with mature ( Mat ) , extended ( ext ) or trimmed ( Trim ) 3 ' end . + U is number of clones with untemplated 3 ' oligo ( U ) ends . For more details see Figure S2 . ( D ) An example of hybrid reads ( produced by intra - strand ligation of RNaseT1 products during CLIP protocol ) for RNU5D snRNA . E . U + reads mapped to the rDNA locus by using the artificial chromosome U13369 . 1 .

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

Figure 2A. Types of uridylated ncRNAs identified by DIS3L2 CLIP-seq.B. U+ read coverage in downstream regions of mature U12 snRNA, Vault1 and 7SL ncRNAs. The full dark box is representing mature ncRNA, the thin line indicates the region downstream of the mature 3' end (extension).C. Quantification of DIS3L2 D391N RIP analysis of U12 and U5D snRNAs showing number of snRNA clones with mature (Mat), extended (ext) or trimmed (Trim) 3' end. +U is number of clones with untemplated 3' oligo(U) ends. For more details see Figure S2. (D) An example of hybrid reads (produced by intra-strand ligation of RNaseT1 products during CLIP protocol) for RNU5D snRNA.E. U+ reads mapped to the rDNA locus by using the artificial chromosome U13369.1.


Figure 3B . Uridylated tRNA reads overlap mainly with 3 ' trailers , T - loop and anticodon loop regions . Heat map showing the coverage of uridylated reads on pre - tRNAs . The pre - tRNA sub - regions ( as indicated in ( A ) ) are in small letters on the x - axis . C . Northern blot analysis of RNAs immunoprecipitated with Flag - tagged WT ( DIS3L2 ) and D391N ( MUT ) DIS3L2 , AGO2 and DIS3 , respectively . The blots were probed with radioactively labeled probes for 5 ' and 3 ' ends of mature tRNAVal , respectively . D . Sequencing of MUT DIS3L2 - bound tRNAVal and tRNALeu confirmed the uridylation status of the tRNA fragments identified by CLIP - seq . Genomic sequence for each tRNA is indicated on top with the 3 ' trailer region separated by a vertical line . Non - templated uridine residues are in red . E . Identification of hybrid reads ( produced by intra - strand ligation of RNaseT1 products during CLIP protocol ) mapping to tRNAPhe ( GAA ) and tRNAAsn ( GTT ) genes . To the right : the schematic representation of selected hybrid reads with corresponding color - code .

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Figure 3B. Uridylated tRNA reads overlap mainly with 3' trailers, T-loop and anticodon loop regions. Heat map showing the coverage of uridylated reads on pre-tRNAs. The pre-tRNA sub-regions (as indicated in (A)) are in small letters on the x-axis.C. Northern blot analysis of RNAs immunoprecipitated with Flag-tagged WT (DIS3L2) and D391N (MUT) DIS3L2, AGO2 and DIS3, respectively. The blots were probed with radioactively labeled probes for 5' and 3' ends of mature tRNAVal, respectively.D. Sequencing of MUT DIS3L2-bound tRNAVal and tRNALeu confirmed the uridylation status of the tRNA fragments identified by CLIP-seq. Genomic sequence for each tRNA is indicated on top with the 3' trailer region separated by a vertical line. Non-templated uridine residues are in red.E. Identification of hybrid reads (produced by intra-strand ligation of RNaseT1 products during CLIP protocol) mapping to tRNAPhe(GAA) and tRNAAsn(GTT) genes. To the right: the schematic representation of selected hybrid reads with corresponding color-code.


Figure 4A . DIS3L2 - bound uridylated mRNA reads map to the 5 ' and 3 ' UTRs . B . Most 3 ' UTR U + reads match the stem - loop structure of histone mRNAs . Heat map representation of the uridylation position . C . The uridylated aberrant RNAs are stabilized in cells with nonfunctional DIS3L2 . Small RNAs were modified with 3 ' end linker , and linker - specific primer was used for cDNA synthesis . Uridylated RNAs were PCR amplified in a semi - quantitative way with the same amount of input RNA used for RT reaction . We used a combination of the gene - and linker - specific primers , - RT ctrl is RT - PCR control , where no reverse transcriptase was added ; PCR control is a reaction with no cDNA added . The graph on the right summarizes the percentage of uridylated and nonuridylated 5 ' fragments of OAT mRNA in HEK293T - Rex cells ( Ctrl ) and cells overexpressing mutant DIS3L2 ( D391N ) . D . DIS3L2 - bound uridylated RNAs are degraded by DIS3L2 in vivo . RT - PCR amplification of 5 ' mRfs of OAT and RPL12 genes and 3 ' extended forms of U12 snRNA ( RNU12 ext ) from HEK293T cell lines with modified expression of DIS3L2 ( as marked on top ) was performed as described in C . Regions of mature 5S rRNA and 7SL RNA were used as loading controls . DIS3L2 KO is a DIS3L2 knock - out cell line . DIS3L2 rescue is DIS3L2 KO cells with stably integrated mutant ( D391N ) and wild type ( WT ) DIS3L2 expressing constructs , respectively . Bands marked with * are unspecific primer - dimer PCR products . E . Metagene analysis of the position of DIS3L2 D391N clipped U + reads peak maximum around mRNA transcription start sites ( TSS ) and of the ChIP - seq data for RNAP II . F . U + 5 ' mRfs and aberrant forms of U12 snRNA are highly enriched in the cytoplasm . RT - PCR detection of 5 ' mRfs of the OAT and RPL12 and aberrant form of U12 snRNA was performed as described in C with RNA isolated from nuclear and cytoplasmic fractions , respectively . The RT - PCR amplification of the body of 5S rRNA was used as a positive control for RNA isolation and RT reaction in both fractions . The identity and uridylation status of cytoplasmic OAT U + 5 ' mRfs was confirmed by sequencing ( shown on the bottom ) . In red are untemplated nucleotides , in green region corresponding to the first intron of OAT pre - mRNA . The efficiency of the subcellular fractionation was monitored by western blot ( right panel , WB ) . DDX5 is nucleoplasmic , ACO2 is mitochondrial , and DIS3L2 and TUBa are cytoplasmic markers .

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Figure 4A. DIS3L2-bound uridylated mRNA reads map to the 5' and 3' UTRs.B. Most 3' UTR U+ reads match the stem-loop structure of histone mRNAs. Heat map representation of the uridylation position.C. The uridylated aberrant RNAs are stabilized in cells with nonfunctional DIS3L2. Small RNAs were modified with 3' end linker, and linker-specific primer was used for cDNA synthesis. Uridylated RNAs were PCR amplified in a semi-quantitative way with the same amount of input RNA used for RT reaction. We used a combination of the gene- and linker-specific primers, -RT ctrl is RT-PCR control, where no reverse transcriptase was added; PCR control is a reaction with no cDNA added. The graph on the right summarizes the percentage of uridylated and nonuridylated 5' fragments of OAT mRNA in HEK293T-Rex cells (Ctrl) and cells overexpressing mutant DIS3L2 (D391N).D. DIS3L2-bound uridylated RNAs are degraded by DIS3L2 in vivo. RT-PCR amplification of 5'mRfs of OAT and RPL12 genes and 3' extended forms of U12 snRNA (RNU12 ext) from HEK293T cell lines with modified expression of DIS3L2 (as marked on top) was performed as described in C. Regions of mature 5S rRNA and 7SL RNA were used as loading controls. DIS3L2 KO is a DIS3L2 knock-out cell line. DIS3L2 rescue is DIS3L2 KO cells with stably integrated mutant (D391N) and wild type (WT) DIS3L2 expressing constructs, respectively. Bands marked with * are unspecific primer-dimer PCR products.E. Metagene analysis of the position of DIS3L2 D391N clipped U+ reads peak maximum around mRNA transcription start sites (TSS) and of the ChIP-seq data for RNAP II.F. U+5'mRfs and aberrant forms of U12 snRNA are highly enriched in the cytoplasm. RT-PCR detection of 5'mRfs of the OAT and RPL12 and aberrant form of U12 snRNA was performed as described in C with RNA isolated from nuclear and cytoplasmic fractions, respectively. The RT-PCR amplification of the body of 5S rRNA was used as a positive control for RNA isolation and RT reaction in both fractions. The identity and uridylation status of cytoplasmic OAT U+5'mRfs was confirmed by sequencing (shown on the bottom). In red are untemplated nucleotides, in green region corresponding to the first intron of OAT pre-mRNA. The efficiency of the subcellular fractionation was monitored by western blot (right panel, WB). DDX5 is nucleoplasmic, ACO2 is mitochondrial, and DIS3L2 and TUBa are cytoplasmic markers.


Figure 1 ( G ) Immunostaining of pS6 ( Ser240 / 244 ) in skin lesions from control and LL37 - induced mice . Bottom right panels , magnified images of boxed areas . DAPI staining ( blue ) indicates nuclear localization . Scale bar : 50 μm . Right panel , the quantification of relative fluorescence intensity for pS6 ( Ser240 / 244 ) in control and LL37 group ( n = 5 for each group ) . Data represent the mean ± SEM . * * P < 0 . 01 . two - tailed unpaired Student ' s t - test was used .

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Figure 1(G) Immunostaining of pS6 (Ser240/244) in skin lesions from control and LL37-induced mice. Bottom right panels, magnified images of boxed areas. DAPI staining (blue) indicates nuclear localization. Scale bar: 50 μm. Right panel, the quantification of relative fluorescence intensity for pS6 (Ser240/244) in control and LL37 group (n=5 for each group). Data represent the mean ± SEM. **P < 0.01. two-tailed unpaired Student's t-test was used.


Figure 2 ( D ) HE staining of lesional skin sections from WT and Raptor cKO mice injected with LL37 or control vehicle ( n = 6 for each group ) . Scale bar : 50 μm . ( E ) Dermal infiltrating cells were quantified ( n = 6 for each group ) for each high power field ( HPF ) . ( F ) The mRNA expression levels of Tnf - α , Il6 , Mmp9 and Vegf in skin lesions ( n = 6 for each group ) . ( I ) HE staining of lesional skin sections from LL37 or control mice topically applied with RAPA or vehicle . Scale bar : 50 μm . ( J ) Dermal infiltrating cells were quantified ( n = 6 for each group ) . Data represent the mean ± SEM . * * P < 0 . 01 . 1 - way ANOVA with Bonferroni ' s post hoc test was used .

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Figure 2(D) HE staining of lesional skin sections from WT and Raptor cKO mice injected with LL37 or control vehicle (n=6 for each group). Scale bar: 50 μm. (E) Dermal infiltrating cells were quantified (n = 6 for each group) for each high power field (HPF). (F) The mRNA expression levels of Tnf-α, Il6, Mmp9 and Vegf in skin lesions (n = 6 for each group).(I) HE staining of lesional skin sections from LL37 or control mice topically applied with RAPA or vehicle. Scale bar: 50 μm. (J) Dermal infiltrating cells were quantified (n = 6 for each group). Data represent the mean ± SEM. **P < 0.01. 1-way ANOVA with Bonferroni's post hoc test was used.


Figure 3 ( A ) The back skins of WT ( Tsc2 + / + ) and Tsc2 + / - mice were intradermally injected with LL37 or control vehicle ( n = 5 for each group ) . Pictures were taken on day 0 . 5 , 2 , 5 , 8 , 12 after the first LL37 injection . The mouse experiments were repeated for three times , and 5 mice were included in each group for each time . The results of a representative mouse experiment were displayed . Scale bar : 2 mm .

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Figure 3(A) The back skins of WT (Tsc2+/+) and Tsc2+/- mice were intradermally injected with LL37 or control vehicle (n=5 for each group). Pictures were taken on day 0.5, 2, 5, 8, 12 after the first LL37 injection. The mouse experiments were repeated for three times, and 5 mice were included in each group for each time. The results of a representative mouse experiment were displayed. Scale bar: 2 mm.


Figure 4 ( A ) The mRNA expression level of human cathelicidin ( CAMP ) in skin lesions from healthy individuals ( n = 10 ) and patients with rosacea ( n = 15 ) . Data ( G ) Immunostaining of cathelicidin in HaCaT keratinocytes treated with LL37 ( 4 μM ) ± RAPA for 24 h . DAPI staining ( blue ) indicates nuclear localization . Scale bar : 50 μm . All results are representative of at least 3 independent experiments . Data represent the mean ± SEM .

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Figure 4(A) The mRNA expression level of human cathelicidin (CAMP) in skin lesions from healthy individuals (n = 10) and patients with rosacea (n = 15). Data(G) Immunostaining of cathelicidin in HaCaT keratinocytes treated with LL37 (4 μM) ± RAPA for 24 h. DAPI staining (blue) indicates nuclear localization. Scale bar: 50 μm. All results are representative of at least 3 independent experiments. Data represent the mean ± SEM.


Figure 5 ( E ) Primary human keratinocytes expressing TLR2 shRNAs or scramble shRNA were treated with FITC - labeled LL37 or sLL37 ± PKH26 for 30 min followed by PBS washed , then live cells were analyzed by fluorescent microscope . Scale bar : 50 μm . ( F , G ) Primary human keratinocytes were incubated with sLL37 - flag or LL37 - flag for 1 h . Cell lysates were immunoprecipitated with anti - flag or anti - TLR2 antibodies , showing an interaction between LL37 and TLR2 . Medium , the medium of primary human keratinocytes treated with sLL37 - flag or LL37 - flag . All results are representative of at least 3 independent experiments .

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Figure 5(E) Primary human keratinocytes expressing TLR2 shRNAs or scramble shRNA were treated with FITC-labeled LL37 or sLL37 ± PKH26 for 30 min followed by PBS washed, then live cells were analyzed by fluorescent microscope. Scale bar: 50 μm.(F, G) Primary human keratinocytes were incubated with sLL37-flag or LL37-flag for 1 h. Cell lysates were immunoprecipitated with anti-flag or anti-TLR2 antibodies, showing an interaction between LL37 and TLR2. Medium, the medium of primary human keratinocytes treated with sLL37-flag or LL37-flag. All results are representative of at least 3 independent experiments.


Figure 6 ( E ) Immunostaining of phospho - p65 ( p - p65 ) in skin sections . Bottom right panels , magnified images of dotted line areas . Epi , epidermis . Der , dermis . Scale bar : 50 μm . ( F ) Quantification of relative fluorescence intensity for p - p65 in epidermis ( n = 6 ) . ( G ) The mRNA expression levels of NF - κB family of transcription factors ( Nfκb1 , Nfκb1 , Rela and Relb ) in mice skin lesions ( n = 6 for each group ) . ( I ) IHC of p65 on skin sections from HS and rosacea . Scale bar , 50 μm . ( J ) Percentage of nuclear p65 positive cells in the epidermis from HS ( n = 8 ) and rosacea ( n = 13 ) . All results are representative of at least 3 independent experiments . Data represent the mean ± SEM . * * P < 0 . 01 . 1 - way ANOVA with Bonferroni ' s post hoc test ( F and G ) or two - tailed unpaired Student ' s t - test ( J ) was used .

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Figure 6(E) Immunostaining of phospho-p65 (p-p65) in skin sections. Bottom right panels, magnified images of dotted line areas. Epi, epidermis. Der, dermis. Scale bar: 50 μm. (F) Quantification of relative fluorescence intensity for p-p65 in epidermis (n=6). (G) The mRNA expression levels of NF-κB family of transcription factors (Nfκb1, Nfκb1, Rela and Relb) in mice skin lesions (n=6 for each group).(I) IHC of p65 on skin sections from HS and rosacea. Scale bar, 50 μm. (J) Percentage of nuclear p65 positive cells in the epidermis from HS (n = 8) and rosacea (n = 13). All results are representative of at least 3 independent experiments. Data represent the mean ± SEM. **P < 0.01. 1-way ANOVA with Bonferroni's post hoc test (F and G) or two-tailed unpaired Student's t-test (J) was used.


Figure 7 ( B ) The mRNA expression levels of mouse chemokines ( Cxcl11 , Cxcl12 , Ccl2 and Ccl3 ) in skin lesions ( n = 6 for each group ) . ( E ) Heatmap of upregulated chemokines and cytokines in rosacea skin lesions determined by RNA - sequencing . All results are representative of at least 3 independent experiments . Data represent the mean ± SEM . * P < 0 . 05 , * * P < 0 . 01 . 1 - way ANOVA with Bonferroni ' s post hoc test was used .

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Figure 7(B) The mRNA expression levels of mouse chemokines (Cxcl11, Cxcl12, Ccl2 and Ccl3) in skin lesions (n=6 for each group).(E) Heatmap of upregulated chemokines and cytokines in rosacea skin lesions determined by RNA-sequencing. All results are representative of at least 3 independent experiments. Data represent the mean ± SEM. *P < 0.05, **P < 0.01. 1-way ANOVA with Bonferroni's post hoc test was used.


Figure 8Topical rapamycin treatment twice daily for 4 weeks showed an obvious therapeutic effect in rosacea patients . ( C ) The change of patients ' CEA score showed in scatter histogram at 0 week and 4th week . ( D ) The change of patients ' IGA score showed in scatter histogram at 0 week and 4th week . " The Change value < 0 " indicates that skin lesions show positive improvement after treatment ; " The Change value > 0 " indicates skin lesions show exacerbation after treatment ; " The Change value = 0 " indicates that skin lesions show no change after treatment ; Data represent the mean ± SEM , and two - tailed unpaired Student ' s t - test was used ( C , D ) .

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Figure 8Topical rapamycin treatment twice daily for 4 weeks showed an obvious therapeutic effect in rosacea patients. (C) The change of patients' CEA score showed in scatter histogram at 0 week and 4th week. (D) The change of patients' IGA score showed in scatter histogram at 0 week and 4th week. "The Change value < 0" indicates that skin lesions show positive improvement after treatment; "The Change value > 0" indicates skin lesions show exacerbation after treatment; "The Change value = 0" indicates that skin lesions show no change after treatment; Data represent the mean ± SEM, and two-tailed unpaired Student's t-test was used (C, D).


Figure 1RAW 264 . 7 mouse macrophages were treated with either oxLDL ( 50 µg / ml ) or TG ( 300 nM ) for 6 hours . Protein lysates were treated with λ Phosphatase ( PPase ) for 30 minutes and analyzed by western blotting using specific antibodies for pFMRP , FMRP , pIRE1 , IRE1 and β - Actin . pFMRP / FMRP fold induction is depicted above the blots ( n = 6 biological replicates ) . Control - or IRE1 - siRNA transfected HEK293T cells were stimulated by either PA ( 500 µM ) or TG ( 600 nM ) for 4 hours . Protein lysates were analyzed by western blotting using specific antibodies for pFMRP , FMRP , pIRE1 , IRE1 and β - Actin . pFMRP / FMRP fold induction is depicted above the blots ( n = 4 biological replicates ) . MEF cells were transfected with either empty vector , EGFP - FMRP or 3xFLAG - IRE1 plasmids then pre - treated either with vehicle ( dimethyl sulfoxide , DMSO ) or AMG - 18 ( 25 µM ; 1 hour ) followed by TG ( 600 nM ) stimulation for 4 hours . Protein lysates were analyzed by western blotting using specific antibodies for pFMRP , FMRP , pIRE1 , IRE1 and β - Actin . pFMRP / FMRP fold induction is depicted above the blots ( n = 4 biological replicates ) . Purified FMRP and IRE1 kinase ( activated ) proteins were subjected to kinase assay and analyzed by western blotting using specific antibodies for ThioP , IRE1 and FMRP ( n = 3 biological replicates ) and with LC - MS / MS . Identified IRE1 kinase - mediated FMRP phosphorylation sites ( bottom ) .

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Figure 1RAW 264.7 mouse macrophages were treated with either oxLDL (50 µg/ml) or TG (300 nM) for 6 hours. Protein lysates were treated with λ Phosphatase (PPase) for 30 minutes and analyzed by western blotting using specific antibodies for pFMRP, FMRP, pIRE1, IRE1 and β-Actin. pFMRP/FMRP fold induction is depicted above the blots (n=6 biological replicates).Control- or IRE1-siRNA transfected HEK293T cells were stimulated by either PA (500 µM) or TG (600 nM) for 4 hours. Protein lysates were analyzed by western blotting using specific antibodies for pFMRP, FMRP, pIRE1, IRE1 and β-Actin. pFMRP/FMRP fold induction is depicted above the blots (n=4 biological replicates).MEF cells were transfected with either empty vector, EGFP-FMRP or 3xFLAG-IRE1 plasmids then pre-treated either with vehicle (dimethyl sulfoxide, DMSO) or AMG-18 (25 µM; 1 hour) followed by TG (600 nM) stimulation for 4 hours. Protein lysates were analyzed by western blotting using specific antibodies for pFMRP, FMRP, pIRE1, IRE1 and β-Actin. pFMRP/FMRP fold induction is depicted above the blots (n=4 biological replicates).Purified FMRP and IRE1 kinase (activated) proteins were subjected to kinase assay and analyzed by western blotting using specific antibodies for ThioP, IRE1 and FMRP (n=3 biological replicates) and with LC-MS/MS. Identified IRE1 kinase-mediated FMRP phosphorylation sites (bottom).


Figure 2Fmr1 + / + and Fmr1 - / - mice were injected with AAV _ PCSK9 and fed with 16 weeks of WD . Residential PM were stained with Oil Red O ( ORO ) and imaged ( n = 7 mice per group ; Scale bar = 50 µm ) . Apoe - / - mice were fed with WD ( 12 weeks ) and injected with vehicle ( DMSO ) or AMG - 18 ( 30 mg / kg / day ) in the last 4 weeks of WD . Residential PM were stained with ORO and imaged ( n = 5 mice per group ; Scale bar = 50 µm ) . RCT experiment ( I ) plasma cholesterol levels after 24 and 48 hours , ( J ) liver cholesterol levels after 48 hours , and ( K ) feces cholesterol levels after 48 hours ( n = 12 mice per group ) .

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Figure 2Fmr1+/+ and Fmr1-/- mice were injected with AAV_PCSK9 and fed with 16 weeks of WD. Residential PM were stained with Oil Red O (ORO) and imaged (n=7 mice per group; Scale bar = 50 µm).    Apoe-/- mice were fed with WD (12 weeks) and injected with vehicle (DMSO) or AMG-18 (30 mg/kg/day) in the last 4 weeks of WD. Residential PM were stained with ORO and imaged (n=5 mice per group; Scale bar = 50 µm).  RCT experiment (I) plasma cholesterol levels after 24 and 48 hours, (J) liver cholesterol levels after 48 hours, and (K) feces cholesterol levels after 48 hours (n=12 mice per group).


Figure 3A - E In vitro and in vivo efferocytosis experiments , where percentage of macrophages F4 / 80 + ( red ) that ingested apoptotic cells ( AC ) labeled with carboxyfluorescein succinimidyl ester ( CFSE ) + ( green ) were reported as % efferocytosis . ( A ) BMDMs were transfected with Fmr1 - or control - siRNA and incubated CFSE - labeled AC for the indicated hours ( n = 4 biological replicates ) . ( B ) Fmr1 + / + and Fmr1 - / - BMDMs were treated with PA ( 500 µM ) for 6 hours and then incubated with CFSE - labeled ACs for 4 hours ( n = 4 biological replicates ) . ( C ) Fmr1 + / + and Fmr1 - / - mice were fed WD ( 16 weeks ) and injected intraperitoneally with CFSE - labeled AC ( 1 . 5 hours ) , followed by PM elicitation ( n = 4 - 5 mice per group ) . ( D ) BMDM were pre - treated either with vehicle ( DMSO ) or AMG - 18 ( 5 µM ) for 1 hour then incubated with CFSE - labeled ACs for 4 hours ( n = 3 biological replicates ) . ( E ) C57BL / 6 mice were injected with AMG - 18 ( 30 mg / kg ) or vehicle ( DMSO ) for 8 hours , followed by intraperitoneal injection with CFSE - labeled ACs for 1 . 5 hours and PM elicitation ( n = 4 mice per group ) . F - G In vitro continuous efferocytosis experiments , where macrophages were stained for F4 / 80 + ( red ) , AC were labeled with CFSE ( AC - 1 ; green ) or Violet ( AC2 ; violet ) . % continuous efferocytosis was determined by the ratio of F4 / 80 + , CFSE + and Violet + ( triple positive ) cells to total F4 / 80 + and CFSE + ( double positive ) cells . ( F ) Fmr1 + / + and Fmr1 - / - BMDM were incubated with AC - 1 for 2 hours , and after 2 hours interval , incubated with AC - 2 for 2 more hours ( n = 4 - 3 biological replicates ) . ( G ) BMDM were pre - treated either with vehicle ( DMSO ) or AMG - 18 ( 5 µM ) for 1 hour , incubated with CFSE - labeled AC - 1 for 2 hours , followed by incubation with Violet - labeled AC - 2 for 2 hours and PM collection ( n = 4 biological replicates ) .

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Figure 3A-E In vitro and in vivo efferocytosis experiments, where percentage of macrophages F4/80+ (red) that ingested apoptotic cells (AC) labeled with carboxyfluorescein succinimidyl ester (CFSE)+ (green) were reported as % efferocytosis. (A) BMDMs were transfected with Fmr1- or control-siRNA and incubated CFSE-labeled AC for the indicated hours (n=4 biological replicates). (B) Fmr1+/+ and Fmr1-/- BMDMs were treated with PA (500 µM) for 6 hours and then incubated with CFSE-labeled ACs for 4 hours (n=4 biological replicates). (C) Fmr1+/+ and Fmr1-/- mice were fed WD (16 weeks) and injected intraperitoneally with CFSE-labeled AC (1.5 hours), followed by PM elicitation (n=4-5 mice per group). (D) BMDM were pre-treated either with vehicle (DMSO) or AMG-18 (5 µM) for 1 hour then incubated with CFSE-labeled ACs for 4 hours (n=3 biological replicates). (E) C57BL/6 mice were injected with AMG-18 (30 mg/kg) or vehicle (DMSO) for 8 hours, followed by intraperitoneal injection with CFSE-labeled ACs for 1.5 hours and PM elicitation (n=4 mice per group).F-G In vitro continuous efferocytosis experiments, where macrophages were stained for F4/80+ (red), AC were labeled with CFSE (AC-1; green) or Violet (AC2; violet). % continuous efferocytosis was determined by the ratio of F4/80+, CFSE+ and Violet+ (triple positive) cells to total F4/80+ and CFSE+ (double positive) cells. (F) Fmr1+/+ and Fmr1-/- BMDM were incubated with AC-1 for 2 hours, and after 2 hours interval, incubated with AC-2 for 2 more hours (n=4-3 biological replicates). (G) BMDM were pre-treated either with vehicle (DMSO) or AMG-18 (5 µM) for 1 hour, incubated with CFSE-labeled AC-1 for 2 hours, followed by incubation with Violet-labeled AC-2 for 2 hours and PM collection (n=4 biological replicates).


Figure 4RNA lysates from Fmr1 + / + and Fmr1 - / - BMDM that were treated with PA ( 500 µM ; 6 hours ) were fractionated using a 10 % - 50 % sucrose gradient and separated to polysome , monosome / NTR fractions . The absorbance ( 260 nm ) of RNA was measured and plotted as a function of time ( n = 3 biological replicates ) . ( B ) The ratio of the Abca1 , Abcg1 , Mertk , Lrp1 , Cd36 , Cd47 and Rac1 mRNA in polysome to NTR fraction ( n = 3 biological replicates ) . D Fmr1 - / - MEF cells were transfected with EV , WT - FMRP or STSA - FMRP plasmids followed by PA treatment ( 500 µM ; 6 hours ) . Protein lysates were analyzed by western blotting using specific antibodies for ABCA1 , MerTK , LRP1 , pFMRP , FMRP and β - Actin and fold inductions relative to β - Actin are depicted above the blots ( n = 5 biological replicates ) .

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Figure 4RNA lysates from Fmr1+/+ and Fmr1-/- BMDM that were treated with PA (500 µM; 6 hours) were fractionated using a 10%-50% sucrose gradient and separated to polysome, monosome/NTR fractions. The absorbance (260 nm) of RNA was measured and plotted as a function of time (n=3 biological replicates). (B) The ratio of the Abca1, Abcg1, Mertk, Lrp1, Cd36, Cd47 and Rac1 mRNA in polysome to NTR fraction (n=3 biological replicates).D Fmr1-/- MEF cells were transfected with EV, WT-FMRP or STSA-FMRP plasmids followed by PA treatment (500 µM; 6 hours). Protein lysates were analyzed by western blotting using specific antibodies for ABCA1, MerTK, LRP1, pFMRP, FMRP and β-Actin and fold inductions relative to β-Actin are depicted above the blots (n=5 biological replicates).


Figure 5Lesion area calculated from en face aorta , stained with ORO ( n = 12 - 13 mice per group ; Scale bar = 5 mm ) . Lesion area calculated from en face aorta , stained with ORO ( n = 9 mice per group ; Scale bar = 5 mm ) .

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Figure 5Lesion area calculated from en face aorta, stained with ORO (n=12-13 mice per group; Scale bar = 5 mm).Lesion area calculated from en face aorta, stained with ORO (n=9 mice per group; Scale bar = 5 mm).


Figure 6Lesion area calculated from en face aorta , stained with ORO ( n = 5 mice per group ; Scale bar = 5 mm ) . Lesion area calculated from en face aorta , stained with ORO ( n = 6 mice per group ; Scale bar = 5 mm ) .

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Figure 6Lesion area calculated from en face aorta, stained with ORO (n=5 mice per group; Scale bar = 5 mm).Lesion area calculated from en face aorta, stained with ORO (n=6 mice per group; Scale bar = 5 mm).


Figure 1Coronal and axial brain MRI of the patient with compound heterozygote mutations in ECE2 shows nodules of heterotopic neurons lining the lateral ventricles . ECE2 expression on RNA level . In situ hybridisation for ECE2 RNA in 50d old cerebral organoids ( COs ) shows higher signal in the cortical plate - like zone ( CP ' ) ( Scalebar = 100 µm ) . Ventricle - like lumen in COs is marked as V ' . ECE2 expression on protein level . Immunohistochemistry ( IHC ) for ECE2 in 50d old COs shows accumulation in the CP ' and at the apical surface ( Scalebar = 50µm ) . Ventricle - like lumen in COs is marked as V ' . COs transfected with micro - RNAs targeting ECE2 ( KD ) or scrambled negative control ( CTRL ) and GFP and analysed 7 days later reveal an increase in ectopic neurons upon ECE2 KD ( Transfected cells are shown in green , NEUN + neuronal nuclei in magenta . d = day . Scalebar = 25 µm ) . Ventricle - like lumen in COs is marked as V ' . Graph depicting the number of ectopically located NEUN + cells in CTRL and KD electroporated COs using two different micro - RNAs targeting ECE2 . Data shown as box plot ( mean = red line , median = black line , box represents 25th and 75th percentiles , whiskers extend to 10th and 90th percentiles , all outliers are shown ; n = number of ventricles analysed ; * * * P < 0 . 001 in Kruskal - Wallis One Way ANOVA on Ranks and Dunn ' s Pairwise Multiple Comparison ) . Scheme depicting the experimental setup of neuronal differentiation and timelapse imaging under treatment with the ECE2 - inhibitor PHOS [ 42 ] . Graphs showing different parameters of neuronal migration dynamics , tracked via live imaging . Upon ECE2 inhibition , significant decrease in velocity , increase in resting TP and increase in tortuosity are observed . Data shown as z - scores ( CTRL = DMSO control ; PHOS = Phosphoramidon ; Resting TP = Resting timepoints ; N = number of individual neurons analysed from 2 batches ; Resting TP : * P = 0 . 024 ; tortuosiy : * P = 0 . 035 , * * * P < 0 . 001 in 2 - tailed Chi - Sqare test ) .

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Figure 1Coronal and axial brain MRI of the patient with compound heterozygote mutations in ECE2 shows nodules of heterotopic neurons lining the lateral ventricles.ECE2 expression on RNA level. In situ hybridisation for ECE2 RNA in 50d old cerebral organoids (COs) shows higher signal in the cortical plate-like zone (CP') (Scalebar=100 µm). Ventricle-like lumen in COs is marked as V'.ECE2 expression on protein level. Immunohistochemistry (IHC) for ECE2 in 50d old COs shows accumulation in the CP' and at the apical surface (Scalebar=50µm). Ventricle-like lumen in COs is marked as V'.COs transfected with micro-RNAs targeting ECE2 (KD) or scrambled negative control (CTRL) and GFP and analysed 7 days later reveal an increase in ectopic neurons upon ECE2 KD (Transfected cells are shown in green, NEUN+ neuronal nuclei in magenta. d=day. Scalebar=25 µm). Ventricle-like lumen in COs is marked as V'.Graph depicting the number of ectopically located NEUN+ cells in CTRL and KD electroporated COs using two different micro-RNAs targeting ECE2. Data shown as box plot (mean = red line, median = black line, box represents 25th and 75th percentiles, whiskers extend to 10th and 90th percentiles, all outliers are shown; n=number of ventricles analysed; ***P < 0.001 in Kruskal-Wallis One Way ANOVA on Ranks and Dunn's Pairwise Multiple Comparison).Scheme depicting the experimental setup of neuronal differentiation and timelapse imaging under treatment with the ECE2-inhibitor PHOS [42].Graphs showing different parameters of neuronal migration dynamics, tracked via live imaging. Upon ECE2 inhibition, significant decrease in velocity, increase in resting TP and increase in tortuosity are observed. Data shown as z-scores (CTRL = DMSO control; PHOS = Phosphoramidon; Resting TP = Resting timepoints; N=number of individual neurons analysed from 2 batches; Resting TP: *P=0.024; tortuosiy: *P=0.035, ***P < 0.001 in 2-tailed Chi-Sqare test).


Figure 2Quantification of distribution of GFP + transfected cells 1dpe upon CTRL and Ece2 KD by IUE ( n = 3 CTRL and 4 Ece2 KD brains ; * P = 0 . 044 in One - way ANOVA and Tukey ' s Multiple Pairwise Comparison ; data shown as mean ± SEM ) . IHC staining of GFP ( transfected cells ) and Pax6 1 dpe showing ectopic Pax6 + progenitors ( red circle ) and a reduction in their apical localisation ( yellow circle ) . High magnification images of GFP and Pax6 showing that ectopic progenitors are mostly not transfected . D - J , E13 - 16 Ece2 KD . GFP - Pax6 + progenitors delaminate upon Ece2 KD ( red arrows ) , leaving behind a region free of Pax6 + cells in the VZ ( yellow circle ) and forming ectopic rosettes ( yellow box ) . Reduction in apical Arl13b in the electroporation site ( white arrowheads ) indicates loss of apical belt integrity and disruption of apico - basal polarity upon Ece2 KD . Inside the nodule , which is surrounded by thickened GFP + processes ( pink arrows ) , Arl13b staining shows the formation of an ectopic apical surfaceReduction in β - catenin in the electroporation site ( white arrowheads ) indicates loss of apical belt integrity and disruption of apico - basal polarity upon Ece2 KD . , Deep layer and I , upper layer neurons localise next to ectopic rosettes , forming nodules of ectopic neurons ( blue arrowheads for single ectopic neurons and blue circle for large nodule ) . Quantification at 3 dpe , counting the proportion of brains with reduced density of Pax6 in the VZ , ectopic progenitors or neurons , and changes in apico - basal polarity ( including ectopic apical surface formation and reduction in apically localised apical markers ; n = 6 brains per condition ; Exact binomial test : * * * * P < 0 . 0001 ) . Scalebars in A , B , D , E , H , I = 100 µm ; in C , F , G Scalebar = 50 µm . V marks the position of the ventricle .

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Figure 2Quantification of distribution of GFP+ transfected cells 1dpe upon CTRL and Ece2 KD by IUE (n=3 CTRL and 4 Ece2 KD brains; *P=0.044 in One-way ANOVA and Tukey's Multiple Pairwise Comparison; data shown as mean±SEM).IHC staining of GFP (transfected cells) and Pax6 1 dpe showing ectopic Pax6+ progenitors (red circle) and a reduction in their apical localisation (yellow circle).High magnification images of GFP and Pax6 showing that ectopic progenitors are mostly not transfected. D-J, E13-16 Ece2 KD.GFP-Pax6+ progenitors delaminate upon Ece2 KD (red arrows), leaving behind a region free of Pax6+ cells in the VZ (yellow circle) and forming ectopic rosettes (yellow box).Reduction in apical Arl13b in the electroporation site (white arrowheads) indicates loss of apical belt integrity and disruption of apico-basal polarity upon Ece2 KD.Inside the nodule, which is surrounded by thickened GFP+ processes (pink arrows), Arl13b staining shows the formation of an ectopic apical surfaceReduction in β-catenin in the electroporation site (white arrowheads) indicates loss of apical belt integrity and disruption of apico-basal polarity upon Ece2 KD., Deep layer and I, upper layer neurons localise next to ectopic rosettes, forming nodules of ectopic neurons (blue arrowheads for single ectopic neurons and blue circle for large nodule).Quantification at 3 dpe, counting the proportion of brains with reduced density of Pax6 in the VZ, ectopic progenitors or neurons, and changes in apico-basal polarity (including ectopic apical surface formation and reduction in apically localised apical markers; n=6 brains per condition; Exact binomial test: ****P < 0.0001). Scalebars in A, B, D, E, H, I=100 µm; in C, F, G Scalebar = 50 µm. V marks the position of the ventricle.


Figure 3Brightfield images of 60d old CTRL and ECE2 KO COs show normal formation of neuroepithelial structures . ISH for ECE2 confirms the absence of ECE2 mRNA in 60d old ECE2 KO COs ( pink dotted line indicates position of the CP ' ) . , FACS analysis of 60d old ECE2 KO COs reveals an increase in progenitors and decrease in neurons ( n = 7 - 8 samples of 3 pooled organoids each from 3 different batches ; * * P < 0 . 01 ; * * * P < 0 . 001 in One Way ANOVA and Tukey Pairwise Multiple Comparison ) . Example images of IHC for PH3 + cells in 60d old CTRL and ECE2 KO COs . Quantification of PH3 + cycling cells in 60d old COs , normalised to length of the apical surface of ventricle - like structures , shows an increase upon ECE2 KO ( Data shown as box plots with median as black line , mean as red line ; n = number of analysed ventricles from 2 batches of COs ; One Way ANOVA : * * P = 0 . 004 ) . In ECE2 KO COs , more ventricles are disorganised in terms of neuronal localisation as revealed from MAP2 + cell bodies in the progenitor zone ( n = number of ventricles from 2 independent batches ; two - tailed Mann Whitney U ( U = 458 ) and chi - square test : χ2 ( 2 ) = 25 . 42 , * * * * P < 0 . 0001 ) .

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Figure 3Brightfield images of 60d old CTRL and ECE2 KO COs show normal formation of neuroepithelial structures.ISH for ECE2 confirms the absence of ECE2 mRNA in 60d old ECE2 KO COs (pink dotted line indicates position of the CP')., FACS analysis of 60d old ECE2 KO COs reveals an increase in progenitors and decrease in neurons (n=7-8 samples of 3 pooled organoids each from 3 different batches; **P < 0.01; ***P < 0.001 in One Way ANOVA and Tukey Pairwise Multiple Comparison).Example images of IHC for PH3+ cells in 60d old CTRL and ECE2 KO COs. Quantification of PH3+ cycling cells in 60d old COs, normalised to length of the apical surface of ventricle-like structures, shows an increase upon ECE2 KO (Data shown as box plots with median as black line, mean as red line; n=number of analysed ventricles from 2 batches of COs; One Way ANOVA: **P = 0.004).In ECE2 KO COs, more ventricles are disorganised in terms of neuronal localisation as revealed from MAP2+ cell bodies in the progenitor zone (n=number of ventricles from 2 independent batches; two-tailed Mann Whitney U (U = 458) and chi-square test: χ2(2)=25.42, ****P < 0.0001).