( D - G ) GSEA analysis of 327 repressed ( D ) or 330 activated ( F ) Pax5 target genes identified in pro - B cells as compared with the ranked log2 - fold gene expression changes in Pax5Jak2 / + ( PJ ) B - ALLs versus control ( Ctrl ) Pax5 + / - Cdkn2ab + / - B - ALLs ( left ) . NES , normalized enrichment score . The expression of five genes that are upregulated in Pax5Jak2 / + B - ALL cells ( upper row ) and Pax5 - / - ( KO ) pro - B cells ( lower row ) is shown in ( E ) . Likewise , the expression of five genes that are upregulated in control B - ALL cells ( upper row ) and Pax5 + / + ( WT ) pro - B cells ( lower row ) is shown in ( G ) . TPM , transcripts per million . Mean TPM values with SEM are shown for the following RNA - seq experiments : 3 ( WT pro - B ) , 2 ( KO pro - B ) , 2 ( Ctrl B - ALL ) , and 4 ( PJ B - ALL ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-G) GSEA analysis of 327 repressed (D) or 330 activated (F) Pax5 target genes identified in pro-B cells as compared with the ranked log2-fold gene expression changes in Pax5Jak2/+ (PJ) B-ALLs versus control (Ctrl) Pax5+/- Cdkn2ab+/- B-ALLs (left). NES, normalized enrichment score. The expression of five genes that are upregulated in Pax5Jak2/+ B-ALL cells (upper row) and Pax5-/- (KO) pro-B cells (lower row) is shown in (E). Likewise, the expression of five genes that are upregulated in control B-ALL cells (upper row) and Pax5+/+ (WT) pro-B cells (lower row) is shown in (G). TPM, transcripts per million. Mean TPM values with SEM are shown for the following RNA-seq experiments: 3 (WT pro-B), 2 (KO pro-B), 2 (Ctrl B-ALL), and 4 (PJ B-ALL).


( H ) Expression of the Ptprc ( CD45 ) gene from exon 3 to exon 8 , as determined by RNA - seq of Pax5Jak2 / + and Pax5 + / - Cdkn2ab + / - B - ALL cells . The alternatively spliced exons 4 ( A ) , 5 ( B ) and 6 ( C ) are indicated in orange in the respective exon - intron structure of the Ptprc gene . Ptprc transcripts of B220 + Pax5 + / - Cdkn2ab + / - B - ALL cells contain all three exons , thus giving rise to expression of the CD45 isoform RABC ( known as B220 ) . In contrast , reads at exon 4 are barely detectable and reads at exon 6 are strongly reduced in the mRNA of Pax5Jak2 / + B - ALL cells , which likely gives rise to the CD45 isoforms RBC and

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

(H) Expression of the Ptprc (CD45) gene from exon 3 to exon 8, as determined by RNA-seq of Pax5Jak2/+ and Pax5+/- Cdkn2ab+/- B-ALL cells. The alternatively spliced exons 4 (A), 5 (B) and 6 (C) are indicated in orange in the respective exon-intron structure of the Ptprc gene. Ptprc transcripts of B220+ Pax5+/- Cdkn2ab+/- B-ALL cells contain all three exons, thus giving rise to expression of the CD45 isoform RABC (known as B220). In contrast, reads at exon 4 are barely detectable and reads at exon 6 are strongly reduced in the mRNA of Pax5Jak2/+ B-ALL cells, which likely gives rise to the CD45 isoforms RBC and RB


( D ) Flow - cytometric analysis of bone marrow cells from 4 - week - old Pax5ihCd2 / + and Pax5Jak2 / ihCd2 mice . The expression of human ( h ) CD2 from the Pax5ihCd2 allele is shown for CD19 + B220 + ( grey ) and CD19 + B220low ( black ) B cells .

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

(D) Flow-cytometric analysis of bone marrow cells from 4-week-old Pax5ihCd2/+ and Pax5Jak2/ihCd2 mice. The expression of human (h) CD2 from the Pax5ihCd2 allele is shown for CD19+B220+ (grey) and CD19+B220low (black) B cells.


( G ) Kaplan - Meier survival analysis of Cd79a - Cre Ikzf1neo / + Pax5LSL - Jak2 / + ( black ) and Cd79a - Cre Pax5LSL - Jak2 / + ( grey ) mice . Statistical analysis of the survival curves was performed with the log - rank ( Mantel - Cox ) test ; * * * * P < 0 . 0001 . n , number of mice analyzed .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Kaplan-Meier survival analysis of Cd79a-Cre Ikzf1neo/+ Pax5LSL-Jak2/+ (black) and Cd79a-Cre Pax5LSL-Jak2/+ (grey) mice. Statistical analysis of the survival curves was performed with the log-rank (Mantel-Cox) test; ****P < 0.0001. n, number of mice analyzed.


( H ) Flow - cytometric analysis of B220 and CD19 expression in B - ALL tumor cells from the lymph node of a Cd79a - Cre Ikzf1neo / + Pax5LSL - Jak2 / + mouse ( black ; left ) . Pax5 expression in these B - ALL tumor cells ( black line ) and control Pax5 + / + lymph node B cells ( grey filled ) was determined by intracellular Pax5 staining ( right ) .

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

(H) Flow-cytometric analysis of B220 and CD19 expression in B-ALL tumor cells from the lymph node of a Cd79a-Cre Ikzf1neo/+ Pax5LSL-Jak2/+ mouse (black; left). Pax5 expression in these B-ALL tumor cells (black line) and control Pax5+/+ lymph node B cells (grey filled) was determined by intracellular Pax5 staining (right).


( B , Flow - cytometric analysis of bone marrow and spleen from Pax5Jak2 - KD / + ( black ; n = 11 ) and control Pax5 + / + ( grey ; n = 7 ) mice at the age of 6 - 8 weeks . The frequencies of the indicated B cell types are shown for each organ ( B ) . The data are presented as mean percentages with SEM and were statistically analyzed by multiple t - tests ( unpaired and two - tailed with Holm - Šídák ' s correction ) ; ns ( P > 0 . 5 ) , * P < 0 . 05 , * * P < 0 . 01 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Flow-cytometric analysis of bone marrow and spleen from Pax5Jak2-KD/+ (black; n = 11) and control Pax5+/+ (grey; n = 7) mice at the age of 6-8 weeks. The frequencies of the indicated B cell types are shown for each organ (B). The data are presented as mean percentages with SEM and were statistically analyzed by multiple t-tests (unpaired and two-tailed with Holm-Šídák's correction); ns (P > 0.5), *P < 0.05, **P < 0.01.


( D , Tumor progression in mice , transplanted with Pax5Jak2 - Luc / + tumor cells , in the presence or absence of the JAK1 / 2 inhibitor ruxolitinib . At day 14 after cell transfer , the transplanted mice were treated twice daily with ruxolitinib or vehicle , and the tumor mass was monitored by bioluminescence measurements . Images of three representative mice with or without ruxolitinib treatment are

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

(D, Tumor progression in mice, transplanted with Pax5Jak2-Luc/+ tumor cells, in the presence or absence of the JAK1/2 inhibitor ruxolitinib. At day 14 after cell transfer, the transplanted mice were treated twice daily with ruxolitinib or vehicle, and the tumor mass was monitored by bioluminescence measurements. Images of three representative mice with or without ruxolitinib treatment are shown


( F ) Kaplan - Meier survival analysis of the transplanted mice upon prolonged treatment with ruxolitinib ( black ) and vehicle ( grey ) . Statistical analysis of the survival curves ( F ) was performed with the log - rank ( Mantel - Cox ) test ; * * P < 0 . 01 .

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

(F) Kaplan-Meier survival analysis of the transplanted mice upon prolonged treatment with ruxolitinib (black) and vehicle (grey). Statistical analysis of the survival curves (F) was performed with the log-rank (Mantel-Cox) test; **P < 0.01.


( B ) Genome - wide binding of Pax5 - Jak2 in in vitro cultured pro - B cells from Pax5Jak2 / + Rosa26BirA / + mice at the age of 3 weeks ( expressing Pax5 ) and ex vivo Pax5Jak2 / + Rosa26BirA / + B - ALL tumors ( lacking Pax5 ) , as determined by Bio - ChIP - seq analysis The DNA - binding pattern of full - length Pax5 was determined by Bio - ChIP - seq analysis of ex vivo sorted Pax5Bio / Bio pro - B cells , which carried a C ‐ terminal biotin acceptor sequence together with an IRES ‐ BirA gene insertion in the 3 ' untranslated region of Pax5 Two independent Bio - ChIP - seq experiments were performed for each cell type . Representative binding patterns of Pax5 and Pax5 - Jak2 in the three B cell types are shown for a selected genomic region , with horizontal bars indicating Pax5 or Pax5 - Jak2 peaks that were identified by MACS peak calling ( left ) . The number of Pax5 ( white ) and Pax5 - Jak2 ( grey or black ) peaks , which were defined by stringent MACS peak calling with a P value of < 10 - 10 in the three B cell types , are shown to the right .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Genome-wide binding of Pax5-Jak2 in in vitro cultured pro-B cells from Pax5Jak2/+ Rosa26BirA/+ mice at the age of 3 weeks (expressing Pax5) and ex vivo Pax5Jak2/+ Rosa26BirA/+ B-ALL tumors (lacking Pax5), as determined by Bio-ChIP-seq analysis The DNA-binding pattern of full-length Pax5 was determined by Bio-ChIP-seq analysis of ex vivo sorted Pax5Bio/Bio pro-B cells, which carried a C‐terminal biotin acceptor sequence together with an IRES‐BirA gene insertion in the 3' untranslated region of Pax5 Two independent Bio-ChIP-seq experiments were performed for each cell type. Representative binding patterns of Pax5 and Pax5-Jak2 in the three B cell types are shown for a selected genomic region, with horizontal bars indicating Pax5 or Pax5-Jak2 peaks that were identified by MACS peak calling (left). The number of Pax5 (white) and Pax5-Jak2 (grey or black) peaks, which were defined by stringent MACS peak calling with a P value of < 10-10 in the three B cell types, are shown to the right.


( F , G ) Flow - cytometric analysis of the bone marrow and spleen from Pax5Prd * - Jak2 / + ( black ; n = 6 ) and control Pax5 + / + ( grey ; n = 5 ) mice at the age of 6 - 7 weeks . The frequency of the indicated B cell types in each organ is indicated in ( F ) , and the flow - cytometric analysis of CD19 and B220 expression on bone marrow B cells is shown in ( G ) . Statistical data are shown as mean percentages with SEM and were analyzed by multiple t - tests ( unpaired and two - tailed with Holm - Šídák ' s correction ) : ns > 0 . 05 ; * P < 0 . 05 ; * * P < 0 . 01 . Each dot corresponds to one mouse .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, G) Flow-cytometric analysis of the bone marrow and spleen from Pax5Prd*-Jak2 /+ (black; n = 6) and control Pax5+/+ (grey; n = 5) mice at the age of 6-7 weeks. The frequency of the indicated B cell types in each organ is indicated in (F), and the flow-cytometric analysis of CD19 and B220 expression on bone marrow B cells is shown in (G). Statistical data are shown as mean percentages with SEM and were analyzed by multiple t-tests (unpaired and two-tailed with Holm-Šídák's correction): ns > 0.05; *P < 0.05; **P < 0.01. Each dot corresponds to one mouse.


( H ) Kaplan - Meier survival analysis of Pax5Prd * - Jak2 / + ( black ) and Pax5 + / + ( grey ) mice . A P value of < 0 . 0001 was determined for the survival curves by statistical analysis with the log - rank ( Mantel - Cox ) test . n , number of mice analyzed .

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

(H) Kaplan-Meier survival analysis of Pax5Prd*-Jak2 /+ (black) and Pax5+/+ (grey) mice. A P value of < 0.0001 was determined for the survival curves by statistical analysis with the log-rank (Mantel-Cox) test. n, number of mice analyzed.


( C ) Specificity of the anti - H3Y41ph antibody , as shown by immunoblot analysis . One phosphorylated ( pY41 ) or non - phosphorylated peptide was coupled to ubiquitin ( Ubi , 8 . 5 kDa ) , followed by separation of the protein conjugates on SDS - PAGE and immunoblot detection with the anti - H3Y41ph antibody present in the 8B2 - C1 cell supernatant . Following treatment with calf intestinal alkaline phosphatase ( CIP ) , the dephosphorylated Ubi - pY41 peptide conjugate could no longer be detected with the anti - H3Y41ph antibody .

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

(C) Specificity of the anti-H3Y41ph antibody, as shown by immunoblot analysis. One phosphorylated (pY41) or non-phosphorylated peptide was coupled to ubiquitin (Ubi, 8.5 kDa), followed by separation of the protein conjugates on SDS-PAGE and immunoblot detection with the anti-H3Y41ph antibody present in the 8B2-C1 cell supernatant. Following treatment with calf intestinal alkaline phosphatase (CIP), the dephosphorylated Ubi-pY41 peptide conjugate could no longer be detected with the anti-H3Y41ph antibody.


( D ) Immunoblot analysis of whole - cell extracts prepared from Pax5Jak2 / + and control Pax5Etv6 / + Cdkn2ab + / - B - ALL cells as well as from the human HEL , TMD8 and K1106 cell lines . Phosphorylated ( p ) STAT5 was detected with an anti - STAT5 ( pY694 ) antibody and the pY41 - peptide with the purified anti H3Y41ph antibody One to five pY41 - peptides were coupled to ubiquitin , which was added in the range of 50 ng per well . The Gapdh and histone H3 proteins were analyzed as loading control . An unspecific protein is denoted by an asterisk . One representative of 5 immunoblot experiments is shown .

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

(D) Immunoblot analysis of whole-cell extracts prepared from Pax5Jak2/+ and control Pax5Etv6/+ Cdkn2ab+/- B-ALL cells as well as from the human HEL, TMD8 and K1106 cell lines. Phosphorylated (p) STAT5 was detected with an anti-STAT5 (pY694) antibody and the pY41-peptide with the purified anti H3Y41ph antibody One to five pY41-peptides were coupled to ubiquitin, which was added in the range of 50 ng per well. The Gapdh and histone H3 proteins were analyzed as loading control. An unspecific protein is denoted by an asterisk. One representative of 5 immunoblot experiments is shown.


( A , Tumor progression in Il7 + / + ( grey ) , Il7 + / - ( dashed ) and Il7 - / - ( black ) mice transplanted with Pax5Jak2 - Luc / + tumor cells , as determined by bioluminescence measurements at the indicated days after cell transfer . Images of three transplanted mice for each Il7 genotype are shown at the indicated days after 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

(A, Tumor progression in Il7+/+ (grey), Il7+/- (dashed) and Il7-/- (black) mice transplanted with Pax5Jak2-Luc/+ tumor cells, as determined by bioluminescence measurements at the indicated days after cell transfer. Images of three transplanted mice for each Il7 genotype are shown at the indicated days after cell transfer


( C ) Kaplan - Meier survival analysis of Il7 + / + , Il7 + / - and Il7 - / - mice transplanted with Pax5Jak2 - Luc / + tumors cells . Statistical analysis of the survival curves was performed with the log - rank ( Mantel - Cox ) test ; ns P > 0 . 05 , * P < 0 . 05 , * * P < 0 . 01 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Kaplan-Meier survival analysis of Il7+/+, Il7+/- and Il7-/- mice transplanted with Pax5Jak2-Luc/+ tumors cells. Statistical analysis of the survival curves was performed with the log-rank (Mantel-Cox) test; ns P > 0.05, *P < 0.05, **P < 0.01.


( E ) Quantification of the median fluorescence intensity ( MFI ) values of the p - STAT5 levels determined by intracellular staining of splenic FO B cells from Pax5 + / + ( grey ) , Pax5Jak2 / + ( black ) and Pax5Jak2 - KD / + ( white ) mice at the age of 3 - 10 weeks Statistical data are presented as mean values with SEM and were analyzed by the two - tailed unpaired Student ' s t - test : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 . Each dot corresponds to one mouse .

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

(E) Quantification of the median fluorescence intensity (MFI) values of the p-STAT5 levels determined by intracellular staining of splenic FO B cells from Pax5+/+ (grey), Pax5Jak2/+ (black) and Pax5Jak2-KD/+ (white) mice at the age of 3-10 weeks Statistical data are presented as mean values with SEM and were analyzed by the two-tailed unpaired Student's t-test: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Each dot corresponds to one mouse.


( F ) Flow cytometric analysis of p - STAT5 levels in pro - B cells ( B220 + CD19 + Kit + IgM - ) and leukemic B220low B cells of Pax5Jak2 / + mice ( black ) as well as in control pro - B cells of Pax5 + / + ( WT ) mice ( grey ) at the age of 4 - 5 weeks . A representative flow cytometric analysis of the bone marrow of a Pax5Jak2 / + mouse ( left ) and representative intracellular p - STAT5 stainings ( middle ) are shown . The p - STAT5 levels in pro - B and leukemic B220low B cells ( right ) are quantified as MFI values relative to that of Pax5 + / + pro - B cells . Statistical data are presented as mean values with SEM and were analyzed by the two - tailed unpaired Student ' s t - test : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 . Each dot corresponds to one mouse .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Flow cytometric analysis of p-STAT5 levels in pro-B cells (B220+CD19+Kit+IgM-) and leukemic B220low B cells of Pax5Jak2/+ mice (black) as well as in control pro-B cells of Pax5+/+ (WT) mice (grey) at the age of 4-5 weeks. A representative flow cytometric analysis of the bone marrow of a Pax5Jak2/+ mouse (left) and representative intracellular p-STAT5 stainings (middle) are shown. The p-STAT5 levels in pro-B and leukemic B220low B cells (right) are quantified as MFI values relative to that of Pax5+/+ pro-B cells. Statistical data are presented as mean values with SEM and were analyzed by the two-tailed unpaired Student's t-test: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Each dot corresponds to one mouse.


( I ) Upregulation of the genes Cxcr5 , Syndig1l and Sema4a in Pax5Jak2 / + ( PJ ) B - ALL cells . Mean TPM values with SEM are shown for the following RNA - seq experiments : 2 ( Ctrl B - ALL ) , 4 ( PJ B - ALL )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Upregulation of the genes Cxcr5, Syndig1l and Sema4a in Pax5Jak2/+ (PJ) B-ALL cells. Mean TPM values with SEM are shown for the following RNA-seq experiments: 2 (Ctrl B-ALL), 4 (PJ B-ALL)


K ) Identification of Cxcr5 , Syndig1l and Sema4a as activated STAT5 target genes . ChIP - seq analysis identified STAT5 - binding regions at all three loci ( K ) . Horizontal bars below the ChIP - seq track indicate STAT5 - binding regions identified by MACS peak calling . RPM , reads per million .

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

K) Identification of Cxcr5, Syndig1l and Sema4a as activated STAT5 target genes. ChIP-seq analysis identified STAT5-binding regions at all three loci (K). Horizontal bars below the ChIP-seq track indicate STAT5-binding regions identified by MACS peak calling. RPM, reads per million.


MPK4 ‐ HA is detected in PAT1 ‐ Myc immunoprecipitates from double transgenic Arabidopsis plants . Immunoblots of input and anti ‐ Myc IPs probed with anti ‐ HA and anti ‐ Myc antibodies . Left panel , input ; right panel , anti ‐ Myc IP .

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

MPK4‐HA is detected in PAT1‐Myc immunoprecipitates from double transgenic Arabidopsis plants. Immunoblots of input and anti‐Myc IPs probed with anti‐HA and anti‐Myc antibodies. Left panel, input; right panel, anti‐Myc IP.


Yeast pat1Δ mutants are unable to grow at 37 ° C while wild ‐ type ( B4742 ) strain grows at 30 ° C ( left panel ) and 37 ° C ( right ) . Growth at 37 ° C is restored in pat1Δ expressing AtPAT1 . Serial dilutions of each yeast strain were plated on YPAD agar plates and grown at the indicated temperatures .

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

Yeast pat1Δ mutants are unable to grow at 37°C while wild‐type (B4742) strain grows at 30°C (left panel) and 37°C (right). Growth at 37°C is restored in pat1Δ expressing AtPAT1. Serial dilutions of each yeast strain were plated on YPAD agar plates and grown at the indicated temperatures.


Co ‐ IP between PAT1 ‐ HA and LSM1 ‐ GFP . Proteins were transiently co ‐ expressed in N . benthamiana and tissue harvested 3 days post ‐ infiltration . Immunoblots of GFP IPs ( top panel ) and inputs ( 20 μg each , bottom panel ) . Immunoblots were cut in half and probed with anti ‐ HA antibodies and anti ‐ GFP antibodies .

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

Co‐IP between PAT1‐HA and LSM1‐GFP. Proteins were transiently co‐expressed in N. benthamiana and tissue harvested 3 days post‐infiltration. Immunoblots of GFP IPs (top panel) and inputs (20 μg each, bottom panel). Immunoblots were cut in half and probed with anti‐HA antibodies and anti‐GFP antibodies.


Agarose gel electrophoresis of 5 ′ RACE PCR to detect capped transcripts of EIF4A1 , UGT87A2 and EXPL1 in 14 ‐ day ‐ old Col ‐ 0 and pat1 ‐ 1 .

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

Agarose gel electrophoresis of 5′ RACE PCR to detect capped transcripts of EIF4A1, UGT87A2 and EXPL1 in 14‐day‐old Col‐0 and pat1‐1.


MPK4 and MPK6 were immunoprecipitated from extracts of Col ‐ 0 seedlings treated with 200 nM flg22 for 10 min . For the negative control ( − ) , extracts were incubated with agarose beads without antibodies . IPs were incubated with His6 ‐ PAT1 , His6 ‐ PAT1 S208A or MBP for 60 min at 37 ° C before boiling and SDS ‐ PAGE . Autoradiogram ( top panel ) , Coomassie ‐ stained gel for loading control ( bottom ) .

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

MPK4 and MPK6 were immunoprecipitated from extracts of Col‐0 seedlings treated with 200 nM flg22 for 10 min. For the negative control (−), extracts were incubated with agarose beads without antibodies. IPs were incubated with His6‐PAT1, His6‐PAT1 S208A or MBP for 60 min at 37°C before boiling and SDS‐PAGE. Autoradiogram (top panel), Coomassie‐stained gel for loading control (bottom).


pat1 mutants have a serrated leaf , semi ‐ dwarf phenotypePlants photographed at 4 weeks of growth in short day conditions , genotypes as indicated . Col ‐ 0 and eds1 ‐ 2 plants and pat1 ‐ 1 and pat1 ‐ 1 / eds1 ‐ 2 , respectively , were placed in the middle of the same pots before the pictures were taken .

pat1 mutants have a serrated leaf, semi‐dwarf phenotypePlants photographed at 4 weeks of growth in short day conditions, genotypes as indicated. Col‐0 and eds1‐2 plants and pat1‐1 and pat1‐1/eds1‐2, respectively, were placed in the middle of the same pots before the pictures were taken.


PR gene expression is elevated in mpk4 ‐ 2 and pat1 ‐ 1 mutants . Four ‐ week ‐ old plants were used for RNA extraction , followed by qRT ‐ PCR . Fold ‐ change in PR1 ( gray bars ) and PR2 ( black ) expression is relative to Col ‐ 0 , normalized to UBQ10 . Standard error of the mean is indicated by errors bars ( n = 3 ) . Statistical significance between the mean values was determined by ANOVA followed by Fisher ' s LSD test , P ‐ values are only shown for mean values significantly different from Col ‐ 0 .

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

PR gene expression is elevated in mpk4‐2 and pat1‐1 mutants. Four‐week‐old plants were used for RNA extraction, followed by qRT‐PCR. Fold‐change in PR1 (gray bars) and PR2 (black) expression is relative to Col‐0, normalized to UBQ10. Standard error of the mean is indicated by errors bars (n = 3). Statistical significance between the mean values was determined by ANOVA followed by Fisher's LSD test, P‐values are only shown for mean values significantly different from Col‐0.


pat1 is more resistant to colonization by P . syringae pv . tomato DC3000 . Bacteria were syringe ‐ infiltrated and samples taken 3 days post ‐ infiltration . Data are shown as log10 ‐ transformed colony ‐ forming units / cm2 leaf tissue ( cfu / cm2 ) . Standard error of the mean is indicated by errors bars ( n = 4 ) . Statistical significance between the mean values was determined by ANOVA followed by Fisher ′ s LSD test .

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

pat1 is more resistant to colonization by P. syringae pv. tomato DC3000. Bacteria were syringe‐infiltrated and samples taken 3 days post‐infiltration. Data are shown as log10‐transformed colony‐forming units/cm2 leaf tissue (cfu/cm2). Standard error of the mean is indicated by errors bars (n = 4). Statistical significance between the mean values was determined by ANOVA followed by Fisher′s LSD test.


Confocalmicroscopy with root elongation zones . Five ‐ day ‐ old Col ‐ 0 / PAT1 ‐ GFP seedlings were treated with 1 μM flg22 on glass slides for 20 min ( second panel ) followed by treatment with 100 μg / ml cycloheximide for 60 min ( bottom panel ) . The scale bar corresponds to 10 μm .

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

Confocalmicroscopy with root elongation zones. Five‐day‐old Col‐0/PAT1‐GFP seedlings were treated with 1 μM flg22 on glass slides for 20 min (second panel) followed by treatment with 100 μg/ml cycloheximide for 60 min (bottom panel). The scale bar corresponds to 10 μm.


Confocalmicroscopy with 5 ‐ day ‐ old VCS ‐ GFP seedlings treated as in ( A ) . The scale bar corresponds to 10 μm .

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

Confocalmicroscopy with 5‐day‐old VCS‐GFP seedlings treated as in (A). The scale bar corresponds to 10 μm.


PAT1 protein is induced by PAMP treatment . Immunoblot detection of equal amounts of protein from Col ‐ 0 seedlings at times in minutes as indicated following vacuum infiltration with 1 μM flg22 or water ( 180 min after infiltration ) . Immunoblots were probed with anti ‐ PAT1 antibodies . Negative control pat1 ‐ 1 was loaded for comparison . Coomassie brilliant blue protein loading control is indicated by CBB .

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

PAT1 protein is induced by PAMP treatment. Immunoblot detection of equal amounts of protein from Col‐0 seedlings at times in minutes as indicated following vacuum infiltration with 1 μM flg22 or water (180 min after infiltration). Immunoblots were probed with anti‐PAT1 antibodies. Negative control pat1‐1 was loaded for comparison. Coomassie brilliant blue protein loading control is indicated by CBB.


Anti ‐ GFP immunoblotting of proteins extracted from Col ‐ 0 / PAT1 ‐ GFP seedlings treated over a time course with flg22 ( top panel ) and Coomassie brilliant blue ( CBB ) ‐ stained PVDF loading control is shown ( bottom panel ) . No PAT1 ‐ GFP was detected in negative control Col ‐ 0 .

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

Anti‐GFP immunoblotting of proteins extracted from Col‐0/PAT1‐GFP seedlings treated over a time course with flg22 (top panel) and Coomassie brilliant blue (CBB)‐stained PVDF loading control is shown (bottom panel). No PAT1‐GFP was detected in negative control Col‐0.


Elevated PR gene expression in pat1 mutants is suppressed by summ2 ‐ 8 . Four ‐ week ‐ old plants were used for RNA extraction , followed by qRT ‐ PCR . Fold ‐ change in PR1 ( gray bars ) and PR2 ( black bars ) expression is relative to Col ‐ 0 , normalized to UBQ10 . Standard error of the mean is indicated by errors bars ( n = 3 ) .

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

Elevated PR gene expression in pat1 mutants is suppressed by summ2‐8. Four‐week‐old plants were used for RNA extraction, followed by qRT‐PCR. Fold‐change in PR1 (gray bars) and PR2 (black bars) expression is relative to Col‐0, normalized to UBQ10. Standard error of the mean is indicated by errors bars (n = 3).


pat1 resistance to P . syringae pv . tomato DC3000 is suppressed by summ2 ‐ 8 . Bacteria were syringe ‐ infiltrated and samples taken 3 days post ‐ infiltration . Data are log10 ‐ transformed colony ‐ forming units / cm2 leaf tissue ( cfu / cm2 ) . Standard error of the mean is indicated by errors bars ( n = 4 ) .

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

pat1 resistance to P. syringae pv. tomato DC3000 is suppressed by summ2‐8. Bacteria were syringe‐infiltrated and samples taken 3 days post‐infiltration. Data are log10‐transformed colony‐forming units/cm2 leaf tissue (cfu/cm2). Standard error of the mean is indicated by errors bars (n = 4).


14 ‐ day ‐ old seedlings grown on ms plates were used for RNA extraction . Next , 1 μg of RNA from each sample was treated with XRN1 ( NEB ) or mock treated before RT ‐ qPCR . Data were normalized to ACT2 , and transcript levels were compared between XRN1 and mock ‐ treated RNA for each genotype . Standard error of the mean is indicated by error bars ( n = 4 ) . Statistical significance between the mean values was determined by ANOVA followed by Fisher ' s LSD test .

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

14‐day‐old seedlings grown on ms plates were used for RNA extraction. Next, 1 μg of RNA from each sample was treated with XRN1 (NEB) or mock treated before RT‐qPCR. Data were normalized to ACT2, and transcript levels were compared between XRN1 and mock‐treated RNA for each genotype. Standard error of the mean is indicated by error bars (n = 4). Statistical significance between the mean values was determined by ANOVA followed by Fisher's LSD test.


Phosphorylation of the PAT1 peptide SSFVSYPPPGSISPDQR , which include Ser208 , in Col ‐ 0 and mkk1 / 1 ‐ summ2 before and after flg22 treatment . The phosphorylation stoichiometry is illustrated relative to Col PAT1 without flg22 treatment . The ratios were obtained using quantitative iTRAQ mass spectrometry .

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

Phosphorylation of the PAT1 peptide SSFVSYPPPGSISPDQR, which include Ser208, in Col‐0 and mkk1/1‐summ2 before and after flg22 treatment. The phosphorylation stoichiometry is illustrated relative to Col PAT1 without flg22 treatment. The ratios were obtained using quantitative iTRAQ mass spectrometry.


Proteins were transiently co ‐ expressed ( PAT1 ‐ GFP + MPK4 ‐ HA or SUMM2 ‐ HA ) in N . benthamiana and tissue harvested 2 days post ‐ infiltration . Anti ‐ HA immunoblots of inputs ( 20 μg each ) are shown in the bottom panel . Immunoblots of GFP IPs were probed with anti ‐ HA antibodies , then stripped and probed with anti ‐ GFP antibodies . Molecular weights are shown in kDa on the right .

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

Proteins were transiently co‐expressed (PAT1‐GFP + MPK4‐HA or SUMM2‐HA) in N. benthamiana and tissue harvested 2 days post‐infiltration. Anti‐HA immunoblots of inputs (20 μg each) are shown in the bottom panel. Immunoblots of GFP IPs were probed with anti‐HA antibodies, then stripped and probed with anti‐GFP antibodies. Molecular weights are shown in kDa on the right.


Confocalimages of yellow fluorescent protein complementation . PAT1 ‐ Nc was transiently co ‐ expressed with either SUMM2 ‐ Nn or At4g12010 in N . benthamiana . Confocalmicroscopy on leaf disks was conducted 2 days post ‐ infiltration . Merged pictures show overlay of brightfield , chlorophyll and eYFP . The scale bar corresponds to 25 μm .

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

Confocalimages of yellow fluorescent protein complementation. PAT1‐Nc was transiently co‐expressed with either SUMM2‐Nn or At4g12010 in N. benthamiana. Confocalmicroscopy on leaf disks was conducted 2 days post‐infiltration. Merged pictures show overlay of brightfield, chlorophyll and eYFP. The scale bar corresponds to 25 μm.


A , B Bottom panels : gene annotation track ( mm10 ) ; the activated gene is marked in green and the location of the TALE - VP64 target sequence is shown by the vertical dashed green line . Middle panels : DamID tracks of NL interactions in control cells ( " control " , blue line ) and cells expressing TALE - VP64 ( " experimental " , red line ) . n indicates the number of independent biological replicates that were combined . Noise was suppressed by a running mean filter of indicated window size . Shading between the lines corresponds to the color of the sample with the highest value . Arrowheads on the right - hand side mark the 5th and 95th percentiles of genome - wide DamID values . Top panels : domainograms ; for every window of indicated size ( vertical axis ) and centered on a genomic position ( horizontal axis ) the pixel shade indicates the ranking of the change in DamID score ( experimental minus control ) in this window compared to the genome - wide changes in DamID scores across all possible windows of the same size . Blue : DamID score is highest in control samples ; red : DamID score is highest in experimental samples ( color key on the right of panel ( a ) ) . In ( a ) activation of Sox6 was the experimental perturbation , activation of Nrp1 ( which is located on a different chromosome ) served as control ; in ( b ) activation of Nrp1 was the experimental condition and activation of Sox6 served as control .

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

A, B Bottom panels: gene annotation track (mm10); the activated gene is marked in green and the location of the TALE-VP64 target sequence is shown by the vertical dashed green line. Middle panels: DamID tracks of NL interactions in control cells ("control", blue line) and cells expressing TALE-VP64 ("experimental", red line). n indicates the number of independent biological replicates that were combined. Noise was suppressed by a running mean filter of indicated window size. Shading between the lines corresponds to the color of the sample with the highest value. Arrowheads on the right-hand side mark the 5th and 95th percentiles of genome-wide DamID values. Top panels: domainograms; for every window of indicated size (vertical axis) and centered on a genomic position (horizontal axis) the pixel shade indicates the ranking of the change in DamID score (experimental minus control) in this window compared to the genome-wide changes in DamID scores across all possible windows of the same size. Blue: DamID score is highest in control samples; red: DamID score is highest in experimental samples (color key on the right of panel (a)). In (a) activation of Sox6 was the experimental perturbation, activation of Nrp1 (which is located on a different chromosome) served as control; in (b) activation of Nrp1 was the experimental condition and activation of Sox6 served as control.


( a - b ) Plots as in Figure 1 , showing changes in Lamin B1 DamID signals upon CRISPRa activation of NLGN1 ( a ) and SOX6 ( b ) . Control cells were treated either without sgRNA or with one of various sgRNAs targeting promoters on different chromosomes . Vertical green dotted lines mark the position of the sgRNA target sequence .

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

(a-b) Plots as in Figure 1, showing changes in Lamin B1 DamID signals upon CRISPRa activation of NLGN1 (a) and SOX6 (b). Control cells were treated either without sgRNA or with one of various sgRNAs targeting promoters on different chromosomes. Vertical green dotted lines mark the position of the sgRNA target sequence.


( c ) Domainograms showing regions with reduced NL interactions around 12 genes individually activated by CRISPRa . Genomic regions are aligned by the respective sgRNA target positions , and oriented so that the activated genes are all transcribed from left to right .

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

(c) Domainograms showing regions with reduced NL interactions around 12 genes individually activated by CRISPRa. Genomic regions are aligned by the respective sgRNA target positions, and oriented so that the activated genes are all transcribed from left to right.


Average DamID values plotted against average expression levels of eight genes activated by CRISPRa ( red ; n = 2 ) or in control cells that were treated either without sgRNA or with one of various sgRNAs targeting promoters on different chromosomes ( blue ; n ranging from 19 to 27 ) .

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

Average DamID values plotted against average expression levels of eight genes activated by CRISPRa (red; n = 2) or in control cells that were treated either without sgRNA or with one of various sgRNAs targeting promoters on different chromosomes (blue; n ranging from 19 to 27).


A - C CRISPRa activation of ADAM22 ( a ) , ABCB1 ( b ) and PTN ( c ) in human RPE - 1 cells . Top panels visualize DamID data similar to Figure 2a - b . Middle panels show maps of replication timing at the same resolution and in the same plotting style as panels , except that different colors are used as indicated . Bottom panels show gene track , with activated gene highlighted in green .

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A-C CRISPRa activation of ADAM22 (a), ABCB1 (b) and PTN (c) in human RPE-1 cells. Top panels visualize DamID data similar to Figure 2a-b. Middle panels show maps of replication timing at the same resolution and in the same plotting style as panels, except that different colors are used as indicated. Bottom panels show gene track, with activated gene highlighted in green.


Changes in NL interactions after CRISPRa activation of MLK4 in human RPE - 1 cells . Visualization of DamID data as in Figure 2a - 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

Changes in NL interactions after CRISPRa activation of MLK4 in human RPE-1 cells. Visualization of DamID data as in Figure 2a-b.


( a ) DamID domainograms of NL interactions after activation of CCSER1 , GRID2 , or both . All data are from human RPE - 1 cells .

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

(a) DamID domainograms of NL interactions after activation of CCSER1, GRID2, or both. All data are from human RPE-1 cells.


( b ) DamID domainograms after activation of ADAM22 , ABCB1 , or the genes ABCB4 , ABCB1 , ADAM22 and RUNC3B simultaneously . All data are from human RPE - 1 cells . DamID data of activation of ADAM22 and ABCB1 alone are same as in Figure 4a , 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

(b) DamID domainograms after activation of ADAM22, ABCB1, or the genes ABCB4, ABCB1, ADAM22 and RUNC3B simultaneously. All data are from human RPE-1 cells. DamID data of activation of ADAM22 and ABCB1 alone are same as in Figure 4a, b.


( a ) DamID profiles of the 129Sv allele of the Morc1 locus with deletions of the promoters of Morc , Morc1 and DppA2 ( deletions marked by yellow vertical boxes ) , and in control cells . ( b ) Same as ( a ) , but for the non - mutated Cast alleles .

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

(a) DamID profiles of the 129Sv allele of the Morc1 locus with deletions of the promoters of Morc, Morc1 and DppA2 (deletions marked by yellow vertical boxes), and in control cells. (b) Same as (a), but for the non-mutated Cast alleles.


( c - d ) Same design as ( a - b ) , but with only a single mono - allelic deletion of the Morc promoter ( vertical yellow box ) .

O O O O O O O O O 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-d) Same design as (a-b), but with only a single mono-allelic deletion of the Morc promoter (vertical yellow box).


( e - f ) Effect of PAS insertion on NL interactions of Cobl gene locus . Same design as ( a - b ) but with insertion of a PAS ( located at red triangle and vertical dotted line ) that truncates the 129Sv allele of the Cobl transcription unit . 129Sv allele is shown in ( e ) , non - mutated Cast allele in ( f ) . Clones with Morc1 locus mutations ( each assayed in four independent biological replicates ) served collectively as control in ( e - f )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-f) Effect of PAS insertion on NL interactions of Cobl gene locus. Same design as (a-b) but with insertion of a PAS (located at red triangle and vertical dotted line) that truncates the 129Sv allele of the Cobl transcription unit. 129Sv allele is shown in (e), non-mutated Cast allele in (f). Clones with Morc1 locus mutations (each assayed in four independent biological replicates) served collectively as control in (e-f)


( c ) Average DamID profiles across 17 transgene integration sites inside LADs . Blue curve shows alleles without integrations , red curve shows the corresponding alleles with integrations . Shading between the lines shows which curve has the highest value .

O O O O O O O O O O O O O O O O O O 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) Average DamID profiles across 17 transgene integration sites inside LADs. Blue curve shows alleles without integrations, red curve shows the corresponding alleles with integrations. Shading between the lines shows which curve has the highest value.


( d ) Relative expression levels of individual barcoded transgenes in LADs and iLADs .

(d) Relative expression levels of individual barcoded transgenes in LADs and iLADs.


( e ) Estimated expression levels of integrated transgenes in LADs ( vertical orange lines ; median value indicated by arrowhead ) , compared to the distribution of expression levels of all active endogenous genes ( black curve ) .

O O O O O O O O O O O O O O O O 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) Estimated expression levels of integrated transgenes in LADs (vertical orange lines; median value indicated by arrowhead), compared to the distribution of expression levels of all active endogenous genes (black curve).


A The family pedigree , where circles denote female members , squares male members , solid symbols affected members , and white symbols asymptomatic members with normal physical exam ; the dots indicate heterozygous carriers and double line denotes a consanguineous marriage . The pictures show scapular winging which is a consistent clinical sign in affected individuals .

A The family pedigree, where circles denote female members, squares male members, solid symbols affected members, and white symbols asymptomatic members with normal physical exam; the dots indicate heterozygous carriers and double line denotes a consanguineous marriage. The pictures show scapular winging which is a consistent clinical sign in affected individuals.


B Hematoxylin and eosin staining ( H & amp ; amp ; E ) of skeletal muscle from patient II . 1 shows histological features of moderate to severe dystrophic pattern . ( Scale bar , 50 μm ) .

B Hematoxylin and eosin staining (H&amp;E) of skeletal muscle from patient II.1 shows histological features of moderate to severe dystrophic pattern. (Scale bar, 50 μm).


C T1 - weighted MRI axial images at thigh and calf levels show that the fatty degeneration is more prominent in thigh muscles , equally affecting posterior and anterior compartment , with relative sparing of the rectus femoris , sartorius and gracilismuscles until late stages ( 4 , 10 and 11 , respectively ) . Strikingly , the fatty tissue is located in the internal parts of almost all the affected muscles in thigh ( 1 , 2 , 3 , 5 - 9 ) , while the external regions are spared . At calf level , only the gastrocnemius medialis muscle ( 12 ) shows this pattern , while the soleus ( 13 ) is diffusely involved . Patient II . 2 ( PII . 2 ) shows late - stage thigh muscles with an unusual involvement of the tibialis posteriormuscle ( 14 ) in the lower leg .

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C T1-weighted MRI axial images at thigh and calf levels show that the fatty degeneration is more prominent in thigh muscles, equally affecting posterior and anterior compartment, with relative sparing of the rectus femoris, sartorius and gracilismuscles until late stages (4, 10 and 11, respectively). Strikingly, the fatty tissue is located in the internal parts of almost all the affected muscles in thigh (1, 2, 3, 5-9), while the external regions are spared. At calf level, only the gastrocnemius medialis muscle (12) shows this pattern, while the soleus (13) is diffusely involved. Patient II.2 (PII.2) shows late-stage thigh muscles with an unusual involvement of the tibialis posteriormuscle (14) in the lower leg.


A Muscle sections show variable labeling using an antibody against glycosylated α - dystroglycan ( αDG - IIH6 ) , whereas labeling using antibodies against α - dystroglycan core protein ( αDG - Core ) , β - dystroglycan ( βDG ) and laminin - 2 is similar to control ( Scale bar , 100 μm ) .

A Muscle sections show variable labeling using an antibody against glycosylated α-dystroglycan (αDG-IIH6), whereas labeling using antibodies against α-dystroglycan core protein (αDG-Core), β-dystroglycan (βDG) and laminin-2 is similar to control (Scale bar, 100 μm).


B Western blots and ligand - overlay ( O / L ) of wheat - germ agglutinin - enriched muscle and fibroblasts lysates from PII . 2 , PII . 5 , the healthy sibling ( HII . 3 ) and healthy controls ( Ctr , Ctr1 , and Ctr2 ) ( Muscle : 250μg protein / lane , Fibroblasts : 800μg / lane ) . In muscle , expression of αDG - IIH6 is reduced but the αDG - Core shows similar expression as controls , with a slight reduction in molecular weight ; laminin overlay assay detected some binding activity but was diminished compared to controls , whereas agrin - binding activity showed no difference with controls . In fibroblasts , both αDG - IIH6 and αDG - Core expression and laminin - binding activity were normal , suggesting that unlike other muscular dystrophies , α - dystroglycan glycosylation defect is muscle - specific in POGLUT1 patients .

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

B Western blots and ligand-overlay (O/L) of wheat-germ agglutinin-enriched muscle and fibroblasts lysates from PII.2, PII.5, the healthy sibling (HII.3) and healthy controls (Ctr, Ctr1, and Ctr2) (Muscle: 250μg protein/lane, Fibroblasts: 800μg/lane). In muscle, expression of αDG-IIH6 is reduced but the αDG-Core shows similar expression as controls, with a slight reduction in molecular weight; laminin overlay assay detected some binding activity but was diminished compared to controls, whereas agrin-binding activity showed no difference with controls. In fibroblasts, both αDG-IIH6 and αDG-Core expression and laminin-binding activity were normal, suggesting that unlike other muscular dystrophies, α-dystroglycan glycosylation defect is muscle-specific in POGLUT1 patients.


C No ultrastructural alterations are observed in muscle by electron microscopy ( 6 - 7 fields were captured from PII . 1 , PII . 3 , and PII . 5 ) . Note normal basement membrane compactation ( arrowheads ) . Original magnification : 26 , 700 .

O O O O O O O O O O O O O O O O O O O O O O O O O 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 No ultrastructural alterations are observed in muscle by electron microscopy (6-7 fields were captured from PII.1, PII.3, and PII.5). Note normal basement membrane compactation (arrowheads). Original magnification: 26,700.


A Protein O - glucosyltransferase activity of wild - type ( WT ) or D233E mutant POGLUT1 protein toward human factor IX EGF repeat ( hFIX - EGF ) . Wild - type shows higher O - glucosyltransferase activity than D233E , and this activity is dependent on the concentration of the acceptor substrate hFIX - EGF repeat ( left ) and on the concentration of donor substrate UDP - glucose ( UDP - Glc ) ( right ) . Values indicate mean ± SEM from three independent assays .

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

A Protein O-glucosyltransferase activity of wild-type (WT) or D233E mutant POGLUT1 protein toward human factor IX EGF repeat (hFIX-EGF). Wild-type shows higher O-glucosyltransferase activity than D233E, and this activity is dependent on the concentration of the acceptor substrate hFIX-EGF repeat (left) and on the concentration of donor substrate UDP-glucose (UDP-Glc) (right). Values indicate mean ± SEM from three independent assays.


B O - glucosyltransferase activity towards five different single EGF repeats from mouse Notch1 . Wild - type POGLUT1 shows higher activity toward all EGF repeats tested than POGLUT1D233E . The Y - axis shows the normalized activity relative to wild - type POGLUT1 . EGF repeats were at 10 μM . Values indicate mean ± SEM from three independent assays .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O-glucosyltransferase activity towards five different single EGF repeats from mouse Notch1. Wild-type POGLUT1 shows higher activity toward all EGF repeats tested than POGLUT1D233E. The Y-axis shows the normalized activity relative to wild-type POGLUT1. EGF repeats were at 10 μM. Values indicate mean ± SEM from three independent assays.


C Elution profiles of the POGLUT1 reaction products on reverse phase HPLC . hFIX - EGF repeat was incubated with wild - type or D233E mutant POGLUT1 and donor substrate , UDP - Glc or UDP - xylose ( UDP - Xyl ) , at 37ºC overnight . Values on top of the peaks indicate the measured masses . Addition of Glc ( 162 Da ) or xylose ( 132 Da ) to hFIX - EGF ( 5696 . 2 Da ) by wild - type POGLUT1 caused a shift to an earlier retention time ( left ) . Similarly , the products exhibited a similar shift after incubation with POGLUT1D233E ( right ) . These results indicate that POGLUT1D233E can add a single glucose or xylose to hFIX - EGF repeats and thus has residual enzymatic activity .

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

C Elution profiles of the POGLUT1 reaction products on reverse phase HPLC. hFIX-EGF repeat was incubated with wild-type or D233E mutant POGLUT1 and donor substrate, UDP-Glc or UDP-xylose (UDP-Xyl), at 37ºC overnight. Values on top of the peaks indicate the measured masses. Addition of Glc (162 Da) or xylose (132 Da) to hFIX-EGF (5696.2 Da) by wild-type POGLUT1 caused a shift to an earlier retention time (left). Similarly, the products exhibited a similar shift after incubation with POGLUT1D233E (right). These results indicate that POGLUT1D233E can add a single glucose or xylose to hFIX-EGF repeats and thus has residual enzymatic activity.


A Western blot of muscle homogenates shows reduced expression of Notch1 intracellular domain ( NICD ) in patients .

A Western blot of muscle homogenates shows reduced expression of Notch1 intracellular domain (NICD) in patients.


B - C qRT - PCR on RNA extracted from skeletal - muscle shows that expression of HES1 ( B ) and PAX7 ( C ) was significantly lower in D233E patients compared to healthy controls . Mean ± SEM , Student ´ s t - test ( B ) and Mann - Whitney U test ( C ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 qRT-PCR on RNA extracted from skeletal-muscle shows that expression of HES1 (B) and PAX7 (C) was significantly lower in D233E patients compared to healthy controls. Mean ± SEM, Student´s t-test (B) and Mann-Whitney U test (C).


D PAX7 + cells in skeletal muscle sections , demonstrating that satellite cells are less abundant in D233E patients than in controls ( n = 3 muscles with 10 - 15 fields analyzed per muscle ) . Mean ± SEM ; Mann - Whitney U test ; Scale bar , 50 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 PAX7+ cells in skeletal muscle sections, demonstrating that satellite cells are less abundant in D233E patients than in controls (n = 3 muscles with 10-15 fields analyzed per muscle). Mean ± SEM; Mann-Whitney U test; Scale bar, 50 μm.


A - A " Staining of Drosophila indirect flight muscles at 25 % pupal development with 22C10 antibody ( myotube marker ) and anti - Twist antibody ( myoblast marker ) indicates that in wild - type animals , indirect flight muscles are formed from 6 myotubes , and a large number of Twist + myoblasts are present . B - B " rumi79 / 79 mutants raised at 18C show no obvious defects in myoblast number or myotube morphology . C - D " rumi79 / 79 mutants exhibit decreased number of myoblasts at 25 ° - 30 ° C and either an incomplete set of short myotubes when raised at 25C or aberrant morphology of myotubes when raised at 30C . E - E " rumi79 / 79 animals overexpressing FLAG - tagged POGLUT1WT in the muscle compartment using Mef2 - GAL4 and raised at 25C during the pupal stage show a rescue of the rumi79 / 79 muscle phenotype . F - G " Overexpression of FLAG - tagged POGLUT1D233E only partially rescues the rumi79 / 79 muscle phenotype , with some variation in the myotube length and the number of restored Twist + cells ( compare G and F ) . Note that the number of Twist + cells restored by POGLUT1D233E is much smaller than that restored by POGLUT1WT . Scale bar in A ' is 50 µm and applies to A - G " . H Quantification of myotube lengths in rumi79 / 79 mutants with or without POGLUT1 expression indicates a weak rescue of the phenotypes by POGLUT1D233E compared to POGLUT1WT . Mean ± standard deviation is shown . The differences in myotube lengths are statistically significant ( n = 22 - 54 ) . Mean ± standard deviation is shown ; One - way Anova with Bonferroni ´ s multiple comparisons test .

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

A-A" Staining of Drosophila indirect flight muscles at 25% pupal development with 22C10 antibody (myotube marker) and anti-Twist antibody (myoblast marker) indicates that in wild-type animals, indirect flight muscles are formed from 6 myotubes, and a large number of Twist+myoblasts are present.B-B" rumi79/79 mutants raised at 18C show no obvious defects in myoblast number or myotube morphology.C-D" rumi79/79 mutants exhibit decreased number of myoblasts at 25°-30°C and either an incomplete set of short myotubes when raised at 25C or aberrant morphology of myotubes when raised at 30C.E-E" rumi79/79 animals overexpressing FLAG-tagged POGLUT1WT in the muscle compartment using Mef2-GAL4 and raised at 25C during the pupal stage show a rescue of the rumi79/79 muscle phenotype.F-G" Overexpression of FLAG-tagged POGLUT1D233E only partially rescues the rumi79/79 muscle phenotype, with some variation in the myotube length and the number of restored Twist+cells (compare G and F). Note that the number of Twist+cells restored by POGLUT1D233E is much smaller than that restored by POGLUT1WT. Scale bar in A' is 50 µm and applies to A-G".H Quantification of myotube lengths in rumi79/79 mutants with or without POGLUT1 expression indicates a weak rescue of the phenotypes by POGLUT1D233E compared to POGLUT1WT. Mean ± standard deviation is shown. The differences in myotube lengths are statistically significant (n = 22-54). Mean ± standard deviation is shown; One-way Anova with Bonferroni´s multiple comparisons test.


A - F Proliferation assays examined proliferating ( PAX7 + MyoD + , arrows ) , self - renewing ( PAX7 + MyoD − , open arrows ) , and differentiating ( PAX7 − MyoD + , arrowheads ) cells from D233E patients ( n = 2 ) , healthy controls ( n = 3 ) and disease controls ( n = 3 ) at 1 , 4 , and 8 days growing in proliferation medium . ( A ) shows representative images from day 8 . The number of patientmyoblasts is lower than that of controls at all three time points , indicating a slower proliferation rate in patientmyoblasts ( B ) , and accordingly , the percentage of proliferating cells ( C ) is smaller in the patient . ( D ) The percentage of self - renewing cells is small in patient ´ s culture , reflecting a poor capacity for maintaining the pool of quiescent SCs . In addition , the percentage of PAX7 + cells ( E ) is smaller in patient ´ s culture , similarly to what was found in adult D233E muscle ( shown in Fig 5D ) . ( F ) The percentage of differentiating cells is higher in the patient . We examine proliferation capturing 7 - 12 randomly fields per day and condition ( mean ± SEM is shown ; Kruskal - Wallis with Dunn ´ s multiple comparisons test ; Scale bar , 50 µm ) .

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

A-F Proliferation assays examined proliferating (PAX7+MyoD+, arrows), self-renewing (PAX7+ MyoD−, open arrows), and differentiating (PAX7− MyoD+, arrowheads) cells from D233E patients (n = 2), healthy controls (n = 3) and disease controls (n = 3) at 1, 4, and 8 days growing in proliferation medium. (A) shows representative images from day 8. The number of patientmyoblasts is lower than that of controls at all three time points, indicating a slower proliferation rate in patientmyoblasts (B), and accordingly, the percentage of proliferating cells (C) is smaller in the patient. (D) The percentage of self-renewing cells is small in patient´s culture, reflecting a poor capacity for maintaining the pool of quiescent SCs. In addition, the percentage of PAX7+ cells (E) is smaller in patient´s culture, similarly to what was found in adult D233E muscle (shown in Fig 5D). (F) The percentage of differentiating cells is higher in the patient. We examine proliferation capturing 7-12 randomly fields per day and condition (mean ± SEM is shown; Kruskal-Wallis with Dunn´s multiple comparisons test; Scale bar, 50 µm).


G - I When myoblasts started to be confluent , the proliferation medium was replaced with differentiation medium , and the myoblasts started to fuse yielding myotubes , which were analyzed after four days of differentiation . ( H ) The fusion index , which measures the percentage of nuclei in myotubes with respect to the total number of nuclei in myogenic cells , was higher in D233E cultures than in controls . ( I ) Besides , myogenin showed increased expression in D233E myoblast cultures compared to controls . The data support that differentiation process is facilitated in patient ´ s muscle . Data was collected from 3 - 10 randomly chosen areas per condition ( mean ± SEM is shown ; Kruskal - Wallis with Dunn ´ s multiple comparisons test ( H ) and One - way Anova with Bonferroni ´ s multiple comparisons test ( I ) ; Scale bar , 50 µm ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-I When myoblasts started to be confluent, the proliferation medium was replaced with differentiation medium, and the myoblasts started to fuse yielding myotubes, which were analyzed after four days of differentiation. (H) The fusion index, which measures the percentage of nuclei in myotubes with respect to the total number of nuclei in myogenic cells, was higher in D233E cultures than in controls. (I) Besides, myogenin showed increased expression in D233E myoblast cultures compared to controls. The data support that differentiation process is facilitated in patient´s muscle. Data was collected from 3-10 randomly chosen areas per condition (mean ± SEM is shown; Kruskal-Wallis with Dunn´s multiple comparisons test (H) and One-way Anova with Bonferroni´s multiple comparisons test (I); Scale bar, 50 µm).


A - C We overexpressed GFP - tagged NICD in immortalized primary myoblasts to rescue the features displayed by immortalized D233E myoblasts . ( B ) We analyzed the proliferation at 1 , 3 and 5 days , and proliferation rate in patient - LV - NICD - GFP reached the level observed in control - LV - GFP , which were higher than in patient - LV - GFP . ( C ) However , the percentage of PAX7 + cells in patient - LV - NICD - GFP showed the same reduced value as in patient - LV - GFP , which were significantly lower than in control - LV - GFP . The majority of control myoblasts were proliferating cells ( PAX7 + MyoD + , arrows ) , with no quiescent cells and few differentiating cells detected . Data were collected from n = 2 independent assays ( 5 - 8 images per condition ) . Mean ± SEM is shown ; One - way Anova with Tukey multiple comparisions test ( B ) and Kruskal - Wallis with Dunn ´ s multiple comparisons test ( C ) ; Scale bar , 50 µm ) .

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

A-C We overexpressed GFP-tagged NICD in immortalized primary myoblasts to rescue the features displayed by immortalized D233E myoblasts. (B) We analyzed the proliferation at 1, 3 and 5 days, and proliferation rate in patient-LV-NICD-GFP reached the level observed in control-LV-GFP, which were higher than in patient-LV-GFP. (C) However, the percentage of PAX7+cells in patient-LV-NICD-GFP showed the same reduced value as in patient-LV-GFP, which were significantly lower than in control-LV-GFP. The majority of control myoblasts were proliferating cells (PAX7+ MyoD+, arrows), with no quiescent cells and few differentiating cells detected. Data were collected from n = 2 independent assays (5-8 images per condition). Mean ± SEM is shown; One-way Anova with Tukey multiple comparisions test (B) and Kruskal-Wallis with Dunn´s multiple comparisons test (C); Scale bar, 50 µm).


D - F As previously described in patient ' s primary myoblasts , patient - LV - GFPmyoblasts showed a striking facilitated differentiation compared to control - LV - GFP - myoblasts . This phenotype fully disappeared in patient - LV - GFP - NICD myoblasts , as quantification of the fusion index ( E ) , and myogenin expression ( F ) demonstrated . The data support a pathogenic role for Notch inhibition on the altered differentiation in our patients . Data were collected from n = 3 independent assays ( 13 - 17 images per condition ) . Mean ± SEM is shown ; Kruskal - Wallis with Dunn ´ s multiple comparisons test ; Scale bar , 50 µm ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-F As previously described in patient's primary myoblasts, patient-LV-GFPmyoblasts showed a striking facilitated differentiation compared to control-LV-GFP-myoblasts. This phenotype fully disappeared in patient-LV-GFP-NICD myoblasts, as quantification of the fusion index (E), and myogenin expression (F) demonstrated. The data support a pathogenic role for Notch inhibition on the altered differentiation in our patients. Data were collected from n = 3 independent assays (13-17 images per condition). Mean ± SEM is shown; Kruskal-Wallis with Dunn´s multiple comparisons test; Scale bar, 50 µm).


A Immortalized primary myoblasts from a healthy control showed a progressive pattern of α - dystroglycan glycosylation during five days of differentiation ( D0 - D5 ) , as has been previously described by Goddeeris et al . Immortalized primary myoblasts from patients displayed an irregular pattern of glycosylation , with a final level of glycosylated α - dystroglycan that was lower than control .

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

A Immortalized primary myoblasts from a healthy control showed a progressive pattern of α-dystroglycan glycosylation during five days of differentiation (D0-D5), as has been previously described by Goddeeris et al. Immortalized primary myoblasts from patients displayed an irregular pattern of glycosylation, with a final level of glycosylated α-dystroglycan that was lower than control.


B α - dystroglycan glycosylation of infected immortalized myoblasts from patient - LV - NICD - GFP at differentiation day 5 ( D5 ) showed a striking reduction , in line with the strong inhibition of myotubes formation and differentiation induced by NICD overexpression . α - dystroglycan in myoblasts from patient - LV - GFP showed a reduction of glycosylation compared to control - LV - GFP at D5 , similar to the results obtained in the adult muscle from patients ( Fig . 2 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 α-dystroglycan glycosylation of infected immortalized myoblasts from patient-LV-NICD-GFP at differentiation day 5 (D5) showed a striking reduction, in line with the strong inhibition of myotubes formation and differentiation induced by NICD overexpression. α-dystroglycan in myoblasts from patient-LV-GFP showed a reduction of glycosylation compared to control-LV-GFP at D5, similar to the results obtained in the adult muscle from patients (Fig. 2).


C - D C2C12 myogenic cells showed a reduced level of glycosylated α - dystroglycan when the Notch signaling inhibitor DAPT or LY3039478 was added to the differentiation medium . This indicates that reducing Notch signaling can affect α - dystroglycan glycosylation in wild - type C2C12 myoblasts cells , which do not harbor any known mutations in α - dystroglycan glycosyltransferases or Poglut1 .

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

C-D C2C12 myogenic cells showed a reduced level of glycosylated α-dystroglycan when the Notch signaling inhibitor DAPT or LY3039478 was added to the differentiation medium. This indicates that reducing Notch signaling can affect α-dystroglycan glycosylation in wild-type C2C12 myoblasts cells, which do not harbor any known mutations in α-dystroglycan glycosyltransferases or Poglut1.


E - F qRT - PCR experiments show that upon DAPT or LY3039478 treatment , a remarkable decrease in Hes1 mRNA is induced in C2C12 cells during differentiation .

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

E-F qRT-PCR experiments show that upon DAPT or LY3039478 treatment, a remarkable decrease in Hes1 mRNA is induced in C2C12 cells during differentiation.


Relative expression of Rab39a in siRNA - treated cells after 48 hours of knockdown . Rab39a expression in I - Ab or Rab39a siRNA - treated cells was normalized to expression in cells treated with control β2m siRNA . Error bars show SD between three independent experiments . * p < 0 . 05 using two - way Student ' s t - test .

Relative expression of Rab39a in siRNA-treated cells after 48 hours of knockdown. Rab39a expression in I-Ab or Rab39a siRNA-treated cells was normalized to expression in cells treated with control β2m siRNA. Error bars show SD between three independent experiments. * p < 0.05 using two-way Student's t-test.


XPT of siRNA treated DC3 . 2R cells after 48 hours of knockdown . Treated cells were fed with the indicated amounts of Ova - Fe The cells were then exposed to RF33 . 70 - Luc Reporter CD8 T cells overnight . RLU indicates relative luminescence units produced by reporter T cell stimulation . Error bars show SD of > 3 replicate wells . * p < 0 . 05 vs control I - Ab using two - way ANOVA . Representative plot of 3 independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O B-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

XPT of siRNA treated DC3.2R cells after 48 hours of knockdown. Treated cells were fed with the indicated amounts of Ova-Fe The cells were then exposed to RF33.70-Luc Reporter CD8 T cells overnight. RLU indicates relative luminescence units produced by reporter T cell stimulation. Error bars show SD of >3 replicate wells. * p < 0.05 vs control I-Ab using two-way ANOVA. Representative plot of 3 independent experiments.


XPT of siRNA treated DC3 . 2R cells after 48 hours of knockdown . Treated cells were fed with the indicated amounts of Ova - latex The cells were then exposed to RF33 . 70 - Luc Reporter CD8 T cells overnight . RLU indicates relative luminescence units produced by reporter T cell stimulation . Error bars show SD of > 3 replicate wells . * p < 0 . 05 vs control I - Ab using two - way ANOVA . Representative plot of 3 independent experiments .

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

XPT of siRNA treated DC3.2R cells after 48 hours of knockdown. Treated cells were fed with the indicated amounts of Ova-latex The cells were then exposed to RF33.70-Luc Reporter CD8 T cells overnight. RLU indicates relative luminescence units produced by reporter T cell stimulation. Error bars show SD of >3 replicate wells. * p < 0.05 vs control I-Ab using two-way ANOVA. Representative plot of 3 independent experiments.


XPT of siRNA treated DC3 . 2R cells after 48 hours of knockdown . Treated cells were fed with the indicated amounts of unconjugated soluble Ova The cells were then exposed to RF33 . 70 - Luc Reporter CD8 T cells overnight . RLU indicates relative luminescence units produced by reporter T cell stimulation . Error bars show SD of > 3 replicate wells . * p < 0 . 05 vs control I - Ab using two - way ANOVA . Representative plot of 3 independent experiments .

XPT of siRNA treated DC3.2R cells after 48 hours of knockdown. Treated cells were fed with the indicated amounts of unconjugated soluble Ova The cells were then exposed to RF33.70-Luc Reporter CD8 T cells overnight. RLU indicates relative luminescence units produced by reporter T cell stimulation. Error bars show SD of >3 replicate wells. * p < 0.05 vs control I-Ab using two-way ANOVA. Representative plot of 3 independent experiments.


DC3 . 2 - Rab39a cells ( Rab39a KO with dox - inducible Rab39a ) were incubated overnight with or without 1 µg / ml dox to induce Rab39a expression . 1x105 Cells were then fed with Ova - Fe and exposed to reporter T cells as in ( B ) . Error bars show SD of duplicate wells . * p < 0 . 05 using two - way ANOVA . Representative plot of 3 independent experiments .

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

DC3.2-Rab39a cells (Rab39a KO with dox-inducible Rab39a) were incubated overnight with or without 1 µg/ml dox to induce Rab39a expression. 1x105 Cells were then fed with Ova-Fe and exposed to reporter T cells as in (B). Error bars show SD of duplicate wells. * p < 0.05 using two-way ANOVA. Representative plot of 3 independent experiments.


MHC - II presentation of siRNA treated DC3 . 2R cells after 48 hours of knockdown . Treated cells were fed with the indicated amounts of Ova - Fe . The cells were then exposed to MF2 . 2D9 - Luc Reporter CD4 T cells overnight . Error bars show SD of > 3 replicate wells . * p < 0 . 05 vs negative control β2m using two - way ANOVA . None of the bead concentrations are p < 0 . 05 for Rab39a siRNA vs β2m . Representative plot of 3 independent

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

MHC-II presentation of siRNA treated DC3.2R cells after 48 hours of knockdown. Treated cells were fed with the indicated amounts of Ova-Fe. The cells were then exposed to MF2.2D9-Luc Reporter CD4 T cells overnight. Error bars show SD of >3 replicate wells. * p < 0.05 vs negative control β2m using two-way ANOVA. None of the bead concentrations are p < 0.05 for Rab39a siRNA vs β2m. Representative plot of 3 independent experiments


DC3 . 2 - Rab39a cells ( Rab39a KO with dox - inducible Rab39a ) were incubated overnight with or without 1 µg / ml dox to induce Rab39a expression . 1x105 cells were then fed with Ova - Fe and exposed to reporter T cells as in ( A ) . Error bars show SD of duplicate wells . None of the bead concentrations are p < 0 . 05 for Rab39a siRNA vs β2m using two - way ANOVA . Representative plot of 3 independent

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

DC3.2-Rab39a cells (Rab39a KO with dox-inducible Rab39a) were incubated overnight with or without 1 µg/ml dox to induce Rab39a expression. 1x105 cells were then fed with Ova-Fe and exposed to reporter T cells as in (A). Error bars show SD of duplicate wells. None of the bead concentrations are p < 0.05 for Rab39a siRNA vs β2m using two-way ANOVA. Representative plot of 3 independent experiments


MHC - I Presentation of SIINFEKL - H2 - Kb complexes on siRNA treated DC3 . 2 NS - Ova ( C ) or DC3 . 2 UbS8L ( D ) cells . 48 hours after siRNA transfection , the indicated amounts of dox was added to the cells to induce Ova expression . After 2 hours at 37oC , RF33 . 70 - Luc CD8 T cells were added along with Brefeldin A ( Golgiplug ) to a final concentration of 1 : 1000 . The cells were incubated overnight for reporter T cell luciferase expression . Error bars show SD of > 3 replicate wells . * p < 0 . 05 vs negative control I - Ab using two - way ANOVA . None of the dox concentrations are p < 0 . 05 for Rab39a siRNA vs I - Ab . Representative plot of 3 independent

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

MHC-I Presentation of SIINFEKL-H2-Kb complexes on siRNA treated DC3.2 NS-Ova (C) or DC3.2 UbS8L (D) cells. 48 hours after siRNA transfection, the indicated amounts of dox was added to the cells to induce Ova expression. After 2 hours at 37oC, RF33.70-Luc CD8 T cells were added along with Brefeldin A (Golgiplug) to a final concentration of 1:1000. The cells were incubated overnight for reporter T cell luciferase expression. Error bars show SD of >3 replicate wells. * p < 0.05 vs negative control I-Ab using two-way ANOVA. None of the dox concentrations are p < 0.05 for Rab39a siRNA vs I-Ab. Representative plot of 3 independent experiments


Flow cytometric analysis of surface H2 - Kb and I - Ab levels of siRNA treated DC3 . 2R cells . Shown are results after 48 hours of knockdown . Representative plot of 3 independent

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Flow cytometric analysis of surface H2-Kb and I-Ab levels of siRNA treated DC3.2R cells. Shown are results after 48 hours of knockdown. Representative plot of 3 independent experiments


Phagocytosis assay after 48 hours of siRNA knockdown . DC3 . 2R cells treated with the indicated siRNAs were incubated on ice for 30 minutes with 4 beads per cell of Alexa Fluor 647 conjugated beads . The mixture was then placed in media at 37oC for 1 hour . Cells were harvested and incubated on ice with 1 mg / ml of Trypan Blue to quench uneaten beads before analysis through flow cytometry . As a control , some cells were incubated with 10 µM Cytochalasin D during the feeding period . Shown are percentages of cells that have taken up beads . Representative plot of two independent experiments .

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Phagocytosis assay after 48 hours of siRNA knockdown. DC3.2R cells treated with the indicated siRNAs were incubated on ice for 30 minutes with 4 beads per cell of Alexa Fluor 647 conjugated beads. The mixture was then placed in media at 37oC for 1 hour. Cells were harvested and incubated on ice with 1 mg/ml of Trypan Blue to quench uneaten beads before analysis through flow cytometry. As a control, some cells were incubated with 10 µM Cytochalasin D during the feeding period. Shown are percentages of cells that have taken up beads. Representative plot of two independent experiments.


Rab39a was induced in DC3 . 2 - Rab39a cells ( Rab39a KO with dox - inducible Rab39a ) via overnight incubation with 1µg / ml dox . KO and rescued cells were then harvested and assayed as in ( A ) for phagocytosis . Representative plot of two independent experiments .

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

Rab39a was induced in DC3.2-Rab39a cells (Rab39a KO with dox-inducible Rab39a) via overnight incubation with 1µg / ml dox. KO and rescued cells were then harvested and assayed as in (A) for phagocytosis. Representative plot of two independent experiments.


DC3 . 2 - Rab39a cells were induced to express Rab39a overnight . Cells on coverslips were washed , fixed and stained according to the listed protocol and antibodies . Green = Rab39a ( HA tag ) , Magenta = Lamp2 . Arrows highlight punctate structures with colocalization . Scale bar = 10 µm . Representative image of two independent experiments .

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DC3.2-Rab39a cells were induced to express Rab39a overnight. Cells on coverslips were washed, fixed and stained according to the listed protocol and antibodies. Green = Rab39a (HA tag), Magenta = Lamp2. Arrows highlight punctate structures with colocalization. Scale bar = 10 µm. Representative image of two independent experiments.


DC3 . 2 Rab39a KO cells were transduced with dox inducible mcherry - tagged Rab39a . Cells were plated in 35 mm glass bottom dishes and induced with 1 µg / ml dox . The next day , 1 µm latex beads conjugated with Alexa 488 - NHS ( 1 bead / cell ) were added . After 2 hours at 37oC , cells were imaged under a Leica TCS SP5 confocal microscope . Green = Alexa 488 labeled beads . Magenta = mcherry - Rab39a . Arrows highlight internalized beads surrounded by Rab39a . Scale bar = 10 µm . Representative image of two independent experiments .

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

DC3.2 Rab39a KO cells were transduced with dox inducible mcherry-tagged Rab39a. Cells were plated in 35 mm glass bottom dishes and induced with 1 µg/ml dox. The next day, 1 µm latex beads conjugated with Alexa 488-NHS (1 bead/cell) were added. After 2 hours at 37oC, cells were imaged under a Leica TCS SP5 confocal microscope. Green = Alexa 488 labeled beads. Magenta = mcherry-Rab39a. Arrows highlight internalized beads surrounded by Rab39a. Scale bar = 10 µm. Representative image of two independent experiments.


5x105 of DC3 . 2 - Rab39a cells were induced to express Rab39a overnight with 1 µg / ml dox in a 6 well plate . Cells were fed at 1 bead / cell of biotinylated - 6µm magnetic beads for the indicated lengths of time . Magnetic - bead - phagosomes were isolated using the listed protocol . Phagosomes were washed and stained for Rab39a ( HA - tag ) . Representative data for 3 independent experiments .

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5x105 of DC3.2-Rab39a cells were induced to express Rab39a overnight with 1 µg/ml dox in a 6 well plate. Cells were fed at 1 bead/cell of biotinylated-6µm magnetic beads for the indicated lengths of time. Magnetic-bead-phagosomes were isolated using the listed protocol. Phagosomes were washed and stained for Rab39a (HA-tag). Representative data for 3 independent experiments.


1 . 25 x 105 of DC3 . 2 - Rab39a cells were incubated in a 12 well plate overnight ± dox ( 1µg / ml ) to induce Rab39a . Cells were fed at 4 beads / cell of biotinylated - ova - 6µm - magnetic - beads for 3 hours with or without 1 : 1000 BFA ( golgiplug , BD ) . Phagosomes were isolated using the listed protocol . Isolated phagosomes were washed , permeabilized and stained . Shown are histograms of indicated stains as well as % of Rab39a positive phagosomes . Representative plot of two independent experiments .

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 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

1.25 x 105 of DC3.2-Rab39a cells were incubated in a 12 well plate overnight ± dox (1µg/ml) to induce Rab39a. Cells were fed at 4 beads/cell of biotinylated-ova-6µm-magnetic-beads for 3 hours with or without 1:1000 BFA (golgiplug, BD). Phagosomes were isolated using the listed protocol. Isolated phagosomes were washed, permeabilized and stained. Shown are histograms of indicated stains as well as % of Rab39a positive phagosomes. Representative plot of two independent experiments.


XPT of siRNA treated DC3 . 2R cells after 48 hours of knockdown . Treated cells were fed with the indicated amounts of C8L peptid conjugated to iron oxide beads via a disulfide bond . The cells were then exposed to RF33 . 70 - Luc Reporter CD8 T cells overnight . Error bars show SD of > 3 replicate wells . * p < 0 . 05 for siRNA vs control I - Ab using two - way ANOVA . Representative plot of 3 independent

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

XPT of siRNA treated DC3.2R cells after 48 hours of knockdown. Treated cells were fed with the indicated amounts of C8L peptid conjugated to iron oxide beads via a disulfide bond. The cells were then exposed to RF33.70-Luc Reporter CD8 T cells overnight. Error bars show SD of >3 replicate wells. * p < 0.05 for siRNA vs control I-Ab using two-way ANOVA. Representative plot of 3 independent experiments


XPT of siRNA treated DC3 . 2R cells after 48 hours of knockdown . Treated cells were fed with the indicated amounts o Ova ( B ) conjugated to iron oxide beads via a disulfide bond . The cells were then exposed to RF33 . 70 - Luc Reporter CD8 T cells overnight . Error bars show SD of > 3 replicate wells . * p < 0 . 05 for siRNA vs control I - Ab using two - way ANOVA . Representative plot of 3 independent

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

XPT of siRNA treated DC3.2R cells after 48 hours of knockdown. Treated cells were fed with the indicated amounts o Ova (B) conjugated to iron oxide beads via a disulfide bond. The cells were then exposed to RF33.70-Luc Reporter CD8 T cells overnight. Error bars show SD of >3 replicate wells. * p < 0.05 for siRNA vs control I-Ab using two-way ANOVA. Representative plot of 3 independent experiments


DC3 . 2 - Rab39a cells ( Rab39a KO with dox - inducible Rab39a ) were incubated overnight with or without 1 µg / ml dox to induce Rab39a expression . 1x105 cells were then fed with C8L peptide beads and exposed to reporter T cells as in ( A ) . Error bars show SD of duplicate wells . * p < 0 . 05 between no dox and + dox using two - way ANOVA . Representative plot of 3 independent

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

DC3.2-Rab39a cells (Rab39a KO with dox-inducible Rab39a) were incubated overnight with or without 1 µg/ml dox to induce Rab39a expression. 1x105 cells were then fed with C8L peptide beads and exposed to reporter T cells as in (A). Error bars show SD of duplicate wells. * p < 0.05 between no dox and + dox using two-way ANOVA. Representative plot of 3 independent experiments


Same as in ( A ) but cells were fed with 8 µg of A9M peptide bead and exposed to 12 . 64 - Luc Reporter T cells . Experiments were combined by normalizing RLU values to the average of the negative control I - Ab . Error bars show SD of 3 independent experiments . * p < 0 . 05 for Rab39a or β2m siRNA vs negative control I - Ab using

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Same as in (A) but cells were fed with 8 µg of A9M peptide bead and exposed to 12.64-Luc Reporter T cells. Experiments were combined by normalizing RLU values to the average of the negative control I-Ab. Error bars show SD of 3 independent experiments. * p < 0.05 for Rab39a or β2m siRNA vs negative control I-Ab using ANOVA


DC3 . 2 - Rab39a - H2 - Ld cells ( Rab39a KO with dox - inducible Rab39a ) were incubated with or without 1 µg / ml dox overnight to induce Rab39a expression . 1x106 cells were fed at 4 beads / cell of biotinylated - magnetic - beads for 4 hours . Magnetic bead phagosomes were isolated using the listed protocol . Phagosomes were washed , fixed , permeabilized and stained for Open ( 64 - 3 - 7 ) and Closed ( 30 - 5 - 7s ) forms of H2 - Ld . Representative histograms shown in ( B ) . The background fluorescence of unstained beads was subtracted from the resulting phagosome gMFI and the fold change of open or closed H2 - Ld ( dox / no dox ) was calculated . Three independent experiments were combined for analysis and error bars show the SD between experiments . Open H2 - Ld , but not Closed H2 - Ld was statistically significant ( p < 0 . 05 ) between KO and rescued cells using two - tailed ratio - paired Student ' s t - test .

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DC3.2-Rab39a-H2-Ld cells (Rab39a KO with dox-inducible Rab39a) were incubated with or without 1 µg/ml dox overnight to induce Rab39a expression. 1x106 cells were fed at 4 beads/cell of biotinylated-magnetic-beads for 4 hours. Magnetic bead phagosomes were isolated using the listed protocol. Phagosomes were washed, fixed, permeabilized and stained for Open (64-3-7) and Closed (30-5-7s) forms of H2-Ld. Representative histograms shown in (B). The background fluorescence of unstained beads was subtracted from the resulting phagosome gMFI and the fold change of open or closed H2-Ld (dox/no dox) was calculated. Three independent experiments were combined for analysis and error bars show the SD between experiments. Open H2-Ld, but not Closed H2-Ld was statistically significant (p<0.05) between KO and rescued cells using two-tailed ratio-paired Student's t-test.


DC3 . 2 - Rab39a - H2 - Ld cells ( Rab39a KO with dox - inducible Rab39a ) were incubated with or without 1 µg / ml dox overnight to induce Rab39a expression . 1x106 cells were fed at 4 beads / cell of biotinylated - magnetic - beads for 4 hours . Magnetic bead phagosomes were isolated using the listed protocol . Phagosomes were washed , fixed , permeabilized and stained for Open ( 64 - 3 - 7 ) and Closed ( 30 - 5 - 7s ) forms of H2 - Ld . H2 - Ld cell surface levels of KO and induced cells as in ( A ) .

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 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

DC3.2-Rab39a-H2-Ld cells (Rab39a KO with dox-inducible Rab39a) were incubated with or without 1 µg/ml dox overnight to induce Rab39a expression. 1x106 cells were fed at 4 beads/cell of biotinylated-magnetic-beads for 4 hours. Magnetic bead phagosomes were isolated using the listed protocol. Phagosomes were washed, fixed, permeabilized and stained for Open (64-3-7) and Closed (30-5-7s) forms of H2-Ld. H2-Ld cell surface levels of KO and induced cells as in (A).


Phagosomes from KO and induced cells were isolated as in ( A ) . Permeabilized , unfixed phagosomes were incubated with 10 µM of the indicated peptides for 3 hours at 25oC in Perm buffer . Phagosomes were then washed and stained for H2 - Ld . Shown are histograms of indicated stains .

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Phagosomes from KO and induced cells were isolated as in (A). Permeabilized, unfixed phagosomes were incubated with 10 µM of the indicated peptides for 3 hours at 25oC in Perm buffer. Phagosomes were then washed and stained for H2-Ld. Shown are histograms of indicated stains.


Phagosomes from KO and induced cells were isolated as in ( A ) . Permeabilized , unfixed phagosomes were incubated with 10 µM of the indicated peptides for 3 hours at 25oC in Perm buffer . Phagosomes were then washed and stained for H2 - Ld . Shown are histograms of indicated stains . Flow cytometry plots of ( D ) .

Phagosomes from KO and induced cells were isolated as in (A). Permeabilized, unfixed phagosomes were incubated with 10 µM of the indicated peptides for 3 hours at 25oC in Perm buffer. Phagosomes were then washed and stained for H2-Ld. Shown are histograms of indicated stains. Flow cytometry plots of (D).


Analysis of phagosomes shown in ( D ) and ( E ) . The background fluorescence of unstained beads was subtracted from the resulting phagosome gMFI and the fold change of pulsed / unpulsed phagosomes was calculated .

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Analysis of phagosomes shown in (D) and (E). The background fluorescence of unstained beads was subtracted from the resulting phagosome gMFI and the fold change of pulsed / unpulsed phagosomes was calculated.


DC3 . 2 Rab39a - H2 - Ld cells were incubated with or without 1 µg / ml dox overnight to induce Rab39a expression . 1x106 cells were fed at 4 beads / cell of biotinylated - Ova - magnetic - beads for 4 hours in the presence or absence of BFA . Isolated phagosomes were stained for H2 - Ld . Representative of two independent experiments .

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

DC3.2 Rab39a-H2-Ld cells were incubated with or without 1 µg/ml dox overnight to induce Rab39a expression. 1x106 cells were fed at 4 beads/cell of biotinylated-Ova-magnetic-beads for 4 hours in the presence or absence of BFA. Isolated phagosomes were stained for H2-Ld. Representative of two independent experiments.


DC3 . 2 - Rab39a - H2 - Ld cells ( Rab39a KO with dox - inducible Rab39a ) were incubated with 1 µg / ml dox overnight to induce Rab39a expression . 1x106 cells were fed at 4 beads / cell of biotinylated - Ova - magnetic - beads for 4 hours with the indicated inhibitors . Magnetic - bead phagosomes were isolated using the listed protocol . Phagosomes were washed , fixed , permeabilized and stained for Open and Closed ( 30 - 5 - 7s ) forms of H2 - Ld . Representative histograms of two independent experiments shown .

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DC3.2-Rab39a-H2-Ld cells (Rab39a KO with dox-inducible Rab39a) were incubated with 1 µg/ml dox overnight to induce Rab39a expression. 1x106 cells were fed at 4 beads/cell of biotinylated-Ova-magnetic-beads for 4 hours with the indicated inhibitors. Magnetic-bead phagosomes were isolated using the listed protocol. Phagosomes were washed, fixed, permeabilized and stained for Open and Closed (30-5-7s) forms of H2-Ld. Representative histograms of two independent experiments shown.