( D ) Left panel , representative images of immunofluorescence labeling of transfected constructs ( Green ) and endogenous P62 ( Sqstm1 , red ) on GT1 - 7 neuronal cells transfected with siRNA targeting C9orf72 and plasmids expressing control GFP , HA tagged wild type or mutant RAB39b ( CA , Q68L or CN , S22N ) , RAB8a or RAB7 . Right panel , quantification of P62 aggregates . Scale bars , 10 µm . Nuclei were counterstained with DAPI . Error bars indicate s . e . m . Student T - test , * indicates p < 0 . 05 , * * * indicates p < 0 . 001 . n = 3 .

O O O O O O O O O B-EXP_ASSAY O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O B-SUBCELLULAR O O O B-EXP_ASSAY O O O O O O O O O O O O O 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) Left panel, representative images of immunofluorescence labeling of transfected constructs (Green) and endogenous P62 (Sqstm1, red) on GT1-7 neuronal cells transfected with siRNA targeting C9orf72 and plasmids expressing control GFP, HA tagged wild type or mutant RAB39b (CA, Q68L or CN, S22N), RAB8a or RAB7. Right panel, quantification of P62 aggregates. Scale bars, 10 µm. Nuclei were counterstained with DAPI. Error bars indicate s.e.m. Student T-test, * indicates p<0.05, *** indicates p<0.001. n=3.


( A ) Immunoblot analysis of HA - immunoprecipitated proteins and lysate of HEK293 cells co - expressing HA - tagged C9ORF72 and HA - tagged SMCR8 with Flag - tagged TBK1 , NAP1 , TANK or SINTBAD .

O O O B-EXP_ASSAY O O B-GENEPROD O B-EXP_ASSAY O O O O B-CELL_LINE O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O

(A) Immunoblot analysis of HA-immunoprecipitated proteins and lysate of HEK293 cells co-expressing HA-tagged C9ORF72 and HA-tagged SMCR8 with Flag-tagged TBK1, NAP1, TANK or SINTBAD.


( B ) Immunoprecipitated HA - tagged C9ORF72 , HA - tagged SMCR8 and HA - tagged WDR41 expressed in HEK293 were subjected to in vitro TBK1 kinase assay in the presence of γP32 - radiolabelled ATP . Proteins were separated by SDS page migration and phosphorylation was detected by autoradiography ( upper panel ) , while expression was detected by western blotting ( lower panel ) .

O O O B-EXP_ASSAY B-GENEPROD O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O B-CELL_LINE O O O O O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-EXP_ASSAY O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O

(B) Immunoprecipitated HA-tagged C9ORF72, HA-tagged SMCR8 and HA-tagged WDR41 expressed in HEK293 were subjected to in vitro TBK1 kinase assay in the presence of γP32-radiolabelled ATP. Proteins were separated by SDS page migration and phosphorylation was detected by autoradiography (upper panel), while expression was detected by western blotting (lower panel).


( C ) Mass spectrometry identification of SMCR8 phosphorylation sites by in vitro TBK1 kinase assay of HIS - tagged C9ORF72 : SMCR8 : WDR41 complex purified from baculovirus infected insect cells .

O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-ORGANISM O O O O

(C) Mass spectrometry identification of SMCR8 phosphorylation sites by in vitro TBK1 kinase assay of HIS-tagged C9ORF72:SMCR8:WDR41 complex purified from baculovirus infected insect cells.


( D ) Left panel , representative images of immunofluorescence labeling of transfected constructs ( Green ) and endogenous P62 ( Sqstm1 , red ) on GT1 - 7 neuronal cells transfected with siRNA targeting the 3 ' UTR of Smcr8 mRNA and plasmids expressing control GFP , HA tagged wild type or mutant SMCR8 ( TA , S402A and T796A ; TD , S402D and T796D ; UD , S400D , S492D , S562D and T666D ) . Right panel , quantification of P62 aggregates .

O O O O O O O O O B-EXP_ASSAY O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O O O O 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-GENEPROD O O

(D) Left panel, representative images of immunofluorescence labeling of transfected constructs (Green) and endogenous P62 (Sqstm1, red) on GT1-7 neuronal cells transfected with siRNA targeting the 3'UTR of Smcr8 mRNA and plasmids expressing control GFP, HA tagged wild type or mutant SMCR8 (TA, S402A and T796A; TD, S402D and T796D; UD, S400D, S492D, S562D and T666D). Right panel, quantification of P62 aggregates.


( E ) Left panel , representative images of immunofluorescence labeling of transfected constructs ( Green ) and endogenous P62 ( Sqstm1 , red ) on GT1 - 7 neuronal cells transfected with siRNA targeting Tbk1 and plasmids expressing control GFP , HA tagged wild type or mutant SMCR8 or RAB39b . Right panel , quantification of P62 aggregates . Scale bars , 10 µm . Nuclei were counterstained with DAPI . Error bars indicate s . e . m . Student T - test , * indicates p < 0 . 05 , * * * indicates p < 0 . 001 . n = 3 .

O O O O O O O O O B-EXP_ASSAY O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O B-SUBCELLULAR O O O B-EXP_ASSAY O O O O O O O O O O O O O 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) Left panel, representative images of immunofluorescence labeling of transfected constructs (Green) and endogenous P62 (Sqstm1, red) on GT1-7 neuronal cells transfected with siRNA targeting Tbk1 and plasmids expressing control GFP, HA tagged wild type or mutant SMCR8 or RAB39b. Right panel, quantification of P62 aggregates. Scale bars, 10 µm. Nuclei were counterstained with DAPI. Error bars indicate s.e.m. Student T-test, * indicates p<0.05, *** indicates p<0.001. n=3.


( A ) Immunoblot analysis of endogenous Tdp - 43 , C9orf72 and control Gapdh of E18 mouse cortical neurons transduced with lentivirus expressing either control shRNA or shRNA targeting C9orf72 mRNA .

O O O B-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O B-GENEPROD O O B-ORGANISM B-CELL_TYPE I-CELL_TYPE O O B-ORGANISM O O O O O O O B-GENEPROD O O

(A) Immunoblot analysis of endogenous Tdp-43, C9orf72 and control Gapdh of E18 mouse cortical neurons transduced with lentivirus expressing either control shRNA or shRNA targeting C9orf72 mRNA.


( B ) Left panel , representative images of immunofluorescence labeling of endogenous phosphorylated Ser409 / 410 - Tdp - 43 on primary cultures of E18 mouse cortical neurons transduced with lentiviral particles expressing either control shRNA or shRNA targeting C9orf72 . Right panel , quantification of cytoplasmic Tdp - 43 aggregates .

O O O O O O O O O B-EXP_ASSAY O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-ORGANISM B-CELL_TYPE I-CELL_TYPE O O B-ORGANISM O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O

(B) Left panel, representative images of immunofluorescence labeling of endogenous phosphorylated Ser409/410-Tdp-43 on primary cultures of E18 mouse cortical neurons transduced with lentiviral particles expressing either control shRNA or shRNA targeting C9orf72. Right panel, quantification of cytoplasmic Tdp-43 aggregates.


( C ) Left panel , representative images of immunofluorescence labeling of D169G mutant GFP - tagged TDP - 43 on primary cultures of E18 mouse cortical neurons transfected with either HA - tagged control or HA - C9ORF72 plasmid . Right panel , quantification of cells with cytoplasmic aggregates of TDP - 43 . Scale bars , 10 µm . Nuclei were counterstained with DAPI . Error bars indicate s . e . m . Student T - test , * * * indicates p < 0 . 001 . n = 3 .

O O O O O O O O O B-EXP_ASSAY O O O O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-ORGANISM B-CELL_TYPE I-CELL_TYPE O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-SUBCELLULAR O O O B-EXP_ASSAY O O O O O 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) Left panel, representative images of immunofluorescence labeling of D169G mutant GFP-tagged TDP-43 on primary cultures of E18 mouse cortical neurons transfected with either HA-tagged control or HA-C9ORF72 plasmid. Right panel, quantification of cells with cytoplasmic aggregates of TDP-43. Scale bars, 10 µm. Nuclei were counterstained with DAPI. Error bars indicate s.e.m. Student T-test, *** indicates p<0.001. n=3.


( A ) Left panel , representative images of organotypic cultures of E18 mouse cortical neurons co - transfected with HA - tagged ATXN2 with control ( Q22x ) or intermediate ( Q30x ) polyQ size and transduced with lentivirus expressing either control shRNA or shRNA targeting C9orf72 mRNA . Right panel , quantification of ATXN2 aggregates . Scale bars , 10 µm . Nuclei were counterstained with DAPI .

O O O O O O O O O O O O O B-ORGANISM B-CELL_TYPE I-CELL_TYPE O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O B-SUBCELLULAR O O O B-EXP_ASSAY O

(A) Left panel, representative images of organotypic cultures of E18 mouse cortical neurons co-transfected with HA-tagged ATXN2 with control (Q22x) or intermediate (Q30x) polyQ size and transduced with lentivirus expressing either control shRNA or shRNA targeting C9orf72 mRNA. Right panel, quantification of ATXN2 aggregates. Scale bars, 10 µm. Nuclei were counterstained with DAPI.


( B ) Cell viability ( tetrazolium assay ) of GT1 - 7 neuronal cells co - transfected with HA - tagged ATXN2 with control ( Q22x ) or intermediate ( Q30x ) polyQ size and control siRNA or siRNA targeting C9orf72 mRNA . Error bars indicate s . e . m . Student T - test , * * * indicates p < 0 . 001 . n = 3 .

O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O 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) Cell viability (tetrazolium assay) of GT1-7 neuronal cells co-transfected with HA-tagged ATXN2 with control (Q22x) or intermediate (Q30x) polyQ size and control siRNA or siRNA targeting C9orf72 mRNA. Error bars indicate s.e.m. Student T-test, *** indicates p<0.001. n=3.


( C ) Tracing of the swimming trajectories of 48 hours post - fertilization zebrafish larvae following light touch . ( D - F ) Quantification of the touch - evoked swimming distance ( D ) , average velocity ( E ) , and maximum velocity attained ( F ) shows significant functional impairment of the zebrafish injected with HA - tagged ATXN2 with intermediate length of polyQ ( Q30x ) and antisense morpholino oligonucleotides ( AMOs ) against C9orf72 compared to control HA - tagged ATXN2 ( Q22x ) or to the sole injection of AMO against C9orf72 .

O O O O O O B-EXP_ASSAY O O O O O O O B-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O B-EXP_ASSAY 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-ORGANISM O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O

(C) Tracing of the swimming trajectories of 48 hours post-fertilization zebrafish larvae following light touch. (D-F) Quantification of the touch-evoked swimming distance (D), average velocity (E), and maximum velocity attained (F) shows significant functional impairment of the zebrafish injected with HA-tagged ATXN2 with intermediate length of polyQ (Q30x) and antisense morpholino oligonucleotides (AMOs) against C9orf72 compared to control HA-tagged ATXN2 (Q22x) or to the sole injection of AMO against C9orf72.


( G ) Representative images of motor neurons visualized with anti - Sv2 immunohistochemistry show severe axonopathy in fish injected with both ATXN2 Q30x and AMO against C9orf72 compared to zebrafish injected with control ATXN2 Q22x or with AMO against C9orf72 alone . ( H ) Quantification of the motor neuron axonal length demonstrates a significant decrease of axonal length in 48 hours post - fertilization zebrafish larvae injected with both ATXN2 Q30x and AMO against C9orf72 compared to controls . Error bars indicate s . e . m . One - way ANOVA , * * indicates p < 0 . 01 . n = 3 .

O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O B-GENEPROD B-EXP_ASSAY O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O B-ORGANISM O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O B-CELL_TYPE I-CELL_TYPE B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O O O O O B-ORGANISM I-ORGANISM O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O 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) Representative images of motor neurons visualized with anti-Sv2 immunohistochemistry show severe axonopathy in fish injected with both ATXN2 Q30x and AMO against C9orf72 compared to zebrafish injected with control ATXN2 Q22x or with AMO against C9orf72 alone. (H) Quantification of the motor neuron axonal length demonstrates a significant decrease of axonal length in 48 hours post-fertilization zebrafish larvae injected with both ATXN2 Q30x and AMO against C9orf72 compared to controls. Error bars indicate s.e.m. One-way ANOVA, ** indicates p<0.01. n=3.


Cell surface H2 - Kb levels of wild type and TAP1 - / - BMDC transduced with control vector or a vector expressing hβ2m were analyzed .

O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD O O O B-CELL_TYPE O O O O O O O O O O O O

Cell surface H2-Kb levels of wild type and TAP1-/- BMDC transduced with control vector or a vector expressing hβ2m were analyzed.


Cross - presentation of OVA by wild type and TAP1 - / - BMDC expressing hβ2m were compared to control BMDC , by measuring IL - 2 production by a Kb - SIINFEKL specific T - cell hybridoma ( B3Z ) cultured with paraformaldehyde fixed BMDC that were incubated with varying numbers of OVA - coated latex beads for 6hr , prior to fixation .

O O O O B-GENEPROD O O O O B-GENEPROD O O O B-CELL_TYPE O O O O O O B-CELL_TYPE O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O B-CELL_LINE O O O B-SMALL_MOLECULE O B-CELL_TYPE O O O O O O O B-GENEPROD O O O O O O O O O O O

Cross-presentation of OVA by wild type and TAP1-/- BMDC expressing hβ2m were compared to control BMDC, by measuring IL-2 production by a Kb-SIINFEKL specific T-cell hybridoma (B3Z) cultured with paraformaldehyde fixed BMDC that were incubated with varying numbers of OVA-coated latex beads for 6hr, prior to fixation.


The effect of hβ2m on the presentation of peptides derived from endogenous antigen was assessed by measuring IL - 2 levels in the culture supernatant of the B3Z hybridoma incubated with fixed VV - OVA infected WT and TAP1 - / - BMDC transduced with control vector or hβ2m .

O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-CELL_LINE O O O O O O B-GENEPROD O O O B-GENEPROD O O O B-CELL_TYPE O O O O O O O

The effect of hβ2m on the presentation of peptides derived from endogenous antigen was assessed by measuring IL-2 levels in the culture supernatant of the B3Z hybridoma incubated with fixed VV-OVA infected WT and TAP1-/- BMDC transduced with control vector or hβ2m.


Surface H2 - Kb levels of wild - type and TAP1 - / - BMDC co - expressing Rab mutants and hβ2m were analyzed by flow cytometry .

O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD O O O B-CELL_TYPE O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O

Surface H2-Kb levels of wild-type and TAP1-/- BMDC co-expressing Rab mutants and hβ2m were analyzed by flow cytometry.


Cross - presentation of OVA by wild - type and TAP1 - / - BMDC co - expressing Rab mutants and hβ2m were analyzed and IL - 2 release is

O O O O B-GENEPROD O O O O O B-GENEPROD O O O B-CELL_TYPE O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O

Cross-presentation of OVA by wild-type and TAP1-/- BMDC co-expressing Rab mutants and hβ2m were analyzed and IL-2 release is shown


TAP1 - / - BMDC transduced with vector control ( A ) or hβ2m ( C ) were incubated with varying concentrations of Epoxomicin for 6hr in absence or presence of exogenously added Kb - binding SIINFEKL peptide and analyzed for surface expression of Kb by flow cytometry . Representative plots are shown ( A , C ) . The impact of Epoxomicin on cell surface Kb was analyzed by plotting Kb surface expression on cells incubated with Epoxomicin , normalized to that on cell incubated without Epoxomicin ( n = 3 ) ( B , D ) .

B-GENEPROD O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-GENEPROD O O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-GENEPROD O O O O B-GENEPROD O B-EXP_ASSAY O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O

TAP1-/- BMDC transduced with vector control (A) or hβ2m (C) were incubated with varying concentrations of Epoxomicin for 6hr in absence or presence of exogenously added Kb-binding SIINFEKL peptide and analyzed for surface expression of Kb by flow cytometry. Representative plots are shown (A, C). The impact of Epoxomicin on cell surface Kb was analyzed by plotting Kb surface expression on cells incubated with Epoxomicin, normalized to that on cell incubated without Epoxomicin (n=3) (B, D).


The effect of Epoxomicin on cross - presentation of OVA - coated beads by TAP1 - / - BMDC expressing hβ2m was determined by incubating the BMDC with OVA coated latex beads in the presence of varying doses of Epoxomicin and 10μM gB peptide . After 6hrs the cells were fixed , incubated with B3Z cells and IL - 2 production was

O O O B-SMALL_MOLECULE O O O O O B-GENEPROD O O O O B-GENEPROD O O O B-CELL_TYPE O O O O O O O B-CELL_TYPE O B-GENEPROD O O O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O B-GENEPROD O O O O O O O O O O O B-CELL_LINE O O B-GENEPROD I-GENEPROD I-GENEPROD O O

The effect of Epoxomicin on cross-presentation of OVA-coated beads by TAP1-/- BMDC expressing hβ2m was determined by incubating the BMDC with OVA coated latex beads in the presence of varying doses of Epoxomicin and 10μM gB peptide. After 6hrs the cells were fixed, incubated with B3Z cells and IL-2 production was measured


293T - FcR - Kb cells co - expressing empty vector or Rab mutants ( Rab5ACA , Rab22ACA , Rab7ADN ) individually , with either LacZ as a control or US6 were analyzed for cross - presentation of OVA using B3Z cells . The effects of US6 on cross - presentation were analyzed by plotting the mean percentage of IL - 2 release by cells co - expressing US6 ( A ) with control vector or Rab mutants compared to cells co - expressing LacZ with control vector or Rab mutants , respectively .

B-CELL_LINE O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O B-GENEPROD O B-CELL_LINE O O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O

293T-FcR-Kb cells co-expressing empty vector or Rab mutants (Rab5ACA, Rab22ACA, Rab7ADN) individually, with either LacZ as a control or US6 were analyzed for cross-presentation of OVA using B3Z cells. The effects of US6 on cross-presentation were analyzed by plotting the mean percentage of IL-2 release by cells co-expressing US6 (A) with control vector or Rab mutants compared to cells co-expressing LacZ with control vector or Rab mutants, respectively.


293T - FcR - Kb cells co - expressing empty vector or Rab mutants ( Rab5ACA , Rab22ACA , Rab7ADN ) individually , with either LacZ as a control or ICP47 ( B ) , were analyzed for cross - presentation of OVA using B3Z cells . The effects of ICP47 on cross - presentation were analyzed by plotting the mean percentage of IL - 2 release by cells co - expressing ICP47 ( B ) with control vector or Rab mutants compared to cells co - expressing LacZ with control vector or Rab mutants , respectively .

B-CELL_LINE O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O B-CELL_LINE O O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O

293T-FcR-Kb cells co-expressing empty vector or Rab mutants (Rab5ACA, Rab22ACA, Rab7ADN) individually, with either LacZ as a control or ICP47 (B), were analyzed for cross-presentation of OVA using B3Z cells. The effects of ICP47 on cross-presentation were analyzed by plotting the mean percentage of IL-2 release by cells co-expressing ICP47 (B) with control vector or Rab mutants compared to cells co-expressing LacZ with control vector or Rab mutants, respectively.


293T - FcR - Kb cells expressing Rab5ACA were incubated with opsonized OVA coated latex beads in the presence of varying doses of Epoxomicin . After 6hrs the cells were fixed , incubated with B3Z cells and IL - 2 production was measured .

B-CELL_LINE O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O B-CELL_LINE O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O

293T-FcR-Kb cells expressing Rab5ACA were incubated with opsonized OVA coated latex beads in the presence of varying doses of Epoxomicin. After 6hrs the cells were fixed, incubated with B3Z cells and IL-2 production was measured.


293T - FcR - Kb cells expressing Rab22ACA were incubated with opsonized OVA coated latex beads in the presence of varying doses of Epoxomicin . After 6hrs the cells were fixed , incubated with B3Z cells and IL - 2 production was measured .

293T-FcR-Kb cells expressing Rab22ACA were incubated with opsonized OVA coated latex beads in the presence of varying doses of Epoxomicin. After 6hrs the cells were fixed, incubated with B3Z cells and IL-2 production was measured.


293T - FcR - Kb cells expressing Rab7ADN ( E ) were incubated with opsonized OVA coated latex beads in the presence of varying doses of Epoxomicin . After 6hrs the cells were fixed , incubated with B3Z cells and IL - 2 production was measured .

B-CELL_LINE O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O B-CELL_LINE O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O

293T-FcR-Kb cells expressing Rab7ADN (E) were incubated with opsonized OVA coated latex beads in the presence of varying doses of Epoxomicin. After 6hrs the cells were fixed, incubated with B3Z cells and IL-2 production was measured.


EM micrographs of double immuno - gold labeling using antibodies against immunoproteasome subunit LMP2 and the endolysosomal membrane marker LAMP1 in BMDC , WT MEF and LMP2 KO MEF . Large gold particles ( 15 nm , black arrow head ) labels LMP2 and small gold particles labels LAMP1 ( 5 nm , white arrow head ) ( Scale bars = 500nm ) . The insets are the magnification of region of interest containing LAMP1 positive vacuole ( Scale bars = 100nm ) , marked by the black rectangle .

B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-SUBCELLULAR O B-GENEPROD O O B-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD O B-CELL_TYPE O O B-CELL_TYPE O B-GENEPROD O B-CELL_TYPE O O B-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD O O B-SMALL_MOLECULE O O B-GENEPROD O 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-GENEPROD O B-SUBCELLULAR O O O O O O O O O O O O O

EM micrographs of double immuno-gold labeling using antibodies against immunoproteasome subunit LMP2 and the endolysosomal membrane marker LAMP1 in BMDC, WT MEF and LMP2 KO MEF. Large gold particles (15 nm, black arrow head) labels LMP2 and small gold particles labels LAMP1 (5 nm, white arrow head) (Scale bars = 500nm). The insets are the magnification of region of interest containing LAMP1 positive vacuole (Scale bars = 100nm), marked by the black rectangle.


The distribution of LMP2 positive signals ( n = 22 images ) assessed by plotting the number of gold particles labeling LMP2 within the LAMP1 positive membrane compartment versus those outside the LAMP1 positive organelles .

O O O B-GENEPROD O O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-GENEPROD O O B-GENEPROD O B-SUBCELLULAR O O O O O B-GENEPROD O B-SUBCELLULAR O

The distribution of LMP2 positive signals (n = 22 images) assessed by plotting the number of gold particles labeling LMP2 within the LAMP1 positive membrane compartment versus those outside the LAMP1 positive organelles.


Immuno - gold labeling with antibodies against constitutive proteasome subunits , α5 , β5 and 19 S2 ( 15 nm , black arrow head ) , co - labeled with an anti - LAMP1 antibody ( 5 nm , white arrow head ) in BMDC ( Scale bars = 500nm ) . Regions of interest containing LAMP1 positive vacuoles are marked by the black rectangles and magnified in the insets ( Scale bars = 100nm ) .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-SUBCELLULAR O O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O B-GENEPROD O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O

Immuno-gold labeling with antibodies against constitutive proteasome subunits, α5, β5 and 19 S2 (15 nm, black arrow head), co-labeled with an anti-LAMP1 antibody (5 nm, white arrow head) in BMDC (Scale bars = 500nm). Regions of interest containing LAMP1 positive vacuoles are marked by the black rectangles and magnified in the insets (Scale bars = 100nm).


Immuno - electron microscopy images ( n = 23 ) were analyzed by plotting the total number of gold particles labeling proteasome subunits inside and outside the LAMP1 positive membrane compartments ( D ) . These data are also presented as the percentage of the total gold particles that are within the LAMP1 positive organelle ( E ) .

B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-SUBCELLULAR O O O O O B-GENEPROD O B-SUBCELLULAR O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-GENEPROD O B-SUBCELLULAR O O O O

Immuno-electron microscopy images (n = 23) were analyzed by plotting the total number of gold particles labeling proteasome subunits inside and outside the LAMP1 positive membrane compartments (D). These data are also presented as the percentage of the total gold particles that are within the LAMP1 positive organelle (E).


BMDC transduced with genes encoding GFP , GFP - Rab5ACA and GFP - Rab22ACA under the control of an inducible promoter were fixed and stained for LMP2 and LAMP1 and analyzed by confocal microscopy 24hr after doxycycline induction and 4hr after Alexa 647 - OVA coated bead uptake . A single optical section of 10 optical sections of a representative cell are shown ( Scale Bar 10μm ) . Region of interest containing LAMP1 positive vacuole containing beads coated with Alexa - 647 conjugated OVA , marked by white rectangles , are magnified as inset ( Scale bars = 1μm )

B-CELL_TYPE O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-GENEPROD O O O O 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-GENEPROD O B-SUBCELLULAR O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-GENEPROD O O O O O O O O O O O O O O O O

BMDC transduced with genes encoding GFP, GFP-Rab5ACA and GFP-Rab22ACA under the control of an inducible promoter were fixed and stained for LMP2 and LAMP1 and analyzed by confocal microscopy 24hr after doxycycline induction and 4hr after Alexa 647-OVA coated bead uptake. A single optical section of 10 optical sections of a representative cell are shown (Scale Bar 10μm). Region of interest containing LAMP1 positive vacuole containing beads coated with Alexa-647 conjugated OVA, marked by white rectangles, are magnified as inset (Scale bars = 1μm)


BMDC transduced with genes encoding GFP , GFP - Rab5ACA and GFP - Rab22ACA under the control of an inducible promoter were fixed and stained for LMP2 and LAMP1 and analyzed by confocal microscopy 3D - rendering of 10 optical sections of a representative cell are shown ( Scale Bar 10μm ) . Region of interest containing LAMP1 positive vacuole containing beads coated with Alexa - 647 conjugated OVA , marked by white rectangles , are magnified as inset ( Scale bars = 1μm )

B-CELL_TYPE O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-SUBCELLULAR O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-GENEPROD O O O O O O O O O O O O O O O O

BMDC transduced with genes encoding GFP, GFP-Rab5ACA and GFP-Rab22ACA under the control of an inducible promoter were fixed and stained for LMP2 and LAMP1 and analyzed by confocal microscopy 3D-rendering of 10 optical sections of a representative cell are shown (Scale Bar 10μm). Region of interest containing LAMP1 positive vacuole containing beads coated with Alexa-647 conjugated OVA, marked by white rectangles, are magnified as inset (Scale bars = 1μm)


The same cells were analyzed for LMP2 fluorescence intensity within the phagosomal lumen as defined by a region of interest positive for Alexa 647 - OVA enclosed within a limiting membrane positive for LAMP1 . The fluorescence intensity of LMP2 within the phagosomal lumen normalized to Alexa647 - OVA was calculated and data compiled from independent experiments ( n = 3 ) are plotted ( H ) . For each transduced cell type , images of at the least 27 cells acquired across three independent experiments were analyzed and each cell is represented as a data point .

O O O O O O B-GENEPROD O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-GENEPROD O O O O B-SUBCELLULAR O O B-GENEPROD O O O O O B-GENEPROD O O B-SUBCELLULAR I-SUBCELLULAR O O B-SMALL_MOLECULE O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

The same cells were analyzed for LMP2 fluorescence intensity within the phagosomal lumen as defined by a region of interest positive for Alexa 647-OVA enclosed within a limiting membrane positive for LAMP1. The fluorescence intensity of LMP2 within the phagosomal lumen normalized to Alexa647-OVA was calculated and data compiled from independent experiments (n=3) are plotted (H).For each transduced cell type, images of at the least 27 cells acquired across three independent experiments were analyzed and each cell is represented as a data point.


BMDC co - expressing LMP2 - RFP and GFP - Rab5ACA were live imaged 16hr after doxycycline induction and 30min post uptake of Alexa647 coated latex beads ( Bar 10μm ) . Images were acquired every 3min and images taken at 12min interval are presented ( I - K ) . The arrowheads in panel ( I and J ) mark the phagosome or vacuoles containing LMP2 , respectively .

B-CELL_TYPE O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-EXP_ASSAY O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O 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-SUBCELLULAR O B-SUBCELLULAR O B-GENEPROD O O O

BMDC co-expressing LMP2-RFP and GFP-Rab5ACA were live imaged 16hr after doxycycline induction and 30min post uptake of Alexa647 coated latex beads (Bar 10μm). Images were acquired every 3min and images taken at 12min interval are presented (I-K). The arrowheads in panel (I and J) mark the phagosome or vacuoles containing LMP2, respectively.


BMDC co - expressing LMP2 - RFP and GFP - Rab22ACA ( J , K ) were live imaged 16hr after doxycycline induction and 30min post uptake of Alexa647 coated latex beads ( Bar 10μm ) . Images were acquired every 3min and images taken at 12min interval are presented ( I - K ) . The arrowheads in panel ( I and J ) mark the phagosome or vacuoles containing LMP2 , respectively . The boxed region in panel ( J ) marks a phagosome that has been enlarged in panel ( K ) ( Bar 1μm ) .

B-CELL_TYPE O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O B-EXP_ASSAY O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O 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-SUBCELLULAR O B-SUBCELLULAR O B-GENEPROD O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O

BMDC co-expressing LMP2-RFP and GFP-Rab22ACA (J, K) were live imaged 16hr after doxycycline induction and 30min post uptake of Alexa647 coated latex beads (Bar 10μm). Images were acquired every 3min and images taken at 12min interval are presented (I-K). The arrowheads in panel (I and J) mark the phagosome or vacuoles containing LMP2, respectively. The boxed region in panel (J) marks a phagosome that has been enlarged in panel (K) (Bar 1μm).


Activity of proteasomes within the phagosomes was determined by measuring the fluorescence intensity of fluorogenic proteasome substrate incubated with latex beads from detergent - treated phagosomes treated or untreated with Epoxomicin . The fluorescence intensities were measured at 1hr intervals and values plotted represent the ratio of the signals in the absence versus the presence of

O O B-SUBCELLULAR O O B-SUBCELLULAR O O O O O O O O O B-SUBCELLULAR O O O O O O O O O B-SUBCELLULAR O O O O B-SMALL_MOLECULE O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O

Activity of proteasomes within the phagosomes was determined by measuring the fluorescence intensity of fluorogenic proteasome substrate incubated with latex beads from detergent-treated phagosomes treated or untreated with Epoxomicin. The fluorescence intensities were measured at 1hr intervals and values plotted represent the ratio of the signals in the absence versus the presence of Epoxomicin


Intraluminal proteasome dependent degradation of phagocytosed antigen was measured by flow cytometric analysis of phagocytosed latex beads covalently conjugated with Alexa647 - OVA . Latex beads were extracted by mechanical disruption of BMDC and incubated with ATP , lysosomal protease inhibitor cocktail and varying dose of Epoxomicin at 37oC for 16hrs . Post incubation latex beads were treated with detergent and analyzed by flow cytometry ( B ) . The mean of Alexa647 fluorescence associated with the latex beads incubated with Epoxomicin were normalized to fluorescence of untreated beads from independent experiments were plotted ( n = 4 ) ( C ) .

O B-SUBCELLULAR O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-SMALL_MOLECULE O B-GENEPROD O O O O O O O O O B-CELL_TYPE O O O B-SMALL_MOLECULE O B-SUBCELLULAR O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O

Intraluminal proteasome dependent degradation of phagocytosed antigen was measured by flow cytometric analysis of phagocytosed latex beads covalently conjugated with Alexa647-OVA. Latex beads were extracted by mechanical disruption of BMDC and incubated with ATP, lysosomal protease inhibitor cocktail and varying dose of Epoxomicin at 37oC for 16hrs. Post incubation latex beads were treated with detergent and analyzed by flow cytometry (B). The mean of Alexa647 fluorescence associated with the latex beads incubated with Epoxomicin were normalized to fluorescence of untreated beads from independent experiments were plotted (n=4) (C).


Ubiquitination of phagosomal cargo within the phagosomes was assessed by measuring the acquisition of GFP fluorescence by phagocytosed latex beads conjugated with OVA post - detergent extraction from BMDC expressing GFP or GFP - Ub ( D ) . The mean of fluorescence intensities of GFP on the beads from independent experiments was plotted ( E ) .

B-EXP_ASSAY O B-SUBCELLULAR O O O B-SUBCELLULAR O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O B-CELL_TYPE B-EXP_ASSAY B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O

Ubiquitination of phagosomal cargo within the phagosomes was assessed by measuring the acquisition of GFP fluorescence by phagocytosed latex beads conjugated with OVA post-detergent extraction from BMDC expressing GFP or GFP-Ub (D). The mean of fluorescence intensities of GFP on the beads from independent experiments was plotted (E).


Whole cell extracts of High Five insect cells individually expressing FLAG - SHLD3 , Strep - RIF1 , and 6xHis - REV7 through baculovirus infection were combined and subjected to FLAG immunoprecipitation or streptactin pulldown and immunoblotted for REV7 or the Strep or FLAG epitopes . Results are representative of three biologically independent experiments . IB : immunoblot , FL : full - length . For SHLD3 , N and C correspond to residues 1 - 125 and 126 - 250 , respectively .

O O O O B-CELL_LINE I-CELL_LINE O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-ORGANISM O O O O O O B-GENEPROD B-EXP_ASSAY O B-SMALL_MOLECULE B-EXP_ASSAY O B-EXP_ASSAY O B-GENEPROD O O B-SMALL_MOLECULE O B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O

Whole cell extracts of High Five insect cells individually expressing FLAG-SHLD3, Strep-RIF1, and 6xHis-REV7 through baculovirus infection were combined and subjected to FLAG immunoprecipitation or streptactin pulldown and immunoblotted for REV7 or the Strep or FLAG epitopes. Results are representative of three biologically independent experiments. IB: immunoblot, FL: full-length. For SHLD3, N and C correspond to residues 1-125 and 126-250, respectively.


Representative micrographs of the LacR / LacO assay using mCherry - LacR - RIF1N as bait to evaluate chromatin recruitment of eGFP - tagged SHLD3 variants . SHLD3 : residues 2 - 250 . SHLD3N : residues 2 - 125 . SHLD3C : residues 126 - 250 . RIF1N : residues 1 - 967 .

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O O O O B-SUBCELLULAR O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O

Representative micrographs of the LacR/LacO assay using mCherry-LacR-RIF1N as bait to evaluate chromatin recruitment of eGFP-tagged SHLD3 variants. SHLD3: residues 2-250. SHLD3N: residues 2-125. SHLD3C: residues 126-250. RIF1N: residues 1-967.


Quantification of LacR / LacO assay measuring chromatin recruitment of eGFP - tagged SHLD3 variants to LacO arrays by mCherry - LacR - RIF1N . GFP intensities are presented as a ratio between the average fluorescence intensity within the mCherry - labeled LacR focus and the average nuclear intensity . Bars represent means ( n = 138 , 126 , 129 , 137 for eGFP , eGFP - SHLD3 , eGFP - SHLD3N , eGFP - SHLD3C from 3 biologically independent experiments ) . Analysis was performed by Kruskal - Wallis test followed by Dunn ' s multiple comparisons against empty vector control or full - length eGFP - SHLD3 . * * * * : P < 0 . 0001 . ns : P > 0 . 05 .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR O O B-GENEPROD O O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O 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-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O

Quantification of LacR/LacO assay measuring chromatin recruitment of eGFP-tagged SHLD3 variants to LacO arrays by mCherry-LacR-RIF1N. GFP intensities are presented as a ratio between the average fluorescence intensity within the mCherry-labeled LacR focus and the average nuclear intensity. Bars represent means (n = 138, 126, 129, 137 for eGFP, eGFP-SHLD3, eGFP-SHLD3N, eGFP-SHLD3C from 3 biologically independent experiments). Analysis was performed by Kruskal-Wallis test followed by Dunn's multiple comparisons against empty vector control or full-length eGFP-SHLD3. ****: P < 0.0001. ns: P > 0.05.


Representative micrographs of the LacR - FokI assay to evaluate DNA damage recruitment of eGFP - tagged SHLD3 variants after induction of LacR - FokI expression .

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-SMALL_MOLECULE O O O B-GENEPROD O O B-GENEPROD O O O O O O B-GENEPROD B-EXP_ASSAY O

Representative micrographs of the LacR-FokI assay to evaluate DNA damage recruitment of eGFP-tagged SHLD3 variants after induction of LacR-FokI expression.


Quantification of LacR - FokI assay evaluating recruitment of eGFP - tagged SHLD3 variants to FokI - induced DSBs at LacO arrays . GFP intensities are presented as a ratio between the average fluorescence intensity within the mCherry - labeled LacR - FokI focus and the average nuclear intensity . Bars represent mean ( n = 143 , 147 , 147 , 141 for eGFP , eGFP - SHLD3 , eGFP - SHLD3N , eGFP - SHLD3C from 3 biologically independent experiments ) . Analysis was performed by Kruskal - Wallis test followed by Dunn ' s multiple comparisons against empty vector control or full - length eGFP - SHLD3 . * * * * : P < 0 . 0001 . ns : P > 0 . 05 .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O B-SUBCELLULAR O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O 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-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O

Quantification of LacR-FokI assay evaluating recruitment of eGFP-tagged SHLD3 variants to FokI-induced DSBs at LacO arrays. GFP intensities are presented as a ratio between the average fluorescence intensity within the mCherry-labeled LacR-FokI focus and the average nuclear intensity. Bars represent mean (n = 143, 147, 147, 141 for eGFP, eGFP-SHLD3, eGFP-SHLD3N, eGFP-SHLD3C from 3 biologically independent experiments). Analysis was performed by Kruskal-Wallis test followed by Dunn's multiple comparisons against empty vector control or full-length eGFP-SHLD3. ****: P < 0.0001. ns: P > 0.05.


Whole cell extracts of High Five insect cells individually expressing the wild - type ( WT ) or indicated alanine substitution FLAG - SHLD3C ( residues 126 - 250 ) variants and Strep - RIF1N ( residues 1 - 980 ) through baculovirus infection were combined and subjected to FLAG immunoprecipitation or streptactin pulldown and immunoblotted for the Strep or FLAG epitopes . The results are representative of two biologically independent experiments . IB : immunoblot . IP : immunoprecipitation .

O O O O B-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O B-SMALL_MOLECULE O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-ORGANISM O O O O O O B-GENEPROD B-EXP_ASSAY O B-SMALL_MOLECULE B-EXP_ASSAY O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O B-EXP_ASSAY O B-EXP_ASSAY O

Whole cell extracts of High Five insect cells individually expressing the wild-type (WT) or indicated alanine substitution FLAG-SHLD3C (residues 126-250) variants and Strep-RIF1N (residues 1-980) through baculovirus infection were combined and subjected to FLAG immunoprecipitation or streptactin pulldown and immunoblotted for the Strep or FLAG epitopes. The results are representative of two biologically independent experiments. IB: immunoblot. IP: immunoprecipitation.


Quantification of LacR / LacO assay measuring recruitment of eGFP - SHLD3C wild - type ( WT ) or alanine substitution variants to LacO arrays in U2OS 2 - 6 - 3 cells by mCherry - LacR - RIF1N . GFP intensities are presented as a ratio between the average fluorescence intensity within the mCherry - labeled LacR - RIF1N focus and the average nuclear intensity . Bars represent means ( n = 134 , 125 , 120 , 130 , 104 , 129 , 117 for EV , WT , S131A , W132A , R166A , N201A , D216A from 3 biologically independent experiments ) . EV : empty vector . Analysis was performed by Kruskal - Wallis test followed by Dunn ' s multiple comparisons against empty vector and WT controls . * * * * : P < 0 . 0001 . * * * : P = 0 . 0004 . * * : P = 0 . 002 . * : P = 0 . 02 . ns : P > 0 . 05 .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Quantification of LacR/LacO assay measuring recruitment of eGFP-SHLD3C wild-type (WT) or alanine substitution variants to LacO arrays in U2OS 2-6-3 cells by mCherry-LacR-RIF1N. GFP intensities are presented as a ratio between the average fluorescence intensity within the mCherry-labeled LacR-RIF1N focus and the average nuclear intensity. Bars represent means (n = 134, 125, 120, 130, 104, 129, 117 for EV, WT, S131A, W132A, R166A, N201A, D216A from 3 biologically independent experiments). EV: empty vector. Analysis was performed by Kruskal-Wallis test followed by Dunn's multiple comparisons against empty vector and WT controls. ****: P < 0.0001. ***: P = 0.0004. **: P = 0.002. *: P = 0.02. ns: P > 0.05.


Quantification of LacR - FokI assay measuring recruitment of eGFP - SHLD3C wild - type ( WT ) or alanine substitution variants to sites of DNA double - strand breaks induced by mCherry - LacR - FokI in U2OS 2 - 6 - 3 cells . GFP intensities are presented as a ratio between the average fluorescence intensity within the mCherry - labeled LacR - FokI focus and the average nuclear intensity . Bars represent means ( n = 103 , 136 , 159 , 151 , 139 , 145 , 140 for EV , WT , S131A , W132A , R166A , N201A , D216A from 3 biologically independent experiments ) . Analysis was performed by Kruskal - Wallis test followed by Dunn ' s multiple comparisons against empty vector and WT controls . * * * * : P < 0 . 0001 . ns : P > 0 . 05 .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O B-GENEPROD O O O B-GENEPROD O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Quantification of LacR-FokI assay measuring recruitment of eGFP-SHLD3C wild-type (WT) or alanine substitution variants to sites of DNA double-strand breaks induced by mCherry-LacR-FokI in U2OS 2-6-3 cells. GFP intensities are presented as a ratio between the average fluorescence intensity within the mCherry-labeled LacR-FokI focus and the average nuclear intensity. Bars represent means (n = 103, 136, 159, 151, 139, 145, 140 for EV, WT, S131A, W132A, R166A, N201A, D216A from 3 biologically independent experiments). Analysis was performed by Kruskal-Wallis test followed by Dunn's multiple comparisons against empty vector and WT controls. ****: P < 0.0001. ns: P > 0.05.


Representative micrographs of immunofluorescence experiments ( from 3 biologically independent experiments ) analyzing localization of SHLD3 to 53BP1 bodies in RPE SHLD3 - KO cells . 3xFLAG - SHLD3 - complemented RPE SHLD3 - KO cells were treated with 200 nM aphidicolin ( Aphi ) for 24 h and processed for immunofluorescence microscopy using antibodies against 53BP1 , FLAG , and cyclin A . 53BP1 bodies are defined as distinct foci visible in cyclin A - negative cells . Quantification of FLAG and 53BP1 colocalization from A performed in CellProfiler . Pearson ' s correlation coefficients ( PCC ) were calculated for pixels within each cyclin A - negative , 53BP1 body - positive nucleus between the 53BP1 and FLAG channels . Each point represents the PCC value of an individual nucleus . Bars represent means . ( n = 120 , 130 for EV , SHLD3 from three biologically independent experiments ) . Analysis was performed using two - tailed Mann - Whitney test . * * * * : P < 0 . 0001 .

O O O B-EXP_ASSAY O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O B-CELL_LINE B-GENEPROD O O O O O O B-GENEPROD O O B-CELL_LINE B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD O O O B-GENEPROD O O O B-SUBCELLULAR O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Representative micrographs of immunofluorescence experiments (from 3 biologically independent experiments) analyzing localization of SHLD3 to 53BP1 bodies in RPE SHLD3-KO cells. 3xFLAG-SHLD3-complemented RPE SHLD3-KO cells were treated with 200 nM aphidicolin (Aphi) for 24 h and processed for immunofluorescence microscopy using antibodies against 53BP1, FLAG, and cyclin A. 53BP1 bodies are defined as distinct foci visible in cyclin A-negative cells. Quantification of FLAG and 53BP1 colocalization from A performed in CellProfiler. Pearson's correlation coefficients (PCC) were calculated for pixels within each cyclin A-negative, 53BP1 body-positive nucleus between the 53BP1 and FLAG channels. Each point represents the PCC value of an individual nucleus. Bars represent means. (n = 120, 130 for EV, SHLD3 from three biologically independent experiments). Analysis was performed using two-tailed Mann-Whitney test. ****: P < 0.0001.


Representative micrographs of immunofluorescence experiments analyzing localization of FLAG - SHLD3 alanine substitution variants stably expressed in RPE SHLD3 - KO cells by lentivirus transduction . Cells were treated with 200 nM Aphi for 24 hours and processed for immunofluorescence microscopy using antibodies raised against 53BP1 , FLAG , and cyclin A . 53BP1 bodies are defined as distinct foci visible in cyclin A - negative cells .

O O O B-EXP_ASSAY O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD B-SMALL_MOLECULE O O O O O B-CELL_LINE B-GENEPROD O O O O B-ORGANISM O O O O O O O O B-SMALL_MOLECULE O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD O O O O

Representative micrographs of immunofluorescence experiments analyzing localization of FLAG-SHLD3 alanine substitution variants stably expressed in RPE SHLD3-KO cells by lentivirus transduction. Cells were treated with 200 nM Aphi for 24 hours and processed for immunofluorescence microscopy using antibodies raised against 53BP1, FLAG, and cyclin A. 53BP1 bodies are defined as distinct foci visible in cyclin A-negative cells.


Quantification of the percentage of cells containing the indicated number of 53BP1 bodies in RPE WT and SHLD3 - KO cells with or without 24 - hour 200 nM Aphi treatment . Bars represent means ± s . d . ( n = 3 biologically independent experiments with ≥ 30 nuclei imaged each ) .

O O O O O O O O O O O B-GENEPROD O O B-CELL_LINE O O B-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O

Quantification of the percentage of cells containing the indicated number of 53BP1 bodies in RPE WT and SHLD3-KO cells with or without 24-hour 200 nM Aphi treatment. Bars represent means ± s.d. (n = 3 biologically independent experiments with ≥ 30 nuclei imaged each).


Left : Representation of the region of the RIF1 N - terminal HEAT repeats that is predicted to bind the SHLD3 eIF4E - like domain . Residues 1 - 173 are highlighted . Right : Quantification of LacR / LacO assay measuring eGFP - SHLD3 recruitment to LacO arrays in U2OS 2 - 6 - 3 cells by the indicated truncated mCherry - LacR - RIF1N variants . GFP intensities are presented as a ratio between the average fluorescence intensity within the mCherry - labeled LacR - RIF1N focus and the average nuclear intensity . Bars represent means ( n = 148 , 123 , 122 for EV , 1 - 567 , 174 - 567 from 3 biologically independent experiments ) . Analysis was performed using Mann - Whitney two - tailed test . * * * * : P < 0 . 001 .

O O O O O O O O B-GENEPROD O O O O O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Left: Representation of the region of the RIF1 N-terminal HEAT repeats that is predicted to bind the SHLD3 eIF4E-like domain. Residues 1-173 are highlighted. Right: Quantification of LacR/LacO assay measuring eGFP-SHLD3 recruitment to LacO arrays in U2OS 2-6-3 cells by the indicated truncated mCherry-LacR-RIF1N variants. GFP intensities are presented as a ratio between the average fluorescence intensity within the mCherry-labeled LacR-RIF1N focus and the average nuclear intensity. Bars represent means (n = 148, 123, 122 for EV, 1-567, 174-567 from 3 biologically independent experiments). Analysis was performed using Mann-Whitney two-tailed test. ****: P < 0.001.


Whole cell extracts of High Five insect cells individually expressing the FLAG - SHLD3C ( residues 126 - 250 ) and the indicated alanine substitution Strep - RIF1 ( residues 1 - 980 ) variants through baculovirus infection were combined and subjected to FLAG immunoprecipitation or streptactin pulldown and immunoblotted for the Strep or FLAG epitopes . The results are representative of two biologically independent experiments . IB : immunoblot . IP : immunoprecipitation .

O O O O B-CELL_LINE I-CELL_LINE O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-GENEPROD O O O O O O O O B-ORGANISM O O O O O O B-GENEPROD B-EXP_ASSAY O B-SMALL_MOLECULE B-EXP_ASSAY O B-EXP_ASSAY O O B-SMALL_MOLECULE O B-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O B-EXP_ASSAY O B-EXP_ASSAY O

Whole cell extracts of High Five insect cells individually expressing the FLAG-SHLD3C (residues 126-250) and the indicated alanine substitution Strep-RIF1 (residues 1-980) variants through baculovirus infection were combined and subjected to FLAG immunoprecipitation or streptactin pulldown and immunoblotted for the Strep or FLAG epitopes. The results are representative of two biologically independent experiments. IB: immunoblot. IP: immunoprecipitation.


Representative micrographs of UV laser microirradiation experiments measuring DNA damage recruitment of REV7 in U2OS 2 - 6 - 3 cells with endogenously mutated RIF1 . DNA damage was induced in U2OS 2 - 6 - 3 cells through irradiation in the form of linear stripes and analyzed by immunofluorescence microscopy with RIF1 and REV7 antibodies . Quantification of UV laser microirradiation immunofluorescence experiment measuring DNA damage recruitment of REV7 in U2OS 2 - 6 - 3 cells with endogenously mutated RIF1 . REV7 immunofluorescence intensities are presented as a ratio between the average fluorescence intensity within the RIF1 - labeled irradiation stripe and the average nuclear intensity . Only nuclei containing RIF1 stripes are quantified . Bars represent means ( n = 206 , 215 for RIF1WT , RIF1D28N from 2 biologically independent experiments ) . Analysis was performed using Welch ' s t - test . * * * * : P < 0 . 0001 .

O O O O O O O O B-SMALL_MOLECULE O O O B-GENEPROD O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O B-EXP_ASSAY O O B-SMALL_MOLECULE O O O B-GENEPROD O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O B-GENEPROD O O O O O O O B-SUBCELLULAR O O O B-SUBCELLULAR O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Representative micrographs of UV laser microirradiation experiments measuring DNA damage recruitment of REV7 in U2OS 2-6-3 cells with endogenously mutated RIF1. DNA damage was induced in U2OS 2-6-3 cells through irradiation in the form of linear stripes and analyzed by immunofluorescence microscopy with RIF1 and REV7 antibodies. Quantification of UV laser microirradiation immunofluorescence experiment measuring DNA damage recruitment of REV7 in U2OS 2-6-3 cells with endogenously mutated RIF1. REV7 immunofluorescence intensities are presented as a ratio between the average fluorescence intensity within the RIF1-labeled irradiation stripe and the average nuclear intensity. Only nuclei containing RIF1 stripes are quantified. Bars represent means (n = 206, 215 for RIF1WT, RIF1D28N from 2 biologically independent experiments). Analysis was performed using Welch's t-test. ****: P < 0.0001.


Quantification of class switch recombination in stimulated CH12F3 Shld3 - / - cells stably expressing the indicated FLAG - SHLD3 variants through lentiviral transduction . Bars represent means ± s . d . , n = 3 biologically independent experiments . Analysis was performed using one - way ANOVA followed by Dunnett ' s multiple comparisons test against Shld3 - / - cells complemented with wild - type SHLD3 . * * : P < 0 . 01 , * : P < 0 . 05 , ns : P > 0 . 05 .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-CELL_LINE B-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD O O B-ORGANISM O O O O O O O O O O O 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-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O

Quantification of class switch recombination in stimulated CH12F3 Shld3-/- cells stably expressing the indicated FLAG-SHLD3 variants through lentiviral transduction. Bars represent means ± s.d., n = 3 biologically independent experiments. Analysis was performed using one-way ANOVA followed by Dunnett's multiple comparisons test against Shld3-/- cells complemented with wild-type SHLD3. **: P < 0.01, *: P < 0.05, ns: P > 0.05.


Competitive growth assay of RPE BRCA1 - KO SHLD3 - KO cells complemented with 3xFLAG - SHLD3 variants . Growth of the indicated eGFP - expressing cells were compared to mCherry - expressing RPE BRCA1 - KO SHLD3 - KO cells transduced with empty vector after mixing 1 : 1 in 24 - well plates . Plates were imaged 1 , 5 , 9 , and 13 days after plating . Dotted black lines represent an eGFP : mCherry ratio of 1 . Points represent means ± s . d . ( n = 3 biologically independent experiments ) . Analysis was performed using a one sample t - test against a hypothetical eGFP : mCherry ratio value of 1 for the day 13 time point . * * : P < 0 . 01 , ns : P > 0 . 05 .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE B-GENEPROD O O B-GENEPROD O O O O O O O B-GENEPROD O O B-EXP_ASSAY O O O B-GENEPROD O O O O O O B-GENEPROD O O B-CELL_LINE B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O 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-GENEPROD O B-GENEPROD O O O O O O O O O O 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-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Competitive growth assay of RPE BRCA1-KO SHLD3-KO cells complemented with 3xFLAG-SHLD3 variants. Growth of the indicated eGFP-expressing cells were compared to mCherry-expressing RPE BRCA1-KO SHLD3-KO cells transduced with empty vector after mixing 1:1 in 24-well plates. Plates were imaged 1, 5, 9, and 13 days after plating. Dotted black lines represent an eGFP:mCherry ratio of 1. Points represent means ± s.d. (n = 3 biologically independent experiments). Analysis was performed using a one sample t-test against a hypothetical eGFP:mCherry ratio value of 1 for the day 13 time point. **: P < 0.01, ns: P > 0.05.


( B ) IHC for CDC50A and RIM1 on cultured neurons at DIV21 . The left panel shows CDC50A is expressed in both neuronal soma and processes . The right panel exhibits CDC50A colocalize to pre - synapses alongside neuronal processes . MAP2 , Microtubule - associated protein 2 , was used to identify neuronal cells and trace dendritic processes . RIM1 is an active zone protein , which marks pre - synapses . Left scale bar = 50 µm ; right scale bar = 2 µm . ( C ) Quantification of the percentage of RIM1 + pre - synapses colocalizing to CDC50A . N = 3 for biological replicates .

O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O B-CELL_TYPE O O O O O O O B-GENEPROD O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O B-GENEPROD B-EXP_ASSAY O O O B-SUBCELLULAR O B-CELL_TYPE O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-CELL_TYPE I-CELL_TYPE O O B-SUBCELLULAR O O B-GENEPROD O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-SUBCELLULAR B-EXP_ASSAY O B-GENEPROD O O O O O O O O

(B) IHC for CDC50A and RIM1 on cultured neurons at DIV21. The left panel shows CDC50A is expressed in both neuronal soma and processes. The right panel exhibits CDC50A colocalize to pre-synapses alongside neuronal processes. MAP2, Microtubule-associated protein 2, was used to identify neuronal cells and trace dendritic processes. RIM1 is an active zone protein, which marks pre-synapses. Left scale bar = 50 µm; right scale bar = 2 µm. (C) Quantification of the percentage of RIM1+ pre-synapses colocalizing to CDC50A. N = 3 for biological replicates.


( E ) IHC for CDC50A and vGluT2 in the developmental dLGN at P14 , P21 and P30 . The left panels show the overview of immunostaining in the dLGN , scale bar = 200 µm . The right panels are the zoomed - in images , scale bar = 5 µm . Arrows indicate the colocalized CDC50A and vGluT2 . ( F ) Quantification of the percentage of CDC50A + / vGlut2 + synapses . The result shows that a significantly higher percentage of CDC50A + / vGlut2 + synapses at P21 and P30 than P14 . N = 6 for biological replicates .

O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O B-TISSUE O O O O O O O O O O O O O O B-EXP_ASSAY O O B-TISSUE O O 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-EXP_ASSAY B-GENEPROD O B-GENEPROD O O O O O O O O O B-GENEPROD O O B-GENEPROD O B-SUBCELLULAR O O O O O O O O O O B-GENEPROD O O B-GENEPROD O B-SUBCELLULAR O O O O O O O O O O O O O O

(E) IHC for CDC50A and vGluT2 in the developmental dLGN at P14, P21 and P30. The left panels show the overview of immunostaining in the dLGN, scale bar = 200 µm. The right panels are the zoomed-in images, scale bar = 5 µm. Arrows indicate the colocalized CDC50A and vGluT2. (F) Quantification of the percentage of CDC50A+/vGlut2+ synapses. The result shows that a significantly higher percentage of CDC50A+/vGlut2+ synapses at P21 and P30 than P14. N = 6 for biological replicates.


( C ) IHC for CDC50A . CDC50A protein intensities were reduced after neuronal inactivation ( day 7 ) , and was recovered after removing neuronal inhibitors ( day 13 ) . Scale bar , 10 µm . ( D ) Relative CDC50A intensities of neurons . N = 5 for biological replicates , mean ± SD , * p < 0 . 5 , * * * * p < 0 . 0001 by one - way ANOVA with Holm - Sidak ' s multiple comparisons test .

O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O B-CELL_TYPE O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O B-GENEPROD O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O 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) IHC for CDC50A. CDC50A protein intensities were reduced after neuronal inactivation (day 7), and was recovered after removing neuronal inhibitors (day 13). Scale bar, 10 µm. (D) Relative CDC50A intensities of neurons. N = 5 for biological replicates, mean ± SD, * p < 0.5, **** p < 0.0001 by one-way ANOVA with Holm-Sidak's multiple comparisons test.


( E ) CDC50A expression at RGC inputs after 5 - day consecutive TTX treatment . Scale bar , 10 µm . ( F ) Quantification of the percentage of CDC50A + / vGlut2 + synapses . N = 5 for biological replicates , mean ± SD , * * * * p < 0 . 0001 by Student ' s test .

O O O B-GENEPROD O O B-CELL_TYPE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O B-SUBCELLULAR O O O 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) CDC50A expression at RGC inputs after 5-day consecutive TTX treatment. Scale bar, 10 µm. (F) Quantification of the percentage of CDC50A+/vGlut2+ synapses. N = 5 for biological replicates, mean ± SD, **** p < 0.0001 by Student's test.


( A ) Immunostaining of CDC50A on Cdc50a knockdown neurons and controls . Accell Cdc50a siRNA pool was used to knock down Cdc50a in neurons for 7 days . CDC50A expression alongside neuronal processed was reduced after Cdc50a siRNA treatment . Scale bar , 10 µm .

O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-CELL_TYPE O O O O B-GENEPROD O O O O O O O B-GENEPROD O B-CELL_TYPE O O O O B-GENEPROD O O B-CELL_TYPE O O O O B-GENEPROD O O O O O O O O O

(A) Immunostaining of CDC50A on Cdc50a knockdown neurons and controls. Accell Cdc50a siRNA pool was used to knock down Cdc50a in neurons for 7 days. CDC50A expression alongside neuronal processed was reduced after Cdc50a siRNA treatment. Scale bar, 10 µm.


( D ) IHC of Cdc50a - RNAi neurons exhibited pSIVA colocalized with synapsin I , a general synapse marker . The dotted square was zoomed - in shown on the right side . Arrows indicated where pSIVA colocalized with synapsin I . Scale bar , 5 µm .

O O O B-EXP_ASSAY O B-GENEPROD O O B-CELL_TYPE O B-SMALL_MOLECULE B-EXP_ASSAY O B-GENEPROD I-GENEPROD O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD O O O O O O O

(D) IHC of Cdc50a-RNAi neurons exhibited pSIVA colocalized with synapsin I, a general synapse marker. The dotted square was zoomed-in shown on the right side. Arrows indicated where pSIVA colocalized with synapsin I. Scale bar, 5 µm.


( D ) Representative IHC images of CDC50A expression at synapses . Scale bar , 5 µm . Arrows indicate VGluT2 + RGC inputs . ( E ) Relative CDC50A expression at mCherry + synapses in Cdc50a RNAi mice and controls . N = 3 for biological replicates , mean ± SD , * p < 0 . 05 by Student ' s t - test .

O O O O B-EXP_ASSAY O O B-GENEPROD O O B-SUBCELLULAR O O O O O O O O O B-GENEPROD O B-CELL_TYPE O O O O O O B-GENEPROD O O B-GENEPROD O B-SUBCELLULAR O B-GENEPROD O B-ORGANISM O O O O 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) Representative IHC images of CDC50A expression at synapses. Scale bar, 5 µm. Arrows indicate VGluT2+ RGC inputs. (E) Relative CDC50A expression at mCherry+ synapses in Cdc50a RNAi mice and controls. N = 3 for biological replicates, mean ± SD, * p < 0.05 by Student's t-test.


( F ) PSIVA staining in the dLGN . Exposed PS was detected by pSIVA on RGC inputs in Cdc50a - RNAi mice . Scale bar , 5 µm . Arrows indicate pSIVA + / mCherry + RGC inputs . ( G ) Quantification of the percentage of pSIVA + / mCherry + RGC inputs in total mCherry + RGC inputs . N = 4 for biological replicates , mean ± SEM , * * p < 0 . 01 by Student ' s t - test .

O O O B-SMALL_MOLECULE B-EXP_ASSAY O O B-TISSUE O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O B-CELL_TYPE O O B-GENEPROD O O B-ORGANISM O O O O O O O O O B-SMALL_MOLECULE O O B-GENEPROD O B-CELL_TYPE O O O O O O O O O O B-SMALL_MOLECULE O O B-GENEPROD O B-CELL_TYPE O O O B-GENEPROD O B-CELL_TYPE O O O O 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) PSIVA staining in the dLGN. Exposed PS was detected by pSIVA on RGC inputs in Cdc50a-RNAi mice. Scale bar, 5 µm. Arrows indicate pSIVA+/mCherry+ RGC inputs. (G) Quantification of the percentage of pSIVA+/mCherry+ RGC inputs in total mCherry+ RGC inputs. N = 4 for biological replicates, mean ± SEM, **p < 0.01 by Student's t-test.


( F ) Maximum intensity z - projection images of vGluT2 + synapses in the core region of Cdc50a RNAi mice and controls . Right panel showed enlarged synapses in the boxed region . Scale bar , 20 µm .

O O O O O O O O O O B-GENEPROD O B-SUBCELLULAR O O O O O B-GENEPROD O B-ORGANISM O O O O O O O B-SUBCELLULAR O O O O O O O O O O O

(F) Maximum intensity z-projection images of vGluT2+ synapses in the core region of Cdc50a RNAi mice and controls. Right panel showed enlarged synapses in the boxed region. Scale bar, 20 µm.


( H ) Maximum intensity z - projection images of vGluT2 + synapses in the shell region . Enlarged images ( right panel ) shows large " complex encapsulated " boutons . Scale bar , 20 µm . ( I ) Quantification of vGluT2 + synapse density . N = 3 for biological replicates , mean ± SD , Student ' s t - test .

O O O O O O O O O O B-GENEPROD O B-SUBCELLULAR O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O B-GENEPROD O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O

(H) Maximum intensity z-projection images of vGluT2+ synapses in the shell region. Enlarged images (right panel) shows large "complex encapsulated" boutons. Scale bar, 20 µm. (I) Quantification of vGluT2+ synapse density. N = 3 for biological replicates, mean ± SD, Student's t-test.


( D ) Images of CTB488 and CTB647 labeling of RGC inputs in the dLGN . Scale bar , 20 µm . ( E ) Quantification of CTB488 + inputs ( non - infected areas ) versus CTB647 + inputs ( infected areas ) in Cdc50a RNAi mice and controls . N = 3 for biological replicates , mean ± SEM , * * * * p < 0 . 0001 , two - way ANOVA with Sidak ' s multiple comparisons test .

O O O O O B-GENEPROD O B-GENEPROD O O B-CELL_TYPE O O O B-TISSUE O O O O O O O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O B-ORGANISM O O O O O O O O O O O O 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) Images of CTB488 and CTB647 labeling of RGC inputs in the dLGN. Scale bar, 20 µm. (E) Quantification of CTB488+ inputs (non-infected areas) versus CTB647+ inputs (infected areas) in Cdc50a RNAi mice and controls. N = 3 for biological replicates, mean ± SEM, **** p < 0.0001, two-way ANOVA with Sidak's multiple comparisons test.


( A ) The confocal images , 3D - rendered images and orthogonal views of microglia engulfing vGluT2 + synapses in controls and Cdc50a - RNAi mice . Scale bar , 10 µm .

O O O O B-EXP_ASSAY O O O O O O O O O O B-CELL_TYPE O B-GENEPROD O B-SUBCELLULAR O O O B-GENEPROD O O B-ORGANISM O O O O O O O

(A) The confocal images, 3D-rendered images and orthogonal views of microglia engulfing vGluT2+ synapses in controls and Cdc50a-RNAi mice. Scale bar, 10 µm.


( A ) IHC images for vGluT2 staining in the dLGN of microglial Gpr56 conditional knockout mice ( Gpr56fl / fl ; Cx3cr1cre / + ) and controls ( Gpr56 + / + ; Cx3cr1cre / + ) . Both mice were intravitreally injected with AAV2 carrying Cdc50a shRNA . The yellow squares indicated the AAV2 non - infected areas and the dotted outlines indicated the AAV2 infected areas . Scale bar , 200 µm .

O O O B-EXP_ASSAY O O B-GENEPROD B-EXP_ASSAY O O B-TISSUE O B-CELL_TYPE B-GENEPROD O O B-ORGANISM O B-GENEPROD O O O B-GENEPROD O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O B-ORGANISM O O O O B-ORGANISM O B-GENEPROD O O O O O O O B-ORGANISM O O O O O O O O O O B-ORGANISM O O O O O O O O O

(A) IHC images for vGluT2 staining in the dLGN of microglial Gpr56 conditional knockout mice (Gpr56fl/fl; Cx3cr1cre/+) and controls (Gpr56+/+; Cx3cr1cre/+). Both mice were intravitreally injected with AAV2 carrying Cdc50a shRNA. The yellow squares indicated the AAV2 non-infected areas and the dotted outlines indicated the AAV2 infected areas. Scale bar, 200 µm.


( B ) Confocal images of vGluT2 + synapses in the AAV2 non - infected areas and infected areas of the dLGN . Scale bar , 20 µm . ( C ) Quantification of vGluT2 + synapses in the non - infected areas and infected areas of microglial GPR56 conditional knockout mice and controls . N = 4 for biological replicates , mean ± SD , * p < 0 . 05 , * * * * p < 0 . 0001 by two - way ANOVA with Sidak ' s multiple comparisons test .

O O O B-EXP_ASSAY O O B-GENEPROD O B-SUBCELLULAR O O B-ORGANISM O O O O O O O O O B-TISSUE O O O O O O O O O O O O B-GENEPROD O B-SUBCELLULAR O O O O O O O O O O B-CELL_TYPE B-GENEPROD O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Confocal images of vGluT2+ synapses in the AAV2 non-infected areas and infected areas of the dLGN. Scale bar, 20 µm. (C) Quantification of vGluT2+ synapses in the non-infected areas and infected areas of microglial GPR56 conditional knockout mice and controls. N = 4 for biological replicates, mean ± SD, * p<0.05, **** p < 0.0001 by two-way ANOVA with Sidak's multiple comparisons test.


RNAPII - RBP - RNA complex was immunoprecipitated by anti - RNAPII antibody or control antibody ( cont ) , and subjected to immunoblotting . The input lanes contained 0 . 4 % of lysates used in the immunoprecipitation experiments .

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RNAPII-RBP-RNA complex was immunoprecipitated by anti-RNAPII antibody or control antibody (cont), and subjected to immunoblotting. The input lanes contained 0.4% of lysates used in the immunoprecipitation experiments.


Generation of tRIP - seq libraries of RNAPII - RBP - RNA complexes . After UV crosslinking , protein - RNA complexes were immunoprecipitated with anti - RNAPII ( 1st IP ) . RNA of the isolated RNAPII - RBP - RNA complexes was partially digested with RNase on beads , and RBP - RNA complexes dissociated from the bead were further immunoprecipitated with anti - FUS or anti - U1C antibody bound to the other beads ( 2nd IP ) . The immunoprecipitated FUS - RNA or U1C - RNA complex was treated with TEX , and then subjected to tRIP - seq library generation . The bottom panels show PCR - amplification of tRIP - seq libraries generated from RNAPII - FUS - RNA complex ( left panel ) and RNAPII - U1C - RNA complex ( right panel ) .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-EXP_ASSAY O O O B-GENEPROD O O O O O B-SUBCELLULAR O O O B-GENEPROD O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O B-SUBCELLULAR O B-GENEPROD O B-SUBCELLULAR O O O O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR O B-GENEPROD O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O B-GENEPROD O B-SUBCELLULAR O O O O O O

Generation of tRIP-seq libraries of RNAPII-RBP-RNA complexes. After UV crosslinking, protein-RNA complexes were immunoprecipitated with anti-RNAPII (1st IP). RNA of the isolated RNAPII-RBP-RNA complexes was partially digested with RNase on beads, and RBP-RNA complexes dissociated from the bead were further immunoprecipitated with anti-FUS or anti-U1C antibody bound to the other beads (2nd IP). The immunoprecipitated FUS-RNA or U1C-RNA complex was treated with TEX, and then subjected to tRIP-seq library generation. The bottom panels show PCR-amplification of tRIP-seq libraries generated from RNAPII-FUS-RNA complex (left panel) and RNAPII-U1C-RNA complex (right panel).


PolyA - seq was performed using N2A cells to examine the effect of downregulation of FUS and U1 snRNP on polyA site usage . Cells were treated with siRNA against Fus ( siFus ) and / or U1 antisense morpholino ( U1AS ) . Control cells ( Cont ) were treated with both control siRNA and control antisense morpholino . A . Correlation of changes in polyA site usage between U1 - inhibition and Fus - silencing . Fold change in the RPM of a polyA site between siFus and Cont ( siFus / Cont ) was plotted against that between U1AS and Cont ( U1AS / Cont ) . Pearson ' s correlation coefficient ( r ) and the slope of the regression line are shown . B . Correlation of changes in polyA site usage between U1 - inhibition alone and the combination of Fus - silencing and U1 - inhibition . Fold change in the RPM of a polyA site between siFus + U1AS and Cont ( siFus + U1AS / Cont ) was plotted against that between U1AS and Cont ( U1AS / Cont ) . Pearson ' s correlation coefficient ( r ) and the slope of the regression line are shown . A slope of 1 . 0 indicates that Fus - silencing has no additive effect on U1 - inhibition .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-CELL_LINE O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O 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-GENEPROD O O O B-GENEPROD O O 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-GENEPROD O O O B-GENEPROD O O O O O O O O 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-GENEPROD O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O 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-GENEPROD O O O O O O O O O O O

PolyA-seq was performed using N2A cells to examine the effect of downregulation of FUS and U1 snRNP on polyA site usage. Cells were treated with siRNA against Fus (siFus) and/or U1 antisense morpholino (U1AS). Control cells (Cont) were treated with both control siRNA and control antisense morpholino. A. Correlation of changes in polyA site usage between U1-inhibition and Fus-silencing. Fold change in the RPM of a polyA site between siFus and Cont (siFus/Cont) was plotted against that between U1AS and Cont (U1AS/Cont). Pearson's correlation coefficient (r) and the slope of the regression line are shown. B. Correlation of changes in polyA site usage between U1-inhibition alone and the combination of Fus-silencing and U1-inhibition. Fold change in the RPM of a polyA site between siFus+U1AS and Cont (siFus+U1AS/Cont) was plotted against that between U1AS and Cont (U1AS/Cont). Pearson's correlation coefficient (r) and the slope of the regression line are shown. A slope of 1.0 indicates that Fus-silencing has no additive effect on U1-inhibition.


Localization of FUS in R495X _ N2A cells ( R495X ) , and wild - type N2A cells ( WT ) . Cells were fractionated to separate nucleus and cytoplasm . Equal proportion of cell extracts from nuclear and cytoplasmic fractions were immunoblotted with indicated antibodies .

B-EXP_ASSAY O B-GENEPROD O O O B-CELL_LINE O O O O O O O O O B-CELL_LINE O O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O B-EXP_ASSAY O O O O

Localization of FUS in R495X_N2A cells (R495X), and wild-type N2A cells (WT). Cells were fractionated to separate nucleus and cytoplasm. Equal proportion of cell extracts from nuclear and cytoplasmic fractions were immunoblotted with indicated antibodies.


Interactions between FUS and U1 snRNP in the R495X _ N2A cells ( R495X ) and wild - type N2A cells ( WT ) . Total cell lysates were immunoprecipitated with anti - U1 - 70K antibody or a control antibody ( cont ) , and immunoblotted with indicated antibodies .

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Interactions between FUS and U1 snRNP in the R495X_N2A cells (R495X) and wild-type N2A cells (WT). Total cell lysates were immunoprecipitated with anti-U1-70K antibody or a control antibody (cont), and immunoblotted with indicated antibodies.


Effects of the R495X mutation on four categories of polyA sites . Four categories of polyA sites are comprised of ( 1 ) FUS - repressed ( rep ) / U1 - repressed sites , ( 2 ) FUS - activated ( act ) / U1 - repressed sites , ( 3 ) FUS - repressed / U1 - activated sites , and ( 4 ) FUS - activated / U1 - activated sites , according to Fig 4A . Fold change of each polyA site usage by the R495X mutation ( R495X / WT ) is shown in violin plots . Bars indicate the median and 95 % confidential intervals for each category . * * * p < 0 . 001 , * * * * p < 0 . 0001 , based on the Steel - Dwass test for comparing all 6 pairs of categories .

O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Effects of the R495X mutation on four categories of polyA sites. Four categories of polyA sites are comprised of (1) FUS-repressed (rep) /U1-repressed sites, (2) FUS-activated (act) /U1-repressed sites, (3) FUS-repressed/U1-activated sites, and (4) FUS-activated/U1-activated sites, according to Fig 4A. Fold change of each polyA site usage by the R495X mutation (R495X/WT) is shown in violin plots. Bars indicate the median and 95% confidential intervals for each category. *** p < 0.001, **** p < 0.0001, based on the Steel-Dwass test for comparing all 6 pairs of categories.


( A ) Representative confocal photomicrographs showing coronal slices from 8W hGFAP ‐ tTA ; H2B ‐ GFP mice with and without doxycycline treatment , upon immunofluorescent staining with the stem cell marker Nestin ( red ) . DAPI was used for nuclear counterstain . Cross indicates slice directions : M = medial , D = dorsal , V = ventral , L = lateral . Scale bar = 20 µm . ( B ) Quantification of Nestin expression in apical and basal cells in doxycycline - treated and untreated animals . N = 3 ( - Doxy ) , N = 4 ( + Doxy ) . Data information : N number refers to biological replicates ; for each biological replicate , at least three technical replicates were performed . Data is represented as mean ± SEM . * and # indicate significance : between apical and basal ( * ) and between doxycycline treatments ( # ) ; * / # p < 0 . 05 , * * p < 0 . 01 , by one - way ANOVA with Tukey ' s multiple comparisons test ( B )

O O O O B-EXP_ASSAY I-EXP_ASSAY O B-TISSUE O O O O O O O B-GENEPROD O B-GENEPROD B-ORGANISM O O O B-SMALL_MOLECULE O O O B-EXP_ASSAY I-EXP_ASSAY O O B-CELL_TYPE I-CELL_TYPE O B-GENEPROD O O O O B-SMALL_MOLECULE O O O B-SUBCELLULAR B-EXP_ASSAY O O O O O O O O O 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-GENEPROD B-EXP_ASSAY O O O B-CELL_TYPE I-CELL_TYPE O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O 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-CELL_TYPE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O 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) Representative confocal photomicrographs showing coronal slices from 8W hGFAP‐tTA;H2B‐GFP mice with and without doxycycline treatment, upon immunofluorescent staining with the stem cell marker Nestin (red). DAPI was used for nuclear counterstain. Cross indicates slice directions: M = medial, D = dorsal, V = ventral, L = lateral. Scale bar = 20 µm. (B) Quantification of Nestin expression in apical and basal cells in doxycycline-treated and untreated animals. N = 3 (- Doxy), N = 4 (+ Doxy). Data information: N number refers to biological replicates; for each biological replicate, at least three technical replicates were performed. Data is represented as mean ± SEM. * and # indicate significance: between apical and basal (*) and between doxycycline treatments (#); */#p<0.05, **p<0.01, by one-way ANOVA with Tukey's multiple comparisons test (B)


( C ) Same as ( A ) , with addition of the primary cilia marker AC3 ( yellow ) . Arrows indicate Nestin - positive ( white ) and GFP - positive ( green ) cells with primary cilium . Scale bar = 10 µm . ( D ) Quantification of ciliated GFP + NSCs in apical and basal cell layer as percentage of total apical / basal GFP + cells . N = 7 . ( E ) Quantification of primary cilia on cells in the apical or basal cell layer according to stem cell marker ; N = 7 . Data information : N number refers to biological replicates ; for each biological replicate , at least three technical replicates were performed . Data is represented as mean ± SEM . * and # indicate significance : between apical and basal ( * ) ; * / # p < 0 . 05 , * * p < 0 . 01 , by two - tailed unpaired student ' s t - test ( D ) .

O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O B-GENEPROD O B-CELL_TYPE O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O B-CELL_TYPE B-GENEPROD O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O B-CELL_TYPE I-CELL_TYPE O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O 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-CELL_TYPE O O O O O O O O O O O O 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) Same as (A), with addition of the primary cilia marker AC3 (yellow). Arrows indicate Nestin-positive (white) and GFP-positive (green) cells with primary cilium. Scale bar = 10 µm. (D) Quantification of ciliated GFP+ NSCs in apical and basal cell layer as percentage of total apical/basal GFP+ cells. N = 7. (E) Quantification of primary cilia on cells in the apical or basal cell layer according to stem cell marker; N = 7. Data information: N number refers to biological replicates; for each biological replicate, at least three technical replicates were performed. Data is represented as mean ± SEM. * and # indicate significance: between apical and basal (*) ; */#p<0.05, **p<0.01, by two-tailed unpaired student's t-test (D).


( A ) Repesentative confocal photomicrographs of coronal sections of the SVZ of 8W hGFAP - H2BGFP mice , which had been administered doxycycline ( Doxy ) as indicated . Nuclear GFP expression and Ki67 immunoreactivity are shown in green and red , respectively . DAPI counterstaining of the nuclei is blue . Scale bar indicates 20 µm .

O O O O B-EXP_ASSAY I-EXP_ASSAY O B-TISSUE O O O B-TISSUE O O O O B-GENEPROD B-ORGANISM O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-SUBCELLULAR B-GENEPROD B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE B-EXP_ASSAY O O B-SUBCELLULAR O O O O O O O O O

(A) Repesentative confocal photomicrographs of coronal sections of the SVZ of 8W hGFAP-H2BGFP mice, which had been administered doxycycline (Doxy) as indicated. Nuclear GFP expression and Ki67 immunoreactivity are shown in green and red, respectively. DAPI counterstaining of the nuclei is blue. Scale bar indicates 20 µm.


( B , C ) Quantitative analyses of the number of apical and basal cells expressing GFP ( G + ) or not ( G - ) ; N = 8 ( B ) , and Ki67 immunoreactivity in these populations ; N ≥ 4 ( C ) . Data information : N number refers to biological replicates ; for each biological replicate at least three technical replicates were performed . Bars represent mean ± SEM . * indicates significance between doxycycline treated and nontreated specimens with apical or basal cell populations . * p < 0 . 05 , * * * p < 0 . 001 , determined by two - way ANOVA with Sidak ' s multiple comparisons test .

O O O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O O O O O O O O O O O O O O O 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) Quantitative analyses of the number of apical and basal cells expressing GFP (G+) or not (G-); N = 8 (B), and Ki67 immunoreactivity in these populations; N ≥ 4 (C). Data information: N number refers to biological replicates; for each biological replicate at least three technical replicates were performed. Bars represent mean ± SEM. * indicates significance between doxycycline treated and nontreated specimens with apical or basal cell populations. * p<0.05, ***p<0.001, determined by two-way ANOVA with Sidak's multiple comparisons test.


( B ) Representative micrographs of the OB of AAV - GFP - injected animals , at 14 days ( 14D ) and 6 weeks ( 6W ) after virus injection . Scale bar indicates 20 µm . ( C ) Quantification of GFP + and / or DCX + cells in the OB , as percentage of total DAPI cells N ≥ 5 . Data information : N number refers to biological replicates ; for each biological replicate at least three technical replicates were performed . Bars represent mean ± SEM . * indicates significance : * p < 0 . 05 , determined by two - way ANOVA with Sidak ' s multiple comparisons test .

O O O O O O O B-TISSUE O B-ORGANISM O B-GENEPROD O O O O O O O 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-GENEPROD O O O O B-GENEPROD O O O O B-TISSUE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative micrographs of the OB of AAV-GFP-injected animals, at 14 days (14D) and 6 weeks (6W) after virus injection. Scale bar indicates 20 µm. (C) Quantification of GFP+ and/or DCX+ cells in the OB, as percentage of total DAPI cells N ≥ 5. Data information: N number refers to biological replicates; for each biological replicate at least three technical replicates were performed. Bars represent mean ± SEM.* indicates significance: *p<0.05, determined by two-way ANOVA with Sidak's multiple comparisons test.


Representative confocal images of coronal slices of the core of the olfactory bulb ( OB ) obtained from RiboTag mice injected with AVV - hGFAP - mycCRE particles . In ( E ) mice were analysed 16 weeks after injection in the lateral ventricle or in the lateral corner as indicated . Scale bars = 20 µm . ( F ) Quantification of RiboTag - HA ( HA ) + and / or DCX + cells in the OB in N ≥ 2 . Data information : N number refers to biological replicates ; for each biological replicate at least three technical replicates were performed . Bars represent mean ± SEM . * indicates significance : * p < 0 . 05 , determined by two - way ANOVA with Sidak ' s multiple comparisons test .

O B-EXP_ASSAY O O B-TISSUE O O O O O O B-TISSUE I-TISSUE O B-TISSUE O O O O B-ORGANISM O O B-ORGANISM O O O B-GENEPROD O O O O O O B-ORGANISM O O O O O O O O B-TISSUE I-TISSUE O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Representative confocal images of coronal slices of the core of the olfactory bulb (OB) obtained from RiboTag mice injected with AVV-hGFAP-mycCRE particles. In (E) mice were analysed 16 weeks after injection in the lateral ventricle or in the lateral corner as indicated. Scale bars = 20 µm. (F) Quantification of RiboTag-HA (HA)+ and/or DCX+ cells in the OB in N ≥ 2. Data information: N number refers to biological replicates; for each biological replicate at least three technical replicates were performed. Bars represent mean ± SEM.* indicates significance: *p<0.05, determined by two-way ANOVA with Sidak's multiple comparisons test.


( A , B ) Representative micrographs of the V - SVZ of mice injected with an AAV overexpressing GFP and a shRNA against Hes1 ( AAV Hes1 - sh ) or a scrambled sequence ( AAV sc - sh ) . Coronal sections were stained with either notch intracellular domain ( NICD , A ) or Ki67 to visualize cycling cells ( B ) . DAPI was used for nuclear counterstain , scale bars indicate 20 µm . Arrows indicate apical GFP + ( white ) and basal GFP - ( yellow ) cells displaying NICD staining . Quantification of fold change in NICD - levels ; N = 3 ( C ) , Ki67 + cells ; N = 5 ( D ) Data information : N number refers to biological replicates ; for each biological replicate at least three technical replicates were performed . Bars represent mean ± SEM . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , determined by two - way ANOVA with Sidak ' s multiple comparisons

O O O O O O O O O B-TISSUE I-TISSUE I-TISSUE O B-ORGANISM O O O B-ORGANISM O B-GENEPROD O O O O B-GENEPROD O B-ORGANISM B-GENEPROD O O O O O O O O B-ORGANISM O O O O O B-TISSUE O O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O O O B-SUBCELLULAR B-EXP_ASSAY O O O O O O O O O O B-GENEPROD O O O O O B-CELL_TYPE B-GENEPROD O O O O O O B-GENEPROD B-EXP_ASSAY O O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative micrographs of the V-SVZ of mice injected with an AAV overexpressing GFP and a shRNA against Hes1 (AAV Hes1-sh) or a scrambled sequence (AAV sc-sh). Coronal sections were stained with either notch intracellular domain (NICD, A) or Ki67 to visualize cycling cells (B). DAPI was used for nuclear counterstain, scale bars indicate 20 µm. Arrows indicate apical GFP+ (white) and basal GFP- (yellow) cells displaying NICD staining. Quantification of fold change in NICD-levels; N = 3 (C), Ki67+ cells; N = 5 (D) Data information: N number refers to biological replicates; for each biological replicate at least three technical replicates were performed. Bars represent mean ± SEM. *p<0.05, **p<0.01, ***p<0.001, determined by two-way ANOVA with Sidak's multiple comparisons test


A , Misreading ( A ) were measured by dual luciferase gain - of - function assays using wild - type mitohybrid ribosomes ( n = 8 misreading , mutant A1555G mitohybrid ribosomes ( n = 8 misreading , E . coli wild - type ribosomes ( n = 9 ) , E . coli mutant A127Y ribosomes ( n = 9 ) , M . smegmatis merodiploid wild - type ribosomes ( n = 9 ) , and M . smegmatis merodiploid mutant S152Y ribosomes ( n = 9 ) . Mistranslation was determined by assessing the misreading at His245 ( CAC codon ) of the F - luc gene that was replaced by the near - cognate codon CGC or the non - cognate codon AGA , both encoding for Arg . Results are derived by calculating mutant hFluc / hRluc activity related to wild - type hFluc / hRluc activity .

O O B-EXP_ASSAY O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-SUBCELLULAR O O O O B-EXP_ASSAY O O O O B-SUBCELLULAR O O O O B-EXP_ASSAY O B-ORGANISM I-ORGANISM I-ORGANISM O O O B-SUBCELLULAR O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O B-SUBCELLULAR O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O B-SUBCELLULAR O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O B-SUBCELLULAR O O O O O O B-EXP_ASSAY O O O O O B-EXP_ASSAY O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-EXP_ASSAY O O O O O O O O B-EXP_ASSAY O

A, Misreading (A) were measured by dual luciferase gain-of-function assays using wild-type mitohybrid ribosomes (n=8 misreading, mutant A1555G mitohybrid ribosomes (n=8 misreading, E. coli wild-type ribosomes (n=9), E.coli mutant A127Y ribosomes (n=9), M. smegmatis merodiploid wild-type ribosomes (n=9), and M. smegmatis merodiploid mutant S152Y ribosomes (n=9). Mistranslation was determined by assessing the misreading at His245 (CAC codon) of the F-luc gene that was replaced by the near-cognate codon CGC or the non-cognate codon AGA, both encoding for Arg. Results are derived by calculating mutant hFluc/hRluc activity related to wild-type hFluc/hRluc activity.


read - through ( B ) were measured by dual luciferase gain - of - function assays using wild - type mitohybrid ribosomes n = 4 read - through ) , mutant A1555G mitohybrid ribosomes n = 4 read - through ) , E . coli wild - type ribosomes ( n = 9 ) , E . coli mutant A127Y ribosomes ( n = 9 ) , M . smegmatis merodiploid wild - type ribosomes ( n = 9 ) , and M . smegmatis merodiploid mutant S152Y ribosomes ( n = 9 ) . To determine read - through the Asp357 ( GAC codon ) of the F - luc gene was replaced by the nonsense TGA stop codon . Results are derived by calculating mutant hFluc / hRluc activity related to wild - type hFluc / hRluc activity .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-SUBCELLULAR O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O B-SUBCELLULAR O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O B-SUBCELLULAR O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O B-SUBCELLULAR O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O B-EXP_ASSAY O

read-through (B) were measured by dual luciferase gain-of-function assays using wild-type mitohybrid ribosomes n=4 read-through), mutant A1555G mitohybrid ribosomes n=4 read-through), E. coli wild-type ribosomes (n=9), E.coli mutant A127Y ribosomes (n=9), M. smegmatis merodiploid wild-type ribosomes (n=9), and M. smegmatis merodiploid mutant S152Y ribosomes (n=9). To determine read-through the Asp357 (GAC codon) of the F-luc gene was replaced by the nonsense TGA stop codon. Results are derived by calculating mutant hFluc/hRluc activity related to wild-type hFluc/hRluc activity.