( F ) The bead - autophagosome fraction was lysed and subjected to immunoprecipitation with an anti - Ub IgG antibody ( FK2 ) or control IgG . Co - immunoprecipitated molecules were examined by Western blotting using the indicated antibodies .

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(F) The bead-autophagosome fraction was lysed and subjected to immunoprecipitation with an anti-Ub IgG antibody (FK2) or control IgG. Co-immunoprecipitated molecules were examined by Western blotting using the indicated antibodies.


( G and H ) NIH3T3 cells stably expressing mStrawberry ( mStr ) - Gal3 and GFP - LC3 , mStr - Gal3 , and GFP - p62 , or mStr - Gal3 and GFP - Ub were transfected with Effectene - coated latex beads for 30 min and then washed . Live cells were observed at 1 - min intervals by fluorescence microscopy . Bar , 3 µm . The time after galectin3 localization was measured for at least 30 cases for each combination ( H ) . Statistical analysis was performed by Student ' s unpaired t test . * , P < 0 . 05 ; NS , not significant .

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

(G and H) NIH3T3 cells stably expressing mStrawberry (mStr)-Gal3 and GFP-LC3, mStr-Gal3, and GFP-p62, or mStr-Gal3 and GFP-Ub were transfected with Effectene-coated latex beads for 30 min and then washed. Live cells were observed at 1-min intervals by fluorescence microscopy. Bar, 3 µm. The time after galectin3 localization was measured for at least 30 cases for each combination (H). Statistical analysis was performed by Student's unpaired t test. *, P < 0.05; NS, not significant.


( A and B ) NIH3T3 cells stably expressing mStr - Ub and GFP - tagged LC3 , Atg5 , WIPI - 1 , Atg14L1 , or ULK1 were transfected with Effectene - coated latex beads for 30 min . Then , live cells were observed at 1 - min intervals by fluorescence microscopy . Bar , 3 µm . The time after Ub localization was measured for at least 30 cases for each combination . Each value represents the mean ± SD . Statistical analysis was performed by Student ' s unpaired t test . * , P < 0 . 05 ; NS , not significant .

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(A and B) NIH3T3 cells stably expressing mStr-Ub and GFP-tagged LC3, Atg5, WIPI-1, Atg14L1, or ULK1 were transfected with Effectene-coated latex beads for 30 min. Then, live cells were observed at 1-min intervals by fluorescence microscopy. Bar, 3 µm. The time after Ub localization was measured for at least 30 cases for each combination. Each value represents the mean ± SD. Statistical analysis was performed by Student's unpaired t test. *, P < 0.05; NS, not significant.


( C ) NIH3T3 cells stably expressing GFP - tagged Ub , LC3 , Atg5 , WIPI - 1 , Atg14L1 , Atg9L1 , or ULK1 were transfected with Effectene - coated beads for 3 h in the presence or absence ( mock ) of 30 µM UBEI - 41 ( a ubiquitin E1 - specific inhibitor ) and subjected to immunocytochemistry for galectin3 . The percentages of GFP - positive per galectin3 - positive beads were enumerated . At least 30 beads were counted ( n = 3 ) . The values are the mean ± SD . Statistical analysis was performed by Student ' s unpaired t test . * , P < 0 . 05 .

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

(C) NIH3T3 cells stably expressing GFP-tagged Ub, LC3, Atg5, WIPI-1, Atg14L1, Atg9L1, or ULK1 were transfected with Effectene-coated beads for 3 h in the presence or absence (mock) of 30 µM UBEI-41 (a ubiquitin E1-specific inhibitor) and subjected to immunocytochemistry for galectin3. The percentages of GFP-positive per galectin3-positive beads were enumerated. At least 30 beads were counted (n = 3). The values are the mean ± SD. Statistical analysis was performed by Student's unpaired t test. *, P < 0.05.


( D and E ) Parent NIH3T3 cells , Atg4B mutant overexpressing NIH3T3 cells stably expressing GFP - tagged LC3 , Atg5 , WIPI - 1 , Atg14L1 , Atg9L1 , or ULK1 were transfected with Effectene - coated latex beads for 3 h and subjected to immunocytochemistry for galectin3 . The percentages of Atg - positive per galectin3 - positive beads were enumerated . At least 30 beads were counted ( n = 3 ) . The values are the mean ± SD .

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

(D and E) Parent NIH3T3 cells, Atg4B mutant overexpressing NIH3T3 cells stably expressing GFP-tagged LC3, Atg5, WIPI-1, Atg14L1, Atg9L1, or ULK1 were transfected with Effectene-coated latex beads for 3 h and subjected to immunocytochemistry for galectin3. The percentages of Atg-positive per galectin3-positive beads were enumerated. At least 30 beads were counted (n = 3). The values are the mean ± SD.


( E ) wild - type MEFs , and Atg5 - KO MEFs stably expressing GFP - tagged LC3 , Atg5 , WIPI - 1 , Atg14L1 , Atg9L1 , or ULK1 were transfected with Effectene - coated latex beads for 3 h and subjected to immunocytochemistry for galectin3 . The percentages of Atg - positive per galectin3 - positive beads were enumerated . At least 30 beads were counted ( n = 3 ) . The values are the mean ± SD .

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

(E) wild-type MEFs, and Atg5-KO MEFs stably expressing GFP-tagged LC3, Atg5, WIPI-1, Atg14L1, Atg9L1, or ULK1 were transfected with Effectene-coated latex beads for 3 h and subjected to immunocytochemistry for galectin3. The percentages of Atg-positive per galectin3-positive beads were enumerated. At least 30 beads were counted (n = 3). The values are the mean ± SD.


( A ) HeLa cells were lysed with 1 % Triton X - 100 lysis buffer and subjected to immunoprecipitation with FK2 - or control IgG - immobilized beads . The coimmunoprecipitated molecules were examined by Western blotting using the indicated antibodies .

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

(A) HeLa cells were lysed with 1% Triton X-100 lysis buffer and subjected to immunoprecipitation with FK2- or control IgG-immobilized beads. The coimmunoprecipitated molecules were examined by Western blotting using the indicated antibodies.


( C ) Purified recombinant GST or GST - Ub - immobilized glutathione Sepharose beads were incubated with lysates from HEK293T cells transiently expressing FLAG - tagged Atg16L1 constructs for 1 h at 25 ° C with gentle agitation . The beads were washed three times with ice - cold PBS and the bound complexes were eluted with 50 mM reduced glutathione and then subjected to Western blotting for FLAG .

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

(C) Purified recombinant GST or GST-Ub-immobilized glutathione Sepharose beads were incubated with lysates from HEK293T cells transiently expressing FLAG-tagged Atg16L1 constructs for 1 h at 25°C with gentle agitation. The beads were washed three times with ice-cold PBS and the bound complexes were eluted with 50 mM reduced glutathione and then subjected to Western blotting for FLAG.


( D ) Purified GST or GST - Ub - immobilized glutathione Sepharose beads were incubated with lysates from bacteria expressing trigger factor ( TF ) or TF - FLAG - WD β - propellers for 1 h at 25 ° C with gentle agitation . Washing and elution were performed as described in C . The eluted samples were subjected to SDS - PAGE and Western blotting for FLAG .

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(D) Purified GST or GST-Ub-immobilized glutathione Sepharose beads were incubated with lysates from bacteria expressing trigger factor (TF) or TF-FLAG-WD β-propellers for 1 h at 25°C with gentle agitation. Washing and elution were performed as described in C. The eluted samples were subjected to SDS-PAGE and Western blotting for FLAG.


( A ) Yeast two - hybrid interactions of the ULK1 complexes with other human Atg proteins . ULK1 , ULK2 , Atg13 , FIP200 , and Atg101 - AD fusions ( rows 2 - 6 ) or control AD constructs ( row 1 , Empty ) were coexpressed with control DBD constructs ( column 1 , Empty ) or Atg protein - DBD fusions ( columns 2 - 35 ) and tested for positive yeast two - hybrid interactions ( top ) or cotransformation ( control , bottom ) . White lines divide images derived from the same plate , and red lines divide images from different plates . Atg16L1 positively interacted with FIP200 in this assay .

O O O B-EXP_ASSAY I-EXP_ASSAY I-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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Yeast two-hybrid interactions of the ULK1 complexes with other human Atg proteins. ULK1, ULK2, Atg13, FIP200, and Atg101-AD fusions (rows 2-6) or control AD constructs (row 1, Empty) were coexpressed with control DBD constructs (column 1, Empty) or Atg protein-DBD fusions (columns 2-35) and tested for positive yeast two-hybrid interactions (top) or cotransformation (control, bottom). White lines divide images derived from the same plate, and red lines divide images from different plates. Atg16L1 positively interacted with FIP200 in this assay.


( B ) FIP200 binds to exogenously expressed Atg16L1 . Myc - Atg16L1 coprecipitations with an empty vector control ( lane 1 ) or One - STrEP - FLAG ( OSF ) - FIP200 ( lane 2 ) .

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(B) FIP200 binds to exogenously expressed Atg16L1. Myc-Atg16L1 coprecipitations with an empty vector control (lane 1) or One-STrEP-FLAG (OSF)-FIP200 (lane 2).


( C ) FIP200 binds endogenous Atg16L1 . Atg16L1 coprecipitations with an empty vector control ( lane 1 ) or OSF - FIP200 ( lane 2 ) .

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(C) FIP200 binds endogenous Atg16L1. Atg16L1 coprecipitations with an empty vector control (lane 1) or OSF-FIP200 (lane 2).


( D ) Wild - type or FIP200 - KO MEFs stably expressing the empty vector , full - length Atg16L1 , or ΔWD mutant were incubated in growth medium ( − ) or Earle ' s balanced salt solution ( EBSS ) ( + ) for 1 h and examined by Western blotting using the indicated antibodies . We previously reported that stable expression of an exogenous Atg16L1 construct can be used to replace the endogenous Atg16L1 protein with an exogenous Atg16L1 protein ( Fujita et al . , 2009 ) because free Atg16L1 molecules not complexed with the Atg12 - Atg5 conjugate are preferentially degraded by the ubiquitin - proteasome system . In control cells ( Empty ) , two Atg16L1 splicing variants , the α - and β - forms , were detected . In full - length β - form - replaced cells ( Full ) , the α - form was not detected , whereas in ΔWD - replaced cells ( ΔWD ) both the α - and β - forms were minimally detected . Thus , we successfully obtained cells that express only endogenous levels of the full - length or ΔWD mutant .

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

(D) Wild-type or FIP200-KO MEFs stably expressing the empty vector, full-length Atg16L1, or ΔWD mutant were incubated in growth medium (−) or Earle's balanced salt solution (EBSS) (+) for 1 h and examined by Western blotting using the indicated antibodies. We previously reported that stable expression of an exogenous Atg16L1 construct can be used to replace the endogenous Atg16L1 protein with an exogenous Atg16L1 protein (Fujita et al., 2009) because free Atg16L1 molecules not complexed with the Atg12-Atg5 conjugate are preferentially degraded by the ubiquitin-proteasome system. In control cells (Empty), two Atg16L1 splicing variants, the α- and β-forms, were detected. In full-length β-form-replaced cells (Full), the α-form was not detected, whereas in ΔWD-replaced cells (ΔWD) both the α- and β-forms were minimally detected. Thus, we successfully obtained cells that express only endogenous levels of the full-length or ΔWD mutant.


( E - L ) Atg16L1 - replaced wild - type or FIP200 - KO MEFs were infected with Salmonella ( MOI = 10 ) for 1 h and subjected to immunocytochemistry for Atg16L1 and Ub ( E and F ) or LC3 and Ub ( I and J ) . The percentages of Atg16L1 - positive ( G and H ) or LC3 - positive per Ub - positive bacteria were enumerated ( K and L ) . At least 50 Salmonella were counted . The average ± SD is shown for three independent experiments . Statistical analysis was performed by Student ' s t test . * , P < 0 . 05 ; NS , not significant . Bar , 5 µm .

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

(E-L) Atg16L1-replaced wild-type or FIP200-KO MEFs were infected with Salmonella (MOI = 10) for 1 h and subjected to immunocytochemistry for Atg16L1 and Ub (E and F) or LC3 and Ub (I and J). The percentages of Atg16L1-positive (G and H) or LC3-positive per Ub-positive bacteria were enumerated (K and L). At least 50 Salmonella were counted. The average ± SD is shown for three independent experiments. Statistical analysis was performed by Student's t test. *, P < 0.05; NS, not significant. Bar, 5 µm.


( A ) Mapping of the Atg16L1 - binding site on FIP200 . Yeast two - hybrid mapping experiments showing that Atg16L1 binds to both the N - terminal and C - terminal coiled - coil regions of FIP200 .

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

(A) Mapping of the Atg16L1-binding site on FIP200. Yeast two-hybrid mapping experiments showing that Atg16L1 binds to both the N-terminal and C-terminal coiled-coil regions of FIP200.


( B ) Mapping of the FIP200 - binding site within Atg16L1 . Yeast two - hybrid mapping experiments showing that the 1 - 840 fragment of FIP200 binds to the C - terminal 247 - 607 region , while the 1276 - 1591 fragment of FIP200 binds to the N - terminal 1 - 264 region of Atg16L1 .

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

(B) Mapping of the FIP200-binding site within Atg16L1. Yeast two-hybrid mapping experiments showing that the 1-840 fragment of FIP200 binds to the C-terminal 247-607 region, while the 1276-1591 fragment of FIP200 binds to the N-terminal 1-264 region of Atg16L1.


( C and D ) Additional yeast two - hybrid mapping experiments showing that the binding site of the FIP200 1276 - 1591 fragment maps to residues 239 - 246 of Atg16L1 . White lines divide images derived from the same plate , and red lines divide images from different plates .

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

(C and D) Additional yeast two-hybrid mapping experiments showing that the binding site of the FIP200 1276-1591 fragment maps to residues 239-246 of Atg16L1. White lines divide images derived from the same plate, and red lines divide images from different plates.


( E ) The N - terminal 1 - 249 fragment of Atg16L1 is sufficient to interact with FIP200 . Full - length ( lanes 1 and 2 ) or the N - terminal 1 - 249 fragments ( lanes 3 and 4 ) of Myc - Atg16L1 coprecipitations with empty vector controls ( lanes 1 and 3 ) or OSF - FIP200 1276 - 1591 ( lanes 2 and 4 ) .

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

(E) The N-terminal 1-249 fragment of Atg16L1 is sufficient to interact with FIP200. Full-length (lanes 1 and 2) or the N-terminal 1-249 fragments (lanes 3 and 4) of Myc-Atg16L1 coprecipitations with empty vector controls (lanes 1 and 3) or OSF-FIP200 1276-1591 (lanes 2 and 4).


( F ) Co - precipitation experiments confirming that OSF - FIP200 1276 - 1591 coprecipitates with wild - type ( WT ) Myc - Atg16L1 ( 1 - 249 ) fragments , but not with analogous fragments containing a mutant FIP200 - binding site ( Myc - Atg16L1 ΔWD 239 - 242A ) .

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(F) Co-precipitation experiments confirming that OSF-FIP200 1276-1591 coprecipitates with wild-type (WT) Myc-Atg16L1 (1-249) fragments, but not with analogous fragments containing a mutant FIP200-binding site (Myc-Atg16L1 ΔWD 239-242A).


( B - E ) Atg16L1 - Δ / Δ MEFs stably expressing the indicated constructs were infected with Salmonella ( MOI = 10 ) for 1 h and then analyzed by immunocytochemistry for LC3 ( B ) or Atg16L1 ( D ) . Bar , 5 µm . The percentage of LC3 - or Atg16L1 - positive Salmonella per Ub - positive Salmonella was enumerated by fluorescence microscopy ( C and E ) . At least 50 bacteria were counted . The average ± SD is shown for three independent experiments .

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

(B-E) Atg16L1-Δ/Δ MEFs stably expressing the indicated constructs were infected with Salmonella (MOI = 10) for 1 h and then analyzed by immunocytochemistry for LC3 (B) or Atg16L1 (D). Bar, 5 µm. The percentage of LC3- or Atg16L1-positive Salmonella per Ub-positive Salmonella was enumerated by fluorescence microscopy (C and E). At least 50 bacteria were counted. The average ± SD is shown for three independent experiments.


( F ) Atg16L1 - Δ / Δ MEFs stably expressing the indicated constructs were cultured in growth medium ( Fed ) or EBSS ( Starved ) for 1 h and then subjected to immunocytochemistry using an anti - LC3 antibody . The number of LC3 puncta in each cell was counted for more than 50 cells . The average ± SD is shown for three independent experiments .

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(F) Atg16L1-Δ/Δ MEFs stably expressing the indicated constructs were cultured in growth medium (Fed) or EBSS (Starved) for 1 h and then subjected to immunocytochemistry using an anti-LC3 antibody. The number of LC3 puncta in each cell was counted for more than 50 cells. The average ± SD is shown for three independent experiments.


A ) Representative size - exclusion chromatography ( SEC ) chromatogram with absorbance measured at nanometers ( nm ) 260 or 280 ( for this chromatogram and the following ones , absorbances are in units of 1000 - 1 ( mAU ) ) of reconstituted K . lactis COMA - Nkp1 - Nkp2 , and image of Coomassie Blue - stained SDS - PAGE gel with fractions from principal SEC peaks . Molecular masses of protein standards ( Mm . S . ) are in kiloDalton ( kDa ) ; L : sample loaded on column . † : Escherichia Coli DnaK ; ╪ : proteolysed Nkp1 - Nkp2 ; Nkp1 - Nkp2 is less prone to spontaneous proteolysis when associated with COMA .

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

A) Representative size-exclusion chromatography (SEC) chromatogram with absorbance measured at nanometers (nm) 260 or 280 (for this chromatogram and the following ones, absorbances are in units of 1000-1 (mAU)) of reconstituted K. lactis COMA-Nkp1-Nkp2, and image of Coomassie Blue-stained SDS-PAGE gel with fractions from principal SEC peaks. Molecular masses of protein standards (Mm. S.) are in kiloDalton (kDa); L: sample loaded on column. †: Escherichia Coli DnaK; ╪: proteolysed Nkp1-Nkp2; Nkp1-Nkp2 is less prone to spontaneous proteolysis when associated with COMA.


B , C ) Nanoflow mass - spectra of COMA - Nkp1 - Nkp2 ( B ) acquired with different injected concentrations , showing an increase in COMA - Nkp1 - Nkp2 dimers or COMA dimers with increasing injectant concentration .

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B,C) Nanoflow mass-spectra of COMA-Nkp1-Nkp2 (B) acquired with different injected concentrations, showing an increase in COMA-Nkp1-Nkp2 dimers or COMA dimers with increasing injectant concentration.


B , C ) Nanoflow mass - spectra of COMA ( C ) , acquired with different injected concentrations , showing an increase in COMA - Nkp1 - Nkp2 dimers or COMA dimers with increasing injectant concentration .

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

B,C) Nanoflow mass-spectra of COMA (C), acquired with different injected concentrations, showing an increase in COMA-Nkp1-Nkp2 dimers or COMA dimers with increasing injectant concentration.


D ) Graph with measured differential refractive index ( relative scale ) and molar mass calculated with multi - angle light scattering ( MALS ) data of COMA eluting from SEC ( SEC - MALS ) . We injected COMA at a concentration of ~ 43 μM ( COMA dimer ) on the column . Dashed lines indicate expected molar masses for monomeric COMA or dimeric COMA : 142337 g mol − 1 or 284674 g mol − 1 ; our experimentally determined molar mass from the principal SEC elution peak : 299100 g mol − 1 ± 5683 g mol − 1 ( mean ± standard deviation from a single SEC - MALS experiment ) ; max . dimeric COMA concentration , from refractive index measurement , in principal peak : 2 . 4 μM .

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

D) Graph with measured differential refractive index (relative scale) and molar mass calculated with multi-angle light scattering (MALS) data of COMA eluting from SEC (SEC-MALS). We injected COMA at a concentration of ~ 43 μM (COMA dimer) on the column. Dashed lines indicate expected molar masses for monomeric COMA or dimeric COMA: 142337 g mol−1 or 284674 g mol−1; our experimentally determined molar mass from the principal SEC elution peak: 299100 g mol−1 ± 5683 g mol−1 (mean ± standard deviation from a single SEC-MALS experiment); max. dimeric COMA concentration, from refractive index measurement, in principal peak: 2.4 μM.


A ) Excerpt from a representative nanoflow mass - spectrum of COMA ( full spectrum and enlarged spectrum areas are in Appendix Fig S2A ) ; B ) mass - spectrum from tandem mass - spectrometry of the isolated 28 + charge state of COMA ( from spectrum shown in A ) , after its collision - induced dissociation in the gas phase . Charge states of assigned masses are indicated above peaks . For this spectrum and our other spectra of this type , charge states of assigned masses are indicated above peaks . Inset table shows identified proteins or protein assemblies , their measured mass values as mean ± standard deviation from multiple peaks in a peak series assigned to the same molecular species ; and their respective expected mass .

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

A) Excerpt from a representative nanoflow mass-spectrum of COMA (full spectrum and enlarged spectrum areas are in Appendix Fig S2A); B) mass-spectrum from tandem mass-spectrometry of the isolated 28+ charge state of COMA (from spectrum shown in A), after its collision-induced dissociation in the gas phase. Charge states of assigned masses are indicated above peaks. For this spectrum and our other spectra of this type, charge states of assigned masses are indicated above peaks. Inset table shows identified proteins or protein assemblies, their measured mass values as mean ± standard deviation from multiple peaks in a peak series assigned to the same molecular species; and their respective expected mass.


A , B ) Plots showing hydrogen - deuterium exchanged peptides , after 10 sec of deuterium exchange of Okp1 ( A ) each in COMA or COMA - Nkp1 - Nkp2 . For this representation , and all following of this type , detected peptides are colour - coded according to their measured deuterium - exchange in % of the maximum measured exchange ( 1 - 25 % : dark blue ; 26 - 50 % : light blue ; 51 - 75 % : yellow ; 76 - 100 % : red ) . Peptides are represented as bars . Each bar is plotted row - wise corresponding to its position in the amino - acid sequence of the protein it derives from . Amino - acid sequence scheme is illustrated at the top with thin or thick lines ; thin line : flexible element : thick line : structured segment . Positions of predicted coiled coils are shown as helices . Plots for full time courses are in Appendix Fig S3C , E .

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

A,B) Plots showing hydrogen-deuterium exchanged peptides, after 10 sec of deuterium exchange of Okp1 (A) each in COMA or COMA-Nkp1-Nkp2. For this representation, and all following of this type, detected peptides are colour-coded according to their measured deuterium-exchange in % of the maximum measured exchange (1-25 %: dark blue; 26-50 %: light blue; 51-75 %: yellow; 76-100 %: red). Peptides are represented as bars. Each bar is plotted row-wise corresponding to its position in the amino-acid sequence of the protein it derives from. Amino-acid sequence scheme is illustrated at the top with thin or thick lines; thin line: flexible element: thick line: structured segment. Positions of predicted coiled coils are shown as helices. Plots for full time courses are in Appendix Fig S3C,E.


A , B ) Plots showing hydrogen - deuterium exchanged peptides , after 10 sec of deuterium exchange of Ame1 ( B ) , each in COMA or COMA - Nkp1 - Nkp2 . For this representation , and all following of this type , detected peptides are colour - coded according to their measured deuterium - exchange in % of the maximum measured exchange ( 1 - 25 % : dark blue ; 26 - 50 % : light blue ; 51 - 75 % : yellow ; 76 - 100 % : red ) . Peptides are represented as bars . Each bar is plotted row - wise corresponding to its position in the amino - acid sequence of the protein it derives from . Amino - acid sequence scheme is illustrated at the top with thin or thick lines ; thin line : flexible element : thick line : structured segment . Positions of predicted coiled coils are shown as helices . Plots for full time courses are in Appendix Fig S3C , E .

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

A,B) Plots showing hydrogen-deuterium exchanged peptides, after 10 sec of deuterium exchange of Ame1 (B), each in COMA or COMA-Nkp1-Nkp2. For this representation, and all following of this type, detected peptides are colour-coded according to their measured deuterium-exchange in % of the maximum measured exchange (1-25 %: dark blue; 26-50 %: light blue; 51-75 %: yellow; 76-100 %: red). Peptides are represented as bars. Each bar is plotted row-wise corresponding to its position in the amino-acid sequence of the protein it derives from. Amino-acid sequence scheme is illustrated at the top with thin or thick lines; thin line: flexible element: thick line: structured segment. Positions of predicted coiled coils are shown as helices. Plots for full time courses are in Appendix Fig S3C,E.


B , C ) Representative images of Coomassie Blue - stained SDS - PAGE gels with Ni2 + - affinity purification eluates . B ) of full - length COMA ( lane 11 ) with full - length polyhistidine - tagged Mcm21 ( His - Mcm21 ) and Ctf19 , or COMA variants with polyhistidine - tagged Mcm21 D - RWD domain ( His - Mcm21 _ D - RWD ; K . lactis Mcm21 residues 108 - 293 ) and Ctf19 D - RWD domain ( Ctf19 _ D - RWD ; K . lactis Ctf19 residues 107 - 270 ) We show one - letter identifiers for protein versions in A . Lanes between lanes 5 and 6 are not relevant .

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

B,C) Representative images of Coomassie Blue-stained SDS-PAGE gels with Ni2+-affinity purification eluates. B) of full-length COMA (lane 11) with full-length polyhistidine-tagged Mcm21 (His-Mcm21) and Ctf19, or COMA variants with polyhistidine-tagged Mcm21 D-RWD domain (His-Mcm21_D-RWD; K. lactis Mcm21 residues 108-293) and Ctf19 D-RWD domain (Ctf19_D-RWD; K. lactis Ctf19 residues 107-270) We show one-letter identifiers for protein versions in A. Lanes between lanes 5 and 6 are not relevant.


B , C ) Representative images of Coomassie Blue - stained SDS - PAGE gels with Ni2 + - affinity purification eluates . C ) of Ame1 - Okp1 variants with polyhistidine - tagged Okp1 variants ( His - Okp1 variants ) . We show one - letter identifiers for protein versions in A . Lanes between lanes 5 and 6 are not relevant .

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

B,C) Representative images of Coomassie Blue-stained SDS-PAGE gels with Ni2+-affinity purification eluates. C) of Ame1-Okp1 variants with polyhistidine-tagged Okp1 variants (His-Okp1 variants). We show one-letter identifiers for protein versions in A. Lanes between lanes 5 and 6 are not relevant.


D ) Image of SDS - PAGE gel with fractions of two purified Ctf19 - Mcm21 - Okp1 variants ( after SEC ) ; expected migrating position of non - proteolysed Okp1 variant ( compare with its position in SDS - PAGE images of COMA or Ame1 - Okp1 variants in B or C ) is marked ; 2 and 3 : SEC - purified fractions of samples from different elution fractions from ion - exchange chromatography . When we co - expressed full - length Okp1 with Ctf19 and Mcm21 , which has a coding regions for an N - terminal polyhistidine - tag , we did not co - purify substantial amounts of full - length Okp1 with Ctf19 - Mcm21 , probably because Okp1 was prone to proteolysis ( in the absence of Ame1 ) .

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

D) Image of SDS-PAGE gel with fractions of two purified Ctf19-Mcm21-Okp1 variants (after SEC); expected migrating position of non-proteolysed Okp1 variant (compare with its position in SDS-PAGE images of COMA or Ame1-Okp1 variants in B or C) is marked; 2 and 3: SEC-purified fractions of samples from different elution fractions from ion-exchange chromatography. When we co-expressed full-length Okp1 with Ctf19 and Mcm21, which has a coding regions for an N-terminal polyhistidine-tag, we did not co-purify substantial amounts of full-length Okp1 with Ctf19-Mcm21, probably because Okp1 was prone to proteolysis (in the absence of Ame1).


A , B ) Overlayed chromatograms showing absorbance at 280 nm from SEC of Ctf19 - Mcm21 - Okp1 variants without Okp1 segment 3 ( A ) or with Okp1 segment 3 ( B ) ; Ctf19 - Mcm21 - Okp1 variants : CMO , Nkp1 - Nkp2 : NN , Ctf19 - Mcm21 - Okp1 variants combined with Nkp1 - Nkp2 : CMO - NN ; and images of SDS - PAGE gels with equivalent elution fractions from five SEC experiments ; L : loaded on column . The elution - volume range that we analysed fractions of on SDS - PAGE is indicated below the chromatograms .

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

A,B) Overlayed chromatograms showing absorbance at 280 nm from SEC of Ctf19-Mcm21-Okp1 variants without Okp1 segment 3 (A) or with Okp1 segment 3 (B); Ctf19-Mcm21-Okp1 variants: CMO, Nkp1-Nkp2: NN, Ctf19-Mcm21-Okp1 variants combined with Nkp1-Nkp2: CMO-NN; and images of SDS-PAGE gels with equivalent elution fractions from five SEC experiments; L: loaded on column. The elution-volume range that we analysed fractions of on SDS-PAGE is indicated below the chromatograms.


C ) Representative microscopy - images of living haploid S . cerevisiae cells with Okp1 _ fl ( integrated full - length Okp1 ) or our Okp1 version that lacks the coding region for segment 3 - Okp1 _ nnΔ , with Nkp1 - GFP or Nkp2 - GFP . Fluorescence images are maximum intensity projected , and we show those from Okp1 _ fl or Okp1 _ nnΔ with the same fluorophor - fusion kinetochore protein on the same intensity scale ; scale bar : 2 μm .

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

C) Representative microscopy-images of living haploid S. cerevisiae cells with Okp1_fl (integrated full-length Okp1) or our Okp1 version that lacks the coding region for segment 3-Okp1_nnΔ, with Nkp1-GFP or Nkp2-GFP. Fluorescence images are maximum intensity projected, and we show those from Okp1_fl or Okp1_nnΔ with the same fluorophor-fusion kinetochore protein on the same intensity scale; scale bar: 2 μm.


D , E ) Plots showing hydrogen - deuterium exchanged peptides , after 10 sec of deuterium exchange of Nkp1 ( D ) each in Nkp1 - Nkp2 or COMA - Nkp1 - Nkp2 . Nkp1 has an N - terminal SNA residual . Plots for full time courses are in Appendix Fig S5A , B .

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

D,E) Plots showing hydrogen-deuterium exchanged peptides, after 10 sec of deuterium exchange of Nkp1 (D) each in Nkp1-Nkp2 or COMA-Nkp1-Nkp2. Nkp1 has an N-terminal SNA residual. Plots for full time courses are in Appendix Fig S5A,B.


D , E ) Plots showing hydrogen - deuterium exchanged peptides , after 10 sec of deuterium exchange of Nkp2 ( E ) , each in Nkp1 - Nkp2 or COMA - Nkp1 - Nkp2 . Nkp1 has an N - terminal SNA residual . Plots for full time courses are in Appendix Fig S5A , B .

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

D,E) Plots showing hydrogen-deuterium exchanged peptides, after 10 sec of deuterium exchange of Nkp2 (E), each in Nkp1-Nkp2 or COMA-Nkp1-Nkp2. Nkp1 has an N-terminal SNA residual. Plots for full time courses are in Appendix Fig S5A,B.


A ) Plots showing deuterium - exchanged peptides , after deuterium exchange of 10 sec of Okp1 in Ctf19D - RWD - Mcm21D - RWD - Okp1229 - 336 , in full - length COMA , or in Ame11 - 260 - Okp1123 - 336 . Plots for full time course are in Appendix Fig S6C .

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

A) Plots showing deuterium-exchanged peptides, after deuterium exchange of 10 sec of Okp1 in Ctf19D-RWD-Mcm21D-RWD-Okp1229-336, in full-length COMA, or in Ame11-260-Okp1123-336. Plots for full time course are in Appendix Fig S6C.


C ) Semi - transparent surface representation of our Ctf19D - RWD - Mcm21D - RWD - Okp1295 - 360 crystal structure , with secondary structure cartoon - diagram underneath ; left : in a similar orientation as Ctf19 - Mcm21 in B .

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

C) Semi-transparent surface representation of our Ctf19D-RWD-Mcm21D-RWD-Okp1295-360 crystal structure, with secondary structure cartoon-diagram underneath; left: in a similar orientation as Ctf19-Mcm21 in B.


D ) Close - up view of the Okp1 binding site of Ctf19 - Mcm21 with Ctf19 - Mcm21 binding motif of Okp1 ; Okp1 residues , and some Ctf19 or Mcm21 sidechains , are shown as sticks ( nitrogen : blue , oxygen : red ) ; Ctf19 - Mcm21 shown as secondary structure cartoon - diagram .

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

D) Close-up view of the Okp1 binding site of Ctf19-Mcm21 with Ctf19-Mcm21 binding motif of Okp1; Okp1 residues, and some Ctf19 or Mcm21 sidechains, are shown as sticks (nitrogen: blue, oxygen: red); Ctf19-Mcm21 shown as secondary structure cartoon-diagram.


F ) Representative baseline - subtracted raw isothermal calorimetry data and derived binding isotherm from titration of Okp1 - derived peptide , with the Ctf19 - Mcm21 binding motif , to full - length Ctf19 - Mcm21 . Mean value for dissociation constant ( Kd ) and its standard error of regression are from three independent experiments .

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

F) Representative baseline-subtracted raw isothermal calorimetry data and derived binding isotherm from titration of Okp1-derived peptide, with the Ctf19-Mcm21 binding motif, to full-length Ctf19-Mcm21. Mean value for dissociation constant (Kd) and its standard error of regression are from three independent experiments.


G ) Plots from microscale - thermophoresis measurements with fluorescently labeled full - length Ctf19 - Mcm21 and with Okp1 - derived synthetic peptide ( same as in F ) , or with Okp1 - peptide variants that differ in one or two residue positions from the native Okp1 sequence ( e . g . N330A : N330A ) . Normalized fluorescence , from which the fluorescence of unbound Ctf19 - Mcm21 had been subtracted ( ΔFnorm ) , is plotted against peptide concentration ( logarithmic scale ) . For dissociation constants ( Kd ) , we calculated mean value and confidence interval ( 68 % probability that Kd is within given range ) with data from three independent experiments . Variant N330A - K332P had no detectable binding in the peptide concentration - range that we measured .

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD 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 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 B-GENEPROD O B-GENEPROD O O 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 O O O O O O O O O O O O O O O O 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) Plots from microscale-thermophoresis measurements with fluorescently labeled full-length Ctf19-Mcm21 and with Okp1-derived synthetic peptide (same as in F), or with Okp1-peptide variants that differ in one or two residue positions from the native Okp1 sequence (e.g. N330A: N330A). Normalized fluorescence, from which the fluorescence of unbound Ctf19-Mcm21 had been subtracted (ΔFnorm), is plotted against peptide concentration (logarithmic scale). For dissociation constants (Kd), we calculated mean value and confidence interval (68 % probability that Kd is within given range) with data from three independent experiments. Variant N330A-K332P had no detectable binding in the peptide concentration-range that we measured.


B ) Representative images of Western - blots of immunoprecipitated fractions from S . cerevisiae extracts with Okp1 - 6 × flag and Ctf19 - 4 × myc ; fl : Okp1 _ fl ; cmΔ : Okp1 variant without the Ctf19 - Mcm21 binding motif ( Okp1 _ cmΔ ) ; IP : immunoprecipitated samples . Protein G : beads coated with Protein G only ( used as control ) ; positions of molecular masses of standards are indicated ; uncropped images are in Appendix Fig S8B , C .

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

B) Representative images of Western-blots of immunoprecipitated fractions from S. cerevisiae extracts with Okp1-6×flag and Ctf19-4×myc; fl: Okp1_fl; cmΔ: Okp1 variant without the Ctf19-Mcm21 binding motif (Okp1_cmΔ); IP: immunoprecipitated samples. Protein G: beads coated with Protein G only (used as control); positions of molecular masses of standards are indicated; uncropped images are in Appendix Fig S8B,C.


C ) Representative microscopy images of living haploid S . cerevisiae cells with Nuf2 - mCherry , and Ctf19 - GFP or Mcm21 - GFP , either with Okp1 _ fl or with Okp1 _ cmΔ . We show merged green fluorescence signals and red fluorescence signals as pseudo - colours . We show fluorescence images of cells with Okp1 _ fl or Okp1 _ cmΔ with the same fluorophor - fusion kinetochore protein on the same intensity scale ( also applies to images in D ) . Fluorescence images and those in D are deconvoluted and maximum intensity projected ; scale bar : 2 μm .

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

C) Representative microscopy images of living haploid S. cerevisiae cells with Nuf2-mCherry, and Ctf19-GFP or Mcm21-GFP, either with Okp1_fl or with Okp1_cmΔ. We show merged green fluorescence signals and red fluorescence signals as pseudo-colours. We show fluorescence images of cells with Okp1_fl or Okp1_cmΔ with the same fluorophor-fusion kinetochore protein on the same intensity scale (also applies to images in D). Fluorescence images and those in D are deconvoluted and maximum intensity projected; scale bar: 2 μm.


D ) Representative microscopy images of living haploid S . cerevisiae cells with Okp1 _ fl or Okp1 _ cmΔ , with GFP - tagged kinetochore subunits . Image pixels for images of cells with GFP - fusion proteins of Chl4 , Iml3 , Mif2 , Ctf3 , Cnn1 , Nkp1 , or Nkp2 are 2 × 2 binned ; scale bar : 2 μm . The low GFP signal - intensity that we observe for Chl4 - GFP and Iml3 - GFP in Okp1 _ fl cells is consistent with the low GFP signal for Chl4 - GFP and Iml3 - GFP at centromeres that was previously reported ( Joglekar et al , 2008 ; Lawrimore et al , 2011 ) .

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D) Representative microscopy images of living haploid S. cerevisiae cells with Okp1_fl or Okp1_cmΔ, with GFP-tagged kinetochore subunits. Image pixels for images of cells with GFP-fusion proteins of Chl4, Iml3, Mif2, Ctf3, Cnn1, Nkp1, or Nkp2 are 2×2 binned; scale bar: 2 μm. The low GFP signal-intensity that we observe for Chl4-GFP and Iml3-GFP in Okp1_fl cells is consistent with the low GFP signal for Chl4-GFP and Iml3-GFP at centromeres that was previously reported (Joglekar et al, 2008; Lawrimore et al, 2011).


A ) Representative images of dilution - series growth assay of haploid S . cerevisiae clones with cnn1Δ , Okp1 _ fl , Okp1 _ cmΔ , Okp1 _ fl cnn1Δ , or Okp1 _ cmΔ cnn1Δ , grown for two or three days on solid YPD agar or on solid YPD agar with 20 μg ml − 1 benomyl . We show two unique clones each for Okp1 _ fl cnn1Δ or Okp1 _ cmΔ cnn1Δ . Arrow indicates decreasing cell - density . Spots are from cell samples that we sequentially diluted four fold .

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A) Representative images of dilution-series growth assay of haploid S. cerevisiae clones with cnn1Δ, Okp1_fl, Okp1_cmΔ, Okp1_fl cnn1Δ, or Okp1_cmΔ cnn1Δ, grown for two or three days on solid YPD agar or on solid YPD agar with 20 μg ml−1 benomyl. We show two unique clones each for Okp1_fl cnn1Δ or Okp1_cmΔ cnn1Δ. Arrow indicates decreasing cell-density. Spots are from cell samples that we sequentially diluted four fold.


B ) Representative images of haploid S . cerevisiae spores from dissection of three tetrads ( we placed the four spores from each tetrad row - wise ) from heterozygous diploid cells with Okp1 _ fl mad1Δ or Okp1 _ cmΔ mad1Δ grown for four and three days , respectively on solid YPD agar at 25 ° C . Coloured circles indicate spore genotypes ( see bottom ) .

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B) Representative images of haploid S. cerevisiae spores from dissection of three tetrads (we placed the four spores from each tetrad row-wise) from heterozygous diploid cells with Okp1_fl mad1Δ or Okp1_cmΔ mad1Δ grown for four and three days, respectively on solid YPD agar at 25 °C. Coloured circles indicate spore genotypes (see bottom).


C ) Representative images of haploid S . cerevisiae with mad1Δ , Okp1 _ fl mad1Δ , or Okp1 _ cmΔ mad1Δ ; native : S . cerevisiae S288C type ( identifier DDY904 in Table EV6 ) . Dilution is as we describe for A . We show two unique clones for Okp1 _ fl mad1Δ .

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C) Representative images of haploid S. cerevisiae with mad1Δ, Okp1_fl mad1Δ, or Okp1_cmΔ mad1Δ; native: S. cerevisiae S288C type (identifier DDY904 in Table EV6). Dilution is as we describe for A. We show two unique clones for Okp1_fl mad1Δ.


D ) Quantification of spore viability after meiosis , of spores of dissected tetrads from homozygous diploid S . cerevisiae clones with either Ctf19 _ 1 - 954Δ ( without Ctf19 basepairs 1 - 954 ; leaving the 3 ' end of Ctf19 , and , on the complementary DNA strand , the IRC15 gene , which encodes a microtubule binding protein ( Keyes & Burke , 2009 ) intact ) , Ctf19Δ ( that lacks the entire Ctf19 ) , Okp1 _ fl , Okp1 _ cmΔ , or Okp1 _ cmΔnnΔ . Quantification is from spores from multiple tetrad dissections .

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

D) Quantification of spore viability after meiosis, of spores of dissected tetrads from homozygous diploid S. cerevisiae clones with either Ctf19_1-954Δ (without Ctf19 basepairs 1-954; leaving the 3' end of Ctf19, and, on the complementary DNA strand, the IRC15 gene, which encodes a microtubule binding protein (Keyes & Burke, 2009) intact), Ctf19Δ (that lacks the entire Ctf19), Okp1_fl, Okp1_cmΔ, or Okp1_cmΔnnΔ. Quantification is from spores from multiple tetrad dissections.


Representative calcium signature enriched in P - EMT tumors ( comparing ECAD - vs . ECAD + tumor cells ) . NES , nominal P - value , and FDR are shown . Statistical analysis performed in GSEA .

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Representative calcium signature enriched in P-EMT tumors (comparing ECAD- vs. ECAD+ tumor cells). NES, nominal P-value, and FDR are shown. Statistical analysis performed in GSEA.


Representative image of tumor cells transduced with GCaMP6 ( Left ) . Yellow arrows ( mesenchymal cells ) , red arrows ( epithelial cells ) , scale bar = 50μm . GCaMP6 mean fluorescent intensity ( MFI ) in ECADHIGH or ECADLOW cells cultured in indicated medium ( 0 Ca2 + or 2 Ca2 + ) for 1h ( Right ) . Representative of 3 independent murine PDA cell lines run in triplicate . Statistical analysis by Student ' s unpaired t - test ( * , p < 0 . 05 ; non - significant ( NS ) ; ± SD ) .

O O O O O O O B-GENEPROD O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O B-ORGANISM B-DISEASE O O O O O O O O O O O O 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 image of tumor cells transduced with GCaMP6 (Left). Yellow arrows (mesenchymal cells), red arrows (epithelial cells), scale bar = 50μm. GCaMP6 mean fluorescent intensity (MFI) in ECADHIGH or ECADLOW cells cultured in indicated medium (0 Ca2+ or 2 Ca2+) for 1h (Right). Representative of 3 independent murine PDA cell lines run in triplicate. Statistical analysis by Student's unpaired t-test (*, p<0.05; non-significant (NS); ±SD).


Flow cytometry analysis of ECADHIGH or ECADLOW cells loaded with Indo - 1 and cultured in indicated medium ( 0 Ca2 + or 2 Ca2 + ) for 1h . Representative of 2 independent cell lines ( n indicates the number of cells analyzed in each group , ECADHIGH 0 Ca2 + n = 4963 , ECADLOW 0 Ca2 + n = 14 , ECADHIGH 2 Ca2 + n = 6575 , ECADLOW 2 Ca2 + n = 29 ) . Representative of 2 independent murine PDA cell lines run in triplicate . Statistical analysis by Student ' s unpaired t - test ( * * * * , p < 0 . 0001 ; non - significant ( NS ) ; ± SD ) .

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Flow cytometry analysis of ECADHIGH or ECADLOW cells loaded with Indo-1 and cultured in indicated medium (0 Ca2+ or 2 Ca2+) for 1h. Representative of 2 independent cell lines (n indicates the number of cells analyzed in each group, ECADHIGH 0 Ca2+ n=4963, ECADLOW 0 Ca2+ n=14, ECADHIGH 2 Ca2+ n=6575, ECADLOW 2 Ca2+ n=29). Representative of 2 independent murine PDA cell lines run in triplicate. Statistical analysis by Student's unpaired t-test (****, p<0.0001; non-significant (NS); ±SD).


Representative 20X brightfield and fluorescent images of murine YFP + tumor cells treated for 72h with vehicle control ( DMSO ) , 2 . 5μM Ionomycin , or 10ng / ml TGFβ and co - stained for E - cadherin ( Red ) and DAPI ( Blue ) . Scale bar is 100μm .

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Representative 20X brightfield and fluorescent images of murine YFP+ tumor cells treated for 72h with vehicle control (DMSO), 2.5μM Ionomycin, or 10ng/ml TGFβ and co-stained for E-cadherin (Red) and DAPI (Blue). Scale bar is 100μm.


Representative flow cytometry analysis of surface ECAD ( Left ) , MFI of surface ECAD ( Middle ) , and percentage of surface ECAD negative cells ( Right ) in murine PDA cells , after treatment with DMSO , 2 . 5μM Ionomycin ( IONO ) , and 10ng / ml TGFβ for 72h . Representative of 3 independent murine PDA cell lines run in triplicate . Statistical analysis by ANOVA ( * * * * , p < 0 . 0001 ; non - significant ( NS ) ; ± SD ) .

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Representative flow cytometry analysis of surface ECAD (Left), MFI of surface ECAD (Middle), and percentage of surface ECAD negative cells (Right) in murine PDA cells, after treatment with DMSO, 2.5μM Ionomycin (IONO), and 10ng/ml TGFβ for 72h. Representative of 3 independent murine PDA cell lines run in triplicate. Statistical analysis by ANOVA (****, p<0.0001; non-significant (NS); ±SD).


Top : Relative mRNA expression of Ecad after treatment with 2 . 5μM ionomycin or 10ng / ml TGFβ for denoted time . Bottom : Western blot analysis of total E - cadherin and Vimentin protein for denoted time . Experiment was run in duplicate with three different murine PDA cell lines . Statistical analysis by ANOVA ( * * * , p < 0 . 001 ; non - significant ( NS ) ; ± SEM ) .

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Top: Relative mRNA expression of Ecad after treatment with 2.5μM ionomycin or 10ng/ml TGFβ for denoted time. Bottom: Western blot analysis of total E-cadherin and Vimentin protein for denoted time. Experiment was run in duplicate with three different murine PDA cell lines. Statistical analysis by ANOVA (***, p<0.001; non-significant (NS); ±SEM).


Normalized MFI of surface E - cadherin in PDA cells treated with DMSO , 2 . 5μM Ionomycin ( IONO ) , and 10ng / ml TGFβ for 7 days , followed by withdrawal of Ionomycin or TGFβ for 7 days as indicated on the x - axis . Treated cells were analyzed at day 2 , 3 , and 7 . Withdrawn cells were analyzed on days 1 , 2 , and 7 . Experiment was performed in duplicate in two different murine PDA cell lines . Error bars indicate SD .

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Normalized MFI of surface E-cadherin in PDA cells treated with DMSO, 2.5μM Ionomycin (IONO), and 10ng/ml TGFβ for 7 days, followed by withdrawal of Ionomycin or TGFβ for 7 days as indicated on the x-axis. Treated cells were analyzed at day 2, 3, and 7. Withdrawn cells were analyzed on days 1, 2, and 7. Experiment was performed in duplicate in two different murine PDA cell lines. Error bars indicate SD.


Quantification of live cellular migration over 4h after treatment with DMSO , 2 . 5μM Ionomycin ( IONO ) , or 10ng / ml TGFβ for 48hrs . ( n = 16 cells per condition . Experiment was run in triplicate in two murine PDA different cell lines ) ( Right ) . Quantification of transwell migration after treatment with DMSO , 2 . 5μM Ionomycin ( IONO ) , or 10ng / ml TGFβ for 72h ( n = 2 cell lines , 3 replicates per cell line with 3 20X images taken per transwell ) ( Left ) . Statistical analysis by ANOVA ( * , p < 0 . 05 ; * * * * , p < 0 . 0001 ; non - significant ( NS ) ; ± SD ) .

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Quantification of live cellular migration over 4h after treatment with DMSO, 2.5μM Ionomycin (IONO), or 10ng/ml TGFβ for 48hrs. (n=16 cells per condition. Experiment was run in triplicate in two murine PDA different cell lines) (Right). Quantification of transwell migration after treatment with DMSO, 2.5μM Ionomycin (IONO), or 10ng/ml TGFβ for 72h (n= 2 cell lines, 3 replicates per cell line with 3 20X images taken per transwell) (Left). Statistical analysis by ANOVA (*, p<0.05; ****, p<0.0001; non-significant (NS); ±SD).


Schematic for RNA sequencing after EMT induction for 48h . Heatmap illustration of EMT related genes in three independent murine PDA cell lines , sequenced in duplicate , treated with DMSO , 2 . 5μM Ionomycin , or 10ng / ml TGFβ . Scale indicates Z - score .

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Schematic for RNA sequencing after EMT induction for 48h. Heatmap illustration of EMT related genes in three independent murine PDA cell lines, sequenced in duplicate, treated with DMSO, 2.5μM Ionomycin, or 10ng/ml TGFβ. Scale indicates Z-score.


GSEA of ionomycin versus DMSO ( Left ) or TGFβ versus DMSO ( Right ) for EMT signatures . NES , nominal P - value , and FDR are shown . Statistical analysis performed in GSEA .

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GSEA of ionomycin versus DMSO (Left) or TGFβ versus DMSO (Right) for EMT signatures. NES, nominal P-value, and FDR are shown. Statistical analysis performed in GSEA.


GSEA of ionomycin versus DMSO ( Left ) or TGFβ versus DMSO ( Right ) for calcium signatures . NES , nominal P - value , and FDR are shown . Statistical analysis performed in GSEA .

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GSEA of ionomycin versus DMSO (Left) or TGFβ versus DMSO (Right) for calcium signatures. NES, nominal P-value, and FDR are shown. Statistical analysis performed in GSEA.


Leading edge plots showing enrichment of C - EMT gene signatures in TGFβ treated cells ( Left ) or P - EMT gene signatures in ionomycin ( IONO ) treated cells ( Right ) , based on GSEA . Enrichment scores ( ES ) , NES , nominal P - value , and FDR are shown . Statistical analysis performed in GSEA .

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Leading edge plots showing enrichment of C-EMT gene signatures in TGFβ treated cells (Left) or P-EMT gene signatures in ionomycin (IONO) treated cells (Right), based on GSEA. Enrichment scores (ES), NES, nominal P-value, and FDR are shown. Statistical analysis performed in GSEA.


Fura - 2 Ca2 + measurements after addition of 5μM CNO ( Top ) or 10μM ATP ( Bottom ) in murine PDA cells expressing a Gαq - DREADD . Blue or red line indicates average of n = 22 cells . ± SD . Tracings are representative of experiments performed twice in 2 independent cell lines .

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Fura-2 Ca2+ measurements after addition of 5μM CNO (Top) or 10μM ATP (Bottom) in murine PDA cells expressing a Gαq-DREADD. Blue or red line indicates average of n=22 cells. ±SD. Tracings are representative of experiments performed twice in 2 independent cell lines.


Representative 20X brightfield images of Gαq - DREADD - expressing cells 48h after addition of CNO ( Left ) . Scale bar = 100μm . MFI of surface E - cadherin in cells expressing a Gαq - DREADD after treatment with DMSO , 5μM CNO , or 2 . 5μM ionomycin ( Right ) . Both experiments performed in triplicate in 2 independent cell lines . Statistical analysis by ANOVA ( * * , p < 0 . 01 ; ± SD ) .

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

Representative 20X brightfield images of Gαq-DREADD-expressing cells 48h after addition of CNO (Left). Scale bar = 100μm. MFI of surface E-cadherin in cells expressing a Gαq-DREADD after treatment with DMSO, 5μM CNO, or 2.5μM ionomycin (Right). Both experiments performed in triplicate in 2 independent cell lines. Statistical analysis by ANOVA (**, p<0.01; ±SD).


MFI of surface E - cadherin in CnB knockout lines after 48h treatment with DMSO , 2 . 5μM ionomycin , or 10ng / ml TGFβ . Experiment was repeated in two different murine PDA cell lines in triplicate . Statistical analysis by ANOVA ( * * * , p < 0 . 001 ; * * * * , p < 0 . 00001 ; ± SD ) .

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MFI of surface E-cadherin in CnB knockout lines after 48h treatment with DMSO, 2.5μM ionomycin, or 10ng/ml TGFβ. Experiment was repeated in two different murine PDA cell lines in triplicate. Statistical analysis by ANOVA (***, p<0.001; ****, p<0.00001; ±SD).


MFI of surface E - cadherin of murine PDA cells pretreated with 20μM W7 for 24h and then DMSO , 2 . 5μM ionomycin , or 20μM W7 with 2 . 5μM ionomycin for 24h . Experiment was repeated in two different murine PDA cell lines in triplicate . Statistical analysis by ANOVA ( * * , p < 0 . 01 ; * * * , p < 0 . 001 ; non - significant ( NS ) ; ± SD ) .

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MFI of surface E-cadherin of murine PDA cells pretreated with 20μM W7 for 24h and then DMSO, 2.5μM ionomycin, or 20μM W7 with 2.5μM ionomycin for 24h. Experiment was repeated in two different murine PDA cell lines in triplicate. Statistical analysis by ANOVA (**, p<0.01; ***, p<0.001; non-significant (NS); ±SD).


MFI of surface E - cadherin of shRNA non - targeting ( shNT ) ( Left ) or shCAMK2B ( Right ) after 48h treatment with DMSO , 2 . 5μM ionomycin , or 10ng / ml TGFβ . Experiment was repeated in two different murine PDA cell lines . Statistical analysis by ANOVA ( * * * , p < 0 . 001 ; * * * * , p < 0 . 00001 ; ± SD ) .

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 B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O B-GENEPROD O O O O O O O B-ORGANISM B-DISEASE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

MFI of surface E-cadherin of shRNA non-targeting (shNT) (Left) or shCAMK2B (Right) after 48h treatment with DMSO, 2.5μM ionomycin, or 10ng/ml TGFβ. Experiment was repeated in two different murine PDA cell lines. Statistical analysis by ANOVA (***, p<0.001; ****, p<0.00001; ±SD).


( a ) [ 3H ] thymidine incorporation at 72 h by wild - type ( WT ) and Irgm1 - / - CD4 + T cells activated with soluble mAb to CD3 and irradiated syngenic wild - type splenocytes .

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(a) [3H]thymidine incorporation at 72 h by wild-type (WT) and Irgm1-/- CD4+ T cells activated with soluble mAb to CD3 and irradiated syngenic wild-type splenocytes.


( b ) [ 3H ] thymidine incorporation at 72 h by OT - II and Irgm1 - / - OT - II CD4 + T cells stimulated with OVA protein ( OVA ) or OVA peptide of amino acids 323 - 339 ( OVA ( 323 - 339 ) ) in the presence of syngenic splenic DCs .

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

(b) [3H]thymidine incorporation at 72 h by OT-II and Irgm1-/- OT-II CD4+ T cells stimulated with OVA protein (OVA) or OVA peptide of amino acids 323-339 (OVA(323-339)) in the presence of syngenic splenic DCs.


( c , d ) Kinetic analysis of [ 3H ] thymidine incorporation ( c ) and cell numbers ( d ) in cultures of OT - II and Irgm1 − / − OT - II spleen cells stimulated with OVA peptide .

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

(c,d) Kinetic analysis of [3H]thymidine incorporation (c) and cell numbers (d) in cultures of OT-II and Irgm1−/− OT-II spleen cells stimulated with OVA peptide.


( e ) Flow cytometric analysis of the activation and proliferation of OVA peptide - stimulated OT - II and Irgm1 - / - OT - II CD4 + T cells pulsed with BrdU during the final 4 h of 72 h of culture and then stained with mAb to CD25 and BrdU . Numbers in quadrants indicate percent viable CD4 + cells .

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

(e) Flow cytometric analysis of the activation and proliferation of OVA peptide-stimulated OT-II and Irgm1-/- OT-II CD4+ T cells pulsed with BrdU during the final 4 h of 72 h of culture and then stained with mAb to CD25 and BrdU. Numbers in quadrants indicate percent viable CD4+ cells.


( f ) Enzyme - linked immunosorbent assay of IL - 2 produced by OT - II and Irgm1 - / - OT - II CD4 + T cells stimulated with OVA peptide in the presence of splenic DCs , measured at 72 h . Data are representative of more than five ( a , b ) or three ( c - f ) independent experiments with similar results ( mean and s . d . of triplicate cultures , a - d , f ) .

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

(f) Enzyme-linked immunosorbent assay of IL-2 produced by OT-II and Irgm1-/- OT-II CD4+ T cells stimulated with OVA peptide in the presence of splenic DCs, measured at 72 h. Data are representative of more than five (a,b) or three (c-f) independent experiments with similar results (mean and s.d. of triplicate cultures, a-d,f).


( a ) Immunoblot analysis of Irgm1 in lysates of flow cytometry - sorted naive wild - type CD4 + lymphocytes activated for 48 h with immobilized agonistic mAb to CD3 ( 10 μg / ml ) alone or with mAb to CD28 ( 2 μg / ml ) in the presence or absence of IFN - γ - neutralizing mAb . Actin , loading control .

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

(a) Immunoblot analysis of Irgm1 in lysates of flow cytometry-sorted naive wild-type CD4+ lymphocytes activated for 48 h with immobilized agonistic mAb to CD3 (10 μg/ml) alone or with mAb to CD28 (2 μg/ml) in the presence or absence of IFN-γ-neutralizing mAb. Actin, loading control.


( b ) Enzyme - linked immunosorbent assay of IFN - γ produced by OT - II and Irgm1 − / − OT - II CD4 + T cells stimulated with OVA peptide in the presence of splenic DC in polarized conditions ( TH1 or TH2 ) or nonpolarized conditions ( N ) , assessed in 72 - hour cultures . ND , not detected .

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

(b) Enzyme-linked immunosorbent assay of IFN-γ produced by OT-II and Irgm1−/− OT-II CD4+ T cells stimulated with OVA peptide in the presence of splenic DC in polarized conditions (TH1 or TH2) or nonpolarized conditions (N), assessed in 72-hour cultures. ND, not detected.


( c ) Accumulation of polarized and nonpolarized OT - II ( open symbols ) or Irgm1 − / − OT - II ( filled symbols ) CD4 + T cells after peptide stimulation , quantified by counting of viable cells .

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

(c) Accumulation of polarized and nonpolarized OT-II (open symbols) or Irgm1−/− OT-II (filled symbols) CD4+ T cells after peptide stimulation, quantified by counting of viable cells.


( d ) Viable OT - II and Irgm1 − / − OT - II CD4 + T cells 72 h after restimulation with OVA peptide in the presence of T cell - depleted APC samples .

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

(d) Viable OT-II and Irgm1−/− OT-II CD4+ T cells 72 h after restimulation with OVA peptide in the presence of T cell-depleted APC samples.


( e ) Flow cytometry of the viability and division of OT - II and Irgm1 − / − OT - II CD4 + T cells stimulated with peptide , allowed to ' rest ' for 7 d in IL - 2 , then labeled with CFSE and restimulated with OVA peptide in the presence of T cell - depleted APC samples and assessed 72 h later .

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

(e) Flow cytometry of the viability and division of OT-II and Irgm1−/− OT-II CD4+ T cells stimulated with peptide, allowed to 'rest' for 7 d in IL-2, then labeled with CFSE and restimulated with OVA peptide in the presence of T cell-depleted APC samples and assessed 72 h later.


( f ) Flow cytometric analysis of the population expansion of CFSE - labeled OT - II and Irgm1 − / − OT - II CD4 + T cells activated with OVA peptide in the presence or absence of IFN - γ - neutralizing mAb . Results in e , f obtained after gating on CD4 + T cells ; numbers in outlined areas ( e ) and in top left corners ( f ) indicate percent CD4 + cells . PI , propidium iodide .

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

(f) Flow cytometric analysis of the population expansion of CFSE-labeled OT-II and Irgm1−/− OT-II CD4+ T cells activated with OVA peptide in the presence or absence of IFN-γ-neutralizing mAb. Results in e,f obtained after gating on CD4+ T cells; numbers in outlined areas (e) and in top left corners (f) indicate percent CD4+ cells. PI, propidium iodide.


( g ) [ 3H ] thymidine incorporation by wild - type , Irgm1 − / − , Ifng − / − and Irgm1 − / − Ifng − / − CD4 + T cells stimulated for 72 h with soluble agonistic mAb to CD3 and irradiated syngenic Ifng − / − splenocytes . Data are representative of two independent experiments with similar results ( mean and s . d . of triplicate cultures , b - d , g ) .

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

(g) [3H]thymidine incorporation by wild-type, Irgm1−/−, Ifng−/− and Irgm1−/−Ifng−/− CD4+ T cells stimulated for 72 h with soluble agonistic mAb to CD3 and irradiated syngenic Ifng−/− splenocytes. Data are representative of two independent experiments with similar results (mean and s.d. of triplicate cultures, b-d,g).


( a ) Flow cytometric analysis of the survival of CD45 . 2 + Ifng − / − ( open symbols ) or Irgm1 − / − Ifng − / − ( filled symbols ) CD4 + T lymphocytes preactivated with agonistic mAb to CD3 in vitro , then transferred intraperitoneally into congenic CD45 . 1 + C57BL / 6 . SJL recipients left untreated or inoculated intraperitoneally with T . gondii or M . avium 3 d earlier , assessed in peritoneal exudate cell samples collected at day 4 after transfer .

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

(a) Flow cytometric analysis of the survival of CD45.2+ Ifng−/− (open symbols) or Irgm1−/−Ifng−/− (filled symbols) CD4+ T lymphocytes preactivated with agonistic mAb to CD3 in vitro, then transferred intraperitoneally into congenic CD45.1+ C57BL/6.SJL recipients left untreated or inoculated intraperitoneally with T. gondii or M. avium 3 d earlier, assessed in peritoneal exudate cell samples collected at day 4 after transfer.


( b ) Survival of M . avium - infected wild - type , Irgm1 − / − , Ifng − / − and Irgm1 − / − Ifng − / − mice ( n = 5 mice per genotype ) .

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

(b) Survival of M. avium-infected wild-type, Irgm1−/−, Ifng−/− and Irgm1−/−Ifng−/− mice (n = 5 mice per genotype).


( c - e ) Splenic bacterial loads ( c ) , circulating lymphocytes ( d ) and splenic lymphocytes ( e ) , assessed at week 4 after M . avium infection ( n = 5 mice per genotype ) . Each symbol in a , c - e represents an individual mouse . Data are representative of two independent experiments .

O O O O O B-TISSUE O O O O O O O B-CELL_TYPE O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O 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

(c-e) Splenic bacterial loads (c), circulating lymphocytes (d) and splenic lymphocytes (e), assessed at week 4 after M. avium infection (n = 5 mice per genotype). Each symbol in a,c-e represents an individual mouse. Data are representative of two independent experiments.


( a ) [ 3H ] thymidine incorporation by naive CD4 + lymphocytes stimulated for 72 h with agonistic mAb to CD3 and IFN - γ - deficient irradiated APCs , with IL - 2 ( 10 U / ml ) added 24 h after the initiation of culture .

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

(a) [3H]thymidine incorporation by naive CD4+ lymphocytes stimulated for 72 h with agonistic mAb to CD3 and IFN-γ-deficient irradiated APCs, with IL-2 (10 U/ml) added 24 h after the initiation of culture.


( b ) Accumulation of viable CD4 + T cells at 72 h in the presence of medium alone ( Med ) or IFN - γ ( 5 U / ml ) .

O O O O O O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE 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) Accumulation of viable CD4+ T cells at 72 h in the presence of medium alone (Med) or IFN-γ (5 U/ml).


( c ) Flow cytometric analysis of the viability and division of CD4 + T cells labeled with CFSE and activated as described in a with T cell - depleted Ifng − / − APC samples in the presence or absence of IFN - γ ( 5 U / ml ) . Numbers in plots indicate percent CFSE + CD4 + T cell populations .

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

(c) Flow cytometric analysis of the viability and division of CD4+ T cells labeled with CFSE and activated as described in a with T cell-depleted Ifng−/− APC samples in the presence or absence of IFN-γ (5 U/ml). Numbers in plots indicate percent CFSE+CD4+ T cell populations.


( d - f ) Death of Ifng − / − and Irgm1 − / − Ifng − / − T cell populations activated with mAb to CD3 and expanded in IL - 2 , then treated with IFN - γ ( d ) , agonistic mAb to CD3 ( e ) or Fas ligand ( FasL ; f ) , assessed by propidium iodide staining at 48 h after treatment . Open bars , Ifng − / − ; filled bars , Irgm1 − / − Ifng − / − ( a , b , d - f ) . Data are representative of three independent experiments with similar results ( mean and s . d . of triplicate cultures a , b , d - f ) .

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

(d-f) Death of Ifng−/− and Irgm1−/−Ifng−/− T cell populations activated with mAb to CD3 and expanded in IL-2, then treated with IFN-γ (d), agonistic mAb to CD3 (e) or Fas ligand (FasL; f), assessed by propidium iodide staining at 48 h after treatment. Open bars, Ifng−/−; filled bars, Irgm1−/−Ifng−/− (a,b,d-f). Data are representative of three independent experiments with similar results (mean and s.d. of triplicate cultures a,b,d-f).


( a - f ) Electron microscopy analysis of Ifng − / − CD4 + lymphocytes ( a ) and Irgm1 − / − Ifng − / − CD4 + lymphocytes ( b - f ) exposed for 30 h to IFN - γ ( 5 U / ml ) . ( c ) Enlargement of the area outlined in b ; arrowheads indicate two autophagic vacuoles . ( d ) Quantification of vacuoles in 50 randomly selected electron microscopy sections of more than 400 cells . Each symbol represents one section . ( e ) Irgm1 − / − CD4 + T cell with many vacuoles containing dense materials . ( f ) Two Irgm1 − / − CD4 + cells with vacuolated cytoplasma . Original magnification ( a - c , e , f ) , × 1 , 000 . Data are representative of two experiments .

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

(a-f) Electron microscopy analysis of Ifng−/− CD4+ lymphocytes (a) and Irgm1−/−Ifng−/− CD4+ lymphocytes (b-f) exposed for 30 h to IFN-γ (5 U/ml). (c) Enlargement of the area outlined in b; arrowheads indicate two autophagic vacuoles. (d) Quantification of vacuoles in 50 randomly selected electron microscopy sections of more than 400 cells. Each symbol represents one section. (e) Irgm1−/− CD4+ T cell with many vacuoles containing dense materials. (f) Two Irgm1−/− CD4+ cells with vacuolated cytoplasma. Original magnification (a-c,e,f), × 1,000. Data are representative of two experiments.


( g , h ) Death of Ifng − / − and Irgm1 − / − Ifng − / − CD4 + T cells in the presence or absence of wortmannin or Ly294002 ( g ) , assessed by flow cytometry with propidium iodide staining at 48 h after IFN - γ exposure . Data are representative of three independent experiments ( mean and s . d . of triplicate cultures ) .

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

(g,h) Death of Ifng−/− and Irgm1−/−Ifng−/− CD4+ T cells in the presence or absence of wortmannin or Ly294002 (g), assessed by flow cytometry with propidium iodide staining at 48 h after IFN-γ exposure. Data are representative of three independent experiments (mean and s.d. of triplicate cultures).


( g , h ) Death of Ifng − / − and Irgm1 − / − Ifng − / − CD4 + T cells transfected with control or beclin1 - specific small interfering RNA , assessed by flow cytometry with propidium iodide staining at 48 h after IFN - γ exposure . Right ( h ) , immunoblot analysis of beclin1 expression . C , control small interfering RNA ; B , beclin1 - specific small interfering RNA . Data are representative of three independent experiments ( mean and s . d . of triplicate cultures ) .

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

(g,h) Death of Ifng−/− and Irgm1−/−Ifng−/− CD4+ T cells transfected with control or beclin1-specific small interfering RNA, assessed by flow cytometry with propidium iodide staining at 48 h after IFN-γ exposure. Right (h), immunoblot analysis of beclin1 expression. C, control small interfering RNA; B, beclin1-specific small interfering RNA. Data are representative of three independent experiments (mean and s.d. of triplicate cultures).


Testicular maturation arrest in the patient ( WHT3759 ) . Testicular tissue obtained by biopsy was sectioned and stained with hematoxylin / eosin . Seminiferous tubules contained meiotic germ cells such as zygotene ( ZS , arrowheads ) and pachytene spermatocytes ( PS , arrows ) , but no post - meiotic germ cells such as round spermatids were detectable . Scale bar , 25 μm .

B-TISSUE O O O O B-ORGANISM O O O O B-TISSUE O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-TISSUE I-TISSUE O O B-CELL_TYPE I-CELL_TYPE 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-CELL_TYPE I-CELL_TYPE O O O B-CELL_TYPE O O O O O O O O O

Testicular maturation arrest in the patient (WHT3759). Testicular tissue obtained by biopsy was sectioned and stained with hematoxylin/eosin. Seminiferous tubules contained meiotic germ cells such as zygotene (ZS, arrowheads) and pachytene spermatocytes (PS, arrows), but no post-meiotic germ cells such as round spermatids were detectable. Scale bar, 25 μm.


Western blot analysis of TEX11 in the testes of 3 - month - old mice with different Tex11 gene dosages . ACTB serves as a loading control .

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

Western blot analysis of TEX11 in the testes of 3-month-old mice with different Tex11 gene dosages. ACTB serves as a loading control.


Testis weight of 3 - month - old males .

Testis weight of 3-month-old males.


Sperm count of 3-month-old males.


Histological analysis of testes from adult wild - type , Tex11 KI / KO , and Tex11 − / Y males . RS , round spermatids ; ES , elongating spermatids ; Pa , pachytene spermatocytes . Scale bar , 50 μm .

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

Histological analysis of testes from adult wild-type, Tex11 KI/KO, and Tex11−/Y males. RS, round spermatids; ES, elongating spermatids; Pa, pachytene spermatocytes. Scale bar, 50 μm.


The Tex11 knockin allele rescues chromosomal synapsis defects in Tex11 − / Y spermatocytes in 3 - month - old males . Chromosomal synapsis defects were assessed by SYCP1 and SYCP2 immunostaining of spread nuclei from 100 pachytene spermatocytes per male ; for each genotype , three males were analyzed . Abbreviations for Tex11 genotypes : − / Y , Tex11 knockout ( Yang et al , 2008 ) ; KI ; − / Y , Tex11 knockin and knockout ; + / Y , wild type ; KI ; + / Y , Tex11 knockin plus wild - type Tex11 .

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

The Tex11 knockin allele rescues chromosomal synapsis defects in Tex11−/Y spermatocytes in 3-month-old males. Chromosomal synapsis defects were assessed by SYCP1 and SYCP2 immunostaining of spread nuclei from 100 pachytene spermatocytes per male; for each genotype, three males were analyzed. Abbreviations for Tex11 genotypes: −/Y, Tex11 knockout (Yang et al, 2008); KI;−/Y, Tex11 knockin and knockout; +/Y, wild type; KI;+/Y, Tex11 knockin plus wild-type Tex11.


Reduced testis weight in juvenile but not adult Tex11 KI ; − / Y males . The time frames of the first wave and adult waves of spermatogenesis are indicated .

O B-TISSUE B-EXP_ASSAY 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

Reduced testis weight in juvenile but not adult Tex11 KI; −/Y males. The time frames of the first wave and adult waves of spermatogenesis are indicated.


Increased chromosomal asynapsis in spermatocytes and reduced levels of TEX11 protein in the testes of 25 - day - old Tex11 KI / KO males . Chromosomal synapsis defects were assessed by SYCP1 and SYCP2 immunostaining of spread nuclei from 100 pachytene spermatocytes per male ; for each genotype , three males were analyzed .

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

Increased chromosomal asynapsis in spermatocytes and reduced levels of TEX11 protein in the testes of 25-day-old Tex11 KI/KO males. Chromosomal synapsis defects were assessed by SYCP1 and SYCP2 immunostaining of spread nuclei from 100 pachytene spermatocytes per male; for each genotype, three males were analyzed.


Pachytene spermatocytes from 25 - day - old Tex11 KI / KO males contain significantly fewer MLH1 foci compared to wild - type spermatocytes . Values are shown as average ± standard deviation . Error bar is standard deviation .

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

Pachytene spermatocytes from 25-day-old Tex11 KI/KO males contain significantly fewer MLH1 foci compared to wild-type spermatocytes. Values are shown as average ± standard deviation. Error bar is standard deviation.


The number of MLH1 foci in pachytene spermatocytes from 3 - month - old males positively correlates with increasing Tex11 gene dosage .

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

The number of MLH1 foci in pachytene spermatocytes from 3-month-old males positively correlates with increasing Tex11 gene dosage.


Quantification of MLH1 foci in pachytene oocytes from the ovaries of Tex11 − / − , Tex11 + / − , and wild - type embryonic day - 17 . 5 ( E17 . 5 ) fetuses reveals a positive correlation of Tex11 gene dosage and the number of MLH1 foci .

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

Quantification of MLH1 foci in pachytene oocytes from the ovaries of Tex11−/−, Tex11+/−, and wild-type embryonic day-17.5 (E17.5) fetuses reveals a positive correlation of Tex11 gene dosage and the number of MLH1 foci.


A Western blot analysis reveals comparable levels of TEX11 protein in the testes of wild - type and point mutant KI / KO males . ACTB serves as a control .

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

A Western blot analysis reveals comparable levels of TEX11 protein in the testes of wild-type and point mutant KI/KO males. ACTB serves as a control.


B - D Body weight ( B ) , testisweight ( C ) in Tex11 KI ( V749A ) / KO males .

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

B-D Body weight (B), testisweight (C) in Tex11 KI(V749A)/KO males.


B - D Significantly reduced sperm count ( per pair of epididymides ) in Tex11 KI ( V749A ) / KO males ( D ) .

O O O O O B-CELL_TYPE O O O O O B-TISSUE O O B-GENEPROD O O O O O O O O O O O

B-D Significantly reduced sperm count (per pair of epididymides) in Tex11 KI(V749A)/KO males (D).


E Mating test of 3 - to 5 - month - old males .

E Mating test of 3- to 5-month-old males.


F Dramatically increased chromosomal asynapsis in spermatocytes from Tex11 KI ( V749A ) / KO males . 100 pachytene spermatocytes per mouse were examined by surface spread analysis .

O O O O O O B-CELL_TYPE O B-GENEPROD O O O O O O O O O O B-CELL_TYPE O B-ORGANISM O O O O O O O

F Dramatically increased chromosomal asynapsis in spermatocytes from Tex11 KI(V749A)/KO males. 100 pachytene spermatocytes per mouse were examined by surface spread analysis.


G Number of MLH1 foci in pachytene spermatocytes . Note a significantly higher number of MLH1 foci in spermatocytes from KI ( Q173R ) / KO males .

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

G Number of MLH1 foci in pachytene spermatocytes. Note a significantly higher number of MLH1 foci in spermatocytes from KI(Q173R)/KO males.


Cell migration assays were performed using Transwell migration chambers . HCT116 cells were transfected with siRNAs targeting 11 candidate lincRNAs and cell migration activities were evaluated by Transwell migration assays ( n = 3 ) . CALIC is the lincRNA targeted by si - LINC00920 .

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

Cell migration assays were performed using Transwell migration chambers. HCT116 cells were transfected with siRNAs targeting 11 candidate lincRNAs and cell migration activities were evaluated by Transwell migration assays (n = 3). CALIC is the lincRNA targeted by si-LINC00920.


Migration of HCT116 cells transfected with the indicated siRNAs or negative control . ( n = 4 )

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

Migration of HCT116 cells transfected with the indicated siRNAs or negative control. (n = 4)


Migration of colon cancer cells treated with an siRNA targeting CALIC . ( n = 3 )

B-EXP_ASSAY O B-DISEASE I-DISEASE O O O O O O B-GENEPROD O O O O O O

Migration of colon cancer cells treated with an siRNA targeting CALIC. (n = 3)


Subcellular localization analysis of CALIC . RNAs were isolated from the nuclear and cytoplasmic fractions of HCT116 cells and quantified by qRT - PCR ( n = 3 ) . Nuclear controls : U6 , MALAT1 ; Cytoplasmic control : GAPDH .

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

Subcellular localization analysis of CALIC. RNAs were isolated from the nuclear and cytoplasmic fractions of HCT116 cells and quantified by qRT-PCR (n = 3). Nuclear controls: U6, MALAT1; Cytoplasmic control: GAPDH.


CALIC expression levels in cancer tissues . N , normal mucosa ; T , tumor .

B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O B-DISEASE O O O O O B-TISSUE O O O O O

CALIC expression levels in cancer tissues. N, normal mucosa; T, tumor.