C . HEK293 cell lines were treated with doxycycline and Z - VAD - FMK for 24 h . The colonies were fixed then stained with crystal violet . Cell growth was quantitated as total area on ImageJ . Values are mean ± s . e . m . of independent experiments ( n = 3 ) .

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C. HEK293 cell lines were treated with doxycycline and Z-VAD-FMK for 24 h. The colonies were fixed then stained with crystal violet. Cell growth was quantitated as total area on ImageJ. Values are mean ± s.e.m. of independent experiments (n = 3).


C . Cells were treated with doxycycline along with DMSO or Z - VAD - FMK for 24 h . They were fixed with formaldehyde , and stained with NPC antibody Mab414 and DAPI . The images were captured by confocal microscopy . Scale bar : 10 μm .

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

C. Cells were treated with doxycycline along with DMSO or Z-VAD-FMK for 24 h. They were fixed with formaldehyde, and stained with NPC antibody Mab414 and DAPI. The images were captured by confocal microscopy. Scale bar: 10 μm.


D . DMSO or Z - VAD - FMK treated HEK293 / MycBioID - FAM111A cell lines were induced with doxycycline for 24 h . Total protein from an equal number of cells was incubated with magnetic streptavidin beads , and pull downs were analyzed by western blotting using GANP and Myc antibodies .

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

D. DMSO or Z-VAD-FMK treated HEK293/MycBioID-FAM111A cell lines were induced with doxycycline for 24 h. Total protein from an equal number of cells was incubated with magnetic streptavidin beads, and pull downs were analyzed by western blotting using GANP and Myc antibodies.


E . HEK293 / MycBioID - FAM111A cell lines were induced with doxycycline for 24 h , while growing in the presence or absence of 50 mM supplemental biotin . Total protein from whole cell lysates was incubated with magnetic streptavidin beads . Pulldowns were analyzed by western blotting using GANP and Myc antibodies .

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

E. HEK293/MycBioID-FAM111A cell lines were induced with doxycycline for 24 h, while growing in the presence or absence of 50 mM supplemental biotin. Total protein from whole cell lysates was incubated with magnetic streptavidin beads. Pulldowns were analyzed by western blotting using GANP and Myc antibodies.


F . DMSO or Z - VAD - FMK treated HEK293 / MycBioID - FAM111A cell lines were induced with doxycycline for 24 h . Total protein from ~ 105 cells was resolved by SDS - PAGE , and probed in western blots using antibodies against NPC ( Mab414 ) , PARP , cleaved Caspase 3 , Myc , or Tubulin .

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

F. DMSO or Z-VAD-FMK treated HEK293/MycBioID-FAM111A cell lines were induced with doxycycline for 24 h. Total protein from ~ 105 cells was resolved by SDS-PAGE, and probed in western blots using antibodies against NPC (Mab414), PARP, cleaved Caspase 3, Myc, or Tubulin.


A . Representative immunofluorescence images of SV40 LT localization 72 h after transfection into U2OS cells infected with control or FAM111A shRNA . Images were taken with a Zeiss Axio Imager . LT is shown in green , and DAPI blue . Scale bar : 100 μm . B . Quantification of LT positive cells described in A and fixed at indicated times post transfection . Five hundred or more cells were counted for each condition . C . Quantification of RFP marker distribution upon co - transfection with SV40 LT variants into U2OS cells infected with control or FAM111A shRNA . Cells were fixed at the indicated times . Five hundred or more cells were counted for each condition . In B & C , values are mean ± s . d . of independent experiments ( n = 3 ) . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 ( two - tailed unpaired t - test ) .

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

A. Representative immunofluorescence images of SV40 LT localization 72 h after transfection into U2OS cells infected with control or FAM111A shRNA. Images were taken with a Zeiss Axio Imager. LT is shown in green, and DAPI blue. Scale bar: 100 μm. B. Quantification of LT positive cells described in A and fixed at indicated times post transfection. Five hundred or more cells were counted for each condition. C. Quantification of RFP marker distribution upon co-transfection with SV40 LT variants into U2OS cells infected with control or FAM111A shRNA. Cells were fixed at the indicated times. Five hundred or more cells were counted for each condition. In B & C, values are mean ± s.d. of independent experiments (n = 3). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 (two-tailed unpaired t-test).


D . Western blot analysis of control or FAM111A - depleted U2OS cells transfected with SV40 LT variants . Cells were collected and lysed at indicated timepoints after SV40 transfection , immunoblotted with FAM111A , LT , or PARP antibodies . Cdc2 - PSTAIR was used as a loading control .

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

D. Western blot analysis of control or FAM111A-depleted U2OS cells transfected with SV40 LT variants. Cells were collected and lysed at indicated timepoints after SV40 transfection, immunoblotted with FAM111A, LT, or PARP antibodies. Cdc2-PSTAIR was used as a loading control.


B . Binding assay using MBP - fused Apc1 - loop500 fragments . MBP - fused Apc1 - loop500 WT or its derivatives ( T532A , T539A , 2A ( T532A / T539A ) and S558A ) was incubated with anaphase extracts supplemented with non - degradable cyclin B at 23 ˚ C for 1 h . The bound proteins were recovered by amylose beads , separated by SDS - PAGE and detected by immunoblotting with Plx1 antibody and Ponceau S staining .

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

B. Binding assay using MBP-fused Apc1-loop500 fragments. MBP-fused Apc1-loop500 WT or its derivatives (T532A, T539A, 2A (T532A/T539A) and S558A) was incubated with anaphase extracts supplemented with non-degradable cyclin B at 23˚C for 1 h. The bound proteins were recovered by amylose beads, separated by SDS-PAGE and detected by immunoblotting with Plx1 antibody and Ponceau S staining.


D . Specific binding of Plx1 - PBD to Apc1 - loop500 . MBP - fused Apc1 - loop500 WT or its derivatives 2A ( T532A / T539A ) was incubated in anaphase extracts supplemented with non - degradable cyclin B at 23 ˚ C for 1 h , and further incubated in the presence of 10 µg of WT Plx1 - PBD for 15 min . The bound proteins were recovered by amylose beads , separated by SDS - PAGE and detected by

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

D. Specific binding of Plx1-PBD to Apc1-loop500. MBP-fused Apc1-loop500 WT or its derivatives 2A (T532A/T539A) was incubated in anaphase extracts supplemented with non-degradable cyclin B at 23˚C for 1 h, and further incubated in the presence of 10 µg of WT Plx1-PBD for 15 min. The bound proteins were recovered by amylose beads, separated by SDS-PAGE and detected by Coomassie


A . Cdc20 binding assay in Xenopus egg extracts . The purified recombinant WT or Apc - loop500 mutant APC / Cs with mutations ( 1 - T532A , 1 - T539A , 1 - T532A / T539A ( 1 - 2A ) and 1 - S558A ) was incubated with APC / C depleted ( ΔAPC ) interphase extract ( Inter ) or ΔAPC anaphase extracts supplemented with non - degradable cyclin B ( Ana ) at 23 ˚ C for 1 h . The APC / C was recovered with Apc3 monoclonal antibody ( AF3 . 1 ) beads , and the bound proteins were analysed by SDS - PAGE and immunoblotting with indicated antibodies .

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

A. Cdc20 binding assay in Xenopus egg extracts. The purified recombinant WT or Apc-loop500 mutant APC/Cs with mutations (1-T532A, 1-T539A, 1-T532A/T539A (1-2A) and 1-S558A) was incubated with APC/C depleted (ΔAPC) interphase extract (Inter) or ΔAPC anaphase extracts supplemented with non-degradable cyclin B (Ana) at 23˚C for 1 h. The APC/C was recovered with Apc3 monoclonal antibody (AF3.1) beads, and the bound proteins were analysed by SDS-PAGE and immunoblotting with indicated antibodies.


E . In vitro phosphorylation by Plx1 . Apc3 - loop fragment , α - Casein , N - terminus of Cdc20 ( N159 ) or MBP protein was incubated with purified Plx1 in the presence of [ γ32P ] - ATP at 23 ˚ C for indicated times , separated by SDS - PAGE and detected by autoradiography and Coomassie brilliant blue ( CBB ) staining .

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

E. In vitro phosphorylation by Plx1. Apc3-loop fragment, α-Casein, N-terminus of Cdc20 (N159) or MBP protein was incubated with purified Plx1 in the presence of [γ32P]-ATP at 23˚C for indicated times, separated by SDS-PAGE and detected by autoradiography and Coomassie brilliant blue (CBB) staining.


C . Plx1 - binding assay using MBP - fused Apc1 - loop500 fragments with mutations in B56 - binding site . The Plx1 - binding to Apc1 - loop500 WT or its derivatives ( L557A / V560A or E562A ) was analysed as described in Fig 1B . D . Quantification of ( C ) . The bar graph is quantification of bound Plx1 . The intensities of WT were arbitrarily set to 1 . 0 . Error bars , SEM from three independent experiments .

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

C. Plx1-binding assay using MBP-fused Apc1-loop500 fragments with mutations in B56-binding site. The Plx1-binding to Apc1-loop500 WT or its derivatives (L557A/V560A or E562A) was analysed as described in Fig 1B. D. Quantification of (C). The bar graph is quantification of bound Plx1. The intensities of WT were arbitrarily set to 1.0. Error bars, SEM from three independent experiments.


E . Binding assay using MBP fused Apc1 - loop500 fragments and B56γ . MBP - fused Apc1 - loop500 WT or its derivatives ( T532A , T539A , T532A / T539A ( 2A ) and S558A ) was incubated with the 35S - labelled Flag - B56γ in anaphase extracts supplemented with non - degradable cyclin B at 23 ˚ C for 1 h . The bound proteins were recovered by amylose beads , separated by SDS - PAGE and detected by autoradiography or Coomassie brilliant blue ( CBB ) staining . The recovery of WT ( x1 / 2 and x1 ) was run as a standard . F . Quantification of ( E ) . The bar graph is quantification of bound B56γ . The intensities of WT were arbitrarily set to 1 . 0 . Error bars , SEM from three independent experiments .

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

E. Binding assay using MBP fused Apc1-loop500 fragments and B56γ. MBP-fused Apc1-loop500 WT or its derivatives (T532A, T539A, T532A/T539A (2A) and S558A) was incubated with the 35S-labelled Flag-B56γ in anaphase extracts supplemented with non-degradable cyclin B at 23˚C for 1 h. The bound proteins were recovered by amylose beads, separated by SDS-PAGE and detected by autoradiography or Coomassie brilliant blue (CBB) staining. The recovery of WT (x1/2 and x1) was run as a standard. F. Quantification of (E). The bar graph is quantification of bound B56γ. The intensities of WT were arbitrarily set to 1.0. Error bars, SEM from three independent experiments.


A . Plx1 and PP2A - B56 competes for Apc1 - loop500 . MBP - fused Apc1 - loop500 WT or its derivatives ( ∆ 11 , deletion of 11 residues including the B56 - binding motif ) was incubated with the 35S - labelled Flag - B56γ in anaphase extracts supplemented with non - degradable cyclin B at 23 ˚ C for 1 h , and further incubated with indicated amounts of WT Plx1 - PBD or Plx1 - PBD Pincer mutant ( Pin ) for 15 min . The bound proteins were recovered by amylose beads , separated by SDS - PAGE and detected by autoradiography or Coomassie brilliant blue ( CBB ) staining .

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

A. Plx1 and PP2A-B56 competes for Apc1-loop500. MBP-fused Apc1-loop500 WT or its derivatives (∆11, deletion of 11 residues including the B56-binding motif) was incubated with the 35S-labelled Flag-B56γ in anaphase extracts supplemented with non-degradable cyclin B at 23˚C for 1 h, and further incubated with indicated amounts of WT Plx1-PBD or Plx1-PBD Pincer mutant (Pin) for 15 min. The bound proteins were recovered by amylose beads, separated by SDS-PAGE and detected by autoradiography or Coomassie brilliant blue (CBB) staining.


C . Dephosphorylation of Apc1 - loop500 by PP2A - B56 in the presence of Plx1 - PBD . MBP - fused Apc1 - loop500 WT or its derivatives ( L557A / V560A or E562A ) was incubated in anaphase extracts supplemented with non - degradable cyclin B at 23 ˚ C for 1 h , and further incubated with 10 µg of WT Plx1 - PBD for 15 min . The complexes were isolated by amylose beads and incubated in the presence ( 78 nM ) or absence of purified PP2A - B56γ at 23 ˚ C for 20 min . The proteins were recovered by amylose beads , and analysed as described in ( B ) .

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

C. Dephosphorylation of Apc1-loop500 by PP2A-B56 in the presence of Plx1-PBD. MBP-fused Apc1-loop500 WT or its derivatives (L557A/V560A or E562A) was incubated in anaphase extracts supplemented with non-degradable cyclin B at 23˚C for 1 h, and further incubated with 10 µg of WT Plx1-PBD for 15 min. The complexes were isolated by amylose beads and incubated in the presence (78 nM) or absence of purified PP2A- B56γ at 23˚C for 20 min. The proteins were recovered by amylose beads, and analysed as described in (B).


A . Phosphorylation kinetics of T532 , T539 and S558 on Apc1 - loop500 in anaphase extracts . MBP - fused Apc1 - loop500 WT or its derivatives ( ∆ 11 , deletion of 11 residues including the B56 - binding motif ) was incubated with Xenopus egg extracts in the presence of non - degradable cyclin B at 23 ˚ C . The samples taken as indicated were analysed by SDS - PAGE and immunoblotting using phospho - specific antibodies ( pT532 , pT539 and pS558 ) or MBP antibody . To monitor CDK activity , Apc3 antibody was used ( bottom ) . The signals of a phospho - specific antibody and MBP antibody on the same membrane were detected in the 800 - and 700 - nm channels , respectively . The two channels were pseudo - coloured ( green ; pT532 , pT539 and pS558 , red ; MBP ) and overlaid in the bottom panel ( Overlay ) . The signal of pS558 was not detected in ∆ 11 mutant because ∆ 11 mutation includes S558 .

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

A. Phosphorylation kinetics of T532, T539 and S558 on Apc1-loop500 in anaphase extracts. MBP-fused Apc1-loop500 WT or its derivatives (∆11, deletion of 11 residues including the B56-binding motif) was incubated with Xenopus egg extracts in the presence of non-degradable cyclin B at 23˚C. The samples taken as indicated were analysed by SDS-PAGE and immunoblotting using phospho-specific antibodies (pT532, pT539 and pS558) or MBP antibody. To monitor CDK activity, Apc3 antibody was used (bottom). The signals of a phospho-specific antibody and MBP antibody on the same membrane were detected in the 800- and 700-nm channels, respectively. The two channels were pseudo-coloured (green; pT532, pT539 and pS558, red; MBP) and overlaid in the bottom panel (Overlay). The signal of pS558 was not detected in ∆11 mutant because ∆11 mutation includes S558.


A . Cdc20 binding assay in Xenopus egg extracts . The Cdc20 - binding activities of WT APC / C and Apc - loop500 - mutant APC / Cs with mutations ( 1 - L557A / V560A or 1 - E562A ) were analysed as described in Fig 2A . B . Quantification of ( A ) . The bar graph is quantification of bound Cdc20 . The intensities of WT control at Ana - 45 min were arbitrarily set to 1 . 0 . Error bars , SEM from three independent experiments .

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

A. Cdc20 binding assay in Xenopus egg extracts. The Cdc20-binding activities of WT APC/C and Apc-loop500-mutant APC/Cs with mutations (1-L557A/V560A or 1-E562A) were analysed as described in Fig 2A. B. Quantification of (A). The bar graph is quantification of bound Cdc20.The intensities of WT control at Ana-45 min were arbitrarily set to 1.0. Error bars, SEM from three independent experiments.


D . APC / C dephosphorylation after inactivation of CDK . The purified recombinant WT or Apc - loop500 - mutant APC / Cs with mutations ( 1 - L557A / V560A or 1 - E562A ) was incubated with APC / C depleted ( ΔAPC ) in anaphase extracts supplemented with non - degradable cyclin B at 23 ˚ C for 1h ( Ana ) . After addition of p27 ( 0 . 3 µM ) , the samples taken at time points shown were analysed by SDS - PAGE and immunoblotting with indicated antibodies . ' Inter ' denotes interphase .

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

D. APC/C dephosphorylation after inactivation of CDK. The purified recombinant WT or Apc-loop500-mutant APC/Cs with mutations (1-L557A/V560A or 1-E562A) was incubated with APC/C depleted (ΔAPC) in anaphase extracts supplemented with non-degradable cyclin B at 23˚C for 1h (Ana). After addition of p27 (0.3 µM), the samples taken at time points shown were analysed by SDS-PAGE and immunoblotting with indicated antibodies. 'Inter' denotes interphase.


A . The mutations in CDK sites on Apc3 - loop reduces phosphorylation of T532 on Apc1 . The purified recombinant WT or a mutant APC / C with mutations at nine CDK sites in Apc3 - loop ( 3 - 9A ) was incubated with APC / C - depleted ( ΔAPC ) anaphase extracts supplemented with non - degradable cyclin B at 23 ˚ C for 60 min . The APC / C was recovered with Apc3 monoclonal antibody ( AF3 . 1 ) beads , and the phosphorylation status of T532 in Apc1 was analysed by SDS - PAGE and immunoblotting with phospho - specific ( pT532 ) or Apc1 ( pan - Apc1 ) antibodies .

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

A. The mutations in CDK sites on Apc3-loop reduces phosphorylation of T532 on Apc1. The purified recombinant WT or a mutant APC/C with mutations at nine CDK sites in Apc3-loop (3-9A) was incubated with APC/C-depleted (ΔAPC) anaphase extracts supplemented with non-degradable cyclin B at 23˚C for 60 min. The APC/C was recovered with Apc3 monoclonal antibody (AF3.1) beads, and the phosphorylation status of T532 in Apc1 was analysed by SDS-PAGE and immunoblotting with phospho-specific (pT532) or Apc1 (pan-Apc1) antibodies.


( B - E ) Immunostaining of germaria from 7 day - old females with anti - Vasa ( green , B - D ) or anti - GFP ( green , E ) , and anti - Hts ( red ) . DAPI ( blue ) was used to visualize DNA . ( B ) aubHN2 / + was used as a control . ( C , D ) examples of aubHN2 / QC42 germ cell loss and tumor , respectively . ( E ) Phenotypic rescue of aubHN2 / QC42 with UASp - GFP - Aub expressed using nos - Gal4 . White arrowheads indicate GSCs ; the white arrow indicates GSC loss .

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

(B-E) Immunostaining of germaria from 7 day-old females with anti-Vasa (green, B-D) or anti-GFP (green, E), and anti-Hts (red). DAPI (blue) was used to visualize DNA. (B) aubHN2/+ was used as a control. (C, D) examples of aubHN2/QC42 germ cell loss and tumor, respectively. (E) Phenotypic rescue of aubHN2/QC42 with UASp-GFP-Aub expressed using nos-Gal4. White arrowheads indicate GSCs; the white arrow indicates GSC loss.


( F ) Quantification of mutant germaria with 0 to 1 GSC , or with GSC tumors shown in ( B - E ) . The number of scored germaria ( n ) is indicated on the right graph . * * * p - value < 0 . 001 , ns : non significant , using the χ2 test .

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

(F) Quantification of mutant germaria with 0 to 1 GSC, or with GSC tumors shown in (B-E). The number of scored germaria (n) is indicated on the right graph. *** p-value <0.001, ns: non significant, using the χ2 test.


( G - H ' ) Germaria containing control ( G , G ' ) or aubHN2 mutant ( H , H ' ) clonal GSCs stained with anti - GFP ( green ) and anti - Hts ( red ) , 14 days after clone induction . DAPI ( blue ) was used to stain DNA . Clonal cells are marked by the lack of GFP . Clonal GSCs and cysts are outlined with dashed line . White arrowheads show clonal GSCs in the control . aub mutant clonal GSCs have been lost ( H , H ' ) . Scale bar : 10 μm in B - E and G - H ' .

O O O O O O B-TISSUE O O O O O O O O O B-GENEPROD O O O O O O O O B-CELL_TYPE O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SUBCELLULAR O O O O O O O O O B-GENEPROD O O B-CELL_TYPE O B-CELL_TYPE O O O O O O O O O O B-CELL_TYPE O O O O B-GENEPROD O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O

(G-H') Germaria containing control (G, G') or aubHN2 mutant (H, H') clonal GSCs stained with anti-GFP (green) and anti-Hts (red), 14 days after clone induction. DAPI (blue) was used to stain DNA. Clonal cells are marked by the lack of GFP. Clonal GSCs and cysts are outlined with dashed line. White arrowheads show clonal GSCs in the control. aub mutant clonal GSCs have been lost (H, H'). Scale bar: 10 μm in B-E and G-H'.


( I ) Quantification of germaria containing at least one clonal GSC at 7 , 14 and 21 days after clonal induction .

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

(I) Quantification of germaria containing at least one clonal GSC at 7, 14 and 21 days after clonal induction.


( J ) Division rate of wild - type and aubHN2 clonal GSCs . The number of scored germaria ( n ) is indicated . * * * p - value < 0 . 001 using the two - tailed Student ' s t test .

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

(J) Division rate of wild-type and aubHN2 clonal GSCs. The number of scored germaria (n) is indicated. *** p-value <0.001 using the two-tailed Student's t test.


( A , B ) Immunostaining of germaria with anti - Vasa ( green ) and anti - Hts ( red ) . DAPI ( blue ) was used to visualize DNA . Examples of mnkP6 and mnkP6 aubHN2 / QC42 germaria are shown . White arrowheads indicate GSCs ; the white arrow indicates GSC loss .

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

(A, B) Immunostaining of germaria with anti-Vasa (green) and anti-Hts (red). DAPI (blue) was used to visualize DNA. Examples of mnkP6 and mnkP6 aubHN2/QC42 germaria are shown. White arrowheads indicate GSCs; the white arrow indicates GSC loss.


( C ) Quantification of the number of GSCs per non - tumorous germarium , and of germaria with GSC tumors , in the indicated genotypes . The number of scored germaria ( n ) is indicated . The two - tailed Student ' s t test and the χ2 test were used in the left and right panels , respectively . * * * p - value < 0 . 001 , * * p - value < 0 . 01 , * p - value < 0 . 05 .

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

(C) Quantification of the number of GSCs per non-tumorous germarium, and of germaria with GSC tumors, in the indicated genotypes. The number of scored germaria (n) is indicated. The two-tailed Student's t test and the χ2 test were used in the left and right panels, respectively. *** p-value <0.001, ** p-value <0.01, * p-value <0.05.


( D - F ) Immunostaining of germaria from 7 day - old females with anti - Vasa or anti - GFP ( green ) and anti - Hts ( red ) . DAPI ( blue ) was used to visualize DNA . aubHN2 / QC42 ; nos - Gal4 / + was used as a negative control . Examples of rescue in aubHN2 / QC42 ; nos - Gal4 / UASp - GFP - Aub germarium ( E ) , and of lack of rescue in aubHN2 / QC42 ; nos - Gal4 / UASp - GFP - AubAA germarium ( F ) . White arrowheads indicate GSCs ; white arrows indicate GSC loss in ( D ) and germ cell loss in ( F ) . Scale bar : 10 μm in A , B and D - F .

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

(D-F) Immunostaining of germaria from 7 day-old females with anti-Vasa or anti-GFP (green) and anti-Hts (red). DAPI (blue) was used to visualize DNA. aubHN2/QC42; nos-Gal4/+ was used as a negative control. Examples of rescue in aubHN2/QC42; nos-Gal4/UASp-GFP-Aub germarium (E), and of lack of rescue in aubHN2/QC42; nos-Gal4/UASp-GFP-AubAA germarium (F). White arrowheads indicate GSCs; white arrows indicate GSC loss in (D) and germ cell loss in (F). Scale bar: 10 μm in A, B and D-F.


( G ) Quantification of mutant germaria with 0 to 1 GSC shown in ( D - F ) . The number of scored germaria ( n ) is indicated . * * * p - value < 0 . 001 , ns : non - significant , using the χ2 test .

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

(G) Quantification of mutant germaria with 0 to 1 GSC shown in (D-F). The number of scored germaria (n) is indicated. *** p-value <0.001, ns: non-significant, using the χ2 test.


( A - B ' ' ) Immunostaining of wild - type ( A - A ' ' ) or nos - Gal / UASp - CCR4 - HA ( B - B ' ' ) germaria with anti - Aub ( red ) , and anti - CCR4 or anti - HA ( green ) . GSCs are shown in ( A - A ' ' ) . White arrowheads in ( B - B ' ' ) indicate cytoplasmic accumulation of CCR4 - HA colocalized with Aub in GSCs .

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

(A-B'') Immunostaining of wild-type (A-A'') or nos-Gal/UASp-CCR4-HA (B-B'') germaria with anti-Aub (red), and anti-CCR4 or anti-HA (green). GSCs are shown in (A-A''). White arrowheads in (B-B'') indicate cytoplasmic accumulation of CCR4-HA colocalized with Aub in GSCs.


( C ) Co - immunoprecipitation ( IP ) of NOT1 , NOT3 and CCR4 with GFP - Aub in ovaries . Wild - type ( WT , mock IP ) or nos - Gal4 / UASp - GFP - Aub ( GFP - Aub ) ovarian extracts were immunoprecipitated with anti - GFP , either in the absence or the presence of RNase A . Western blots were revealed with anti - GFP , anti - NOT1 , anti - NOT3 and anti - CCR4 . Inputs are extracts prior to IP .

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

(C) Co-immunoprecipitation (IP) of NOT1, NOT3 and CCR4 with GFP-Aub in ovaries. Wild-type (WT, mock IP) or nos-Gal4/UASp-GFP-Aub (GFP-Aub) ovarian extracts were immunoprecipitated with anti-GFP, either in the absence or the presence of RNase A. Western blots were revealed with anti-GFP, anti-NOT1, anti-NOT3 and anti-CCR4. Inputs are extracts prior to IP.


( D ) Co - IP of NOT1 and NOT3 with GFP - Aub or GFP - AubAA in ovaries . Wild - type ( WT , mock IP ) , nos - Gal4 / UASp - GFP - Aub ( GFP - Aub ) or nos - Gal4 / UASp - GFP - AubAA ( GFP - AubAA ) ovarian extracts were immunoprecipitated with anti - GFP in the presence of RNase A . Western blots were revealed with anti - GFP , anti - NOT1 and anti - NOT3 . Inputs are extracts prior to IP .

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

(D) Co-IP of NOT1 and NOT3 with GFP-Aub or GFP-AubAA in ovaries. Wild-type (WT, mock IP), nos-Gal4/UASp-GFP-Aub (GFP-Aub) or nos-Gal4/UASp-GFP-AubAA (GFP-AubAA) ovarian extracts were immunoprecipitated with anti-GFP in the presence of RNase A. Western blots were revealed with anti-GFP, anti-NOT1 and anti-NOT3. Inputs are extracts prior to IP.


( E - G ) Genetic interaction between aub and twin in Vasa self - renewal . Immunostaining of germaria with anti - DNA ( green ) and anti - Hts ( red ) . DAPI ( blue ) was used to visualize DNA . Examples of aubHN2 / + , twinDG24102 and aubHN2 / + ; twinDG24102 germaria are shown . White arrowheads indicate GSCs ; the white arrow indicates GSC loss . Scale bar : 10 μm in A - B ' ' and E - G .

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

(E-G) Genetic interaction between aub and twin in Vasa self-renewal. Immunostaining of germaria with anti-DNA (green) and anti-Hts (red). DAPI (blue) was used to visualize DNA. Examples of aubHN2/+, twinDG24102 and aubHN2/+; twinDG24102 germaria are shown. White arrowheads indicate GSCs; the white arrow indicates GSC loss. Scale bar: 10 μm in A-B'' and E-G.


( H ) Quantification of mutant germaria with no GSC in 3 , 7 and 14 day - old females of the genotypes shown in ( D - F ) . The number of scored germaria ( n ) is indicated . * * * p - value < 0 . 001 using the χ2 test .

O O O O O O B-TISSUE 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 B-TISSUE O O O O O O O O O O O O O O O O O O O O O

(H) Quantification of mutant germaria with no GSC in 3, 7 and 14 day-old females of the genotypes shown in (D-F). The number of scored germaria (n) is indicated. *** p-value <0.001 using the χ2 test.


( B - C ' ' ) Immunostaining of mosaic germaria with anti - GFP ( green ) , to identify clonal cells by the lack of GFP , and either 8C4 ( B - B ' ' ) or 10F1 ( C - C ' ' ) monoclonal anti - Cbl ( red ) . DAPI was used to visualize DNA . White arrowheads indicate aubHN2 / + control GSCs ; yellow arrowheads indicate clonal mutant aubHN2 GSCs . Scale bar : 10 μm .

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

(B-C'') Immunostaining of mosaic germaria with anti-GFP (green), to identify clonal cells by the lack of GFP, and either 8C4 (B-B'') or 10F1 (C-C'') monoclonal anti-Cbl (red). DAPI was used to visualize DNA. White arrowheads indicate aubHN2/+ control GSCs; yellow arrowheads indicate clonal mutant aubHN2 GSCs. Scale bar: 10 μm.


( D - E ) Quantification of Cbl protein levels in aubHN2 / + and aubHN2 mutant GSCs using fluorescence intensity of immunostaining with 8C4 or 10F1 . Fluorescence intensity was measured in arbitrary units using the ImageJ software . Horizontal bars correspond to the mean and standard deviation . * * * p - value < 0 . 001 using the two - tailed Student ' s t test .

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

(D-E) Quantification of Cbl protein levels in aubHN2/+ and aubHN2 mutant GSCs using fluorescence intensity of immunostaining with 8C4 or 10F1. Fluorescence intensity was measured in arbitrary units using the ImageJ software. Horizontal bars correspond to the mean and standard deviation. *** p-value <0.001 using the two-tailed Student's t test.


( F ) RNA immunoprecitation ( IP ) with anti - GFP antibody in wild - type ( mock IP ) and nos - Gal4 / UASp - GFP - Aub ovarian extracts . Cbl mRNA was quantified using RT - qPCR . Normalization was with RpL32 mRNA . Mean of three biological replicates . The error bar represents standard error to the mean . * p - value < 0 . 05 using the two - tailed Student ' s t test .

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

(F) RNA immunoprecitation (IP) with anti-GFP antibody in wild-type (mock IP) and nos-Gal4/UASp-GFP-Aub ovarian extracts. Cbl mRNA was quantified using RT-qPCR. Normalization was with RpL32 mRNA. Mean of three biological replicates. The error bar represents standard error to the mean. * p-value <0.05 using the two-tailed Student's t test.


( G ) ePAT assay of CblL mRNA . Ovaries from 1 - day - old ( germarium to stage 8 ) wild - type , aub and twin mutant females , and from 4 - to 7 - day - old bam mutant females were used .

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

(G) ePAT assay of CblL mRNA. Ovaries from 1-day-old (germarium to stage 8) wild-type, aub and twin mutant females, and from 4- to 7-day-old bam mutant females were used.


( D , E ) Coverage of CblL ( D ) and CblS ( E ) mRNAs with ovarian and embryonic piRNAs . Significant crosslink clusters are indicated in red at the top of the graphs .

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

(D, E) Coverage of CblL (D) and CblS (E) mRNAs with ovarian and embryonic piRNAs. Significant crosslink clusters are indicated in red at the top of the graphs.


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


( A - E ) Cbl expression in germaria . ( A ) Immunostaining of wild - type germaria with anti - Cbl 8C4 .

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

(A-E) Cbl expression in germaria. (A) Immunostaining of wild-type germaria with anti-Cbl 8C4.


( A - E ) Cbl expression in germaria . ( B - B ' ' ) Immunostaining of bamP - GFP germaria that express GFP under the bam promoter , with anti - GFP ( green ) and anti - Cbl 8C4 ( red ) .

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

(A-E) Cbl expression in germaria. (B-B'') Immunostaining of bamP-GFP germaria that express GFP under the bam promoter, with anti-GFP (green) and anti-Cbl 8C4 (red).


( A - E ) Immunostaining expression in germaria . ( C - C ' ) Immunostaining of germaria expressing GFP - Par1 to label spectrosomes with anti - GFP ( green ) and anti - Cbl 8C4 ( red ) . DAPI ( blue ) was used to visualize DNA . White arrowheads indicate GSCs ; yellow arrowheads indicate cystoblasts .

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

(A-E) Immunostaining expression in germaria. (C-C') Immunostaining of germaria expressing GFP-Par1 to label spectrosomes with anti-GFP (green) and anti-Cbl 8C4 (red). DAPI (blue) was used to visualize DNA. White arrowheads indicate GSCs; yellow arrowheads indicate cystoblasts.


( F ) Western blots of protein extracts from wild - type and Cbl mutant ovaries showing CblL ( left panel ) and CblS ( right panel ) revealed with 8C4 and 10F1 antibodies , respectively . A low level of CblS remained , while no CblL was present in the CblEY11427 / MB05683 combination . The 10F1 antibody recognizes very poorly CblL in western blot .

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

(F) Western blots of protein extracts from wild-type and Cbl mutant ovaries showing CblL (left panel) and CblS (right panel) revealed with 8C4 and 10F1 antibodies, respectively. A low level of CblS remained, while no CblL was present in the CblEY11427/MB05683 combination. The 10F1 antibody recognizes very poorly CblL in western blot.


( G - J ) Cbl is required for germline differentiation . Immunostaining of wild - type and CblEY11427 / MB05683 germaria with anti - Hts to visualize spectrosomes and fusomes . White arrowheads indicate GSCs ; the white arrow indicates increased number of spectrosomes .

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

(G-J) Cbl is required for germline differentiation. Immunostaining of wild-type and CblEY11427/MB05683 germaria with anti-Hts to visualize spectrosomes and fusomes. White arrowheads indicate GSCs; the white arrow indicates increased number of spectrosomes.


( G - J ) Cbl is required for germline differentiation . Immunostaining of wild - type and CblEY11427 / MB05683 germaria with anti - Hts to visualize spectrosomes and fusomes . White arrowheads indicate GSCs ; the white arrow indicates increased number of spectrosomes . ( I ) Quantification of germaria with increased number of

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

(G-J) Cbl is required for germline differentiation. Immunostaining of wild-type and CblEY11427/MB05683 germaria with anti-Hts to visualize spectrosomes and fusomes. White arrowheads indicate GSCs; the white arrow indicates increased number of spectrosomes. (I) Quantification of germaria with increased number of spectrosomes


( G - J ) Cbl is required for germline differentiation . Immunostaining of wild - type and CblEY11427 / MB05683 germaria with anti - Hts to visualize spectrosomes and fusomes . White arrowheads indicate GSCs ; the white arrow indicates increased number of spectrosomes . ( J ) quantification of spectrosomes per germarium . The number of scored germaria ( n ) is indicated in ( I ) . * * * p - value < 0 . 001 using the χ2 test in ( I ) , and the two - tailed Student ' s t test in ( J ) .

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

(G-J) Cbl is required for germline differentiation. Immunostaining of wild-type and CblEY11427/MB05683 germaria with anti-Hts to visualize spectrosomes and fusomes. White arrowheads indicate GSCs; the white arrow indicates increased number of spectrosomes. (J) quantification of spectrosomes per germarium. The number of scored germaria (n) is indicated in (I). ***p-value <0.001 using the χ2 test in (I), and the two-tailed Student's t test in (J).


( K - M ) Cbl induces GSC differentiation . Immunostaining of germaria overexpressing Cbl with Hsp83 - CblL ( K ) White arrows indicate a cyst in the GSC niche ( K , outlined )

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

(K-M) Cbl induces GSC differentiation. Immunostaining of germaria overexpressing Cbl with Hsp83-CblL (K)White arrows indicate a cyst in the GSC niche (K, outlined)


( K - M ) Cbl induces GSC differentiation . Immunostaining of germaria overexpressing Cbl with Hsp83 - CblS ( L ) with anti - Vasa ( green ) and anti - Hts ( red ) . DNA ( blue ) was revealed with DAPI . White arrows indicatethe loss of GSCs and germ cells ( L ) .

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

(K-M) Cbl induces GSC differentiation. Immunostaining of germaria overexpressing Cbl with Hsp83-CblS (L) with anti-Vasa (green) and anti-Hts (red). DNA (blue) was revealed with DAPI. White arrows indicatethe loss of GSCs and germ cells (L).


( K - M ) Cbl induces GSC differentiation . ( M ) Quantification of germaria with 0 to 1 GSC in the indicated genotypes . The number of scored germaria ( n ) is indicated . * * * p - value < 0 . 001 using the χ2 test . Scale bar : 10 μm in A - C ' , G , H , K , L .

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(K-M) Cbl induces GSC differentiation.(M) Quantification of germaria with 0 to 1 GSC in the indicated genotypes. The number of scored germaria (n) is indicated. *** p-value <0.001 using the χ2 test. Scale bar: 10 μm in A-C', G, H, K, L.


( A - B ) Immunostaining of germaria from aubHN2 / QC42 ( A ) females with anti - Vasa ( green ) and anti - Hts ( red ) . DAPI ( blue ) was used to visualize DNA . The white arrow indicates the lack of GSCs ; white arrowheads indicate GSCs . Scale bar : 10 μm .

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(A-B) Immunostaining of germaria from aubHN2/QC42 (A) females with anti-Vasa (green) and anti-Hts (red). DAPI (blue) was used to visualize DNA. The white arrow indicates the lack of GSCs; white arrowheads indicate GSCs. Scale bar: 10 μm.


( A - B ) Immunostaining of germaria from aubHN2 / QC42 ; CblMB / + ( B ) females with anti - Vasa ( green ) and anti - Hts ( red ) . DAPI ( blue ) was used to visualize DNA . The white arrow indicates the lack of GSCs ; white arrowheads indicate GSCs . Scale bar : 10 μm .

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

(A-B) Immunostaining of germaria from aubHN2/QC42; CblMB/+ (B) females with anti-Vasa (green) and anti-Hts (red). DAPI (blue) was used to visualize DNA. The white arrow indicates the lack of GSCs; white arrowheads indicate GSCs. Scale bar: 10 μm.


( C ) Quantification of mutant germaria with 0 to 1 GSC in the indicated genotypes . The number of scored germaria ( n ) is indicated . * * * p - value < 0 . 001 , * * p - value < 0 . 01 , * p - value < 0 . 05 , using the χ2 test .

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(C) Quantification of mutant germaria with 0 to 1 GSC in the indicated genotypes. The number of scored germaria (n) is indicated. *** p-value <0.001, ** p-value <0.01, * p-value <0.05, using the χ2 test.


( b ) N2a cells coexpressing YFP - PrP and HLA - A2 - CFP were subjected to 6 mM trypsin for 100 s . Images were taken before and after trypsin treatment as indicated . Note that the membrane signal of YFP - PrP was destroyed by the protease , whereas HLA - A2 - CFP ' s membrane signal remained unaffected .

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

(b) N2a cells coexpressing YFP-PrP and HLA-A2-CFP were subjected to 6 mM trypsin for 100 s. Images were taken before and after trypsin treatment as indicated. Note that the membrane signal of YFP-PrP was destroyed by the protease, whereas HLA-A2-CFP's membrane signal remained unaffected.


( d ) Digitonin treatment permeabilizes the plasma membrane and releases first cytosolic and later nucleoplasmicGFP . N2a cells expressing GFP were treated with 20 μM digitonin . Images were taken before and after digitonin application at the indicated time points . Scale bars , 10 μm .

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

(d) Digitonin treatment permeabilizes the plasma membrane and releases first cytosolic and later nucleoplasmicGFP. N2a cells expressing GFP were treated with 20 μM digitonin. Images were taken before and after digitonin application at the indicated time points. Scale bars, 10 μm.


( b ) FPP assay of N2a cells expressing RFP and the type I membrane protein CD3δ - CFP . Images were taken before and after 1 - min treatment with 20 μM digitonin and 4 mM trypsin as indicated . Scale bar , 10 μm .

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(b) FPP assay of N2a cells expressing RFP and the type I membrane protein CD3δ-CFP. Images were taken before and after 1-min treatment with 20 μM digitonin and 4 mM trypsin as indicated. Scale bar, 10 μm.


( b ) NRK cells expressing CD3δ - CFP and YFP - CD3δ were subjected to the FPP assay . Images were taken before and after 1 - min treatment with 20 μM digitonin and 4 mM trypsin as indicated . Scale bar , 10 μm .

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(b) NRK cells expressing CD3δ-CFP and YFP-CD3δ were subjected to the FPP assay. Images were taken before and after 1-min treatment with 20 μM digitonin and 4 mM trypsin as indicated. Scale bar, 10 μm.


c ) Biochemical protease protection assay of NRK cells expressing YFP - CD3δ , CD3δ - CFP and GFP . Transfected cells were subjected to SDS - PAGE and Western blot analysis before ( lane 1 ) and after separation of the cytosolic ( lane 2 ) and membrane ( lane 3 ) fractions by ultracentrifugation . Equal amounts of the membrane fraction were treated with trypsin in the absence ( lane 4 ) or presence ( lane 5 ) of Triton X - 100 . Protein extracts were analyzed by immunoblotting with monoclonal anti - GFP antibody JL - 8 .

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

c) Biochemical protease protection assay of NRK cells expressing YFP-CD3δ, CD3δ-CFP and GFP. Transfected cells were subjected to SDS-PAGE and Western blot analysis before (lane 1) and after separation of the cytosolic (lane 2) and membrane (lane 3) fractions by ultracentrifugation. Equal amounts of the membrane fraction were treated with trypsin in the absence (lane 4) or presence (lane 5) of Triton X-100. Protein extracts were analyzed by immunoblotting with monoclonal anti-GFP antibody JL-8.


( a ) Cartoon of the FPP assay illustrating the cellular localization of the Golgi marker GalT - YFP ( red ) and ER - bound CD3δ - CFP ( green ) , and the position of the fluorescent protein tag relative to its respective membrane . ( b ) N2a cells coexpressing GalT - YFP and CD3δ - CFP were subjected to the FPP assay , as in Figure

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

(a) Cartoon of the FPP assay illustrating the cellular localization of the Golgi marker GalT-YFP (red) and ER-bound CD3δ-CFP (green), and the position of the fluorescent protein tag relative to its respective membrane. (b) N2a cells coexpressing GalT-YFP and CD3δ-CFP were subjected to the FPP assay, as in Figure 2b


c ) Cartoon illustrating localization of mitochondrial EYFP - Mito ( red ) and Cd3δ - CFP ( green ) . ( d ) N2a cells coexpressing EYFP - Mito and CD3δ - CFP were subjected to the FPP assay , as in b . Scale bars , 10 μm .

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

c) Cartoon illustrating localization of mitochondrial EYFP-Mito (red) and Cd3δ-CFP (green). (d) N2a cells coexpressing EYFP-Mito and CD3δ-CFP were subjected to the FPP assay, as in b. Scale bars, 10 μm.


( a ) Cartoon of the FPP assay illustrating the cellular localization of RFP - SKL ( red ) and Pex3p - GFP ( green ) and the position of the fluorescent protein tag relative to its respective membrane . ( b ) N2a cells coexpressing RFP - SKL and Pex3p - GFP were subjected to the FPP assay . Images were taken before and after 1 - min treatment with 20 μM digitonin and 100 - s treatment with 4 mM trypsin as indicated . Scale bar , 10 μm .

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

(a) Cartoon of the FPP assay illustrating the cellular localization of RFP-SKL (red) and Pex3p-GFP (green) and the position of the fluorescent protein tag relative to its respective membrane. (b) N2a cells coexpressing RFP-SKL and Pex3p-GFP were subjected to the FPP assay. Images were taken before and after 1-min treatment with 20 μM digitonin and 100-s treatment with 4 mM trypsin as indicated. Scale bar, 10 μm.


( a ) Cartoon of the FPP assay illustrating the cellular localization of the proteins and the position of the fluorescent protein tag relative to its respective membrane . GAPDH - RFP is shown in red and CFP - LC3 is shown in green . ( b ) NRK cells stably expressing CFP - LC3 were transiently transfected to express GAPDH - RFP and subjected to the FPP assay . Images were taken before and after 1 - min treatment with 20 μM digitonin and 100 - s treatment with 4 mM trypsin . The inset shows the overlap of the GAPDH - RFP and CFP - LC3 signals ( 4 × magnification of the central area ( outlined by a box ) of the micrographs . Scale bar , 10 μm .

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

(a) Cartoon of the FPP assay illustrating the cellular localization of the proteins and the position of the fluorescent protein tag relative to its respective membrane. GAPDH-RFP is shown in red and CFP-LC3 is shown in green. (b) NRK cells stably expressing CFP-LC3 were transiently transfected to express GAPDH-RFP and subjected to the FPP assay. Images were taken before and after 1-min treatment with 20 μM digitonin and 100-s treatment with 4 mM trypsin. The inset shows the overlap of the GAPDH-RFP and CFP-LC3 signals (4 × magnification of the central area (outlined by a box) of the micrographs. Scale bar, 10 μm.


A . Left column , an active summer squirrel ; right column , a torpid squirrel .

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A. Left column, an active summer squirrel; right column, a torpid squirrel.


B . The morphology of quadriceps is unchanged by hibernation as seen in haematoxylin and eosin ( H & amp ; amp ; E ) stained sections ( scale bar 90 µm ) .

B. The morphology of quadriceps is unchanged by hibernation as seen in haematoxylin and eosin (H&amp;E) stained sections (scale bar 90 µm).


C . Dystrophin staining was performed to outline the sarcolemma to determine percentage distribution of minimum Feret ' s diameter .

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

C. Dystrophin staining was performed to outline the sarcolemma to determine percentage distribution of minimum Feret's diameter.


D . Average ± SD of minimum Feret ' s diameter in quadriceps ( p = 0 . 26 ) and tibialis anterior ( p = 0 . 33 ) muscles is not significantly different between summer and hibernation .

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D. Average ± SD of minimum Feret's diameter in quadriceps (p = 0.26) and tibialis anterior (p = 0.33) muscles is not significantly different between summer and hibernation.


A . Western blot of quadriceps muscle from summer active ( S ) and hibernating ( H ) squirrels using antibodies against the proteins indicated . An increased abundance of P − Foxo3a ( serine − 253 ) is accompanied by a decrease in P − Akt ( serine − 478 ) . Corresponding densitometry of P − Akt and P − Foxo3a as a function of total Akt and Foxo3a .

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

A. Western blot of quadriceps muscle from summer active (S) and hibernating (H) squirrels using antibodies against the proteins indicated. An increased abundance of P−Foxo3a (serine−253) is accompanied by a decrease in P−Akt (serine−478). Corresponding densitometry of P−Akt and P−Foxo3a as a function of total Akt and Foxo3a.


B . Relative mRNA levels for four Foxo3a downstream targets show no significant changes during hibernation .

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

B. Relative mRNA levels for four Foxo3a downstream targets show no significant changes during hibernation.


C . Western blots analysis of mTOR downstream targets shows significant upregulation of P − P70 and P − 4E − BP1 during hibernation .

O O B-EXP_ASSAY I-EXP_ASSAY 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

C. Western blots analysis of mTOR downstream targets shows significant upregulation of P−P70 and P−4E−BP1 during hibernation.


A . Western blot analyses and densitometry of quadriceps muscles from non − hibernating and hibernating squirrels demonstrates significant increases in total SGK1 and phosphorylated SGK1 . Immunostaining of quadriceps muscle for total SGK1 shows upregulation during hibernation .

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

A. Western blot analyses and densitometry of quadriceps muscles from non−hibernating and hibernating squirrels demonstrates significant increases in total SGK1 and phosphorylated SGK1. Immunostaining of quadriceps muscle for total SGK1 shows upregulation during hibernation.


B . Levels of phosphorylated Foxo3a in S315 and T32 are significantly increased during hibernation . Densitometric analysis of P ‐ Foxo3a as a function of Foxo3a .

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B. Levels of phosphorylated Foxo3a in S315 and T32 are significantly increased during hibernation. Densitometric analysis of P‐Foxo3a as a function of Foxo3a.


A . The morphology of tibialis anterior muscle as revealed by haematoxylin - eosin staining shows increased variation in muscle fiber size with numerous atrophic muscle fibers ( scale bar 90 µm ) .

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

A.The morphology of tibialis anterior muscle as revealed by haematoxylin-eosin staining shows increased variation in muscle fiber size with numerous atrophic muscle fibers (scale bar 90 µm).


B . Percentage distribution and mean minimum Feret ' s diameter in tibialis anterior muscle ( p = 0 . 02 ) .

O O O O O O O O O O O O B-TISSUE I-TISSUE O O O O O O O O O O O

B. Percentage distribution and mean minimum Feret's diameter in tibialis anterior muscle (p = 0.02).


C . Increased levels of phosphorylated Akt ( S478 ) in skeletal muscles of sgk1 − / − mice .

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C. Increased levels of phosphorylated Akt (S478) in skeletal muscles of sgk1−/− mice.


D . sgk1 − / − mice exhibit decreased running distance after 36 days of exposure to a running wheel , ( p = 0 . 01 ; n = 6 animals per group ) .

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D. sgk1−/− mice exhibit decreased running distance after 36 days of exposure to a running wheel, (p = 0.01; n = 6 animals per group).


E . Isometric force from soleus muscles of WT and sgk1 - / - mice during repetitive electrical stimulations for 500 ms . Amplitudes of isometric force from soleus muscles at two stimulation frequencies . Specific force is plotted in all cases . Mean values ± SD from n = 9 ( sgk1 − / − ) and n = 10 ( WT ) independent animals ( p = 0 . 01 ) .

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E. Isometric force from soleus muscles of WT and sgk1-/- mice during repetitive electrical stimulations for 500 ms. Amplitudes of isometric force from soleus muscles at two stimulation frequencies. Specific force is plotted in all cases. Mean values ± SD from n = 9 (sgk1−/−) and n = 10 (WT) independent animals (p = 0.01).


A . H & amp ; amp ; E staining of tibialis anterior sections from 2 ‐ month old control and transgenic mice shows no differences ( scale bar 90 µm ) .

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

A. H&amp;E staining of tibialis anterior sections from 2‐month old control and transgenic mice shows no differences (scale bar 90 µm).


B . Morphometric analyses of minimum Feret ' s diameter of TA muscle reveal no changes in fiber size distribution or mean fiber size .

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

B. Morphometric analyses of minimum Feret's diameter of TA muscle reveal no changes in fiber size distribution or mean fiber size.


C . Western blots and densitometric analysis of muscle from transgenic and control mice . Downstream targets of the mTOR signalling cascade ( p70S6K and 4EBP1 ) demonstrate significant upregulation of their phosphorylated forms in sgk1tg mice .

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C. Western blots and densitometric analysis of muscle from transgenic and control mice. Downstream targets of the mTOR signalling cascade (p70S6K and 4EBP1) demonstrate significant upregulation of their phosphorylated forms in sgk1tg mice.


D . Western blot analyses and densitometry of phosphorylated FOXO3a and total FOXO3a of tibialis anterior muscles from control and transgenic mice ( n = 4 ) show an increase of phosphorylation at S315 and T32 .

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

D. Western blot analyses and densitometry of phosphorylated FOXO3a and total FOXO3a of tibialis anterior muscles from control and transgenic mice (n = 4) show an increase of phosphorylation at S315 and T32.


A . H & amp ; amp ; E staining of tibialis anterior sections reveals no changes in muscle architecture ( scale bar 90 µm ) .

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

A. H&amp;E staining of tibialis anterior sections reveals no changes in muscle architecture (scale bar 90 µm).


B . Morphometric analysis demonstrates a decrease in muscle fiber size of the wild − type control mice compared with sgk1tg transgenic littermates ( p = 0 . 0015 ) .

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

B. Morphometric analysis demonstrates a decrease in muscle fiber size of the wild−type control mice compared with sgk1tg transgenic littermates (p = 0.0015).


C . Western blot and densitometry analysis shows that levels of phosphorylated Foxo3a at S315 and T32 are increased in sgk1tg .

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C. Western blot and densitometry analysis shows that levels of phosphorylated Foxo3a at S315 and T32 are increased in sgk1tg.


A . Transfection of constitutively active Sgk1 ( CA ) into immobilized tibialis anterior muscle ( green ) reveals increased fiber size diameter when compared to control , EGFP only transfected muscle fibers of immobilized tibialis anterior muscles ( 100 µm ) . Laminin γ − 1 staining ( red ) outlines the basement membrane and blue staining marks nuclei ( DAPI ) .

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A. Transfection of constitutively active Sgk1 (CA) into immobilized tibialis anterior muscle (green) reveals increased fiber size diameter when compared to control, EGFP only transfected muscle fibers of immobilized tibialis anterior muscles (100 µm). Laminin γ−1 staining (red) outlines the basement membrane and blue staining marks nuclei (DAPI).


B . Representation of the minimum Feret ' s diameter average ± SD ( control and immobilized ; n = 9 - 10 per group ; 800 - 950 fibers were measured ) ( * p = 0 . 029 , * p = 0 . 034 , * p = 0 . 047 , p = 0 . 43 , respectively ) .

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B. Representation of the minimum Feret's diameter average ± SD (control and immobilized; n = 9-10 per group; 800-950 fibers were measured) (*p = 0.029, *p = 0.034, *p = 0.047, p = 0.43, respectively).


C . Percentage distribution of the minimum Feret ' s diameter of tibialis anterior muscle immobilized and transfected with eGFP ( GFP ) , wild − type Sgk1 ( WT ) , kinase dead Sgk1 ( KD ) and constitutively active Sgk1 ( CA ) compared to non − immobilized control TA muscle ( black dotted line ) .

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C. Percentage distribution of the minimum Feret's diameter of tibialis anterior muscle immobilized and transfected with eGFP (GFP), wild−type Sgk1 (WT), kinase dead Sgk1 (KD) and constitutively active Sgk1 (CA) compared to non−immobilized control TA muscle (black dotted line).


( B ) Representative confocal images show the distribution of the P4M2x reporter in HEK293 - AT1 cells before and after the addition of GSK - A1 ( 30 nM ) . Note the signal disappearing from the PM and strongly highlighting the endosomes and Golgi . Scale bars are 20

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(B) Representative confocal images show the distribution of the P4M2x reporter in HEK293-AT1 cells before and after the addition of GSK-A1 (30 nM). Note the signal disappearing from the PM and strongly highlighting the endosomes and Golgi. Scale bars are 20 µ


( C ) Representative result from a BRET experiment detecting PI4P in various endosomes ( Rab7 , Rab5 and Rab11 ) following addition of GSK - A1 . The graph shows normalized BRET values where the averages of triplicate measurements of A1 - treated cells were divided by the averages of triplicates obtained in DMSO - treated controls . Note that these signals originate from thousands of cells .

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(C) Representative result from a BRET experiment detecting PI4P in various endosomes (Rab7, Rab5 and Rab11) following addition of GSK-A1. The graph shows normalized BRET values where the averages of triplicate measurements of A1-treated cells were divided by the averages of triplicates obtained in DMSO-treated controls. Note that these signals originate from thousands of cells.


( D ) Similar BRET experiment showing the Rab7 - pool of PI4P both in control HEK293 - AT1 cells and in two clones of cells with PI4K2A deletion ( # 20 and # 26 ) . Means ± S . E . M . are shown from three experiments performed in triplicates .

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(D) Similar BRET experiment showing the Rab7-pool of PI4P both in control HEK293-AT1 cells and in two clones of cells with PI4K2A deletion (#20 and #26). Means ± S.E.M. are shown from three experiments performed in triplicates.


( A ) BRET measurement of PI4P in Rab7 compartment in parental and PI4K2A K / O cells after expression of HA - tagged PI4K2A or its kinase - dead mutant . Means ± S . E . M . are shown from three experiments performed in triplicates .

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(A) BRET measurement of PI4P in Rab7 compartment in parental and PI4K2A K/O cells after expression of HA-tagged PI4K2A or its kinase-dead mutant. Means ± S.E.M. are shown from three experiments performed in triplicates.


( B ) Western blot analysis shows that the level of PI4K2A expression was closely matched with that of wild - type cells .

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(B) Western blot analysis shows that the level of PI4K2A expression was closely matched with that of wild-type cells.


( C ) Distribution of GFP - Rab7 in parental ( left panels ) and two clones of PI4K2A K / O cells . Representative pictures show tubulation in the K / O cells reminiscent of those seen in wild - type cells expressing the GTP - locked Rab7Q67L mutant . Scale bars are 10 µm .

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(C) Distribution of GFP-Rab7 in parental (left panels) and two clones of PI4K2A K/O cells. Representative pictures show tubulation in the K/O cells reminiscent of those seen in wild-type cells expressing the GTP-locked Rab7Q67L mutant. Scale bars are 10 µm.


( B ) Representative confocal pictures showing these changes in live cells . Cells were transfected with the indicated constructs and imaged before and after addition of rapamycin ( 100 nM ) for 10 min . Note that the Rab7 recruiter itself is dissociating from the membranes after PI ( 4 , 5 ) P2 generation . Left panels show some cells enlarged . Scale bars : 20 µm .

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(B) Representative confocal pictures showing these changes in live cells. Cells were transfected with the indicated constructs and imaged before and after addition of rapamycin (100 nM) for 10 min. Note that the Rab7 recruiter itself is dissociating from the membranes after PI(4,5)P2 generation. Left panels show some cells enlarged. Scale bars: 20 µm.


( C ) Quantification of the changes in BRET experiments . Here the BRET sensor contained the PLCδ1PH - fused to Sluc and the Venus targeted with Rab7 expressed from a single vector and it was co - transfected with the Rab7 - targeted FRB ( tagged with iRFP ) and the FKBP12 - fused PIP5Kγ in which the CFP was mutated to eliminate its fluorescence ( CFP * ) . Recruitment of the PIP5Kγ but not its kinase - dead version caused an increase in the BRET signal indicating the increased PI ( 4 , 5 ) P2 in this compartment ( means ± S . E . M . , from three separate experiments performed in triplicates ) .

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(C) Quantification of the changes in BRET experiments. Here the BRET sensor contained the PLCδ1PH-fused to Sluc and the Venus targeted with Rab7 expressed from a single vector and it was co-transfected with the Rab7-targeted FRB (tagged with iRFP) and the FKBP12-fused PIP5Kγ in which the CFP was mutated to eliminate its fluorescence (CFP*). Recruitment of the PIP5Kγ but not its kinase-dead version caused an increase in the BRET signal indicating the increased PI(4,5)P2 in this compartment (means ± S.E.M., from three separate experiments performed in triplicates).


( D ) PI ( 4 , 5 ) P2 increases were larger when the Venus part of the BRET sensor was targeted with the GTP - locked form of Rab7 ( Q67L ) and was negligible with the GDP - locked form of Rab7 ( N125I ) in the BRET construct . Also note that the Rab7 wild - type - based BRET signal slowly returned toward the green trace consistent with Rab7 falling off from the membrane both in the BRET and the recruiting constructs ( means ± S . E . M . , from three separate experiments performed in triplicates ) .

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

(D) PI(4,5)P2 increases were larger when the Venus part of the BRET sensor was targeted with the GTP-locked form of Rab7 (Q67L) and was negligible with the GDP-locked form of Rab7 (N125I) in the BRET construct. Also note that the Rab7 wild-type-based BRET signal slowly returned toward the green trace consistent with Rab7 falling off from the membrane both in the BRET and the recruiting constructs (means ± S.E.M., from three separate experiments performed in triplicates).


( E ) The response of PI4K2A K / O cells was significantly reduced Means ± S . E . M . are shown from three experiments performed in triplicates .

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(E) The response of PI4K2A K/O cells was significantly reduced Means ± S.E.M. are shown from three experiments performed in triplicates.


( F ) Areas below the curves calculated from the time of rapamycin addition for each of the three separate experiments ( means ± S . E . M . , n = 3 ) . One - way ANOVA with Dunnett ' s multiple comparisons was used for statistical analysis ( * P = 0 . 0478 and 0 . 0287 for # 20 and # 26 clones , respectively ) .

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(F) Areas below the curves calculated from the time of rapamycin addition for each of the three separate experiments (means ± S.E.M., n=3). One-way ANOVA with Dunnett's multiple comparisons was used for statistical analysis (* P = 0.0478 and 0.0287 for #20 and #26 clones, respectively).


( A ) Representative confocal images of HEK293 - AT1 cells , which show that treatment with OSW1 ( 20 nM ) , a drug that inhibits the cholesterol - PI4P transport protein , OSBP , causes accumulation of PI4P in the Golgi and in the endosomes ( upper panels ) . Note that the accumulation is much reduced in the endosome but less so in the Golgi in the PI4K2A K / O cells . Scale bars : 20

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

(A) Representative confocal images of HEK293-AT1 cells, which show that treatment with OSW1 (20 nM), a drug that inhibits the cholesterol-PI4P transport protein, OSBP, causes accumulation of PI4P in the Golgi and in the endosomes (upper panels). Note that the accumulation is much reduced in the endosome but less so in the Golgi in the PI4K2A K/O cells. Scale bars: 20 µm


( B ) Quantification of these changes by BRET analysis . The PI4P - Rab7 BRET sensor construct was transfected into the respective cell lines and the cells treated with OSW1 ( 20 nM ) for the indicated times . The BRET ratios were expressed relative to those of DMSO treated cells . Data information : Means ± S . E . M . are shown from three experiments performed in triplicates .

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

(B) Quantification of these changes by BRET analysis. The PI4P-Rab7 BRET sensor construct was transfected into the respective cell lines and the cells treated with OSW1 (20 nM) for the indicated times. The BRET ratios were expressed relative to those of DMSO treated cells. Data information: Means ± S.E.M. are shown from three experiments performed in triplicates.


( C ) Detection of endosomal PI ( 4 , 5 ) P2 in cells pre - treated with OSW1 for 1 h in serum - free medium using confocal microscopy and the PLCδ1PH - GFP PI ( 4 , 5 ) P2 reporter . Angiotensin II ( AngII , 100 nM ) ( a Gq and PLC - activating agonist ) was used to liberate the PLCδ1PH - GFP PI ( 4 , 5 ) P2 reporter from the PM , which was necessary to detect the endosomal PI ( 4 , 5 ) P2 . The signal is transient as the AngII receptors show desensitization . Note the reduced signal in the two PI4K2A K / O clones . Scale bars : 20 µm .

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

(C) Detection of endosomal PI(4,5)P2 in cells pre-treated with OSW1 for 1 h in serum-free medium using confocal microscopy and the PLCδ1PH-GFP PI(4,5)P2 reporter. Angiotensin II (AngII, 100 nM) (a Gq and PLC-activating agonist) was used to liberate the PLCδ1PH-GFP PI(4,5)P2 reporter from the PM, which was necessary to detect the endosomal PI(4,5)P2. The signal is transient as the AngII receptors show desensitization. Note the reduced signal in the two PI4K2A K/O clones. Scale bars: 20 µm.


( D ) Quantification of the same changes by BRET analysis in wild - type cells using the PI ( 4 , 5 ) P2 sensor with Rab7 - Q67L - targeting . Note the transient signal increase after AngII stimulation only in cells treated with OSW1 . ( E ) This increase is barely detectable in the PI4K2A K / O cells . D Data information : Means ± S . E . M . are shown from three experiments performed in triplicates . The values in ( D ) and ( E ) were measured in the same 96 - well plate at the same time .

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

(D) Quantification of the same changes by BRET analysis in wild-type cells using the PI(4,5)P2 sensor with Rab7-Q67L-targeting. Note the transient signal increase after AngII stimulation only in cells treated with OSW1. (E) This increase is barely detectable in the PI4K2A K/O cells. D Data information: Means ± S.E.M. are shown from three experiments performed in triplicates. The values in (D) and (E) were measured in the same 96-well plate at the same time.


( A ) BRET experiment in parental HEK293 - AT1 cells in OSW1 - pretreated cells after RNAi mediated knock - down of PIP5Kβ or PIP5Kγ . ( means ± S . E . M . from three experiments , each performed in triplicates ) . Note the reduced response in the PIP5Kγ knock - down cells and the slight reduction in the case of PIP5Kβ knock - down . ( B ) Areas below the curves calculated from the time of rapamycin addition for each of the three separate experiments shown in panel A ( means ± S . E . M . , n = 3 ) . One - way ANOVA with Dunnett ' s multiple comparisons was used for statistical analysis ( * P = 0 . 0241 ; * * P = 0 . 0088 ) . (

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

(A) BRET experiment in parental HEK293-AT1 cells in OSW1-pretreated cells after RNAi mediated knock-down of PIP5Kβ or PIP5Kγ. (means ± S.E.M. from three experiments, each performed in triplicates). Note the reduced response in the PIP5Kγ knock-down cells and the slight reduction in the case of PIP5Kβ knock-down. (B) Areas below the curves calculated from the time of rapamycin addition for each of the three separate experiments shown in panel A (means ± S.E.M., n=3). One-way ANOVA with Dunnett's multiple comparisons was used for statistical analysis (* P=0.0241; **P=0.0088). (


( C ) These reductions are proportional to the reduction in PIP5Kγ levels as assessed by Western blot analysis of cell lysates obtained from cells treated with siRNAs at the same time .

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(C) These reductions are proportional to the reduction in PIP5Kγ levels as assessed by Western blot analysis of cell lysates obtained from cells treated with siRNAs at the same time.


( D ) Increased appearance of GFP - LC3 positive vesicles in PI4K2A K / O cells and reduced acidification of the LC3 - positive vesicles using the GFP - mCherry - LC3 reporter . Cells were transfected with the indicated constructs for one day and observed by confocal microscopy . Scale bars : 20 µm . ( E ) Quantification of the LC3 positive vesicles . ( n = 67 , 34 , 26 , 13 , 30 , 27 for the various groups , respectively from left to right ) . * * * designates P < 0 . 0001 using unpaired t - test . The same data set was also analyzed with the non - parametric Mann - Whitney test showing a P = 0 . 0004 and 0 . 0012 for # 20 and # 26 clones relative to control , respectively . ( F ) Co - localization analysis using the Pearson coefficient ( n = 31 , 21 and 14 cells analyzed for the different groups from left to right ) . * * * designates P < 0 . 0001 using unpaired t - test . The same data set was also analyzed with the non - parametric Mann - Whitney test showing a P value < 0 . 0001 for both groups .

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(D) Increased appearance of GFP-LC3 positive vesicles in PI4K2A K/O cells and reduced acidification of the LC3-positive vesicles using the GFP-mCherry-LC3 reporter. Cells were transfected with the indicated constructs for one day and observed by confocal microscopy. Scale bars: 20 µm. (E) Quantification of the LC3 positive vesicles. (n = 67, 34, 26, 13, 30, 27 for the various groups, respectively from left to right). *** designates P < 0.0001 using unpaired t-test. The same data set was also analyzed with the non-parametric Mann-Whitney test showing a P = 0.0004 and 0.0012 for #20 and #26 clones relative to control, respectively. (F) Co-localization analysis using the Pearson coefficient (n = 31, 21 and 14 cells analyzed for the different groups from left to right). *** designates P < 0.0001 using unpaired t-test. The same data set was also analyzed with the non-parametric Mann-Whitney test showing a P value < 0.0001 for both groups.


( A , B ) Distribution of mCherry - GFP - LC3 in HEK293 - AT1 cells ( without starvation ) after treatment with control siRNA ( A ) or siRNA for PIP5Kγ ( PIP5K1C ) . Scale bars : 20 µm . Note the massive tubulation of the LC3 compartment in some of the knocked - down cells ( B , lower images , scale bar : 10 µm ) . ( C ) Comparison of Pearson coefficients from cells treated with control or PIP5Kγ RNAi . For statistical analysis the unpaired t - test was used ( n = 50 and 45 cells for control a PIP5Kγ knock down groups , respectively ; * * * P < 0 . 0001 ) . ( D ) Representative confocal images of live HEK293 - AT1 cells expressing GFP - Rab7 treated with control or PIP5Kγ RNAi . Note the tubulation in the PIP5Kγ RNAi - treated cells . Scale bars : 10 µm .

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(A, B) Distribution of mCherry-GFP-LC3 in HEK293-AT1 cells (without starvation) after treatment with control siRNA (A) or siRNA for PIP5Kγ (PIP5K1C). Scale bars: 20 µm. Note the massive tubulation of the LC3 compartment in some of the knocked-down cells (B, lower images, scale bar: 10 µm). (C) Comparison of Pearson coefficients from cells treated with control or PIP5Kγ RNAi. For statistical analysis the unpaired t-test was used (n = 50 and 45 cells for control a PIP5Kγ knock down groups, respectively; *** P< 0.0001). (D) Representative confocal images of live HEK293-AT1 cells expressing GFP-Rab7 treated with control or PIP5Kγ RNAi. Note the tubulation in the PIP5Kγ RNAi-treated cells. Scale bars: 10 µm.


( A ) HEK293 - AT1 cells were transfected with PLEKHM1 - GFP - Rab7 along with CFP - FKBP - PIP5Kγ and iRFP - FRB - Rab7 constructs for one day and examined live by confocal microscopy . Representative cells show reduced PLEKHM1 localization when PIP5Kγ was recruited to the Rab7 endosomes ( lower row images ) . ( B ) This response is reduced in PI4K2A K / O cells . ( Data information : Scale bars : 20 µm .

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

(A) HEK293-AT1 cells were transfected with PLEKHM1-GFP-Rab7 along with CFP-FKBP-PIP5Kγ and iRFP-FRB-Rab7 constructs for one day and examined live by confocal microscopy. Representative cells show reduced PLEKHM1 localization when PIP5Kγ was recruited to the Rab7 endosomes (lower row images). (B) This response is reduced in PI4K2A K/O cells. ( Data information: Scale bars: 20 µm.


( C ) Quantification of these changes by BRET analysis where the full - length PLEKHM1 was fused to Sluc and Venus was fused to Rab7 in the BRET construct . For PIP5Kγ recruitment a mutant dark CFP ( W66A ) - FKBP - PIP5Kγ was used with an iRFP - FRB - Rab7 . BRET values were normalized to those of DMSO treated cells . Comparisons were made for parental ( blue ) and PI4K2A K / O ( green and orange ) cells . ( D ) Similar BRET analysis where the PLEKHM1 was replaced by either RILP or Vps35 in the BRET construct . D Data information Means ± S . E . M . are shown from three experiments performed in triplicates .

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

(C) Quantification of these changes by BRET analysis where the full-length PLEKHM1 was fused to Sluc and Venus was fused to Rab7 in the BRET construct. For PIP5Kγ recruitment a mutant dark CFP(W66A)-FKBP-PIP5Kγ was used with an iRFP-FRB-Rab7. BRET values were normalized to those of DMSO treated cells. Comparisons were made for parental (blue) and PI4K2A K/O (green and orange) cells. (D) Similar BRET analysis where the PLEKHM1 was replaced by either RILP or Vps35 in the BRET construct. D Data information Means ± S.E.M. are shown from three experiments performed in triplicates.


lines . Analysis of 2019 - nCoV - S expression ( A ) by Western blot . Representative blots from three experiments are shown . ß - Actin ( cell lysates )

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

lines.Analysis of 2019-nCoV-S expression (A) by Western blot. Representative blots from three experiments are shown. ß-Actin (cell lysates)


ines . Analysis of 2019 - nCoV - S pseudotype incorporation ( B ) by Western blot . Representative blots from three experiments are shown . VSV - M ( particles ) served as loading controls .

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

ines.Analysis of 2019-nCoV-S pseudotype incorporation (B) by Western blot. Representative blots from three experiments are shown. VSV-M (particles) served as loading controls.


( C ) Cell lines of human and animal origin were inoculated with pseudotyped VSV harboring VSV - G , SARS - S or 2019 - nCoV - S . At 16 h postinoculation , pseudotype entry was analyzed . Shown are the combined data of three experiments . Error bars indicate SEM .

O O O O O O O O O O O O O B-ORGANISM I-ORGANISM O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD 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) Cell lines of human and animal origin were inoculated with pseudotyped VSV harboring VSV-G, SARS-S or 2019-nCoV-S. At 16 h postinoculation, pseudotype entry was analyzed. Shown are the combined data of three experiments. Error bars indicate SEM.


( C ) 293T cells transiently expressing ACE2 of human ( dark blue ) or bat ( light blue ) origin , human APN ( purple ) or hDPP4 ( green ) were inoculated with pseudotyped VSV harboring VSV - G , SARS - S , 2019 - nCoV - S , MERS - S or 229E - S . At 16 h postinoculation , pseudotype entry was analyzed . The average of three independent experiments is shown . Error bars indicate SEM .

O O O B-CELL_LINE O O O B-GENEPROD O B-ORGANISM O O O O O B-ORGANISM O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O B-ORGANISM I-ORGANISM O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD 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) 293T cells transiently expressing ACE2 of human (dark blue) or bat (light blue) origin, human APN (purple) or hDPP4 (green) were inoculated with pseudotyped VSV harboring VSV-G, SARS-S, 2019-nCoV-S, MERS-S or 229E-S. At 16 h postinoculation, pseudotype entry was analyzed. The average of three independent experiments is shown. Error bars indicate SEM.


priming . Ammonium chloride ( A ) , were added to the indicated target cells before transduction with pseudotypes bearing the indicated glycoproteins .

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priming.Ammonium chloride (A), were added to the indicated target cells before transduction with pseudotypes bearing the indicated glycoproteins.


E64d ( CatB / L inhibitor ) ( B ) and / or camostat ( TMPRSS2 inhibitor ) ( B ) were added to the indicated target cells before transduction with pseudotypes bearing the indicated glycoproteins .

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E64d (CatB/L inhibitor) (B) and/or camostat (TMPRSS2 inhibitor) (B) were added to the indicated target cells before transduction with pseudotypes bearing the indicated glycoproteins.