A . Phosphorylation kinetics of T532 , T539 S558 on Apc1 - loop500 in anaphase extracts . MBP - fused Apc1 - loop500 WT or its derivatives ( ∆ 11 , deletion of 11 residues including B56 - binding motif ) was incubated with Xenopus egg extracts in presence of non - degradable cyclin B at 23 ˚ C . samples taken as indicated were analysed by SDS - PAGE immunoblotting using phospho - specific antibodies ( pT532 , pT539 pS558 ) or MBP antibody . To monitor CDK activity , Apc3 antibody was used ( bottom ) . signals of a phospho - specific antibody MBP antibody on same were detected in 800 - 700 - nm channels , respectively . two channels were pseudo - coloured ( green ; pT532 , pT539 pS558 , red ; MBP ) overlaid in bottom panel ( Overlay ) . 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 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 B-EXP_ASSAY O O O O O B-ORGANISM B-TISSUE O O O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY 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 B-EXP_ASSAY O O O B-GENEPROD O O O O O O O O O O O O O O O O O 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

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

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

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 . 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 ) , samples taken at time points shown were analysed by SDS - PAGE 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 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 B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-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 . mutations in CDK sites on Apc3 - loop reduces phosphorylation of T532 on Apc1 . 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 . APC / C was recovered with Apc3 monoclonal antibody ( AF3 . 1 ) beads , phosphorylation status of T532 in Apc1 was analysed by SDS - PAGE immunoblotting with phospho - specific ( pT532 ) or Apc1 ( pan - Apc1 ) antibodies .

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 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 B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O B-GENEPROD 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 I-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 with anti - Vasa ( green , B - D ) or anti - GFP ( green , E ) , anti - Hts ( red ) . DAPI ( blue ) was used to visualize . ( B ) aubHN2 / + was used as a control . ( C , D ) examples of aubHN2 / QC42 germ loss , respectively . ( E ) Phenotypic rescue of aubHN2 / QC42 with UASp - GFP - Aub expressed using nos - Gal4 . arrowheads indicate GSCs ; arrow indicates GSC loss .

O O O O O B-EXP_ASSAY O B-TISSUE 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 B-GENEPROD 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 B-GENEPROD O O B-CELL_TYPE O 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 B-CELL_TYPE 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 ) of mutant germaria with 0 to 1 GSC , or with GSC tumors shown in ( B - E ) . number of scored germaria ( n ) is indicated on right graph . * * * p - value < 0 . 001 , ns : non significant , using χ2 .

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 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

(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 ) anti - Hts ( red ) , 14 days after clone induction . DAPI ( blue ) was used to stain . Clonal are marked by lack of GFP . Clonal GSCs cysts are outlined with dashed . arrowheads show clonal GSCs in control . aub mutant clonal GSCs have been lost ( H , H ' ) . Scale bar : 10 μm in B - E 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 B-GENEPROD 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 O B-CELL_TYPE I-CELL_TYPE O O O O O O O O B-CELL_TYPE 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

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

O O O O B-TISSUE O O O O O B-CELL_TYPE 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 aubHN2 clonal GSCs . number of scored germaria ( n ) is indicated . * * * p - value < 0 . 001 using two - Student ' s t .

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

(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 ) anti - Hts ( red ) . DAPI ( blue ) was used to visualize . Examples of mnkP6 mnkP6 aubHN2 / QC42 germaria are shown . arrowheads indicate GSCs ; 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 B-GENEPROD O O O O B-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-TISSUE O O O O O B-CELL_TYPE 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 ) of number of GSCs per non - tumorous germarium , of germaria with GSC tumors , in indicated genotypes . number of scored germaria ( n ) is indicated . two - Student ' s t χ2 were used in left right panels , respectively . * * * p - value < 0 . 001 , * * p - value < 0 . 01 , * p - value < 0 . 05 .

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

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

O O O O O B-EXP_ASSAY O B-TISSUE O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD B-TISSUE 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 B-CELL_TYPE 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 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 ) of mutant germaria with 0 to 1 GSC shown in ( D - F ) . number of scored germaria ( n ) is indicated . * * * p - value < 0 . 001 , ns : non - significant , using χ2 .

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 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

(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 ) , anti - CCR4 or anti - HA ( green ) . GSCs are shown in ( A - A ' ' ) . arrowheads in ( B - B ' ' ) indicate 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 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 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 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 absence or presence of RNase A . Western blots were revealed with anti - GFP , anti - NOT1 , anti - NOT3 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 I-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 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 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 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 presence of RNase A . Western blots were revealed with anti - GFP , anti - NOT1 anti - NOT3 . Inputs are extracts prior to IP .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-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 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 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 twin in Vasa self - renewal . Immunostaining of germaria with anti - ( green ) anti - Hts ( red ) . DAPI ( blue ) was used to visualize . Examples of aubHN2 / + , twinDG24102 aubHN2 / + ; twinDG24102 germaria are shown . arrowheads indicate GSCs ; arrow indicates GSC loss . Scale bar : 10 μm in A - B ' ' E - G .

O O O O O O O O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O B-EXP_ASSAY O B-TISSUE O O O O O O O O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD O O O B-GENEPROD B-TISSUE O O O O O B-CELL_TYPE O O O B-CELL_TYPE 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 ) of mutant germaria with no GSC in 3 , 7 14 day - old of genotypes shown in ( D - F ) . number of scored germaria ( n ) is indicated . * * * p - value < 0 . 001 using χ2 .

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 B-TISSUE 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 by lack of GFP , either 8C4 ( B - B ' ' ) or 10F1 ( C - C ' ' ) monoclonal anti - Cbl ( red ) . DAPI was used to visualize . 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 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 O O O O B-SMALL_MOLECULE O O O 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 ) of Cbl in aubHN2 / + aubHN2 mutant GSCs using of immunostaining with 8C4 or 10F1 . was measured in arbitrary units using ImageJ software . Horizontal bars correspond to mean standard deviation . * * * p - value < 0 . 001 using two - Student ' s t .

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

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

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

O O O B-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 I-GENEPROD O O O O O O O O O O O B-GENEPROD 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 ) CblS ( E ) mRNAs with ovarian embryonic piRNAs . Significant crosslink clusters are indicated in red at top of graphs .

O O O O O O O B-GENEPROD O O O O O O O O O B-TISSUE I-TISSUE B-GENEPROD 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 B-GENEPROD O O O O O B-TISSUE I-TISSUE B-GENEPROD 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 bam promoter , with anti - GFP ( green ) anti - Cbl 8C4 ( red ) .

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(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 ) anti - Cbl 8C4 ( red ) . DAPI ( blue ) was used to visualize . arrowheads indicate GSCs ; yellow arrowheads indicate cystoblasts .

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

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(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 CblEY11427 / MB05683 germaria with anti - Hts to visualize spectrosomes fusomes . arrowheads indicate GSCs ; arrow indicates increased number of spectrosomes .

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(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 CblEY11427 / MB05683 germaria with anti - Hts to visualize spectrosomes fusomes . arrowheads indicate GSCs ; arrow indicates increased number of spectrosomes . ( I ) of germaria with increased number of

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(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 CblEY11427 / MB05683 germaria with anti - Hts to visualize spectrosomes fusomes . arrowheads indicate GSCs ; arrow indicates increased number of spectrosomes . ( J ) of spectrosomes per germarium . number of scored germaria ( n ) is indicated in ( I ) . * * * p - value < 0 . 001 using χ2 in ( I ) , two - Student ' s t in ( J ) .

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(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 ) arrows indicate a cyst in GSC niche ( K , outlined )

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(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 ) anti - Hts ( red ) . ( blue ) was revealed with DAPI . arrows indicatethe loss of GSCs germ ( L ) .

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(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 ) of germaria with 0 to 1 GSC in indicated genotypes . number of scored germaria ( n ) is indicated . * * * p - value < 0 . 001 using χ2 . 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 ) with anti - Vasa ( green ) anti - Hts ( red ) . DAPI ( blue ) was used to visualize . arrow indicates lack of GSCs ; 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 ) with anti - Vasa ( green ) anti - Hts ( red ) . DAPI ( blue ) was used to visualize . arrow indicates lack of GSCs ; arrowheads indicate GSCs . Scale bar : 10 μm .

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

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

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(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 plasma releases first later nucleoplasmicGFP . N2a expressing GFP were treated with 20 μM digitonin . were taken before after digitonin application at indicated time points . Scale bars , 10 μm .

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(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 of N2a expressing RFP type I CD3δ - CFP . were taken before after 1 - min treatment with 20 μM digitonin 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 expressing CD3δ - CFP YFP - CD3δ were subjected to FPP . were taken before after 1 - min treatment with 20 μM digitonin 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 of NRK expressing YFP - CD3δ , CD3δ - CFP GFP . Transfected were subjected to SDS - PAGE Western blot before ( lane 1 ) after separation of ( lane 2 ) ( lane 3 ) fractions by ultracentrifugation . Equal amounts of fraction were treated with trypsin in absence ( lane 4 ) or presence ( lane 5 ) of Triton X - 100 . extracts were analyzed by immunoblotting with monoclonal anti - GFP antibody JL - 8 .

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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 FPP illustrating localization of Golgi marker GalT - YFP ( red ) ER - bound CD3δ - CFP ( green ) , position of tag relative to its respective . ( b ) N2a coexpressing GalT - YFP CD3δ - CFP were subjected to FPP , as in Figure

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(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 ) Cd3δ - CFP ( green ) . ( d ) N2a coexpressing EYFP - Mito CD3δ - CFP were subjected to FPP , as in b . Scale bars , 10 μm .

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

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

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

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(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 . of quadriceps is unchanged by hibernation as seen in haematoxylin 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 was performed to outline sarcolemma to determine distribution of minimum Feret ' s diameter .

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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 ) tibialis anterior ( p = 0 . 33 ) is not significantly different between summer 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 ) hibernating ( H ) squirrels using antibodies against indicated . An increased abundance of P − Foxo3a ( serine − 253 ) is accompanied by a decrease in P − Akt ( serine − 478 ) . Corresponding densitometry of P − Akt P − Foxo3a as a function of total Akt Foxo3a .

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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 for four Foxo3a downstream targets show no significant changes during hibernation .

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


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

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


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

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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 . of phosphorylated Foxo3a in S315 T32 are significantly increased during hibernation . Densitometric 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 . of tibialis anterior muscle as revealed by haematoxylin - eosin shows increased variation in muscle fiber with numerous atrophic muscle ( scale bar 90 µm ) .

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

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


C . Increased of phosphorylated Akt ( S478 ) in skeletal 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 after 36 days of exposure to a running wheel , ( p = 0 . 01 ; n = 6 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 of WT sgk1 - / - mice during repetitive electrical stimulations for 500 ms . Amplitudes of isometric force from soleus at two stimulation frequencies . Specific force is plotted in cases . Mean values ± SD from n = 9 ( sgk1 − / − ) n = 10 ( WT ) independent ( 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 of tibialis anterior sections from 2 ‐ month old control transgenic mice shows no differences ( scale bar 90 µm ) .

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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 distribution or mean fiber .

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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 densitometric of muscle from transgenic control mice . Downstream targets of mTOR signalling cascade ( p70S6K 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 densitometry of phosphorylated FOXO3a total FOXO3a of tibialis anterior from control transgenic mice ( n = 4 ) show an increase of phosphorylation at S315 T32 .

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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 of tibialis anterior sections reveals no changes in muscle architecture ( scale bar 90 µm ) .

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


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

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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 densitometry shows that of phosphorylated Foxo3a at S315 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 diameter when compared to control , EGFP only transfected muscle of immobilized tibialis anterior ( 100 µm ) . Laminin γ − 1 ( red ) outlines basement blue 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 minimum Feret ' s diameter average ± SD ( control immobilized ; n = 9 - 10 per group ; 800 - 950 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 . distribution of minimum Feret ' s diameter of tibialis anterior muscle immobilized transfected with eGFP ( GFP ) , wild − type Sgk1 ( WT ) , kinase dead Sgk1 ( KD ) constitutively active Sgk1 ( CA ) compared to non − immobilized control TA muscle ( black dotted ) .

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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 show distribution of P4M2x reporter in HEK293 - AT1 before after addition of GSK - A1 ( 30 nM ) . Note signal disappearing from PM strongly highlighting endosomes 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 Rab11 ) following addition of GSK - A1 . graph shows normalized BRET values where averages of triplicate measurements of A1 - treated were divided by averages of triplicates obtained in DMSO - treated controls . Note that these signals originate from thousands of .

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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 Rab7 - pool of PI4P both in control HEK293 - AT1 in two clones of with PI4K2A deletion ( # 20 # 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 PI4K2A K / O 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 shows that level of PI4K2A expression was closely matched with that of wild - type .

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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 ) two clones of PI4K2A K / O . Representative pictures show tubulation in K / O reminiscent of those seen in wild - type expressing 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 . were transfected with indicated constructs imaged before after addition of rapamycin ( 100 nM ) for 10 min . Note that Rab7 recruiter itself is dissociating from membranes after PI ( 4 , 5 ) P2 generation . Left panels show some enlarged . Scale bars : 20 µm .

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

(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 ) of changes in BRET experiments . Here BRET sensor contained PLCδ1PH - fused to Sluc Venus targeted with Rab7 expressed from a single vector it was co - transfected with Rab7 - targeted FRB ( tagged with iRFP ) FKBP12 - fused PIP5Kγ in which CFP was mutated to eliminate its ( CFP * ) . Recruitment of PIP5Kγ but not its kinase - dead version caused an increase in BRET signal indicating 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 Venus part of BRET sensor was targeted with GTP - locked form of Rab7 ( Q67L ) was negligible with GDP - locked form of Rab7 ( N125I ) in BRET construct . Also note that Rab7 wild - type - BRET signal slowly returned toward green trace consistent with Rab7 falling off from both in BRET recruiting constructs ( means ± S . E . M . , from three separate experiments performed in triplicates ) .

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(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 ) response of PI4K2A K / O 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 curves calculated from time of rapamycin addition for each of three separate experiments ( means ± S . E . M . , n = 3 ) . One - way ANOVA with Dunnett ' s multiple comparisons was used for statistical ( * P = 0 . 0478 0 . 0287 for # 20 # 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 of HEK293 - AT1 , which show that treatment with OSW1 ( 20 nM ) , a drug that inhibits cholesterol - PI4P transport , OSBP , causes accumulation of PI4P in Golgi in endosomes ( upper panels ) . Note that accumulation is much reduced in endosome but less so in Golgi in PI4K2A K / O . Scale bars : 20

O O O O B-EXP_ASSAY O B-CELL_LINE O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-GENEPROD O O B-EXP_ASSAY O B-SMALL_MOLECULE O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O B-EXP_ASSAY O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR O B-GENEPROD 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 ) of these changes by BRET . PI4P - Rab7 BRET sensor construct was transfected into respective lines treated with OSW1 ( 20 nM ) for indicated times . BRET ratios were expressed relative to those of DMSO treated . Data information : Means ± S . E . M . are shown from three experiments performed in triplicates .

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(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 pre - treated with OSW1 for 1 h in serum - free medium using confocal microscopy PLCδ1PH - GFP PI ( 4 , 5 ) P2 reporter . Angiotensin II ( AngII , 100 nM ) ( a Gq PLC - activating agonist ) was used to liberate PLCδ1PH - GFP PI ( 4 , 5 ) P2 reporter from PM , which was necessary to detect endosomal PI ( 4 , 5 ) P2 . signal is transient as AngII receptors show desensitization . Note reduced signal in 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 B-SMALL_MOLECULE O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY 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 I-GENEPROD 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 B-SUBCELLULAR 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 B-SMALL_MOLECULE 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 ) of same changes by BRET in wild - type using PI ( 4 , 5 ) P2 sensor with Rab7 - Q67L - targeting . Note transient signal increase after AngII stimulation only in treated with OSW1 . ( E ) This increase is barely detectable in PI4K2A K / O . D Data information : Means ± S . E . M . are shown from three experiments performed in triplicates . values in ( D ) ( E ) were measured in same 96 - well plate at same time .

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

(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 in OSW1 - pretreated after RNAi mediated knock - down of PIP5Kβ or PIP5Kγ . ( means ± S . E . M . from three experiments , each performed in triplicates ) . Note reduced response in PIP5Kγ knock - down slight reduction in case of PIP5Kβ knock - down . ( B ) Areas below curves calculated from time of rapamycin addition for each of 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 ( * P = 0 . 0241 ; * * P = 0 . 0088 ) . (

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

(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 reduction in PIP5Kγ as assessed by Western blot of lysates obtained from treated with siRNAs at 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 reduced acidification of LC3 - positive vesicles using GFP - mCherry - LC3 reporter . were transfected with indicated constructs for one day observed by confocal microscopy . Scale bars : 20 µm . ( E ) of LC3 positive vesicles . ( n = 67 , 34 , 26 , 13 , 30 , 27 for various groups , respectively from left to right ) . * * * designates P < 0 . 0001 using unpaired t - . same data set was also analyzed with non - parametric Mann - Whitney showing a P = 0 . 0004 0 . 0012 for # 20 # 26 clones relative to control , respectively . ( F ) Co - localization using Pearson coefficient ( n = 31 , 21 14 analyzed for different groups from left to right ) . * * * designates P < 0 . 0001 using unpaired t - . same data set was also analyzed with non - parametric Mann - Whitney 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 ( without starvation ) after treatment with control siRNA ( A ) or siRNA for PIP5Kγ ( PIP5K1C ) . Scale bars : 20 µm . Note massive tubulation of LC3 compartment in some of knocked - down ( B , lower , scale bar : 10 µm ) . ( C ) Comparison of Pearson coefficients from treated with control or PIP5Kγ RNAi . For statistical unpaired t - was used ( n = 50 45 for control a PIP5Kγ knock down groups , respectively ; * * * P < 0 . 0001 ) . ( D ) Representative confocal of live HEK293 - AT1 expressing GFP - Rab7 treated with control or PIP5Kγ RNAi . Note tubulation in PIP5Kγ RNAi - treated . 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 were transfected with PLEKHM1 - GFP - Rab7 along with CFP - FKBP - PIP5Kγ iRFP - FRB - Rab7 constructs for one day examined live by confocal microscopy . Representative show reduced PLEKHM1 localization when PIP5Kγ was recruited to Rab7 endosomes ( lower row ) . ( B ) This response is reduced in PI4K2A K / O . ( Data information : Scale bars : 20 µm .

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

O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD O O O B-GENEPROD O B-EXP_ASSAY O O O B-GENEPROD 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 B-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD 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 . of 2019 - nCoV - S expression ( A ) by Western blot . Representative blots from three experiments are shown . ß - Actin ( lysates )

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

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


ines . 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 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 ) lines of human 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 combined data of three experiments . Error bars indicate .

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

(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 transiently expressing ACE2 of human ( blue ) or bat ( 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 . average of three independent experiments is shown . Error bars indicate .

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

(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 indicated target before transduction with pseudotypes bearing 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 ) / or camostat ( TMPRSS2 inhibitor ) ( B ) were added to indicated target before transduction with pseudotypes bearing 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.


( C ) 293T transiently expressing ACE2 alone or in combination with TMPRSS2 were incubated with CatB / L inhibitor E64d or PBS as control inoculated with pseudotypes bearing indicated viral surface . average of three independent experiments is shown in panels A - C . Error bars indicate . Statistical significance was tested by two - way ANOVA with Dunnett posttest .

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(C) 293T cells transiently expressing ACE2 alone or in combination with TMPRSS2 were incubated with CatB/L inhibitor E64d or PBS as control and inoculated with pseudotypes bearing the indicated viral surface proteins. The average of three independent experiments is shown in panels A-C. Error bars indicate SEM. Statistical significance was tested by two-way ANOVA with Dunnett posttest.


entry . Pseudotypes harboring indicated viral surface were incubated with different dilutions of serum from a convalescent SARS subsequently inoculated onto 293T that transiently express ACE2 in order to evaluate cross - neutralization . results from a representative experiment with triplicate samples are shown were confirmed in a separate experiment . Error bars indicate SD . Statistical significance was tested by two - way ANOVA with Dunnett posttest .

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entry.Pseudotypes harboring the indicated viral surface proteins were incubated with different dilutions of serum from a convalescent SARS patient and subsequently inoculated onto 293T cells that transiently express ACE2 in order to evaluate cross-neutralization. The results from a representative experiment with triplicate samples are shown and were confirmed in a separate experiment. Error bars indicate SD. Statistical significance was tested by two-way ANOVA with Dunnett posttest.


( B ) Structure of FRH from large crystallographic with an accordingly colored domain map below .

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(B) Structure of FRH from the large crystallographic cell with an accordingly colored domain map below.


( A ) FRH large ( cyan ) , Mtr4 ( pdb 2XGJ , green , backbone in orange ) , FRH small alternate ( blue ) differ primarily in orientation of arch domain . FRH - bound structure also crystallizes in small - configuration . two different FRH structures have an overall RMSD of 3 . 9 Å RMSD between Mtr4 FRH is 2 . 9 Å ( over 734 residues , for large FRH ) .

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(A) FRH large cell (cyan), Mtr4 (pdb 2XGJ, green, RNA backbone in orange), and FRH small alternate cell (blue) differ primarily in the orientation of the arch domain. The FRH RNA-bound structure also crystallizes in the small-cell configuration. The two different FRH structures have an overall RMSD of 3.9 Å and the RMSD between Mtr4 and FRH is 2.9 Å (over 734 residues, for large cell FRH).


( B ) SAXS data indicates that FRH - Δ114 is monomeric flexible . crystal structure of large structure predicts solution scattering curve ( left , χ2 = 0 . 92 ) fits well to calculated molecular envelope ( right ) .

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(B) SAXS data indicates that FRH-Δ114 is monomeric and flexible. The crystal structure of the large cell structure predicts the solution scattering curve (left, χ2 = 0.92) and fits well to the calculated molecular envelope (right).


( A ) Comparison of FRH N - terminus ( large structure , magenta ) to that of Mtr4 ( green ) aligned on their RecA domains . Only additional elements of FRH are shown ( cyan ) . FRH N - terminal extension has little ordered secondary structure , although regions 143 - 147 129 - 135 form small turns that interact with helicase . ( B ) very N - terminus of FRH extends away from RecA - 2 domain to interact with a symmetry related molecule in crystal ( below ) , whereas Mtr4 N - terminus turns back to form an outer β − strand . Center panel shows crystal contacts , with close - ups for each region highlighted as insets on left right .

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(A) Comparison of the FRH N-terminus (large cell structure, magenta) to that of Mtr4 (green) aligned on their RecA domains. Only the additional elements of FRH are shown (cyan). The FRH N-terminal extension has little ordered secondary structure, although regions 143-147 and 129-135 form small turns that interact with the helicase core. (B) The very N-terminus of FRH extends away from the RecA-2 domain to interact with a symmetry related molecule in the crystal (below), whereas the Mtr4 N-terminus turns back to form an outer β−strand. Center panel shows the crystal contacts, with close-ups for each region highlighted as insets on the left and right.


( A ) two RecA domains are rotated relative to one another in FRH ( cyan , large ) compared to Mtr4 ( green , backbone in orange ) . change in positioning of RecA - 2 with respect to RecA - 1 results in an overall displacement from Mtr4 domain juxtaposition that ranges from 1 . 1 - 4 . 0 Å throughout RecA - 2 with largest differences at external helices .

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(A) The two RecA domains are rotated relative to one another in FRH (cyan, large cell) compared to Mtr4 (green, RNA backbone in orange). The change in positioning of RecA-2 with respect to RecA-1 results in an overall displacement from the Mtr4 domain juxtaposition that ranges from 1.1 - 4.0 Å throughout RecA-2 with the largest differences at the external helices.


( B ) Comparison of arch domains conformations from Mtr4 ( green ) FRH ( large in cyan , small in marine ) after superposition of most N - terminal helical region . isolated KOW modules of FRH , Mtr4 ( with FRH superimposed ) , Ski2 have considerably different loop configurations .

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(B) Comparison of arch domains conformations from Mtr4 (green) and FRH (large cell in cyan, small cell in marine) after superposition of the most N-terminal helical region. The isolated KOW modules of FRH, Mtr4 (with FRH superimposed), and Ski2 have considerably different loop configurations.


( A ) 3 . 8 Å resolution mFo - DFc omit map density ( green , calculated with sharpened B - factors contoured at 2 . 0  ) reveals presence of ADP a short segment of bound to FRH .

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(A) 3.8 Å resolution mFo-DFc omit map density (green, calculated with sharpened B-factors and contoured at 2.0) reveals the presence of ADP and a short segment of RNA bound to FRH.


( B ) 3 . 8 Å resolution 2mFo - DFc calculated without ADP or contributing to model . Green electron density shown for ADP ( 0 . 6     orange density shown for surrounding ( 0 . 6   .

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(B) 3.8 Å resolution 2mFo-DFc calculated without ADP or RNA contributing to the model. Green electron density shown for ADP and RNA (0.6orange density shown for surrounding protein (0.6.


( C ) Comparison of Mtr4 FRHADP binding ( FRH in cyan with ADP , , F179 in orange bonds ; Mtr4 in green with ADP , , F148 in yellow bonds ) . ( c ) Unwound from Hel308 ( pdb 2PR6 ; magenta , in orange bonds ) overlaid in FRH with ADP ( FRH in cyan with yellow cyan ADP in stick ) after superposition of respective RecA domains .

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(C) Comparison of Mtr4 and FRHADP binding (FRH in cyan with ADP, RNA, and F179 in orange bonds; Mtr4 in green with ADP, RNA, and F148 in yellow bonds). (c) Unwound DNA from Hel308 (pdb 2PR6; magenta, DNA in orange bonds) overlaid in FRH with RNA and ADP (FRH in cyan with yellow RNA and cyan ADP in stick) after superposition of the respective RecA domains.


( d ) Hel308 helicase ( pdb 2PR6 ; backbone in orange , bases in gray bonds ) after aligning RecA domains of Hel308 with those of FRH ( cyan , in yellow ) ; see also Appendix Fig S4 . superposition indicates that KOW module is positioned to interact with double stranded that is being unwound by RecA DHSCT domains .

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(d) Hel308 DNA helicase DNA (pdb 2PR6; backbone in orange, bases in gray bonds) after aligning the RecA domains of Hel308 with those of FRH (cyan, RNA in yellow); see also Appendix Fig S4. The superposition indicates that the KOW module is positioned to interact with double stranded RNA that is being unwound by the RecA and DHSCT domains.


( A ) Key residues for FRH function shown on large - structure : K208 S324 compose ATP binding site , E294 lies between ADP pockets participates in ATP hydrolysis ; P871 resides in elbow region of arch domain . K811 , R806 , R712 K766 contribute to a positively charged surface on KOW domain . R806H disrupts clock . Q131 , G132 V142 in N - terminus provide interactions to helicase RecA domains ( FRH domains are colored according to Fig1 , ADP are in orange bonds ) . ( B ) Same as ( A ) after rotation about a horizontal axis .

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(A) Key residues for FRH function shown on the large-cell structure: K208 and S324 compose the ATP binding site, E294 lies between the ADP and RNA pockets and participates in ATP hydrolysis; P871 resides in the elbow region of the arch domain. K811, R806, R712 and K766 contribute to a positively charged surface on the KOW domain. R806H disrupts the clock. Q131, G132 and V142 in the N-terminus provide interactions to the helicase RecA domains (FRH domains are colored according to Fig1, ADP and RNA are in orange bonds). (B) Same as (A) after rotation about a horizontal axis.


( C ) Close - up of ADP - binding pocket N - terminal interaction for large - structure ( cyan ribbons ) small - structure ( blue ribbons ) .

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(C) Close-up of the ADP-binding pocket and N-terminal interaction for the large-cell structure (cyan ribbons) and the small-cell structure (blue ribbons).


( D ) Electrostatic potential surface of FRH ( blue positive , red negative ) shown with same orientation as in ( B ) .

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(D) Electrostatic potential surface of FRH (blue positive, red negative) shown with the same orientation as in (B).


( E ) Close - up of KOW domain showing large number of basic ( aqua sticks ) acidic side chains ( yellow sticks ) on surface that also includes mutational sites R806 , R712 , K811 K766 ( grey sticks )

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(E) Close-up of the KOW domain showing the large number of basic (aqua sticks) and acidic side chains (yellow sticks) on the surface that also includes the mutational sites R806, R712, K811 and K766 (grey sticks)


( A ) Race tube assays with FRH mutants . Every circadian cycle N . crassa lays down aerial hyphae that appear as fluffy yellow / orange conidial bands . between bands reflects clock period . strains were grown in constant darkness growth fronts were labeled every 24 h ( black vertical lines ) , rhythmicity on right ( τ = period in hours , σ = standard deviation , n = number of race tubes , ARR , arrhythmic ) . race tubes shown here are representative samples from three replicate tubes .

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(A) Race tube assays with FRH mutants. Every circadian cycle N. crassa lays down aerial hyphae that appear as fluffy yellow/orange conidial bands. The distance between the bands reflects the clock period. The strains were grown in constant darkness and growth fronts were labeled every 24 h (black vertical lines), rhythmicity on right (τ= period in hours, σ = standard deviation, n = number of race tubes, ARR, arrhythmic). The race tubes shown here are representative samples from three replicate tubes.


( B ) Pull - down assays evaluating interactions among FRH , FRQ , WC - 1 WC - 2 . of FRQ , WC - 1 WC - 2 binding to V5 tagged FRH mutants as compared to inter - variations in FRH , error bars represent standard deviation for run in triplicate . * P < 0 . 05 ( unpaired two - t - with Gaussian distribution assumption ) . Representative immunoblots are shown in Appendix Fig S5 .

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(B) Pull-down assays evaluating interactions among FRH, FRQ, WC-1 and WC-2. Ratio of FRQ, WC-1 and WC-2 binding to the V5 tagged FRH mutants as compared to the inter-strain variations in FRH levels, error bars represent standard deviation for the assay run in triplicate. *P < 0.05 (unpaired two-tailed t-test with Gaussian distribution assumption). Representative immunoblots are shown in Appendix Fig S5.


( a ) Relative ( RQ ) of CG7630 transcript after RNAi with a 120 - bp inverted - repeat ( act5c - gal4 > UAS - CG7630RNAi ) compared to control ( act5c - gal4 > + ) .

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(a) Relative quantification (RQ) of CG7630 transcript after RNAi with a 120-bp inverted-repeat sequence (act5c-gal4>UAS-CG7630RNAi) compared to control (act5c-gal4>+).


( b ) Relative of egg to adult viability of CG7630 RNAi cross ( act5c - gal4 / CyO . GFP x UAS - CG7630RNAi ) control cross ( act5c - gal4 / CyO . GFP x w1118 ) , calculated at three developmental stages ( eggs , pupae adults ) , ( n > 250 , Chi - square 9 . 920 df ( 2 ) , * * p = 0 . 007 ) .

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(b) Relative percentage of egg to adult viability of CG7630 RNAi cross (act5c-gal4/CyO.GFP x UAS-CG7630RNAi) and control cross (act5c-gal4/CyO.GFP x w1118), calculated at three developmental stages (eggs, pupae and adults), (n > 250, Chi-square test 9.920 df(2), **p = 0.007).


( d ) Kinetic enzyme activity of MRC complexes in CG7630 RNAi control individuals normalized by citrate synthase ( CS ) activity .

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(d) Kinetic enzyme activity of individual MRC complexes in CG7630 RNAi and control individuals normalized by citrate synthase (CS) activity.


( e ) In gel - activity assays for MRC complexes II IV in DDM - solubilized mitochondria from RNAi ( act5c - gal4 > UAS - CG7630RNAi ) control larvae ( act5c - gal4 > + ) .

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(e) In gel-activity assays for MRC complexes II and IV in DDM-solubilized mitochondria from RNAi (act5c-gal4>UAS-CG7630RNAi) and control larvae (act5c-gal4>+).


( f ) BN - PAGE , Western blot immunodetection of MRC complexes from CG7630 RNAi ( act5c - gal4 > UAS - CG7630RNAi ) control ( act5c - gal4 > + ) larvae using antibodies against specific subunits : anti - UQCRC2 ( III ) , anti - NDUFS3 ( I ) , anti - COX4 ( IV ) anti - SDHA ( II ) .

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(f) BN-PAGE, Western blot immunodetection of MRC complexes from CG7630 RNAi (act5c-gal4>UAS-CG7630RNAi) and control (act5c-gal4>+) larvae using antibodies against specific subunits: anti-UQCRC2 (complex III), anti-NDUFS3 (complex I), anti-COX4 (complex IV) and anti-SDHA (complex II).


( g ) Western blot of steady state ( SDS - PAGE ) of MRC subunits COX4 ( IV ) , SDHA ( II ) UQCR - C2 ( III ) in whole lysates from CG7630 RNAi ( act5c - gal4 > UAS - CG7630RNAi ) control ( act5c - gal4 > + ) larvae .

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

(g) Western blot analysis of steady state levels (SDS-PAGE) of MRC subunits COX4 (complex IV), SDHA (complex II) and UQCR-C2 (complex III) in whole lysates from CG7630 RNAi (act5c-gal4>UAS-CG7630RNAi) and control (act5c-gal4>+) larvae.


( h ) Western blot of anti - HA affinity purified samples from S2R + D . melanogaster . EV = samples from carrying empty expression vector . CG7630 - HA = samples from expressing HA - tagged CG7630 . Tot = total mitochondrial - enriched fractions ; unb = unbound material ; IP : HA = immunoprecipitated material . Samples were probed with antibodies against MRC subunits of complexes I ( NDUFS3 ) , II ( SDHA ) , III ( UQCR - C2 ) , IV ( COX4 ) V ( ATP5A ) .

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

(h) Western blot analysis of anti-HA affinity purified samples from S2R+ D. melanogaster cells. EV = samples from cells carrying the empty expression vector. CG7630-HA = samples from cells expressing HA-tagged CG7630. Tot = total mitochondrial-enriched fractions; unb = unbound material; IP:HA = immunoprecipitated material. Samples were probed with antibodies against MRC subunits of complexes I (NDUFS3), II (SDHA), III (UQCR-C2), IV (COX4) and V (ATP5A).


( a ) Relative ( RQ ) of CG7630 transcript in wild - type D . melanogaster S2R + treated with a dsRNA targeting CG7630 ( CG7630 KD ) a mock control dsRNA ( mock ) .

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

(a) Relative quantification (RQ) of CG7630 transcript in wild-type D. melanogaster S2R+ cells treated with a dsRNA targeting CG7630 (CG7630 KD) and a mock control dsRNA (mock).


( b ) Relative ( RQ ) of CG7630 COX7B transcripts in stable S2R + lines carrying empty expression vector ( EV : GFP ; neo ) , expression vectors carrying HA - tagged forms of human COX7B ( hCOX7B - HA ; neo ) CG7630 ( CG7630 - HA ; neo ) . were treated with a dsRNA targeting CG7630 ( striped bars , CG7630 KD ) a mock control dsRNA ( solid bars , mock ) . N / D = not detected .

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

(b) Relative quantification (RQ) of CG7630 and COX7B transcripts in stable S2R+ cell lines carrying the empty expression vector (EV:GFP;neo), and expression vectors carrying HA-tagged forms of human COX7B (hCOX7B-HA;neo) and CG7630 (CG7630-HA;neo). Cells were treated with a dsRNA targeting CG7630 (striped bars, CG7630 KD) and a mock control dsRNA (solid bars, mock). N/D = not detected.


( c ) Immunoblot of stable S2R + lines carrying empty expression vector ( EV : GFP ; neo ) , expression vectors carrying HA - tagged forms of human COX7B ( hCOX7B - HA ; neo ) CG7630 ( CG7630 - HA ; neo ) . CG7630 transcript was knocked - down ( KD ) in by a dsRNA targeting CG7630 compared with treated with a mock control dsRNA ( mock ) . Samples were probed with an antibody anti HA tag with an antibody against Hsp70 as an endogenous control .

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

(c) Immunoblot analysis of stable S2R+ cell lines carrying the empty expression vector (EV:GFP;neo), and expression vectors carrying HA-tagged forms of human COX7B (hCOX7B-HA;neo) and CG7630 (CG7630-HA;neo). The CG7630 transcript was knocked-down (KD) in cells by a dsRNA targeting CG7630 and compared with cells treated with a mock control dsRNA (mock). Samples were probed with an antibody anti HA tag and with an antibody against Hsp70 as an endogenous control.