HAND2 - AS1 deletion in HCC decreases BMPR1A expression inactivates BMP signaling .

HAND2-AS1 deletion in HCC cells decreases BMPR1A expression and inactivates BMP signaling.


ChIRP - immunoblotting of BMPR1A interaction with INO80 in control HAND2 - AS1 - knockout liver CSCs .

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ChIRP-immunoblotting analysis of BMPR1A interaction with the INO80 complex in control and HAND2-AS1-knockout liver CSCs.


EMSA analysis of the interaction of HAND2-AS1, INO80 and RUVBL2 with the BMPR1A promoter region.


BMPR1A promoter binding region to HAND2 - AS1 / INO80 was deleted ( BMPR1A - p - KO ) in liver CSCs using lenti - Cas9 , followed by HAND2 - AS1 INO80 overexpression ( oeHAND2 - AS1 , oeINO80 ) for 36 h . BMPR1A mRNA were examined by realtime PCR . Data are shown as means ± SD . n = 5 . * * P < 0 . 01 by two - Student ' s t .

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BMPR1A promoter binding region to HAND2-AS1/INO80 was deleted (BMPR1A-p-KO) in liver CSCs using lenti-Cas9, followed by HAND2-AS1 and INO80 overexpression (oeHAND2-AS1, oeINO80) for 36 h. BMPR1A mRNA levels were examined by realtime PCR. Data are shown as means ± SD. n=5. ** P < 0.01 by two-tailed Student's t test.


BMP signaling was examined in INO80 - silenced liver CSCs by Western blot .

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BMP signaling was examined in INO80-silenced liver CSCs by Western blot.


BMPR1A is highly expressed in HCC tissues provided by Wang ' s cohort ( GSE14520 ) .

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BMPR1A is highly expressed in HCC tumor tissues provided by Wang's cohort (GSE14520).


Kaplan - Meier analyses of liver cancer outcomes in cancer genome atlas ( TCGA ) . P value for Kaplan - Meier curves was determined using a Mantel - Cox log - rank .

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Kaplan-Meier analyses of liver cancer outcomes in the cancer genome atlas (TCGA). P value for Kaplan-Meier curves was determined using a Mantel-Cox log-rank test.


Representative immunohistochemistry of BMPR1A was detected in HCC samples . Scale bar , 100 μm .

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Representative immunohistochemistry staining of BMPR1A was detected in HCC samples. Scale bar, 100 μm.


BMPR1A overexpression rescues sphere formation ability reduced by HAND2 - AS1 depletion in HCC samples . Representative sphere formation is shown on left panel . Scale bar , 100 μm . Percentages of sphere - forming were calculated as means ± SD ( right panel ) . n = 4 .

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BMPR1A overexpression rescues sphere formation ability reduced by HAND2-AS1 depletion in HCC samples. Representative sphere formation is shown on the left panel. Scale bar, 100 μm. Percentages of sphere-forming cells were calculated as means ± SD (right panel). n=4.


BMPR1A depletion reduces - initiating capacity . n = 12 mice per group .

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BMPR1A depletion reduces tumor-initiating capacity. n=12 mice per group.


Representative whole - imaging of Huh7 - Luc transduced with scramble ( Ctrl ) or BMPR1A shRNAs . for numbers of 7 after orthotopically implanted to B - NSG mice . Calculated data are shown as means ± SD ( n = 5 mice per group ) .

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Representative whole-body imaging of Huh7-Luc cells transduced with scramble (Ctrl) or BMPR1A shRNAs. Quantification for tumor numbers of 7 weeks after tumor cells orthotopically implanted to B-NSG mice. Calculated data are shown as means ± SD (n = 5 mice per group).


Macroscopic appearance of livers from 10 - month - old mice treated by DEN or vehicle . Scale bar , 1 cm . Black arrows indicates tumors .

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Macroscopic appearance of livers from 10-month-old mice treated by DEN or vehicle. Scale bar, 1 cm. Black arrows indicates tumors.


Representative H & E immunohistological with Ki67 antibody in liver sections of 10 - month - old DEN - treated WT Bmpr1a - / - mice . Scale bar , 100 μm . Right : Quantitation of Ki67 - positive in DEN - treated mice . Data are shown as means ± SD . n = 5 .

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Representative H&E and immunohistological staining with Ki67 antibody in liver sections of 10-month-old DEN-treated WT and Bmpr1a-/- mice. Scale bar, 100 μm. Right: Quantitation of Ki67-positive cells in DEN-treated mice. Data are shown as means ± SD. n=5.


BMP signaling inhibitor noggin decreased sphere formation . Scale bar , 100 μm . Right : Data are shown as means ± SD ( n = 4 ) .

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BMP signaling inhibitor noggin decreased tumor sphere formation. Scale bar, 100 μm. Right: Data are shown as means ± SD (n=4).


Representative whole - imaging of Huh7 - Luc in different groups . Mice treated with scramble ASOs / or siRNAs obtained similar results to vehicle treatment . n ≥ 5 mice in each group .

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Representative whole-body imaging of Huh7-Luc cells in different groups. Mice treated with scramble ASOs and/or siRNAs obtained similar results to vehicle treatment. n≥5 mice in each group.


of numbers treated with ASOs / or siRNAs after Huh7 - Luc orthotopically implanted to B - NSG mice . Calculated data are shown as means ± SD ( n = 5 ) .

Quantification of tumor numbers treated with ASOs and/or siRNAs after Huh7-Luc tumor cells orthotopically implanted to B-NSG mice. Calculated data are shown as means ± SD (n = 5).


Tumors were excised from livers of PDX liver cancer models . Scale bar , 0 . 5 cm .

Tumors were excised from livers of PDX liver cancer models. Scale bar, 0.5 cm.


Representative H & E in liver sections from - bearing mice treated with ASO1 siBMPR1A in orthotopic transplants of tumors from HCC # 50 HCC # 51 . Scale bar , 100 μm .

Representative H&E staining in liver sections from tumor-bearing mice treated with ASO1 and siBMPR1A in orthotopic transplants of tumors from HCC#50 and HCC#51. Scale bar, 100 μm.


Representative immunohistological with Ki67 antibody of - bearing mice treated with ASO1 siBMPR1A . Scale bar , 100 μm . Lower panel : Quantitation of Ki67 - positive in different groups . Data are shown as means ± SD ( n = 5 per group ) .

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Representative immunohistological staining with Ki67 antibody of tumor-bearing mice treated with ASO1 and siBMPR1A. Scale bar, 100 μm. Lower panel: Quantitation of Ki67-positive cells in different groups. Data are shown as means ± SD (n=5 per group).


Northern blotting of HAND2 - AS1 immunobloting of BMPR1A in tumors from PDX HCC # 50 after treatment .

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Northern blotting of HAND2-AS1 and immunobloting of BMPR1A in tumors from PDX HCC#50 after treatment.


Kaplan - Meier survival of B - NSG mice orthotopically transplanted with PDXs HCC # 50 ( I ) HCC # 51 ( J ) . Treatment with ASO1 combined siBMPR1A causes synergistic therapeutic effect than each treatment alone ( n = 10 per group ) . P value for Kaplan - Meier curves was determined using a Mantel - Cox log - rank .

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Kaplan-Meier survival analysis of B-NSG mice orthotopically transplanted with PDXs HCC#50 (I) and HCC#51 (J). Treatment with ASO1 combined siBMPR1A causes synergistic therapeutic effect than each treatment alone (n = 10 per group). P value for Kaplan-Meier curves was determined using a Mantel-Cox log-rank test.


Time lapse of T6SS activity in tagged parental ( VipA - mCherry2 ) ∆ tssA mutant background . parental forms multiple dynamic structures per . ∆ tssA mutant predominantly forms dynamic sheath spots ( arrow ) few WT - like sheath structures ( blue arrow ) . Scale bars : 2 µm .

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Time lapse images of T6SS activity in tagged parental strain (VipA-mCherry2) and ∆tssA mutant background. The parental strain forms multiple dynamic structures per cell. ∆tssA mutant predominantly forms dynamic sheath spots (white arrow) and few WT-like sheath structures (blue arrow). Scale bars: 2 µm.


H2 - T6SS dynamics in tagged parental ( ∆ retS TssB2 - mCherry2 , referred to as WT H2 ) ΔtssA2PA . Blue arrow indicates extending contracting T6SS sheath structures , arrow points to dynamic sheath spots . WT H2 harbors multiple dynamic sheath structures per , while ΔtssA2PA displays very few dynamic spots or extended contracting structures . Scale bars : 2 µm .

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H2-T6SS dynamics in tagged parental strain (∆retS TssB2-mCherry2, referred to as WT H2) and ΔtssA2PA strain. Blue arrow indicates extending and contracting T6SS sheath structures, white arrow points to dynamic sheath spots. The WT H2 strain harbors multiple dynamic sheath structures per cell, while the ΔtssA2PA strain displays very few dynamic spots or extended and contracting structures. Scale bars: 2 µm.


Time lapse of T6SS activity in tagged ( VipA - mCherry2 TssAVC - mNeonGreen ) . arrow indicates path of TssAVC localization during one cycle of T6SS dynamics . Scale bar : 1 µm .

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Time lapse images of T6SS activity in tagged strain (VipA-mCherry2 TssAVC-mNeonGreen). White arrow indicates path of TssAVC localization during one cycle of T6SS dynamics. Scale bar: 1 µm.


Kymographs of dynamics observed for TssAVC . Frequency of specific dynamics is indicated in upper right corner of merge image . ( B ) Sheath TssAVC dynamics from A . TssAVC localizes to T6SS initiation site to distal end of growing sheath ( arrow ) . ( C ) Once sheath reaches periphery , TssAVC dissociates from distal end ( arrow ) sheath stays extended a prolonged period of time . ( D ) TssAVC dissociates from growing sheath structure re - associates at a later time point ( arrows ) to continue catalysis of polymerization . ( E ) TssAVC stays attached to sheath distal end after contraction ( arrow ) .

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Kymographs of dynamics observed for TssAVC. Frequency of specific dynamics is indicated in the upper right corner of the merge image. (B) Sheath and TssAVC dynamics from A. TssAVC localizes to T6SS initiation site and to distal end of growing sheath (white arrow). (C) Once sheath reaches cell periphery, TssAVC dissociates from distal end (white arrow) and sheath stays extended a prolonged period of time. (D) TssAVC dissociates from growing sheath structure and re-associates at a later time point (white arrows) to continue catalysis of polymerization. (E) TssAVC stays attached to sheath distal end after contraction (white arrow).


microscopy of T6SS dynamics in a double tagged ( ∆ retS TssB2 - mCherry2 TssA2PA - mNeonGreen ) . arrow indicates full cycle of extending contracting T6SS sheath . Scale bar : 1 µm .

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Fluorescence microscopy of T6SS dynamics in a double tagged strain (∆retS TssB2-mCherry2 TssA2PA-mNeonGreen). White arrow indicates full cycle of extending and contracting T6SS sheath. Scale bar: 1 µm.


Kymographs of TssB2 TssA2PA dynamics . TssA2PA localizes at sheath initiation site on distal end during polymerization ( G ) , dissociates before contraction ( H ) , dissociates re - associated during sheath extension ( I ) or stays attached to distal end after contraction ( J ) .

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Kymographs of TssB2 and TssA2PA dynamics. TssA2PA localizes at sheath initiation site and on distal end during polymerization (G), dissociates before contraction (H), dissociates and re-associated during sheath extension (I) or stays attached to distal end after contraction (J).


Top view ( XY - plane ) side view ( XZ - plane ) of TssAVC Nt2 domain cryo - EM reconstruction ( pink , non transparent ) shown with fitted Nt2 - dimer model ( left ) . Partial Nt2 - CTD Nt1 - Nt2 linker densities are highlighted with black arrows . Top side views of ribbon diagram of Nt2 - dimer ( middle ) . Representative 2D class averages of TssAVC particles with visible connections between Nt2 - dimer CTD ( top right ) , representative 2D class averages of Nt2 - dimer particles , Nt2 - CTD linker Nt1 - Nt2 linker densities are highlighted with blue yellow arrows ( bottom right ) .

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Top view (XY-plane) and side view (XZ-plane) of the TssAVC Nt2 domain cryo-EM reconstruction (pink, non transparent) shown with fitted Nt2-dimer model (left). Partial Nt2-CTD and Nt1-Nt2 linker densities are highlighted with black arrows. Top and side views of ribbon diagram of Nt2-dimer (middle). Representative 2D class averages of TssAVC particles with visible connections between Nt2-dimer and CTD (top right), representative 2D class averages of Nt2-dimer particles, Nt2-CTD linker and Nt1-Nt2 linker densities are highlighted with blue and yellow arrows (bottom right).


∆ tagA mutant displays long bent but still dynamic structures . Scale bar : 2 µm . Frames from time lapse movies showing sheath dynamics in ∆ tagA mutant . Sheath polymerization does not stop when distal end reaches opposing ( arrow ) . Continued polymerization causes sheath to tilt distal end to move towards pole , ultimately effecting bending of whole structure . C . Sheath detaches from its anchor ( orange arrow ) , keeps extending to maximum then contracts . Scale bars : 1 µm .

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The ∆tagA mutant displays long and bent but still dynamic structures. Scale bar: 2 µm. Frames from time lapse movies showing sheath dynamics in the ∆tagA mutant. Sheath polymerization does not stop when the distal end reaches opposing membrane (white arrow). Continued polymerization causes the sheath to tilt and distal end to move towards the bacterial cell pole, ultimately effecting bending of the whole structure. C. Sheath detaches from its membrane anchor (orange arrow), keeps extending to maximum cell length and then contracts. Scale bars: 1 µm.


Time lapse series of T6SS activity in tagged ( VipA - mCherry2 TagAVC - mNeonGreen ) . Orange arrow indicates sheath distal end , arrow indicates initiation site of another T6SS sheath . Scale bar : 1 µm .

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Time lapse series of T6SS activity in tagged strain (VipA-mCherry2 TagAVC-mNeonGreen). Orange arrow indicates sheath distal end, white arrow indicates initiation site of another T6SS sheath. Scale bar: 1 µm.


Kymographs of dynamics observed for TagAVC . E . TagAVC localizes at distal end when sheath reaches periphery . Signal of TagAVC - mNeonGreen disappeared reappeared on this site repeatedly . F . TagAVC localizes at distal end when sheath reaches periphery stays associated to this site until contraction . G . TagAVC briefly localizes at T6SS initiation site . H . TagAVC briefly localizes at T6SS initiation site at distal end , where it stays attached after contraction .

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Kymographs of dynamics observed for TagAVC. E. TagAVC localizes at the distal end when sheath reaches the cell periphery. Signal of TagAVC-mNeonGreen disappeared and reappeared on this site repeatedly. F. TagAVC localizes at the distal end when sheath reaches the cell periphery and stays associated to this site until contraction. G. TagAVC briefly localizes at T6SS initiation site. H. TagAVC briefly localizes at T6SS initiation site and at distal end, where it stays attached after contraction.


H1 - T6SS dynamics in tagged parental ( ∆ retS TssB1 - mCherry2 , referred to as WT H1 ) ΔtssA1PA . Blue arrow indicates extending contracting T6SS sheath structures , arrow points to dynamic sheath spots . Scale bars : 2 µm .

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H1-T6SS dynamics in tagged parental strain (∆retS TssB1-mCherry2, referred to as WT H1) and ΔtssA1PA strain. Blue arrow indicates extending and contracting T6SS sheath structures, white arrow points to dynamic sheath spots. Scale bars: 2 µm.


microscopy of T6SS dynamics in ∆ retS TssB1 - mCherry2 harboring pPSV35 plasmid with TssA1PA - mNeonGreen fusion . Scale bar : 2 µm .

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Fluorescence microscopy of T6SS dynamics in ∆retS TssB1-mCherry2 strain harboring pPSV35 plasmid with TssA1PA-mNeonGreen fusion. Scale bar: 2 µm.


Kymographs of TssB1 TssA1PA dynamics . TssA1PA localizes exclusively at sheath initiation site ( B - E ) , disappears after contraction ( C , D ) or remains in place for longer timeframe ( C , E ) . TssA1PA signal appears in neighboring shortly after T6SS attack ( B , C ) .

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Kymographs of TssB1 and TssA1PA dynamics. TssA1PA localizes exclusively at sheath initiation site (B-E), disappears after contraction (C,D) or remains in place for longer timeframe (C,E). TssA1PA signal appears in neighboring cell shortly after T6SS attack (B,C).


( D - E ) Western blot of RIPA soluble - fraction RIPA - insoluble , urea - soluble fraction of prepared from HEK293T expressing empty plasmid ( control ) , FLAG - KIF5A WT , FLAG - ΔExon27 . Insoluble / soluble KIF5A fractions were detected with anti - FLAG actin was used as a loading control , three biological replicates . Statistical was performed using student t - ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ) . Data represent mean ± s . d .

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(D-E) Western blot analysis of RIPA soluble-fraction and RIPA-insoluble, urea-soluble fraction of proteins prepared from HEK293T cells expressing empty plasmid (control), FLAG-KIF5A WT, FLAG-ΔExon27. Insoluble/soluble KIF5A fractions were detected with anti-FLAG and actin was used as a loading control, three biological replicates. Statistical analysis was performed using student t-test (*p < 0.05, **p<0.01, ***p<0.001). Data represent the mean ± s.d.


( B ) Pull down showing association of ΔExon27 with WT KIF5A , two biological replicates .

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(B) Pull down analysis showing the association of ΔExon27 with WT KIF5A, two biological replicates.


( A ) Sanger sequencing chromatograms of cDNA from control - derived iMNs ( P1 - P3 ) showing effect of mutations on KIF5A transcripts . While c . 3020 + 2T > C mutation causes exon 27 skipping , c . 2993 - 1G > A mutation results in an aberrant transcript with a splice acceptor shifted only by a single nucleotide , leading to a frameshift but containing exon 27 . respective product predicted for both mutations comprise same C - terminal aberrant 39 acid end .

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(A) Sanger sequencing chromatograms of cDNA from control and patient-derived iMNs (P1-P3) showing the effect of mutations on KIF5A transcripts. While the c.3020+2T>C mutation causes exon 27 skipping, the c.2993-1G>A mutation results in an aberrant transcript with a splice acceptor shifted only by a single nucleotide, leading to a frameshift but containing exon 27. The respective protein product predicted for both mutations comprise the same C-terminal aberrant 39 amino acid end.


( B ) Western blot for validation of custom mutant - specific antibody using transfected HEK293T . While an antibody against total KIF5A ( lower panel ) recognized both wild type mutant , custom antibody targeting aberrant " neopeptide " detected ΔExon27 specifically ( upper panel ) . NT : No transfection .

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(B) Western blot analysis for validation of the custom mutant-specific antibody using transfected HEK293T cells. While an antibody against the total KIF5A (lower panel) recognized both wild type and mutant protein, the custom antibody targeting the aberrant "neopeptide" detected ΔExon27 specifically (upper panel). NT: No transfection.


( C ) Mutant KIF5A are endogenously expressed in iMNs , detected by using custom mutant specific antibody directed against C - terminal aberrant 39 acid . CTL : Control , P1 : 1 , P2 : 2 , P3 : 3 , two independent experiments .

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(C) Mutant KIF5A proteins are endogenously expressed in iMNs, detected by using the custom mutant specific antibody directed against the C-terminal aberrant 39 amino acid sequence. CTL: Control, P1: Patient 1, P2: Patient 2, P3: Patient 3, two independent experiments.


( D ) Representative of KIF5A distribution ( arrowheads ) in control iMNs . Scale bar : 20 µm . ( E ) Abundance of KIF5A inclusions after normalizing to total neuron numbers . Graph represents data from n = 146 control 1 ( CTL1 ) , n = 134 control 2 ( CTL2 ) , n = 154 1 ( P1 ) , n = 162 2 ( P2 ) n = 133 3 ( P3 ) iMNs from 3 experiments . Bars indicate mean ± s . d . Statistical was performed using Mann Whitney Wilcoxon ( * p < 0 . 05 ) .

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(D) Representative images of KIF5A distribution (arrowheads) in control and patient iMNs. Scale bar: 20 µm. (E) Abundance of KIF5A inclusions after normalizing to total neuron numbers. Graph represents data from n=146 control 1 (CTL1), n = 134 control 2 (CTL2), n = 154 patient 1 (P1), n = 162 patient 2 (P2) and n = 133 patient 3 (P3) iMNs from 3 individual experiments. Bars indicate mean ± s.d. Statistical analysis was performed using Mann Whitney Wilcoxon Test (*p<0.05).


B , ITC experiment shows CBS - pair domain of CNNM3 binds PRL2 with a nanomolar Kd one - to - one stoichiometry .

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B, ITC experiment shows the CBS-pair domain of CNNM3 binds PRL2 with a nanomolar Kd and one-to-one stoichiometry.


D , Structure of CBS - pair domain of CNNM2 ( PDB 4IY0 ) with residues DPFYE indicated .

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D, Structure of CBS-pair domain of CNNM2 (PDB 4IY0) with the residues DPFYE indicated.


F , ITC data of CBS - pair domain mutants binding to PRL2 identifies aspartic acid 426 as essential for binding .

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F, ITC data of CBS-pair domain mutants binding to PRL2 identifies aspartic acid 426 as essential for binding.


G , Kinetics of hydrolysis of a synthetic substrate by PRL2 shows burst kinetics . Addition of wild - type CBS - pair domain but not D426A mutant inhibits phosphatase activity .

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G, Kinetics of hydrolysis of a synthetic substrate by PRL2 shows burst kinetics. Addition of the wild-type CBS-pair domain but not the D426A mutant inhibits phosphatase activity.


A , Treatment of PRL2 with a synthetic substrate leads to complete phosphorylation of catalytic residue , cysteine 101 . Recombinant PRL2 was treated with 3 - O - methylfluorescein phosphate ( OMFP ) analyzed by SDS - PAGE with Phos - tag reagent . Phosphocysteine is hydrolyzed by boiling .

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A, Treatment of PRL2 with a synthetic substrate leads to complete phosphorylation of the catalytic residue, cysteine 101. Recombinant PRL2 was treated with 3-O-methylfluorescein phosphate (OMFP) and analyzed by SDS-PAGE with the Phos-tag reagent. Phosphocysteine is hydrolyzed by boiling.


B , Detection of phosphorylated by electrospray mass spectrometry . OMFP - treatment increased mass of intact by 79 . 99 amu but had no effect on C101A mutant .

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B, Detection of phosphorylated by electrospray mass spectrometry. OMFP-treatment increased the mass of the intact protein by 79.99 amu but had no effect on the C101A mutant.


C , 31P NMR spectrum of thioPO3 signal from phosphocysteine in PRL2 . broad peak is TCEP in buffer .

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C, 31P NMR spectrum of the thioPO3 signal from phosphocysteine in PRL2. The broad peak is TCEP in the buffer.


D , Cysteine phosphorylated PRL2 does not bind to CNNM3 . Recombinant PRL2 was phosphorylated by treatment with OMFP subjected to pull - down by CBS - pair domain of CNNM3 as a GST fusion . Only unphosphorylated PRL2 was observed in pull - down lanes while phosphorylated PRL2 was in flow - through lanes .

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D, Cysteine phosphorylated PRL2 does not bind to CNNM3. Recombinant PRL2 was phosphorylated by treatment with OMFP and subjected to pull-down by the CBS-pair domain of CNNM3 as a GST fusion. Only unphosphorylated PRL2 was observed in the pull-down lanes while phosphorylated PRL2 was in the flow-through lanes.


E , ITC measurement of affinity of PRL2 for CNNM3 GST - CBS - pair domain . Phosphorylation by OMFP completely blocked binding while oxidization of PRL2 by diamide decreased affinity 200 - fold .

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E, ITC measurement of the affinity of PRL2 for the CNNM3 GST-CBS-pair domain. Phosphorylation by OMFP completely blocked binding while oxidization of PRL2 by diamide decreased the affinity 200-fold.


F , cysteine residues in PRL2 active site are not required for binding CNNM3 CBS - pair domain .

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F, The cysteine residues in the PRL2 active site are not required for binding the CNNM3 CBS-pair domain.


A , Mutations in CBS - pair loop of CNNM4 prevent binding to PRL3 . Lysates of COS7 transfected with indicated constructs were subjected to immunoprecipitation ( IP ) immunoblotting ( IB ) with indicated antibodies .

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A, Mutations in the CBS-pair loop of CNNM4 prevent binding to PRL3. Lysates of COS7 cells transfected with the indicated constructs were subjected to immunoprecipitation (IP) and immunoblotting (IB) with the indicated antibodies.


B C , Wild - type CNNM4 - dependent magnesium efflux is inhibited by co - transfection by PRL3 but efflux by CNNM4 D485A mutant , which is unable to bind PRL3 , is resistant to inhibition . HEK293 transfected with indicated constructs were loaded with Magnesium Green Mg2 + removed from medium at indicated time point ( arrowhead ) . means of relative intensities of 10 are graphed as a function of time in panel B mean s . e . m . at 5 min plotted in panel C . P - values were calculated using one way ANOVA followed by Bonferroni ' s multiple comparison . n . s . , not significant .

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B C, Wild-type CNNM4-dependent magnesium efflux is inhibited by co-transfection by PRL3 but the efflux by the CNNM4 D485A mutant, which is unable to bind PRL3, is resistant to inhibition. HEK293 cells transfected with the indicated constructs were loaded with Magnesium Green and Mg2+ removed from the medium at the indicated time point (arrowhead). The means of relative fluorescence intensities of 10 cells are graphed as a function of time in panel B and the mean and s.e.m. at 5 min plotted in panel C. P-values were calculated using one way ANOVA followed by Bonferroni's multiple comparison test. n.s., not significant.


A , CBS - pair domain ( yellow ) forms a dimer binds two molecules of PRL2 via an extended loop . positions of PRL2 C - terminal CAAX prenylation motif [ 42 ] CNNM3 N - terminus suggest that would sit above .

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A, The CBS-pair domain (yellow) forms a dimer and binds two molecules of PRL2 via an extended loop. The positions of the PRL2 C-terminal CAAX prenylation motif [42] and CNNM3 N-terminus suggest that the membrane would sit above the complex.


B , Contacts between CNNM3 CBS2 loop PRL2 active site .

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B, Contacts between the CNNM3 CBS2 loop and the PRL2 active site.


C , Stereoview of 2FO - FC omit map of PRL2 binding loop of CNNM3 contoured at 1 .

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C, Stereoview of the 2FO-FC omit map of the PRL2 binding loop of CNNM3 contoured at 1 .


D , Detail of with reduced PRL2 , showing how CNNM3 Asp426 inserts into pocket adjacent to catalytic cysteine Cys101 .

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D, Detail of complex with reduced PRL2, showing how CNNM3 Asp426 inserts into the pocket adjacent to the catalytic cysteine Cys101.


E , Model of phosphocysteine preventing CNNM binding . Crystal structure of PRL1 with a bound sulfate shows phosphocysteine would prevent Asp426 binding . Residues are numbered according to PRL1 structure ( PDB 1XM2 ) .

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E, Model of phosphocysteine preventing CNNM binding. Crystal structure of PRL1 with a bound sulfate shows phosphocysteine would prevent Asp426 binding. Residues are numbered according to the PRL1 structure (PDB 1XM2).


A , PRLs expressed in HEK293 are intrinsically phosphorylated on cysteine . Phos - tag of lysates shows close to 50 % of transiently expressed FLAG - PRL1 , - 2 , - 3 are phosphorylated . phosphorylated PRL bands are marked by an asterisk .

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A, PRLs expressed in HEK293 cells are intrinsically phosphorylated on cysteine. Phos-tag analysis of cell lysates shows close to 50% of transiently expressed FLAG-PRL1, -2, and -3 are phosphorylated. The phosphorylated PRL bands are marked by an asterisk.


B , Immunoblots of PRL1 expressed in HEK293 show phosphorylation requires catalytic cysteine ( C104 ) but not adjacent cysteine ( C49 ) in active site .

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B, Immunoblots of PRL1 expressed in HEK293 cells show phosphorylation requires the catalytic cysteine (C104) but not the adjacent cysteine (C49) in the active site.


C , Co - immunoprecipitation of Myc - PRL by FLAG - CNNMs shows only unphosphorylated forms of PRL1 binds CNNM4 .

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C, Co-immunoprecipitation of Myc-PRL by FLAG-CNNMs shows only the unphosphorylated forms of PRL1 binds CNNM4.


D , Endogenous PRLs in HeLa are cysteine phosphorylated .

D, Endogenous PRLs in HeLa cells are cysteine phosphorylated.


E , of unphosphorylated PRL increase in cultured in response to removal of magnesium from cultured medium .

E, Levels of unphosphorylated PRL increase in cultured cells in response to removal of magnesium from the cultured medium.


A ) In our chemoattraction - setup , nematode chemoattraction behavior is quantified using a measurable chemoattraction index ( C . I . ) ; higher C . I . measured in , stronger attraction of to sex pheromone . Data information : We assayed 400 from each by using sex pheromone chemoattraction . Two biological replicates were combined into a single value .

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A) In our chemoattraction-assay setup, nematode chemoattraction behavior is quantified using a measurable chemoattraction index (C.I.); the higher the C.I. measured in the assay, the stronger the attraction of the males to the sex pheromone. Data information: We assayed 400 males from each strain by using the sex pheromone chemoattraction assay. Two biological replicates were combined into a single value.


B ) After vaporization ( VAC ) at 4 ° C for 24 h , C . remanei extract failed to attract wild - type C . elegans . Conversely , a drop of sex pheromone extract placed on lid of a 60 - mm Petri dish evoked chemoattractive behavior in wild - type C . elegans ( volatility ) . Thus , functional components in C . remanei sex pheromone are volatile substances . Data information : We assayed 400 from each by using sex pheromone chemoattraction . Two biological replicates were combined into a single value . Significance was determined using one - way ANOVA with Bonferroni correction : * * * P < 0 . 001 . Means ± ( error bars ) are shown .

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B) After vaporization (VAC) at 4°C for 24 h, the C. remanei female extract failed to attract wild-type C. elegans males. Conversely, a drop of the sex pheromone extract placed on the lid of a 60-mm Petri dish evoked chemoattractive behavior in wild-type C. elegans males (volatility test). Thus, the functional components in C. remanei female sex pheromone are volatile substances. Data information: We assayed 400 males from each strain by using the sex pheromone chemoattraction assay. Two biological replicates were combined into a single value. Significance was determined using one-way ANOVA with Bonferroni correction: ***P<0.001. Means ± SEM (error bars) are shown.


C ) Mutant with established defects in amphid neurons responded to C . remanei sex pheromone to a lesser degree relative to wild - type , demonstrating that some or of amphid neurons are required for sex pheromone perception . Data information : We assayed 400 from each by using sex pheromone chemoattraction . Two biological replicates were combined into a single value . Significance was determined using one - way ANOVA with Bonferroni correction : * * * P < 0 . 001 . Means ± ( error bars ) are shown .

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C) Mutant males with established defects in amphid neurons responded to C. remanei sex pheromone to a lesser degree relative to wild-type, demonstrating that some or all of the amphid neurons are required for sex pheromone perception. Data information: We assayed 400 males from each strain by using the sex pheromone chemoattraction assay. Two biological replicates were combined into a single value. Significance was determined using one-way ANOVA with Bonferroni correction: ***P<0.001. Means ± SEM (error bars) are shown.


A ) Inspection of specific amphid - neuron - defective mutants revealed that AWA neurons are most likely responsible for pheromone perception . Here , an edge connects neurons to a genetic mutant if neurons are documented to be affected in mutant . color code of edges represents average C . I . of multiple genetic mutants with impaired neuronal functions or specified abnormal neuron identities . region colored green shows range of wild - type sex pheromone response in . We assayed 400 from each in sex pheromone chemoattraction . Two biological replicates were combined into a single value . Significance was determined using one - way ANOVA with Bonferroni correction : * * * P < 0 . 001 . Means ± ( error bars ) are shown .

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A) Inspection of specific amphid-neuron-defective mutants revealed that AWA neurons are most likely responsible for pheromone perception. Here, an edge connects neurons to a genetic mutant if the neurons are documented to be affected in the mutant. The color code of edges represents the average C.I. of multiple genetic mutants with impaired neuronal functions or specified abnormal neuron identities. The region colored green shows the range of wild-type sex pheromone response in the male. We assayed 400 males from each strain in the sex pheromone chemoattraction assay. Two biological replicates were combined into a single value. Significance was determined using one-way ANOVA with Bonferroni correction: ***P<0.001. Means ± SEM (error bars) are shown.


B ) AWA - - ablated were not attracted by sex pheromone in single - worm chemoattraction assays . Both AWA - defective mutant ( lin - 11 odr - 7 ) AWA - - ablated failed to reach sex pheromone within 30 min . Wild - type C . elegans responded normally to sex pheromone wild - type C . elegans hermaphrodites were not attracted by same - sex pheromone . single - worm chemoattraction was performed as described in Materials Methods . Sample of in AWA - - ablation experiment : 12 ; other experiments : 100 worms .

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B) AWA-laser-ablated males were not attracted by the sex pheromone in single-worm chemoattraction assays. Both AWA-defective mutant males (lin-11 and odr-7) and AWA-laser-ablated males failed to reach the sex pheromone within 30 min. Wild-type C. elegans males responded normally to the sex pheromone and wild-type C. elegans hermaphrodites were not attracted by same-sex pheromone. The single-worm chemoattraction assay was performed as described in Materials and Methods. Sample size of males in AWA-laser-ablation experiment: 12; all other experiments: 100 worms.


B ) Mutant of 4 GPCR signaling cascade components failed to respond to C . remanei sex pheromone , suggesting that sex pheromone receptor is a GPCR that both aforementioned signaling pathways are involved in pheromone perception . We assayed 400 from each in sex pheromone chemoattraction . Two biological replicates were combined into a single value . Significance was determined using one - way ANOVA with Bonferroni correction : * * * P < 0 . 001 . Means ± ( error bars ) are shown .

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B) Mutant males of 4 GPCR signaling cascade components failed to respond to C. remanei sex pheromone, suggesting that the sex pheromone receptor is a GPCR and that both aforementioned signaling pathways are involved in pheromone perception. We assayed 400 males from each strain in the sex pheromone chemoattraction assay. Two biological replicates were combined into a single value. Significance was determined using one-way ANOVA with Bonferroni correction: ***P<0.001. Means ± SEM (error bars) are shown.


C ) In from both AWA - specific - rescue odr - 3 mutant odr - 3 rescued mutant , sex pheromone response was significantly restored in single worm arrival assays . However , from CEM - specific - rescue odr - 3 mutant failed to reach sex pheromone within 30 min . In odr - 3 him - 5 experiments , sample sizes were 12 20 worms , respectively ; in other experiments , 40 worms were used .

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C) In males from both AWA-specific-rescue odr-3 mutant strain and odr-3 rescued mutant strain, the sex pheromone response was significantly restored in single male worm arrival assays. However, males from the CEM-specific-rescue odr-3 mutant strain failed to reach the sex pheromone within 30 min. In odr-3 and him-5 strain experiments, the sample sizes were 12 and 20 worms, respectively; in all other experiments, 40 worms were used.


A ) srd - 1 mutant were not attracted to sex pheromone but were attracted to positive - control chemoattractants , 1 : 1000 diacetyl 10 mg / mL pyrazine . This demonstrated that SRD - 1 is required specific for sex pheromone perception . Data information : For each transgenic , 3 independent lines were examined . We assayed 400 from each in sex pheromone chemoattraction . Two biological replicates were combined into a single value . Significance was determined using one - way ANOVA with Bonferroni correction : * * * P < 0 . 001 . Means ± ( error bars ) are shown .

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A) srd-1 mutant males were not attracted to the sex pheromone but were attracted to the positive-control chemoattractants, 1:1000 diacetyl and 10 mg/mL pyrazine. This demonstrated that SRD-1 is required and specific for sex pheromone perception. Data information: For each transgenic strain, 3 independent lines were examined. We assayed 400 males from each strain in the sex pheromone chemoattraction assay. Two biological replicates were combined into a single value. Significance was determined using one-way ANOVA with Bonferroni correction: ***P<0.001. Means ± SEM (error bars) are shown.


B ) Summary of srd - 1 expression pattern . transcriptional reporter Psrd - 1 : : gfp was detected in both hermaphrodite ASI neurons in head region , only in AWA ADF neurons .

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B) Summary of srd-1 expression pattern. The transcriptional reporter Psrd-1::gfp was detected in both male and hermaphrodite ASI neurons in the head region, and only in male AWA and ADF neurons.


C ) SRD - 1 : : GFP fusion construct revealed predominant SRD - 1 localization at cilia of AWAs in C . elegans . Scale bar = 5 μm .

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C) SRD-1::GFP fusion construct revealed predominant SRD-1 localization at the cilia of AWAs in C. elegans males. Scale bar = 5 μm.


D ) Examination of srd - 1 transcriptional reporter revealed that srd - 1 is expressed in AWA neurons in but not hermaphrodites . A transcriptional reporter for odr - 7 indicated locations of AWAs ( arrowheads ) . Scale bar = 50 μm .

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D) Examination of srd-1 transcriptional reporter revealed that srd-1 is expressed in AWA neurons in males but not hermaphrodites. A transcriptional reporter for odr-7 indicated the locations of AWAs (arrowheads). Scale bar = 50 μm.


F ) AWA - specific rescue of srd - 1 restored sex pheromone perception ability in both hermaphrodites . Data information : For each transgenic , 3 independent lines were examined . We assayed 400 from each in sex pheromone chemoattraction . Two biological replicates were combined into a single value . Significance was determined using one - way ANOVA with Bonferroni correction : * * * P < 0 . 001 . Means ± ( error bars ) are shown .

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F) AWA-specific rescue of srd-1 restored sex pheromone perception ability in both males and hermaphrodites. Data information: For each transgenic strain, 3 independent lines were examined. We assayed 400 males from each strain in the sex pheromone chemoattraction assay. Two biological replicates were combined into a single value. Significance was determined using one-way ANOVA with Bonferroni correction: ***P<0.001. Means ± SEM (error bars) are shown.


G ) Ectopic expression of srd - 1 in AWB neurons elicited a distinct repulsive behavior toward sex pheromone in both hermaphrodites . Data information : For each transgenic , 3 independent lines were examined . We assayed 400 from each in sex pheromone chemoattraction . Two biological replicates were combined into a single value . Significance was determined using one - way ANOVA with Bonferroni correction : * * * P < 0 . 001 . Means ± ( error bars ) are shown .

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G) Ectopic expression of srd-1 in AWB neurons elicited a distinct repulsive behavior toward the sex pheromone in both males and hermaphrodites. Data information: For each transgenic strain, 3 independent lines were examined. We assayed 400 males from each strain in the sex pheromone chemoattraction assay. Two biological replicates were combined into a single value. Significance was determined using one-way ANOVA with Bonferroni correction: ***P<0.001. Means ± SEM (error bars) are shown.


A ) GCaMP5 reporter was used to visualize activation of SRD - 1 receptor by sex pheromone in AWA neurons . To facilitate real - time imaging of process , heads of transgenic worms were immobilized on soft - agar pads permeable to sex pheromone .

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A) The GCaMP5 reporter was used to visualize activation of the SRD-1 receptor by the sex pheromone in AWA neurons. To facilitate real-time imaging of the process, the heads of the transgenic worms were immobilized on soft-agar pads permeable to sex pheromone.


B ) Excitation of GCaMP5 reporter in AWA neurons could be observed through both lateral top views but top view was clearer more suitable for . Arrowheads : AWA neurons . Scale bar = 50 μm .

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B) Excitation of the GCaMP5 reporter in AWA neurons could be observed through both lateral and top views but the top view was clearer and more suitable for quantification. Arrowheads: AWA neurons. Scale bar = 50 μm.


C ) Relative - change was quantified through calcium imaging . Wild - type showed receptor excitation by sex pheromones from C . elegans hermaphrodites or C . remanei . This response was lost in srd - 1 - mutant - but was restored following AWA - specific rescue of srd - 1 . Red triangles : time points of sex pheromone addition . Three were assayed from each .

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C) Relative fluorescence-intensity change was quantified through calcium imaging. Wild-type males showed receptor excitation by sex pheromones from C. elegans hermaphrodites or C. remanei females. This response was lost in srd-1-mutant-strain males but was restored following AWA-specific rescue of srd-1. Red triangles: time points of sex pheromone addition. Three males were assayed from each strain.


B ) Wild - type C . elegans are less attracted to sex pheromone than wild - type C . remanei . Data information : We assayed 400 from each wild - type in sex pheromone chemoattraction . Two biological replicates were combined into a single value . Significance was determined using one - way ANOVA with Bonferroni correction : * * * P < 0 . 001 . Mean ± ( error bars ) are shown .

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B) Wild-type C. elegans males are less attracted to sex pheromone than wild-type C. remanei males. Data information: We assayed 400 males from each wild-type strain in the sex pheromone chemoattraction assay. Two biological replicates were combined into a single value. Significance was determined using one-way ANOVA with Bonferroni correction: ***P<0.001. Mean ± SEM (error bars) are shown.


C ) Expression level of srd - 1 cDNA in srd - 1 transgenic lines . srd - 1 mutant were rescued using , separately , wild - type C . elegans wild - type C . remanei srd - 1 cDNAs . Transgenic lines expressing molecule at a similar level were selected for use in chemoattraction assays . Four independent transgenic lines were selected , expression - level data were combined into a single bar . expression were measured using qPCR . Data information : For transgenic - studies , each bar represents a total of 1200 from 4 independent transgenic lines ; 20 assays were performed for 2 independent transgenic lines 10 assays were performed for other 2 independent transgenic lines , were conducted over 2 - 4 separate days . Significance was determined using one - way ANOVA with Bonferroni correction : * * * P < 0 . 001 . Mean ± ( error bars ) are shown .

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C) Expression level of srd-1 cDNA in srd-1 transgenic lines. srd-1 mutant males were rescued using, separately, wild-type C. elegans and wild-type C. remanei srd-1 cDNAs. Transgenic lines expressing the molecule at a similar level were selected for use in chemoattraction assays. Four independent transgenic lines were selected, and the expression-level data were combined into a single bar. The expression levels were measured using qPCR. Data information: For transgenic-line studies, each bar represents a total of 1200 males from 4 independent transgenic lines; 20 assays were performed for 2 independent transgenic lines and 10 assays were performed for the other 2 independent transgenic lines, and the tests were conducted over 2-4 separate days. Significance was determined using one-way ANOVA with Bonferroni correction: ***P<0.001. Mean ± SEM (error bars) are shown.


D ) srd - 1 mutant rescued with Ce - srd - 1 cDNA showed lower responsiveness than those rescued with Cre - srd - 1 cDNA , which indicates that sex pheromone perception ability depends primarily on coding region not regulatory sequences in promoter . sex pheromone perception defect of srd - 1 mutants was not rescued in expressing CT - truncated SRD - 1 . findings show that CT region is essential for sex pheromone perception . Data information : We assayed 400 from each wild - type in sex pheromone chemoattraction . Two biological replicates were combined into a single value . Significance was determined using one - way ANOVA with Bonferroni correction : * * * P < 0 . 001 . Mean ± ( error bars ) are shown .

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

D) srd-1 mutant males rescued with Ce-srd-1 cDNA showed lower responsiveness than those rescued with Cre-srd-1 cDNA, which indicates that sex pheromone perception ability depends primarily on the protein coding region and not the regulatory sequences in the promoter. The sex pheromone perception defect of srd-1 mutants was not rescued in males expressing CT-truncated SRD-1 proteins. The findings show that the CT region is essential for sex pheromone perception. Data information: We assayed 400 males from each wild-type strain in the sex pheromone chemoattraction assay. Two biological replicates were combined into a single value. Significance was determined using one-way ANOVA with Bonferroni correction: ***P<0.001. Mean ± SEM (error bars) are shown.


E ) srd - 1 mutant rescued by using Ce - srd - 1 cDNA together with Cre - srd - 1 CT cDNA were more responsive to sex pheromone than those rescued using Cre - srd - 1 cDNA with Ce - srd - 1 CT cDNA . results of CT - swapping experiment demonstrated that sex pheromone perception ability is determined by CT region not of SRD - 1 . Data information : We assayed 400 from each wild - type in sex pheromone chemoattraction . Two biological replicates were combined into a single value . Significance was determined using one - way ANOVA with Bonferroni correction : * * * P < 0 . 001 . Mean ± ( error bars ) are shown .

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

E) srd-1 mutant males rescued by using Ce-srd-1 cDNA together with Cre-srd-1 CT cDNA were more responsive to sex pheromone than those rescued using Cre-srd-1 cDNA with Ce-srd-1 CT cDNA. The results of the CT-swapping experiment demonstrated that sex pheromone perception ability is determined by the CT region and not the rest of the SRD-1 protein. Data information: We assayed 400 males from each wild-type strain in the sex pheromone chemoattraction assay. Two biological replicates were combined into a single value. Significance was determined using one-way ANOVA with Bonferroni correction: ***P<0.001. Mean ± SEM (error bars) are shown.


PS decarboxylase activity in wild - type ( WT , W303 ) indicated mutant strains expressed as ( B ) molecular species composition of 13C315N - labeled PS ( * PS ) 13C215N - labeled PE ( * PE ) , after 20 min incubation with 13C315N - serine .

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

PS decarboxylase activity in wild-type (WT, W303) and the indicated mutant strains expressed as (B) the molecular species composition of 13C315N-labeled PS (*PS) and 13C215N-labeled PE (*PE), after 20 min incubation with 13C315N-serine.


( C ) corresponding molecular signatures of PSD activity represented by label distribution per molecular species between * PS * PE as percentages of total amount of label incorporated in molecular species indicated .

O O O O O O O O O O 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) The corresponding molecular signatures of PSD activity represented by the label distribution per molecular species between *PS and *PE as percentages of the total amount of label incorporated in the molecular species indicated.


PS decarboxylase activity in a psd1Δpsd2Δdpl1Δ mutant expressing Psd1 , Psd1 ( ER ) or Psd1 ( Mt ) expressed as ( B ) molecular species composition of * PS * PE after 20 min incubation with 13C315N -

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

PS decarboxylase activity in a psd1Δpsd2Δdpl1Δ mutant expressing Psd1, Psd1(ER) or Psd1(Mt) expressed as (B) the molecular species composition of *PS and *PE after 20 min incubation with 13C315N-serine


PS decarboxylase activity in a psd1Δpsd2Δdpl1Δ mutant expressing Psd1 , Psd1 ( ER ) or Psd1 ( Mt ) expressed as ; ( C ) corresponding molecular signatures of PSD activity .

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

PS decarboxylase activity in a psd1Δpsd2Δdpl1Δ mutant expressing Psd1, Psd1(ER) or Psd1(Mt) expressed as ; (C) the corresponding molecular signatures of PSD activity.


( B ) Acyl chain composition of WT psd1 ∆ overexpressing SCT1 ( pSCT1 ) vs . empty vector control ( pEmpty ) . Data is presented as mean ± SD ( n > 10 ) . * * * * p < 0 . 0001 , multiple t - .

O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O 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) Acyl chain composition of WT and psd1∆ cells overexpressing SCT1 (pSCT1) vs. empty vector control (pEmpty). Data is presented as mean ± SD (n>10). **** p < 0.0001, multiple t-test.


( C ) of proportions of saturated over unsaturated acyl chains ( SFA / UFA ) of WT , psd1 ∆ , crd1 ∆ psd2 ∆ overexpressing SCT1 vs . empty vector control . Data is presented as mean ± SD ( n ≥ 3 ) . * * p < 0 . 01 , * * * * p < 0 . 0001 , unpaired two - t - .

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

(C) Ratio of the proportions of saturated over unsaturated acyl chains (SFA/UFA) of WT, psd1∆, crd1∆ and psd2∆ overexpressing SCT1 vs. empty vector control. Data is presented as mean ± SD (n≥3). ** p<0.01, **** p < 0.0001, unpaired two-tailed t-test.


( D ) OLE1 transcript in WT psd1 ∆ transformed with pSCT1 vs . pEmpty as determined by RT - qPCR . Data were normalized to endogenous ACT1 mRNA , to average of WT pEmpty ( n = 3 ) .

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

(D) OLE1 transcript levels in WT and psd1∆ transformed with pSCT1 vs. pEmpty as determined by RT-qPCR. Data were normalized to endogenous ACT1 mRNA, and to the average of WT pEmpty (n=3).


( A ) SFA / UFA ratios of WT indicated mutant strains overexpressing SCT1 ( pSCT1 ) vs . control ( pEmpty ) . Data is presented as mean ± SD ( n ≥ 3 ) . * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 , n . s . not significant , unpaired two - t - of indicated bar compared to WT pSCT1 .

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

(A) SFA/UFA ratios of WT and indicated mutant strains overexpressing SCT1 (pSCT1) vs. control (pEmpty). Data is presented as mean ± SD (n≥3). * p < 0.05, ** p < 0.01, **** p < 0.0001, n.s. not significant, unpaired two-tailed t-test of the indicated bar compared to WT pSCT1.


( B ) Growth of WT indicated mutant strains overexpressing SCT1 ( pSCT1 ) vs . pEmpty . Serial dilutions ( 10 - 1 - 10 - 4 ) were spotted on SGal + 0 . 05 % glucose incubated for 3 days at 30 ° C .

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

(B) Growth of WT and indicated mutant strains overexpressing SCT1 (pSCT1) vs. pEmpty. Serial dilutions (10-1 - 10-4) were spotted on SGal + 0.05% glucose and incubated for 3 days at 30°C.


PS decarboxylase activity in wild - type ( WT , BY4741 ) indicated mutant strains transformed with pEmpty , expressed , ( D ) molecular species composition of 13C315N - labeled PS ( * PS ) 13C215N - labeled PE ( * PE ) after 20 min incubation with 13C315N - serine . differences in molecular species profiles of * PS * PE between BY4741 WT shown here those obtained by 2H3 - serine labeling ( Figure 4A ) are attributed to different culture media , lipid extraction procedures mass spectrometry techniques used .

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

PS decarboxylase activity in wild-type (WT, BY4741) and the indicated mutant strains transformed with pEmpty, expressed , and (D) molecular species composition of 13C315N-labeled PS (*PS) and 13C215N-labeled PE (*PE) after 20 min incubation with 13C315N-serine. The differences in molecular species profiles of *PS and *PE between the BY4741 WT shown here and those obtained by 2H3-serine labeling (Figure 4A) are attributed to the different culture media, lipid extraction procedures and mass spectrometry techniques used.


( E ) corresponding molecular signatures of PSD activity represented by label distribution per molecular species between * PS * PE as percentages of total amount of label incorporated in molecular species indicated .

(E) The corresponding molecular signatures of PSD activity represented by the label distribution per molecular species between *PS and *PE as percentages of the total amount of label incorporated in the molecular species indicated.


SFA / UFA - of WT indicated mutant strains overexpressing SCT1 cultured without ( data taken from Fig 5A ) with 1 mM ethanolamine ( etn ) or 1 mM choline ( cho ) as indicated . Data is presented as average ± SD ( n ≥ 3 ) . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , unpaired t - of indicated bar compared to corresponding condition of WT . Underlying data for this figure can be found in Appendix Table S1 .

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

SFA/UFA-ratio of WT and indicated mutant strains overexpressing SCT1 cultured without (data taken from Fig 5A) and with 1 mM ethanolamine (etn) or 1 mM choline (cho) as indicated. Data is presented as average ± SD (n≥3). * p < 0.05, ** p < 0.01, *** p < 0.001, unpaired t-test of the indicated bar compared to the corresponding condition of WT. Underlying data for this figure can be found in Appendix Table S1.


( b ) CD spectra of Beclin 1 CC domain at different temperatures .

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

(b) The CD spectra of the Beclin 1 CC domain at different temperatures.


( c ) . Dynamic exchange of wild - type Beclin 1 . His - tagged untagged Beclin 1 CC domain were co - incubated at indicated temperatures coprecipitated at preset temperatures by Ni2 + - nitrilotriacetic acid ( NTA ) agarose beads . presence of heterodimeric Beclin 1 CC domain , consisting of one monomer with his - tag other monomer without tag , was checked by SDS gel indicated by arrow .

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

(c). Dynamic exchange assay of wild-type Beclin 1. His-tagged and untagged Beclin 1 CC domain were co-incubated at the indicated temperatures and coprecipitated at preset temperatures by Ni2+- nitrilotriacetic acid (NTA) agarose beads. The presence of heterodimeric Beclin 1 CC domain, consisting of one monomer with his-tag and the other monomer without tag, was checked by SDS gel and indicated by the arrow.


( d ) Self - association pattern of Beclin 1 in vivo . Both GFP - AsRed - tagged full - Beclin 1 are co - transfected into HEK 293T their self - association is probed by co - IP using GFP antibody for immunoprecipitation , followed by western blot with anti - AsRed antibody .

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

(d) Self-association pattern of Beclin 1 in vivo. Both GFP- and AsRed-tagged full-length Beclin 1 are co-transfected into HEK 293T cells and their self-association is probed by co-IP using GFP antibody for immunoprecipitation, followed by western blot with anti-AsRed antibody.


( a , b ) ITC titration profile of Beclin 1 CC domain with Atg14L ( a ) or UVRAG ( b ) .

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

(a, b) ITC titration profile of Beclin 1 CC domain with Atg14L (a) or UVRAG (b).


c ) Pull - down to assess competition between Beclin 1 - Atg14L Beclin 1 - UVRAG complexes . Increasing amount of His6 - tagged Beclin 1 CC domain was added to a mixture of Atg14L UVRAG corresponding to their respective CC region . Beclin 1 - Atg14L / UVRAG complexes were pulled down by Ni2 + - nitrilotriacetic acid ( NTA ) agarose beads checked by SDS gel .

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

c) Pull-down assay to assess the competition between Beclin 1-Atg14L and Beclin 1-UVRAG complexes. Increasing amount of His6-tagged Beclin 1 CC domain was added to a mixture of Atg14L and UVRAG proteins corresponding to their respective CC region. The Beclin 1-Atg14L/UVRAG complexes were pulled down by Ni2+- nitrilotriacetic acid (NTA) agarose beads and checked by SDS gel.


( d ) Co - IP experiments to assess competition between Beclin 1 - Atg14L Beclin 1 - UVRAG complexes in vivo . FLAG - or GFP - tagged Atg14L or UVRAG was transfected alone , or both were co - transfected into HEK 293T their interaction with endogenous Beclin 1 was probed by co - IP using GFP antibody , followed by western blot with anti - Beclin 1 antibody .

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

(d) Co-IP experiments to assess the competition between Beclin 1-Atg14L and Beclin 1-UVRAG complexes in vivo. FLAG- or GFP-tagged Atg14L or UVRAG was transfected alone, or both were co-transfected into HEK 293T cells and their interaction with endogenous Beclin 1 was probed by co-IP using GFP antibody, followed by western blot with anti-Beclin 1 antibody.


( c ) Self - association pattern of wild - type , MutM MutStab constructs of Beclin 1 in vivo . For each construct , both GFP - Myc - tagged full - Beclin 1 are co - transfected into HEK 293T their self - association is probed by co - IP using GFP antibody for IP , followed by western blot with anti - Myc antibody .

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

(c) Self-association pattern of wild-type, MutM and MutStab constructs of Beclin 1 in vivo. For each construct, both GFP- and Myc-tagged full-length Beclin 1 are co-transfected into HEK 293T cells and their self-association is probed by co-IP using GFP antibody for IP, followed by western blot with anti-Myc antibody.


( d ) . - dependent CD profile of Beclin 1 CC domain wild - type MutStab constructs at 220 nm .

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

(d). Temperature-dependent CD profile of Beclin 1 CC domain wild-type and MutStab constructs at 220 nm.


( a , b ) Interaction of Beclin 1 wild - type monomeric ( MutM ) mutants with Atg14L or UVRAG in cultured . HEK 293T were transfected with GFP - tagged Beclin 1 variant alone ( a ) or together with FLAG - UVRAG ( b ) . lysates were immunoprecipitated by GFP antibody , followed by western - blot with anti - Atg14L antibody for detecting binding to endogenous Atg14L ( a ) or with anti - UVRAG antibody for detecting binding to FLAG - tagged UVRAG ( b ) .

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

(a,b) Interaction of Beclin 1 wild-type and monomeric (MutM) mutants with Atg14L or UVRAG in cultured cells. HEK 293T cells were transfected with GFP-tagged Beclin 1 variant alone (a) or together with FLAG-UVRAG (b). The lysates were immunoprecipitated by GFP antibody, followed by western-blot analysis with anti-Atg14L antibody for detecting binding to endogenous Atg14L (a) or with anti-UVRAG antibody for detecting binding to the FLAG-tagged UVRAG protein (b).


( a , b ) Interaction of MutStab mutants of Beclin 1 with Atg14L or UVRAG in vivo . HEK 293T were transfected with GFP - tagged Beclin 1 variant alone ( b ) or together with FLAG - UVRAG ( b ) . lysates were immunoprecipitated by GFP antibody , followed by western blot with anti - Atg14L antibody for detecting binding to endogenous Atg14L ( a ) or with anti - UVRAG antibody for detecting binding to FLAG - tagged UVRAG ( b ) .

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

(a, b) Interaction of MutStab mutants of Beclin 1 with Atg14L or UVRAG in vivo. HEK 293T cells were transfected with GFP-tagged Beclin 1 variant alone (b) or together with FLAG-UVRAG (b). The lysates were immunoprecipitated by GFP antibody, followed by western blot analysis with anti-Atg14L antibody for detecting binding to endogenous Atg14L (a) or with anti-UVRAG antibody for detecting binding to the FLAG-tagged UVRAG protein (b).


( a , b ) . Representative of HeLa co - tansfected with GFP - tagged Beclin 1 wild type , MutM L178A / L192A or MutStab E189L / A217L / E224L / A255L ( green ) AsRed - Atg14L ( red ) ( a ) or FLAG - UVRAG ( red ) ( b ) ; Co - localization of AsRed - Atg14L with GFP - tagged Beclin 1 wild type or MutM is shown in discrete .

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

(a,b). Representative fluorescent images of HeLa cells co-tansfected with GFP-tagged Beclin 1 wild type, MutM L178A/L192A or MutStab E189L/A217L/E224L/A255L (green) and AsRed-Atg14L (red) (a) or FLAG-UVRAG (red) (b); Co-localization of AsRed-Atg14L with GFP-tagged Beclin 1 wild type or MutM is shown in discrete puncta.


( c - e ) Representative of HeLa co - transfected with Myc - Beclin 1 wild type , MutM L178A / L192A or MutStab E189L / A217L / E224L / A255L ( blue ) , AsRed - Atg14L ( red ) GFP - tagged Atg12 ( c ) . scale bar is 20 μM .

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

(c-e) Representative fluorescence images of HeLa cells co-transfected with Myc-Beclin 1 wild type, MutM L178A/L192A or MutStab E189L/A217L/E224L/A255L (blue), AsRed-Atg14L (red) and GFP-tagged Atg12 (c). The scale bar is 20 μM.


( c - e ) Representative of HeLa co - transfected with Myc - Beclin 1 wild type , MutM L178A / L192A or MutStab E189L / A217L / E224L / A255L ( blue ) , AsRed - Atg14L ( red ) GFP - tagged LC3 ( d ) DFCP ( e ) ( green ) . scale bar is 20 μM .

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

(c-e) Representative fluorescence images of HeLa cells co-transfected with Myc-Beclin 1 wild type, MutM L178A/L192A or MutStab E189L/A217L/E224L/A255L (blue), AsRed-Atg14L (red) and GFP-tagged LC3 (d) and DFCP (e) (green). The scale bar is 20 μM.


( A ) Confocal of adult brain of a young ( 2 d old ) wild - type fly . Positive for Ref ( 2 ) P ubiquitin is not evident . ( B - D ) Confocal of adult brain of an old ( 8 wk old ) wild - type fly . Ref ( 2 ) P accumulates in that often colocalize with ubiquitin ( arrows ) . rectangle in C indicates shown in D .

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

(A) Confocal micrographs of adult brain of a young (2 d old) wild-type fly. Positive staining for Ref(2)P and ubiquitin is not evident. (B-D) Confocal micrographs of adult brain of an old (8 wk old) wild-type fly. Ref(2)P accumulates in protein aggregates that often colocalize with ubiquitin (arrows). The white rectangle in C indicates the area shown in D.


( E F ) Western blot of total lysates ( E ) insoluble fraction ( F ) of wild - type young old adult heads probed with anti - ref ( 2 ) P anti - ubiquitin antibodies , demonstrating a significant increase in Ref ( 2 ) P insoluble ubiquitinated in old flies .

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

(E and F) Western blot analysis of total cell lysates (E) and insoluble protein fraction (F) of wild-type young and old adult heads probed with anti-ref(2)P and anti-ubiquitin antibodies, demonstrating a significant increase in Ref(2)P and insoluble ubiquitinated protein levels in old flies.


( A - D ) Electron of adult brain of old wild - type flies . Ref ( 2 ) P localizes in electron - dense structures ranging from 50 nm to 1 μm in diameter that are surrounded ( arrows ) or not ( arrowhead ) by a limiting . ( E F ) Electron of wild - type brain of old flies demonstrating that Ref ( 2 ) P forms shell - like structures that surround aggregated filamentous material ( arrows ) . Bars : ( A - C ) 50 nm ; ( D ) 100 nm ; ( E F ) 200 nm .

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(A-D) Electron micrographs of adult brain of old wild-type flies. Ref(2)P localizes in electron-dense structures ranging from 50 nm to 1 μm in diameter that are surrounded (arrows) or not (arrowhead) by a limiting membrane. (E and F) Electron micrographs of wild-type brain of old flies demonstrating that Ref(2)P forms shell-like structures that surround aggregated filamentous material (arrows). Bars: (A-C) 50 nm; (D) 100 nm; (E and F) 200 nm.


( A B ) atg8a mutants exhibiting a striking positive for Ref ( 2 ) P ubiquitin in regions of adult brain . As it is shown in high magnification of optic lobe ( medulla region ; B ) , Ref ( 2 ) P - ubiquitin - positive are observed ( arrows ) . Genotypes : ( A B ) atg8aKG07569 /

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(A and B) atg8a mutants exhibiting a striking positive staining for Ref(2)P and ubiquitin staining in all the regions of the adult brain. As it is shown in high magnification of the optic lobe (medulla region; B), Ref(2)P- and ubiquitin-positive aggregates are observed (arrows). Genotypes: (A and B) atg8aKG07569/Y


( C ) Normal phenotype is observed in β2 6 dominant - negative - sensitive mutants at 25 ° C . ( D ) Accumulation of Ref ( 2 ) P - ubiquitin - positive are observed in β2 6 mutants at 29 ° C ( arrows ) . ( C D ) elavGal4 / + ; UAS - β6 / + ; UAS - β2 / +

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(C) Normal phenotype is observed in β2 and 6 dominant-negative temperature-sensitive mutants at 25°C. (D) Accumulation of Ref(2)P- and ubiquitin-positive aggregates are observed in β2 and 6 mutants at 29°C (arrows). (C and D) elavGal4/+; UAS-β6/+; UAS-β2/+


( E F ) double mutants for atg8a ref ( 2 ) P loss - of - function alleles lacking PB1 ( E ) or UBA ( F ) domain do not form ubiquitinated . Ubiquitin exhibits a pattern ( arrows ) . Genotypes : ( E ) atg8aKG07569 / Y ; ref ( 2 ) Pod2 / ref ( 2 ) Pod2 , ( F ) atg8aKG07569 / Y ; ref ( 2 ) Pod3 / ref ( 2 ) Pod3 .

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(E and F) double mutants for atg8a and ref(2)P loss-of-function alleles lacking the PB1 (E) or UBA (F) domain do not form ubiquitinated protein aggregates. Ubiquitin exhibits a cytoplasmic staining pattern (arrows). Genotypes: (E) atg8aKG07569/Y; ref(2)Pod2/ref(2)Pod2, and (F) atg8aKG07569/Y; ref(2)Pod3/ref(2)Pod3.


( A B ) in atg8aKG07569 mutants exhibit sequestosome are positively labeled for Ref ( 2 ) P ( A ) ubiquitin ( B ) . are surrounded by small electron - dense vesicles ( arrows ) .

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(A and B) Protein aggregates in atg8aKG07569 mutants exhibit sequestosome morphology and are positively labeled for Ref(2)P (A) and ubiquitin (B). Protein aggregates are surrounded by small electron-dense vesicles (arrows).


( C ) Western blot of lysates of wild type atg8aKG07569 young adult heads demonstrating a significant increase of Ref ( 2 ) P in atg8aKG07569 flies .

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(C) Western blot analysis of cell lysates of wild type and atg8aKG07569 young adult heads demonstrating a significant increase of Ref(2)P protein levels in atg8aKG07569 flies.


( D , left ) Ref ( 2 ) P migration profile in ref ( 2 ) P loss - of - function alleles . Ref ( 2 ) P mutant for PB1 domain has an apparent molecular mass of 85 kD ( ref ( 2 ) Pod2 / ref ( 2 ) Pod2 ) Ref ( 2 ) P mutant for UBA domain has an apparent molecular mass of 69 kD ( ref ( 2 ) Pod3 / ref ( 2 ) Pod3 ) . ( D , right ) Western blot of insoluble fractions of adult heads of double mutants for atg8a ref ( 2 ) P probed for ubiquitin . Genotypes : ( 1 ) atg8aKG07569 / Y ; ref ( 2 ) Pod2 / Cyo , ( 2 ) atg8aKG07569 / Y ; ref ( 2 ) Pod2 / ref ( 2 ) Pod2 , ( 3 ) atg8aKG07569 / Y ; ref ( 2 ) Pod3 / CyO , ( 4 ) atg8aKG07569 / Y ; ref ( 2 ) Pod3 / ref ( 2 ) Pod3 . insoluble ubiquitinated profile of double mutants is diminished compared with one of respective control heterozygous for ref ( 2 ) P .

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(D, left) Ref(2)P protein migration profile in ref(2)P loss-of-function alleles. Ref(2)P mutant protein for the PB1 domain has an apparent molecular mass of 85 kD (ref(2)Pod2/ref(2)Pod2) and the Ref(2)P mutant protein for the UBA domain has an apparent molecular mass of 69 kD (ref(2)Pod3/ref(2)Pod3). (D, right) Western blot analysis of insoluble protein fractions of adult heads of double mutants for atg8a and ref(2)P probed for ubiquitin. Genotypes: (1) atg8aKG07569/Y; ref(2)Pod2/Cyo, (2) atg8aKG07569/Y; ref(2)Pod2/ref(2)Pod2, (3) atg8aKG07569/Y; ref(2)Pod3/CyO, and (4) atg8aKG07569/Y; ref(2)Pod3/ref(2)Pod3. The insoluble ubiquitinated protein profile of the double mutants is diminished compared with the one of the respective control heterozygous for ref(2)P.


( E F ) Neuronal mutant for atg1 ( green ) accumulate Ref ( 2 ) P - ( E ) ubiquitin - positive ( F ) structures ( arrows ) , whereas normal surrounding do not exhibit positive . Genotypes : ( E F ) yw / UAS - CD8 - GFP , hs - flp ; tubGal4 / + ; atg1Δ3D , FRT80 / tubGal80 ,

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(E and F) Neuronal cells mutant for atg1 (green) accumulate Ref(2)P- (E) and ubiquitin-positive (F) structures (arrows), whereas the normal surrounding cells do not exhibit positive staining. Genotypes: (E and F) yw/UAS-CD8-GFP, hs-flp; tubGal4/+; atg1Δ3D, FRT80/tubGal80, FRT80


( G ) Confocal of optic lobe ( medulla region ) of young flies expressing a mutated form of human tau exhibiting Ref ( 2 ) P - tau - positive ( arrows ) . Genotypes : ( G ) elav / + ; UAS - E14 / + .

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(G) Confocal micrographs of the optic lobe (medulla region) of young flies expressing a mutated form of human tau protein exhibiting Ref(2)P- and tau-positive aggregates (arrows). Genotypes: (G) elav/+;UAS-E14/+.


B . Identification of highly variable genes ( HVGs ) for time - point ZT2 . trend for global relation ( red ) between CV2 ( variance / mean2 ) mean expression is defined using genes , minus small lowly expressed genes ( see material methods for more detail ) used to identify HVG ( blue ) . For each gene , a corrected CV2 is calculated : log2 ( CV2 / trend ) .

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B. Identification of highly variable genes (HVGs) for the time-point ZT2. The trend for the global relation (red line) between CV2 (variance/mean2) and mean expression is defined using all genes, minus small and lowly expressed genes (see material and methods for more detail) and used to identify HVG (blue). For each gene, a corrected CV2 is calculated: log2(CV2 /trend).


C . Expression profiles ( top ) in 14 seedlings over a 24 hour time course ( with 12 time - points ) for a non - variable gene ( left ) , a highly variable gene ( middle ) a gene with level of variability changing across 24 hour ( right ) . Each dot is mean normalised expression level for a single seedling . Variability profiles ( bottom ) of log2 ( CV2 / trend ) for same genes are also shown . Blue stars indicate time - points for which gene is identified as being highly variable .

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C. Expression profiles (top) in the 14 seedlings over a 24 hour time course (with 12 time-points) for a non-variable gene (left), a highly variable gene (middle) and a gene with the level of variability changing across the 24 hour (right). Each dot is the mean normalised expression level for a single seedling. Variability profiles (bottom) of the log2(CV2 /trend) for the same genes are also shown. Blue stars indicate time-points for which the gene is identified as being highly variable.


A . Number of genes identified as being highly variable for each time - point . These genes are separated between those that are also selected as highly variable in at least one other time - point ( blue ) those highly variable in only one time - point ( blue ) . top bar indicates time - points harvested during day ( orange ) , just before dusk ( red ) , during night ( black ) just before dawn ( blue ) .

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A. Number of genes identified as being highly variable for each time-point. These genes are separated between those that are also selected as highly variable in at least one other time-point (dark blue) and those highly variable in only one time-point (light blue). The top bar indicates time-points harvested during the day (orange), just before dusk (red), during the night (black) and just before dawn (blue).


B . Distribution of number of time - points at which genes are identified as highly variable ( blue ) or lowly variable ( green ) . distribution of average ( black ) 95 % confidence interval ( dotted grey ) for thousand random sets is also represented .

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B. Distribution of the number of time-points at which genes are identified as highly variable (blue) or lowly variable (green). The distribution of average (black) and 95% confidence interval (dotted grey) for the thousand random sets is also represented.


C . of of HVGs shared between time - points . Red indicates a high of HVGs in common between two time - points . top side bars indicates time - points harvested during day ( orange ) , just before dusk ( red ) , during night ( black ) just before dawn ( blue ) . D . Hierarchical clustering of HVGs on log2 ( CV2 / trend ) at each time point . result is represented as a where yellow indicates a high log2 ( CV2 / trend ) . genes were separated into four clusters , indicated by side colored bar . top bar indicates time - points harvested during day ( orange ) , just before dusk ( red ) , during night ( black ) just before dawn ( blue ) . See Appendix Fig S3G for heatmaps of log2 ( CV2 / trend ) with same colour cutoffs for HVGs , LVGs random genes . E . of mean normalised expression level for genes in Fig 2D , keeping same clustering organisation . top bar indicates time - points harvested during day ( orange ) , just before dusk ( red ) , during night ( black ) just before dawn ( blue ) .

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C. Heatmap of the percentage of HVGs shared between time-points. Red indicates a high percentage of HVGs in common between two time-points. The top and side bars indicates time-points harvested during the day (orange), just before dusk (red), during the night (black) and just before dawn (blue). D. Hierarchical clustering of HVGs based on the log2(CV2/trend) at each time point. The result is represented as a heatmap where yellow indicates a high log2(CV2/trend). The genes were separated into four clusters, indicated by the side colored bar. The top bar indicates time-points harvested during the day (orange), just before dusk (red), during the night (black) and just before dawn (blue). See Appendix Fig S3G for heatmaps of the log2(CV2/trend) with the same colour cutoffs for HVGs, LVGs and random genes. E. Heatmap of the mean normalised expression level for the genes in Fig 2D, keeping the same clustering organisation. The top bar indicates time-points harvested during the day (orange), just before dusk (red), during the night (black) and just before dawn (blue).


C . Distribution of number of TFs targeting a gene , on DAP - seq available dataset , for LVG ( green ) HVG ( blue ) . distribution of average ( black ) 95 % confidence interval ( dotted grey ) for thousand random sets is also represented .

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C. Distribution of the number of TFs targeting a gene, based on the DAP-seq available dataset, for LVG (green) and HVG (blue). The distribution of average (black) and 95% confidence interval (dotted grey) for the thousand random sets is also represented.


A . Proportion of genes marked with several marks among genes passing expression level thresholds ( black ) , HVGs ( blue ) , LVGs ( green ) average for thousand random sets ( grey ) . Error bars indicate 95 % confidence interval for thousand random sets . Blue green stars indicate statistical differences in number of marked genes compare to genes , for variable stable genes respectively ( * Indicates a p - value < 0 . 05 , * * * Indicates a p - value < 0 . 001 , Chi - square ) .

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A. Proportion of genes marked with several chromatin marks among all genes passing size and expression level thresholds (black), HVGs (blue), LVGs (green) and the average for the thousand random sets (grey). Error bars indicate the 95% confidence interval for the thousand random sets. Blue and green stars indicate statistical differences in the number of marked genes compare to all genes, for variable and stable genes respectively (* Indicates a p-value < 0.05, *** Indicates a p-value < 0.001, Chi-square test).


B . Average profile for H3K27me3 , H2A . Z , H3K4me3 H3K23ac at HVGs ( blue ) , LVGs ( green ) random genes ( grey ) . C . of enrichment for H3K27me3 , H2A . Z , H3K4me3 H3K23ac for HVGs ( top ) , random genes ( middle ) LVG ( bottom ) . Red means a high level blue means a low level for marks .

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B. Average profile for H3K27me3, H2A.Z, H3K4me3 and H3K23ac at HVGs (blue), LVGs (green) and random genes (grey). C. Heatmap of the enrichment for H3K27me3, H2A.Z, H3K4me3 and H3K23ac for HVGs (top), random genes (middle) and LVG (bottom). Red means a high level and blue means a low level for the chromatin marks.


( b ) shows succinate by NMR from tracheal aspirates collected in mechanically - ventilated with ( pink bar , n = 9 ) or without ( orange bar , n = 7 ) diagnostic of IAV pneumonia . * P < 0 . 05 . ( c ) IL - 6 IL - 8 measurements by ELISA from these same tracheal aspirates . Data information : Data are mean ± statistical was performed using Mann - Whitney U - .

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(b) shows succinate quantification by NMR from tracheal aspirates collected in mechanically-ventilated patients with (pink bar, n=9) or without (orange bar, n=7) diagnostic of IAV pneumonia. *P < 0.05. (c) IL-6 and IL-8 measurements by ELISA from these same tracheal aspirates. Data information: Data are mean ± SEM and statistical analysis was performed using the Mann-Whitney U-test.


8 - week - old C57Bl / 6 mice were infected intranasally with 150 pfu of influenza A / Scotland / 20 / 74 ( H3N2 ) ( IAV ) treated or not simultaneously with 4 mg of succinate ( Suc ; by intranasal route ) . Some mice were euthanized at 4 days post - infection lungs were collected to determine : ; ( b ) viral expression by Western blotting followed by a relative ( c ) , Data information : Statistical was performed using Mann - Whitney c , ( * P < 0 . 05 ) .

8-week-old female C57Bl/6 mice were infected intranasally with 150 pfu of influenza A/Scotland/20/74 (H3N2) virus (IAV) and treated or not simultaneously with 4 mg of succinate (Suc; by the intranasal route). Some mice were euthanized at 4 days post-infection and lungs were collected to determine: ; (b) the viral protein expression by Western blotting followed by a relative quantification (c), Data information: Statistical analysis was performed using the Mann-Whitney test c, (*P < 0.05).


8 - week - old C57Bl / 6 mice were infected intranasally with 150 pfu of influenza A / Scotland / 20 / 74 ( H3N2 ) ( IAV ) treated or not simultaneously with 4 mg of succinate ( Suc ; by intranasal route ) . Some mice were euthanized at 4 days post - infection lungs were collected to determine : ( h ) tissue lesions in lung sections stained with hematoxylin - eosin further assessed by microscopy . Scale bar : x6 / 20 µm . data are represented as mean or mean ± are cumulative of 2 independent experiments with 5 each . Data information : Statistical was performed using Mann - Whitney , h , , ( * P < 0 . 05 ) .

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8-week-old female C57Bl/6 mice were infected intranasally with 150 pfu of influenza A/Scotland/20/74 (H3N2) virus (IAV) and treated or not simultaneously with 4 mg of succinate (Suc; by the intranasal route). Some mice were euthanized at 4 days post-infection and lungs were collected to determine: (h) tissue lesions in lung sections stained with hematoxylin-eosin and further assessed by microscopy. Scale bar: x6/20 µm. All data are represented as the mean or the mean ± SEM and are cumulative of 2 independent experiments with 5 animals each. Data information: Statistical analysis was performed using the Mann-Whitney test , h, , (*P < 0.05).


Human bronchial epithelial BEAS - 2B were infected with A / Scotland / 20 / 74 ( H3N2 ) ( IAV ) at MOI = 1 . After 4 h , were treated or not with succinate ( Suc ; 4 mg / ml ) up to 20 h . expression ( c ) were assessed by Western blotting to detect viral ( β - actin was used as a loading control ) , respectively .

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Human bronchial epithelial BEAS-2B cells were infected with A/Scotland/20/74 (H3N2) virus (IAV) at MOI=1. After 4 h, cells were treated or not with succinate (Suc; 4 mg/ml) up to 20 h. protein expression (c) were assessed by Western blotting to detect viral proteins (β-actin was used as a loading control), respectively.