( A ) Overlay of 15N - 1H HSQC spectra of 15N - labeled Ca2 + / CaM by itself ( red peaks ) after addition of saturating , unlabeled PSD - 95 ( 1 - 21 ) ( blue peaks ) or saturating , unlabeled PSD - 95 _ pT19 ( green peaks ) . NMR experiments were recorded at 600 MHz 1H frequency .

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(A) Overlay of 15N-1H HSQC spectra of 15N-labeled Ca2+/CaM by itself (red peaks) and after addition of saturating, unlabeled PSD-95 (1-21) (blue peaks) or saturating, unlabeled PSD-95_pT19 (green peaks). All NMR experiments were recorded at 600 MHz 1H frequency.


( B ) Chemical shift perturbation of CaM amide resonances in absence vs . presence of PSD - 95 _ pT19 . Chemical shift difference ( CSD ) plotted on vertical axis was defined as CSD = { ( HNA - HNB ) 2 + ( 15NA - 15NB ) 2 } 1 / 2 , where " A " " B " designate free bound states of CaM . HN 15N represent amide 1H 15N chemical shifts , respectively . Perturbations greater than average were used as active ambiguous restraints in Haddock docking calculation ( see Methods ) .

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(B) Chemical shift perturbation of CaM amide resonances in the absence vs. presence of PSD-95_pT19. Chemical shift difference (CSD) plotted on the vertical axis was defined as CSD = {(HNA - HNB)2 + (15NA - 15NB)2}1/2 , where "A" and "B" designate free and bound states of CaM. HN and 15N represent amide 1H and 15N chemical shifts, respectively. Perturbations greater than the average were used as active ambiguous restraints in the Haddock docking calculation (see Methods).


( C ) Representative data from IPAP [ 15N - 1H ] - HSQC spectra of 15N - labeled Ca2 + / CaM bound to PSD - 95 _ pT19 in absence ( blue ) presence of 17 mg / ml filamentous bacteriophage Pf1 ( red ) . Residual dipolar couplings ( RDCs ) for backbone HN resonances are calculated ( in Hertz ) as difference in splitting for 15N - { 1H } doublet components ( red peaks ) relative to isotropic 1JNH coupling ( blue peaks ) . RDC values are calculated to be + 40 , + 30 , + 20 , + 20 , - 20 Hz for I100 , I27 , N137 , D64 , I130 , respectively .

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(C) Representative data from IPAP [15N-1H]-HSQC spectra of 15N-labeled Ca2+/CaM bound to PSD-95_pT19 in the absence (blue) and presence of 17 mg/ml filamentous bacteriophage Pf1 (red). Residual dipolar couplings (RDCs) for backbone HN resonances are calculated (in Hertz) as the difference in splitting for the 15N-{1H} doublet components (red peaks) relative to the isotropic 1JNH coupling (blue peaks). RDC values are calculated to be +40, +30, +20, +20, and -20 Hz for I100, I27, N137, D64, and I130, respectively.


( D ) Correlation between observed RDCs ( panel C ) versus RDCs calculated from final NMR derived structure of Ca2 + / CaM bound to PSD - 95 _ pT19 . correlation coefficient ( R ) equals 0 . 99 Q is 0 . 11 .

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(D) Correlation between observed RDCs (panel C) versus RDCs calculated from the final NMR derived structure of Ca2+/CaM bound to PSD-95_pT19. The correlation coefficient (R) equals 0.99 and Q is 0.11.


( A ) NMR structure of Ca2 + / CaM ( grey ) bound to unphosphorylated PSD - 95 . PSD - 95 residues D2 ( depicted as starting point ) to R13 are resolved form an - helix ( cyan ) ( Zhang et al . , 2014 ) . Residues 14 - 19 of unphosphorylated PSD - 95 are unstructured not shown . Ca2 + ions bound to CaM are depicted by orange spheres .

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(A) NMR structure of Ca2+/CaM (grey) bound to unphosphorylated PSD-95. PSD-95 residues D2 (depicted as starting point) to R13 are resolved and form an -helix (cyan) (Zhang et al., 2014). Residues 14-19 of unphosphorylated PSD-95 are unstructured and not shown. Ca2+ ions bound to CaM are depicted by orange spheres.


( B ) NMR structure of Ca2 + / CaM bound to PSD - 95 phosphorylated at T19 ( PDB accession no . 5J7J ) . Residues 14 - 19 of phosphorylated PSD - 95 adopt a hook - like structure ( highlighted in red ) not seen in unphosphorylated PSD - 95 . This hook - like structure is stabilized by PSD - 95 residues E17 pT19 forming salt bridges with R126 K115 , respectively . Side - chain atoms are depicted by a stick representation for PSD - 95 residues Q15 , E17 T19 ( red ) CaM residues K115 , T117 , R126 ( blue ) .

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(B) NMR structure of Ca2+/CaM bound to PSD-95 phosphorylated at T19 (PDB accession no. 5J7J). Residues 14-19 of phosphorylated PSD-95 adopt a hook-like structure (highlighted in red) not seen in unphosphorylated PSD-95. This hook-like structure is stabilized by PSD-95 residues E17 and pT19 forming salt bridges with R126 and K115, respectively. Side-chain atoms are depicted by a stick representation for PSD-95 residues Q15, E17 and T19 (red) and CaM residues K115, T117, and R126 (blue).


( A ) FP titrations showing binding of wild - type ( WT ) CaM to PSD - 95 ( 1 - 21 ) peptides , either WT ( blue ) , Q15A ( salmon ) , E17R ( black ) , T19E ( green ) , or T19K ( yellow ) . solid lines represent optimal theoretical curves calculated using a one - site binding model described in Methods . calculated dissociation constants in each case are given in Table 1 .

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(A) FP titrations showing the binding of wild-type (WT) CaM to PSD-95 (1-21) peptides, either WT (blue), Q15A (salmon), E17R (black), T19E (green), or T19K (yellow). The solid lines represent optimal theoretical curves calculated using a one-site binding model described in Methods. The calculated dissociation constants in each case are given in Table 1.


( B ) FP titrations showing binding of R126E CaM to PSD - 95 peptides , either WT ( blue ) or E17R ( black ) .

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(B) FP titrations showing the binding of R126E CaM to PSD-95 peptides, either WT (blue) or E17R (black).


( C ) FP titrations showing binding of K115E CaM to PSD - 95 peptides T19K ( yellow ) or T19E ( green ) .

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(C) FP titrations showing the binding of K115E CaM to PSD-95 peptides T19K (yellow) or T19E (green).


Cultured cortical neurons were treated with BIC ( 50 μM ) at 15 DIV for 48h or at 17 DIV for 2 h or left untreated before harvesting , extraction , of palmitoylation by biotin switch method pull down with NeutrAvidin - agarose beads . ( A ) Representative immunoblots of pull down samples ( top two panels ) total lysate ( bottom two panels ) for PSD - 95 , as control , trimeric G subunit G - i3 . Omission of NH2OH before biotinylation resulted in no NeutrAvidin pull down as negative control for non - specific pull down .

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Cultured cortical neurons were treated with BIC (50 μM) at 15 DIV for 48h or at 17 DIV for 2 h or left untreated before harvesting, extraction, and analysis of palmitoylation by the biotin switch method and pull down with NeutrAvidin-agarose beads. (A) Representative immunoblots of pull down samples (top two panels) and total lysate (bottom two panels) for PSD-95 and, as control, the trimeric G protein subunit G-i3. Omission of NH2OH before biotinylation resulted in no NeutrAvidin pull down as negative control for non-specific pull down.


( B ) of PSD - 95 palmitoylation . Seven samples from three independent sets of cultures were analyzed per condition ( * * p < 0 . 01 ; t - ) .

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(B) Quantification of PSD-95 palmitoylation. Seven samples from three independent sets of cultures were analyzed per condition (**p<0.01; t-test).


Cultured hippocampal neurons were infected at 14 DIV with lentivirus for expression of both shRNA against PSD - 95 ( sh95 ) sh - resistant PSD - 95 - EGFP . Thus endogenous PSD - 95 was replaced with either wild - type ( WT ) , E17R , or T19K PSD - 95 . Cultures were treated with or without BIC ( 50 μM ) at 17 DIV for 24 - 48 h or left untreated before fixation for surface GluA1 . ( A ) Representative confocal microscopic of PSD - 95 - EGFP ( green ) surface GluA1 immunostaining ( red ) at low ( top panels ; scale bar : 20 µm ) high enlargement ( bottom panels ; scale bar : 5 µm ) .

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Cultured hippocampal neurons were infected at 14 DIV with lentivirus for expression of both shRNA against PSD-95 (sh95) and sh-resistant PSD-95-EGFP. Thus endogenous PSD-95 was replaced with either wild-type (WT), E17R, or T19K PSD-95. Cultures were treated with or without BIC (50 μM) at 17 DIV for 24-48 h or left untreated before fixation and staining for surface GluA1. (A) Representative confocal microscopic images of PSD-95-EGFP fluorescence (green) and surface GluA1 immunostaining (red) at low (top panels; scale bar: 20 µm) and high enlargement (bottom panels; scale bar: 5 µm).


( B ) of surface GluA1 from ( A ) showed reduction in AMPAR surface during scaling down in neurons expressing WT PSD - 95 but not in those expressing either E17R or T19K mutant . Three dendritic segments from each of 31 - 42 neurons from three independent sets of cultures were analyzed per condition ( * * p < 0 . 01 ; n . s . , not significant ; one - way ANOVA followed by Bonferroni ' s post hoc ) .

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(B) Quantification of surface GluA1 intensity from (A) showed reduction in AMPAR surface levels during scaling down in neurons expressing WT PSD-95 but not in those expressing either E17R or T19K mutant. Three dendritic segments from each of 31-42 neurons from three independent sets of cultures were analyzed per condition (**p<0.01; n.s., not significant; one-way ANOVA followed by Bonferroni's post hoc test).


Cultured hippocampal neurons were co - transfected with mCherry EGFP - tagged PSD - 95 wild - type ( WT ) , E17R , or T19K at 10 - 11 DIV treated with BIC ( 50 μM ) or left untreated at 17 DIV for 24 - 48 h before fixation . ( A ) Representative confocal microscopic of PSD - 95 - EGFP ( green ) mCherry ( red ) used as " fill " to visualize dendrite with spine heads ( scale bar : 2 µm ) after control ( top ) BIC treatment ( bottom ) . channels are shown in grayscale .

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Cultured hippocampal neurons were co-transfected with mCherry and EGFP-tagged PSD-95 wild-type (WT), E17R, or T19K at 10-11 DIV and treated with BIC (50 μM) or left untreated at 17 DIV for 24-48 h before fixation. (A) Representative confocal microscopic images of PSD-95-EGFP (green) and mCherry (red) used as "fill" to visualize the dendrite with spine heads (scale bar: 2 µm) after control (top) and BIC treatment (bottom). Individual channels are shown in grayscale.


( B ) BIC treatment reduced spine enrichment of WT but not E17R or T19K PSD - 95 as quantified as of EGFP in spine heads relative to that in adjacent dendritic shaft . Values were normalized to control treatments , which were set to equal 100 % for each PSD - 95 construct .

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(B) BIC treatment reduced spine enrichment of WT but not E17R or T19K PSD-95 as quantified as the ratio of EGFP intensity in spine heads relative to that in the adjacent dendritic shaft. Values were normalized to control treatments, which were set to equal 100% for each PSD-95 construct.


( C ) mCherry showed no enrichment in spines BIC had no effect on its localization .

(C) mCherry showed no enrichment in spines and BIC had no effect on its localization.


( D - F ) Spine enrichment of PSD - 95 were comparable under basal conditions for WT , E17R , T19K PSD - 95 ( A . U . , arbitrary unit ) .

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(D-F) Spine enrichment and puncta size and intensity of PSD-95 were comparable under basal conditions for WT, E17R, and T19K PSD-95 (A.U., arbitrary unit).


( G - I ) Spine density , head diameter , were comparable under basal conditions for neurons expressing WT , E17R , T19K PSD - 95 .

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(G-I) Spine density, head diameter, and length were comparable under basal conditions for neurons expressing WT, E17R, and T19K PSD-95.


Cultured hippocampal neurons were transfected at 10 - 11 DIV with indicated CaM - IRES - ECFP plasmids infected at 14 DIV with lentivirus expressing shRNA against PSD - 95 ( sh95 ) sh - resistant wild - type ( WT ) or E17R PSD - 95 - EGFP . Cultures were treated with BIC ( 50 μM ) or left untreated at 17 DIV for 24 - 48 h before fixation . ( A ) Representative confocal microscopic of intrinsic ECFP EGFP , surface GluA1 immunostaining at low ( top panels ; scale bar : 20 µm ) high enlargement ( bottom panels ; scale bar : 5 µm ) .

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Cultured hippocampal neurons were transfected at 10-11 DIV with the indicated CaM-IRES-ECFP plasmids and infected at 14 DIV with lentivirus expressing shRNA against PSD-95 (sh95) and sh-resistant wild-type (WT) or E17R PSD-95-EGFP. Cultures were treated with BIC (50 μM) or left untreated at 17 DIV for 24-48 h before fixation. (A) Representative confocal microscopic images of intrinsic ECFP and EGFP fluorescence, and surface GluA1 immunostaining at low (top panels; scale bar: 20 µm) and high enlargement (bottom panels; scale bar: 5 µm).


( B ) of surface GluA1 from ( A ) showed impaired AMPAR downscaling in neurons expressing PSD - 95 - E17R in presence of wild - type CaM ( CaM - WT ) that was rescued by coexpression of CaM - R126E . Three dendritic segments from each of 18 - 26 neurons from three independent sets of cultures were analyzed per condition ( * * p < 0 . 01 , * p < 0 . 05 vs . untreated basal ; one - way ANOVA followed by Bonferroni ' s post hoc ) .

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(B) Quantification of surface GluA1 intensity from (A) showed impaired AMPAR downscaling in neurons expressing PSD-95-E17R in presence of wild-type CaM (CaM-WT) that was rescued by coexpression of CaM-R126E. Three dendritic segments from each of 18-26 neurons from three independent sets of cultures were analyzed per condition (**p<0.01,*p<0.05 vs. untreated basal levels; one-way ANOVA followed by Bonferroni's post hoc test).


Cultured hippocampal neurons were co - transfected at 10 - 11 DIV with RFP indicated CaM - IRES - ECFP plasmids infected at 12 - 13 DIV with lentivirus expressing shRNA against PSD - 95 ( sh95 ) sh - resistant wild - type ( WT ) or E17R PSD - 95 - EGFP . mEPSC recordings were performed at DIV 17 - 19 , following 48 h of control or BIC ( 50 μM ) treatment . ( A ) Representative traces of AMPAR mEPSC recordings ( left ) averages of mEPSC events from single representative neurons ( right ) drawn to scale ( top ) normalized to peak ( bottom ) .

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Cultured hippocampal neurons were co-transfected at 10-11 DIV with RFP and the indicated CaM-IRES-ECFP plasmids and infected at 12-13 DIV with lentivirus expressing shRNA against PSD-95 (sh95) and sh-resistant wild-type (WT) or E17R PSD-95-EGFP. mEPSC recordings were performed at DIV 17-19, following 48 h of control or BIC (50 μM) treatment. (A) Representative traces of AMPAR mEPSC recordings (left) and averages of all mEPSC events from single representative neurons (right) drawn to scale (top) and normalized to peak (bottom).


( B ) of mEPSC amplitude revealed downscaling by BIC in neurons expressing PSD - 95WT / CaMWT or PSD - 95E17R / CaMR126E but not PSD - 95WT / CaMR126E ( left ) over full range of amplitude distributions ( right ) .

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(B) Quantification of mEPSC amplitude revealed downscaling by BIC in neurons expressing PSD-95WT / CaMWT or PSD-95E17R / CaMR126E but not PSD-95WT / CaMR126E (left) over the full range of amplitude distributions (right).


( C , D ) Mini - EPSC frequency ( C ) decay time ( D ) were comparable among conditions ( n = 11 - 25 neurons per condition from 8 - 14 independent sets of cultures prepared from of 8 - 14 dams ; * p < 0 . 05 ; two - way ANOVA followed by Bonferroni ' s ) .

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(C,D) Mini-EPSC frequency (C) and decay time (D) were comparable among all conditions (n=11-25 neurons per condition from 8-14 independent sets of cultures prepared from the embryos of 8-14 dams;* p<0.05; two-way ANOVA followed by Bonferroni's test).


( B ) Confocal microscopy of 293T transfected with 0 . 5 μg pQCXIP encoding IFITM3 variants containing N - terminal FLAG ( pQCXIP - FLAG - IFITM3 ) immunostained with anti - FLAG M2 antibody . Scale bar , 5 μm .

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(B) Confocal fluorescence microscopy of 293T cells transfected with 0.5 μg pQCXIP encoding IFITM3 variants containing N-terminal FLAG (pQCXIP-FLAG-IFITM3) and immunostained with anti-FLAG M2 antibody. Scale bar, 5 μm.


( C ) Flow cytometry of transfected in ( B ) immunostained with anti - FLAG M2 antibody . Corresponding mean flourescence values are shown .

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(C) Flow cytometry analysis of transfected cells in (B) immunostained with anti-FLAG M2 antibody. Corresponding mean flourescence intensity values are shown.


( D ) SDS - PAGE of lysates derived from 293T transfected in ( B ) followed by immunoblotting with anti - FLAG M2 anti - actin antibodies .

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(D) SDS-PAGE of cell lysates derived from 293T cells transfected in (B) followed by immunoblotting with anti-FLAG M2 and anti-actin antibodies.


( E ) SDS - PAGE of Opti - prep purified supernatants ( 25 ng p24 equivalent ) derived from 293T transfected with pQCXIP - FLAG - IFITM3 variants , HIV - 1 pNL4 - 3 , Vpr - Blam plasmid followed by immunoblotting with anti - FLAG M2 anti - p24 Gag ( 183 - H12 - 5C ) .

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(E) SDS-PAGE of Opti-prep purified supernatants (25 ng p24 equivalent) derived from 293T cells transfected with pQCXIP-FLAG-IFITM3 variants, HIV-1 pNL4-3, and Vpr-Blam plasmid followed by immunoblotting with anti-FLAG M2 and anti-p24 Gag (183-H12-5C).


( F ) Opti - prep purified supernatants from ( E ) were incubated with SupT1 target for 4 hours - fusion was scored by flow cytometry . Mean + SD of 5 - 10 experiments ( each performed with produced from an independent transfection ) is shown . ( G ) used in ( E ) were used to infect SupT1 productive infection was scored at 48 - 60 hours by immunostaining with KC57 antibody flow cytometry . Expression of pCMV - MxA served as a negative control . mean + SD of 5 - 10 experiments is shown .

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(F) Opti-prep purified supernatants from (E) were incubated with SupT1 target cells for 4 hours and virus-cell fusion was scored by flow cytometry. Mean + SD of 5-10 experiments (each performed with virus produced from an independent transfection) is shown. (G) Virus used in (E) were used to infect SupT1 cells and productive infection was scored at 48-60 hours by immunostaining with the KC57 antibody and flow cytometry. Expression of pCMV-MxA served as a negative control. The mean + SD of 5-10 experiments is shown.


( H ) Confocal microscopy of 293T transfected with 0 . 5 μg pQCXIP - FLAG - IFITM3 variants immunostained with anti - FLAG M2

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(H) Confocal fluorescence microscopy of 293T cells transfected with 0.5 μg pQCXIP-FLAG-IFITM3 variants and immunostained with anti-FLAG M2 antibody


( I ) SDS - PAGE of lysates derived from 293T transfected in ( H ) followed by immunoblotting with anti - FLAG M2 anti - actin antibodies .

(I) SDS-PAGE of cell lysates derived from 293T cells transfected in (H) followed by immunoblotting with anti-FLAG M2 and anti-actin antibodies.


( J ) SDS - PAGE of Opti - prep purified supernatants ( 25 ng p24 equivalent ) derived from 293T transfected with pQCXIP - FLAG - IFITM3 constructs , HIV - 1 pNL4 - 3 , Vpr - Blam plasmid followed by immunoblotting with anti - FLAG M2 anti - p24 Gag . Scale bar , 5 μm .

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(J) SDS-PAGE of Opti-prep purified supernatants (25 ng p24 equivalent) derived from 293T cells transfected with pQCXIP-FLAG-IFITM3 constructs, HIV-1 pNL4-3, and Vpr-Blam plasmid followed by immunoblotting with anti-FLAG M2 and anti-p24 Gag. Scale bar, 5 μm.


( K ) Opti - prep purified supernatants from ( J ) were incubated with SupT1 target for 4 hours - fusion was scored by flow cytometry . mean + SD of 3 experiments is shown .

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(K) Opti-prep purified supernatants from (J) were incubated with SupT1 target cells for 4 hours and virus-cell fusion was scored by flow cytometry. The mean + SD of 3 experiments is shown.


( A ) Confocal microscopy of SupT1 T transduced with Tet - inducible pQCXIP - FLAG - IFITM3 or pQCXIP - FLAG - IFITM3 Δ1 - 21 ( Tet - ON ) immunostained with anti - FLAG M2 antibody following overnight treatment with 500 ng / mL doxycycline . Scale bar , 10 μm .

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(A) Confocal fluorescent microscopy of SupT1 T cells transduced with Tet-inducible pQCXIP-FLAG-IFITM3 or pQCXIP-FLAG-IFITM3 Δ1-21 (Tet-ON) immunostained with anti-FLAG M2 antibody following overnight treatment with 500 ng/mL doxycycline. Scale bar, 10 μm.


( B ) Tet - ON SupT1 lines were treated or not with 500 ng / mL doxycycline overnight induction of IFITM3 was assessed anti - FLAG M2 immunostaining flow cytometry .

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(B) Tet-ON SupT1 cell lines were treated or not with 500 ng/mL doxycycline overnight and induction of IFITM3 protein was assessed anti-FLAG M2 immunostaining and flow cytometry.


( C ) Tet - ON SupT1 lines were productively infected with NL4 - 3 VSV - G then treated with 500 ng / mL doxycycline overnight to produce from IFITM3 - IFITM3 + . 25 ng p24 equivalents of purified were used to infect fresh Tet - ON SupT1 targets , which were previously treated with 500 ng / mL doxycycline or not . Infection was scored at 72h post - infection by immunostaining with KC57 flow cytometry . Mean + SD of 3 experiments are shown .

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(C) Tet-ON SupT1 cell lines were productively infected with NL4-3 VSV-G and then treated with 500 ng/mL doxycycline overnight to produce virus from IFITM3- and IFITM3+ cells. 25 ng p24 equivalents of purified were used to infect fresh Tet-ON SupT1 cells cell targets, which were previously treated with 500 ng/mL doxycycline or not. Infection was scored at 72h post-infection by immunostaining with KC57 and flow cytometry. Mean + SD of 3 experiments are shown.


( D ) 25 ng p24 equivalents of used in ( C ) was subjected to SDS - PAGE . Immunoblotting was performed using anti - FLAG M2 , anti - p24 Gag , anti - gp120 Env ( NIH # 288 ) antibodies .

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(D) 25 ng p24 equivalents of virus used in (C) was subjected to SDS-PAGE. Immunoblotting was performed using the anti-FLAG M2, anti-p24 Gag, and anti-gp120 Env (NIH #288) antibodies.


( E ) 293T were transfected with indicated amount of pQCXIP - FLAG - IFITM3 or pQCXIP - FLAG - Δ1 - 21 or pCMV - MxA 1 . 0 μg of HIV - 1 pNL4 - 3 . SDS - PAGE was performed on whole lysates followed by immunoblotting with anti - FLAG M2 , anti - p24 Gag , anti - Env gp120 ( NIH # 288 ) , anti - tubulin antibodies . Western blot are representative of 3 independent experiments . Unpaired t - , * p < 0 . 05 , ♯ p = 0 . 12 , non statistically significant . Comparisons were made between condition indicated correponding condition in Tet - ON IFITM3 .

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(E) 293T cells were transfected with the indicated amount of pQCXIP-FLAG-IFITM3 or pQCXIP-FLAG-Δ1-21 or pCMV-MxA and 1.0 μg of HIV-1 pNL4-3. SDS-PAGE was performed on whole cell lysates followed by immunoblotting with anti-FLAG M2, anti-p24 Gag, anti-Env gp120 (NIH #288), and anti-tubulin antibodies. Western blot images are representative of 3 independent experiments. Unpaired t-test, *p<0.05, ♯p=0.12, non statistically significant. Comparisons were made between the condition indicated and the correponding condition in Tet-ON IFITM3 cells.


( A ) Confocal microscopy of 293T transfected with 0 . 5 μg of pQCXIP - FLAG - Human IFITM3 mutated at indicated residues immunostained with anti - FLAG M2 antibody . Scale bars , 5 μm . P17L , P18H , Δ22 - 24 , P18L / L23P P18H / Δ19 - 23 mutations were introduced into human IFITM3 background .

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(A) Confocal fluorescence microscopy of 293T cells transfected with 0.5 μg of pQCXIP-FLAG-Human IFITM3 mutated at indicated residues and immunostained with anti-FLAG M2 antibody. Scale bars, 5 μm. The P17L, P18H, Δ22-24, P18L/L23P and P18H/Δ19-23 mutations were introduced into the human IFITM3 background.


( B ) SDS - PAGE of FLAG - immunoprecipitated fractions from 293T following transfection of 0 . 5 μg of pQCXIP - FLAG - IFITM3 variants 0 . 5 μg of pCI - HA - NEDD4 or pCI - HA - NEDD4 C867A . Immunoblotting was performed with anti - FLAG M2 , anti - IFITM3 ( Abcam , EPR5242 ) , an anti - ubiquitin antibody ( Enzo Life Sciences , recognizing K29 - , K48 - , K63 - linked mono - polyubiquitinylated ) . ' L ' indicates presence of chain antibody derived from FLAG antibody - coupled beads used for immunoprecipitation . * , * * , * * * indicate mono - , di - , tri - ubiquitinated forms of IFITM3 .

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(B) SDS-PAGE of FLAG-immunoprecipitated fractions from 293T cells following transfection of 0.5 μg of pQCXIP-FLAG-IFITM3 variants and 0.5 μg of pCI-HA-NEDD4 or pCI-HA-NEDD4 C867A. Immunoblotting was performed with anti-FLAG M2, anti-IFITM3 (Abcam, EPR5242), and an anti-ubiquitin antibody (Enzo Life Sciences, recognizing K29-, K48-, and K63-linked mono- and polyubiquitinylated proteins). 'L' indicates the presence of light chain antibody derived from the FLAG antibody-coupled beads used for immunoprecipitation. *, **, and *** indicate mono-, di-, and tri-ubiquitinated forms of IFITM3.


( C ) SDS - PAGE of whole lysate input fractions from 293T used in ( B ) . Immunoblotting was performed with anti - FLAG M2 , anti - NEDD4 , anti - actin .

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(C) SDS-PAGE of whole cell lysate input fractions from 293T cells used in (B). Immunoblotting was performed with anti-FLAG M2, anti-NEDD4, and anti-actin.


( D ) 293T were transfected with 1 . 0 μg of pNL4 - 3 0 . 5 μg of pQCXIP - FLAG - IFITM3 variants . - containing supernatants were harvested 10 ng p24 equivalents were used to reinfect fresh SupT1 T . Productive infection was scored by immunostaining with KC57 antibody flow cytometry at 48 hours post - infection . means + SD of 3 experiments are shown .

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(D) 293T cells were transfected with 1.0 μg of pNL4-3 and 0.5 μg of pQCXIP-FLAG-IFITM3 variants. Virus-containing supernatants were harvested and 10 ng p24 equivalents were used to reinfect fresh SupT1 T cells. Productive infection was scored by immunostaining with the KC57 antibody and flow cytometry at 48 hours post-infection. The means + SD of 3 experiments are shown.


( E ) 2 . 0 x 10ˆ5 were seeded challenged with 10 ng of HIV - 1NL4 - 3 in presence of 2 μg / mL DEAE - Dextran . Productive infection was scored by immunostaining with KC57 flow cytometry at 48 hours post - infection . ' Colobus native ' represents full IFITM3 identified in colobus monkey . ( F ) As in ( E ) , except were challenged with Influenza A H1N1 PR / 8 / 34 , ( Charles River Laboratories ) , at a dose that resulted in ˜ 50 % infection of control ( Empty ) ( equivalent to 103 TCID50 / 0 . 2 mL ) . Infection was scored by immunostaining with an anti - IAV NP antibody flow cytometry at 18 hours post - infection . Results are presented in a logarithmic scale . Means + SD of 3 - 5 experiments are shown . Unpaired t - , * p < 0 . 05 , * * p < 0 . 005 .

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(E) 2.0 x 10ˆ5 cells were seeded and challenged with 10 ng of HIV-1NL4-3 in the presence of 2 μg/mL DEAE-Dextran. Productive infection was scored by immunostaining with KC57 and flow cytometry at 48 hours post-infection. The 'Colobus native' sequence represents the full IFITM3 sequence identified in the colobus monkey. (F) As in (E), except cells were challenged with Influenza A virus H1N1 PR/8/34, (Charles River Laboratories), at a dose that resulted in ˜50% infection of control (Empty) cells (equivalent to 103 TCID50 / 0.2 mL). Infection was scored by immunostaining with an anti-IAV NP antibody and flow cytometry at 18 hours post-infection. Results are presented in a logarithmic scale. Means + SD of 3-5 experiments are shown. Unpaired t-test, *p<0.05, **p<0.005.


( E ) Quantitative RT - PCR ( qRT - PCR ) of μGLT , αGLT , AID from siControl - siPhf5a - treated samples . values are presented as mean ± sd ( n = 3 ) .

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(E) Quantitative RT-PCR (qRT-PCR) of μGLT, αGLT, and AID from siControl- and siPhf5a-treated samples. The values are presented as mean ± sd (n=3).


( B ) Southern blots of IgH / c - Myc translocation using genomic extracted from CIT - stimulated CH12F3 - 2A treated with siRNAs shown at bottom of each panel . Numbers on top indicate gel lanes . ( C ) Frequency of IgH / c - Myc chromosomal translocations derived from number of translocations detected in total number of PCR reactions per sample ( Boboila et al . , 2012b ) . values are presented as mean ± sd ( n = 3 ) . Statistical significance was assessed by two unpaired Student ' s t - ( * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 ) .

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(B) Southern blots of the IgH/c-Myc translocation assay using genomic DNA extracted from CIT-stimulated CH12F3-2A cells treated with the siRNAs shown at the bottom of each panel. Numbers on the top indicate gel lanes. (C) Frequency of IgH/c-Myc chromosomal translocations derived from the number of translocations detected in the total number of PCR reactions per sample (Boboila et al., 2012b). The values are presented as mean ± sd (n=3). Statistical significance was assessed by two tailed unpaired Student's t-test (** p ≤ 0.01, *** p ≤ 0.001).


( D - E ) Detection of Sμ double - strand breaks by LM - PCR in CH12F3 - 2A transfected with indicated siRNA combinations . samples were either left untreated ( none ) or treated with indicated end processing enzymes prior to linker ligation .

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(D-E) Detection of Sμ DNA double-strand breaks by LM-PCR in CH12F3-2A cells transfected with the indicated siRNA combinations. The DNA samples were either left untreated (none) or treated with indicated end processing enzymes prior to linker ligation.


( B ) Effect of Phf5a knockdown on I - SceI - induced NHEJ . Plot represents GFP - positive obtained by FACS after co - transfection of I - SceI expression plasmid indicated siRNAs into a harboring NHEJ reporter construct . values are presented as mean ± sd ( n = 3 ) . Statistical significance was evaluated against corresponding siControl by two - unpaired Student ' s t - ( * * * p ≤ 0 . 001 ) .

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(B) Effect of Phf5a knockdown on I-SceI-induced NHEJ. Plot represents the GFP-positive cells obtained by FACS analysis after co-transfection of the I-SceI expression plasmid and the indicated siRNAs into a cell line harboring the NHEJ reporter construct. The values are presented as mean ± sd (n=3). Statistical significance was evaluated against corresponding siControl by two-tailed unpaired Student's t-test (*** p ≤0.001).


( C ) Top : schematic diagram of position of ChIP PCR products specific to S region . Bottom : ChIP of indicated repair using CIT - stimulated CH12F3 - 2A transfected with siControl or siPhf5a . values are presented as mean ± sd ( n = 3 ) . Statistical significance was evaluated in reference to siControl by two - unpaired Student ' s t - ( * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 ) .

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(C) Top: schematic diagram of the position of the ChIP assay PCR products specific to the S region. Bottom: ChIP assay of the indicated DNA repair proteins using CIT-stimulated CH12F3-2A cells transfected with siControl or siPhf5a. The values are presented as mean ± sd (n=3). Statistical significance was evaluated in reference to siControl by two-tailed unpaired Student's t-test (*p ≤ 0.05, ** p ≤0.01, *** p ≤0.001).


( C ) Western blot of whole extract from CIT - stimulated CH12F3 - 2A transfected with indicated siRNAs . Expression of various including AID , variant H2As Ku80 were not decresed by Phf5a KD . ( ns , non - specific )

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(C) Western blot analysis of the whole cell extract from CIT-stimulated CH12F3-2A cells transfected with the indicated siRNAs. Expression of various proteins including AID, variant H2As and Ku80 were not decresed by Phf5a KD. (ns, non-specific)


( C ) CH12F3 - 2A were transfected with WT Phf5a or its mutants ( Phf5aR - MF ) along with siRNA against Phf5a . An anti - Flag antibody was used to immunoprecipitate immunodetect Phf5aR - MF . An anti - Phf5a immunoblot confirmed knockdown efficiency of endogenous Phf5a . antibodies used to detect co - associated are indicated next to respective blot .

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(C) CH12F3-2A cells were transfected with WT Phf5a or its mutants (Phf5aR-MF) along with siRNA against Phf5a. An anti-Flag antibody was used to immunoprecipitate and immunodetect Phf5aR-MF. An anti-Phf5a immunoblot confirmed the knockdown efficiency of the endogenous Phf5a. The antibodies used to detect the co-associated proteins are indicated next to the respective blot.


( B ) Western blot showing expression of endogenous exogenously introduced Phf5a .

(B) Western blot showing expression of endogenous and exogenously introduced Phf5a.


( C ) H2A . Z ChIP was performed using CH12F3 - 2A transfected with indicated siRNA Phf5a expression constructs . data are compiled from 2 experiments presented relative to siControl treated sample , which was set as 100 . error bars represent mean ± sd .

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(C) H2A.Z ChIP assay was performed using CH12F3-2A cells transfected with indicated siRNA and the Phf5a expression constructs. The data are compiled from 2 experiments and presented relative to siControl treated sample, which was set as 100. The error bars represent the mean ± sd.


Mutations in epg - 7 cause defective degradation of SQST - 1 . ( A B ) In wild - type , SQST - 1 : : GFP is weakly expressed diffusely localized in cytoplasm . ( A ) DIC image of shown in B . ( C ) Elevated expression level accumulation of many SQST - 1 : : GFP in epg - 7 mutant .

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Mutations in epg-7 cause defective degradation of SQST-1 aggregates. (A and B) In wild-type embryos, SQST-1::GFP is weakly expressed and diffusely localized in the cytoplasm. (A) DIC image of the embryo shown in B. (C) Elevated expression level and accumulation of many SQST-1::GFP aggregates in epg-7 mutant embryos.


( D ) Compared with wild type epg - 2 mutants , of SQST - 1 are dramatically elevated in atg - 3 epg - 7 mutant in an immunoblotting .

(D) Compared with wild type and epg-2 mutants, levels of SQST-1 are dramatically elevated in atg-3 and epg-7 mutant embryos in an immunoblotting assay.


( E - G ) Compared with wild - type ( E F ) , endogenous SQST - 1 shows dramatically elevated accumulates into a large number of in epg - 7 mutant ( G ) . ∼ 200 - stage are shown in E - G . ( E ) DAPI image of shown in F .

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(E-G) Compared with wild-type embryos (E and F), endogenous SQST-1 shows dramatically elevated levels and accumulates into a large number of aggregates in epg-7 mutant embryos (G). ∼200-cell stage embryos are shown in E-G. (E) DAPI image of the embryo shown in F.


( H ) Number of SQST - 1 per focal plane in wild type , atg - 3 mutants , epg - 7 mutants .

(H) Number of SQST-1 aggregates per focal plane in wild type, atg-3 mutants, and epg-7 mutants.


( I - L ) At ∼ 100 - stage , far fewer SQST - 1 are formed in epg - 7 mutants than in atg - 3 mutants . ( I K ) DAPI of shown in J L , respectively .

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(I-L) At the ∼100-cell stage, far fewer SQST-1 aggregates are formed in epg-7 mutants than in atg-3 mutants. (I and K) DAPI images of the embryos shown in J and L, respectively.


( M ) SQST - 1 directly interacts with ZK1053 . 4 in a GST pull - down .

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(M) SQST-1 directly interacts with ZK1053.4 in a GST pull-down assay.


( N ) Compared with wild - type , of ZK1053 . 4 : : GFP are dramatically elevated in atg - 3 epg - 7 mutants .

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(N) Compared with wild-type animals, levels of ZK1053.4::GFP are dramatically elevated in atg-3 and epg-7 mutants.


( O - R ) ZK1053 . 4 : : GFP is weakly expressed in wild - type ( O - P ) sqst - 1 mutants ( R ) , but forms a large number of in epg - 7 mutants ( Q ) . ( O ) DIC image of shown in P .

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(O-R) ZK1053.4::GFP is weakly expressed in wild-type embryos (O-P) and sqst-1 mutants (R), but forms a large number of aggregates in epg-7 mutants (Q). (O) DIC image of the embryo shown in P.


( S ) Survival curve of L1 larvae under food depletion conditions in wild type , epg - 1 mutants , epg - 7 mutants . This experiment was completed once ( wild type : n = 2 , 964 ; epg - 7 : n = 602 ; epg - 1 : n = 215 ) . Statistical of L1 survival shows that epg - 7 mutants exhibit significant difference to wild type also to epg - 1 mutants ( log - rank , P = 0 . 000 ) .

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(S) Survival curve of L1 larvae under food depletion conditions in wild type, epg-1 mutants, and epg-7 mutants. This experiment was completed once (wild type: n = 2,964; epg-7: n = 602; epg-1: n = 215). Statistical analysis of L1 survival shows that epg-7 mutants exhibit significant difference to wild type and also to epg-1 mutants (log-rank test, P = 0.000).


( T ) Loss of function of epg - 7 does not ameliorate degeneration of mec - 4 : : gfp - labeled touch neurons caused by mec - 4 ( u231 ) .

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(T) Loss of function of epg-7 does not ameliorate the degeneration of mec-4::gfp-labeled touch neurons caused by mec-4(u231).


( U ) Survival curves of wild type various mutants . sqst - 1 ; epg - 7 mutants have a shortened mean maximum life span . ( P = 0 . 000 for epg - 7 or wild type vs . sqst - 1 ; epg - 7 in three repeats ; log - rank ) .

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(U) Survival curves of wild type and various mutants. sqst-1;epg-7 mutants have a shortened mean and maximum life span. (P = 0.000 for epg-7 or wild type vs. sqst-1;epg-7 in all three repeats; log-rank test).


( V ) Survival of hydrogen peroxide - treated . ( In three repeats , P = 0 . 000 for epg - 7 vs . sqst - 1 ; epg - 7 ; P > 0 . 2 for wild type vs . sqst - 1 ; epg - 7 ; log - rank ) .

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

(V) Survival of hydrogen peroxide-treated animals. (In all three repeats, P = 0.000 for epg-7 vs. sqst-1;epg-7; P > 0.2 for wild type vs. sqst-1;epg-7; log-rank test).


( W ) Survival of at indicated time after heat shock treatment . ( P < 0 . 05 for wild type vs . epg - 7 or epg - 7 vs . sqst - 1 ; epg - 7 at 5 , 6 , 7 h ) .

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

(W) Survival of animals at the indicated time after heat shock treatment. (P < 0.05 for wild type vs. epg-7 or epg-7 vs. sqst-1;epg-7 at 5, 6, and 7 h).


( X ) Dauer formation of daf - 2 ( e1370 ) mutants in different backgrounds .

(X) Dauer formation of daf-2(e1370) mutants in different backgrounds.


( D ) A large number of SQST - 1 : : GFP accumulate in epg - 7 mutant carrying an epg - 7 ( del Atg11 ) transgene .

O O O O O O O B-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

(D) A large number of SQST-1::GFP aggregates accumulate in epg-7 mutant embryos carrying an epg-7(del Atg11) transgene.


( E F ) No SQST - 1 are detected in epg - 7 mutants carrying an epg - 7 : : gfp reporter . ( E ) DAPI image of shown in F .

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

(E and F) No SQST-1 aggregates are detected in epg-7 mutants carrying an epg-7::gfp reporter. (E) DAPI image of the embryo shown in F.


( G ) EPG - 7 : : GFP is weakly expressed diffusely localized in cytoplasm in wild - type .

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

(G) EPG-7::GFP is weakly expressed and diffusely localized in the cytoplasm in wild-type embryos.


( H - I ) EPG - 7 : : GFP is expressed in pharyngeal ( H , arrow ) in tail region ( I ) at larval stages .

O O O O O B-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

(H-I) EPG-7::GFP is expressed in pharyngeal muscles (H, arrow) and cells in the tail region (I) at larval stages.


( J ) Compared with wild - type , of EPG - 7 : : GFP are dramatically elevated in epg - 4 mutants , but remain unchanged in sqst - 1 mutants .

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

(J) Compared with wild-type animals, levels of EPG-7::GFP are dramatically elevated in epg-4 mutants, but remain unchanged in sqst-1 mutants.


( K ) expression level of EPG - 7 : : GFP is dramatically elevated many EPG - 7 : : GFP are formed in lgg - 1 mutant .

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

(K) The expression level of EPG-7::GFP is dramatically elevated and many EPG-7::GFP aggregates are formed in lgg-1 mutant embryos.


( L M ) No EPG - 7 , detected by anti - EPG - 7 antibody , are formed in wild - type . ( L ) DAPI image of shown in M . ( N ) of EPG - 7 are dramatically elevated a large number of EPG - 7 are formed in atg - 3 mutants . ( O ) No EPG - 7 are formed in sqst - 1 mutant .

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

(L and M) No EPG-7 aggregates, detected by anti-EPG-7 antibody, are formed in wild-type embryos. (L) DAPI image of the embryo shown in M. (N) Levels of EPG-7 are dramatically elevated and a large number of EPG-7 aggregates are formed in atg-3 mutants. (O) No EPG-7 aggregates are formed in sqst-1 mutant embryos.


( A ) EPG - 7 colocalizes with SQST - 1 in epg - 8 mutant . Colocalization was defined as two showing > 70 % overlap in their signals . Insets show a magnified view .

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

(A) EPG-7 colocalizes with SQST-1 aggregates in epg-8 mutant embryos. Colocalization was defined as two aggregates showing >70% overlap in their fluorescence signals. Insets show a magnified view.


( B ) of EPG - 7 colocalized with SQST - 1 in indicated autophagy mutants .

(B) Percentage of EPG-7 aggregates colocalized with SQST-1 in indicated autophagy mutants.


( C ) A coimmunoprecipitation reveals that EPG - 7 associates with SQST - 1 .

(C) A coimmunoprecipitation assay reveals that EPG-7 associates with SQST-1.


( D ) EPG - 7 interacts with SQST - 1 in a pull - down .

(D) EPG-7 interacts with SQST-1 in a pull-down assay.


( E ) EPG - 7 self - associates in a pull - down .

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

(E) EPG-7 self-associates in a pull-down assay.


( F ) SDS - PAGE blue - native PAGE of purified EPG - 7 ( 520 - 714 ) . Lane M shows molecular marker . Lanes 1 , 2 , 3 , 4 contain 10 , 5 , 2 . 5 , 1 . 25 µg of , respectively .

O O O O O O O O O O O O B-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 O O O O O O O O O O O

(F) SDS-PAGE and blue-native PAGE of purified EPG-7(520-714) proteins. Lane M shows molecular weight marker. Lanes 1, 2, 3, and 4 contain 10, 5, 2.5, and 1.25 µg of proteins, respectively.


( G ) An EPG - 7 fragment containing 531 - 631 self - interacts .

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

(G) An EPG-7 fragment containing amino acids 531-631 self-interacts.


( H I ) SQST - 1 accumulate in epg - 7 mutant carrying an epg - 7 ( del 531 - 631 ) : : gfp transgene . ( H ) DAPI image of shown in I .

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

(H and I) SQST-1 aggregates accumulate in epg-7 mutant embryos carrying an epg-7(del 531-631)::gfp transgene. (H) DAPI image of the embryo shown in I.


( J K ) EPG - 7 ( del 531 - 631 ) : : GFP is weakly expressed diffusely localized in epg - 7 ( tm2508 ) . Its expression is not elevated in lgg - 1 ( RNAi ) . H - K show carrying same transgene . in J K were taken using same exposure time .

O O O O B-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 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

(J and K) EPG-7(del 531-631)::GFP is weakly expressed and diffusely localized in epg-7(tm2508) embryos. Its expression is not elevated in lgg-1(RNAi) animals. H-K show embryos carrying the same transgene. Images in J and K were taken using the same exposure time.


( L M ) SQST - 1 self - interacts ( L ) self - interaction is mediated by a fragment containing 26 - 181 ( M ) .

O O O O B-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

(L and M) SQST-1 self-interacts (L) and the self-interaction is mediated by a fragment containing amino acids 26-181 (M).


( N ) of SQST - 1 ( del 26 - 181 ) : : GFP are dramatically elevated in epg - 7 atg - 3 mutants .

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

(N) Levels of SQST-1(del 26-181)::GFP are dramatically elevated in epg-7 and atg-3 mutants.


( O P ) SQST - 1 ( del 26 - 181 ) : : GFP is weakly expressed diffusely localized in sqst - 1 ( ok2892 ) mutant . ( O ) DIC image of shown in P .

O O O O B-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 O O O O O O O

(O and P) SQST-1(del 26-181)::GFP is weakly expressed and diffusely localized in sqst-1(ok2892) mutant embryos. (O) DIC image of the embryo shown in P.


( Q R ) SQST - 1 ( del 26 - 181 ) : : GFP is expressed at dramatically elevated accumulates into a large number of in lgg - 1 ( Q ) epg - 7 ( R ) mutants .

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

(Q and R) SQST-1(del 26-181)::GFP is expressed at dramatically elevated levels and accumulates into a large number of aggregates in lgg-1 (Q) and epg-7 (R) mutants.


( A - D ) Expression of LGG - 1 in wild - type at ∼ 100 - ( A B ) , ∼ 200 - ( C ) , fourfold ( D ) stages . ( A ) DAPI image of shown in B .

O O O O O O O B-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 O O O O O O O O O O

(A-D) Expression of LGG-1 in wild-type embryos at the ∼100-cell (A and B), the ∼200-cell (C), and the fourfold (D) stages. (A) DAPI image of the embryo shown in B.


( E ) Loss of function of epg - 7 does not increase of LGG - 1 also does not suppress accumulation of LGG - 1 in epg - 3 mutant .

(E) Loss of function of epg-7 does not increase levels of LGG-1 and also does not suppress accumulation of LGG-1 in epg-3 mutant embryos.


( F - H ) Expression of LGG - 1 in epg - 7 mutant before ∼ 100 - stage ( F ) , at ∼ 200 - stage ( G ) , at fourfold stage ( H ) . in B - D F - H were taken using same exposure time .

O O O O O O O B-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 O O O O O O O O O O O O O O O O O O

(F-H) Expression of LGG-1 in epg-7 mutant embryos before the ∼100-cell stage (F), at the ∼200-cell stage (G), and at the fourfold stage (H). Images in B-D and F-H were taken using the same exposure time.


( I ) SQST - 1 are separable from LGG - 1 in epg - 7 mutant . Insets show a magnified view . ( J ) In epg - 6 mutant , LGG - 1 SQST - 1 are enlarged colocalized . ( K ) LGG - 1 SQST - 1 are smaller in largely separable in epg - 6 ; epg - 7 double mutants . ( I - K ) DAPI of shown in same row .

(I) SQST-1 aggregates are separable from LGG-1 puncta in epg-7 mutant embryos. Insets show a magnified view. (J) In epg-6 mutant embryos, LGG-1 puncta and SQST-1 aggregates are enlarged and colocalized. (K) LGG-1 and SQST-1 aggregates are smaller in size and largely separable in epg-6;epg-7 double mutants. (I-K) DAPI images of the embryos shown in the same row.


( L - O ) Enlarged LGG - 1 SQST - 1 are colocalized in epg - 4 mutants ( L M ) . Simultaneously depleting epg - 7 reduces of LGG - 1 SQST - 1 , which become largely separable ( N O ) . ( L N ) DAPI of shown in M O , respectively .

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 B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR 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

(L-O) Enlarged LGG-1 and SQST-1 aggregates are colocalized in epg-4 mutants (L and M). Simultaneously depleting epg-7 reduces the size of LGG-1 puncta and SQST-1 aggregates, which become largely separable (N and O). (L and N) DAPI images of the embryos shown in M and O, respectively.


( P ) In atg - 9 mutant , LGG - 1 largely colocalize with SQST - 1 at comma stage . Notably , there are more SQST - 1 than LGG - 1 . ( Q ) SQST - 1 are separable from LGG - 1 in atg - 9 ; epg - 7 mutants at comma stage .

(P) In atg-9 mutant embryos, LGG-1 puncta largely colocalize with SQST-1 aggregates at the comma stage. Notably, there are more SQST-1 aggregates than LGG-1 puncta. (Q) SQST-1 aggregates are separable from LGG-1 puncta in atg-9;epg-7 mutants at the comma stage.


( R ) of LGG - 1 colocalized with SQST - 1 in indicated autophagy mutants . Comma - stage were examined for atg - 9 atg - 9 ; epg - 7 mutants . ∼ 200 - stage were examined for other autophagy mutants .

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

(R) Percentage of LGG-1 puncta colocalized with SQST-1 aggregates in indicated autophagy mutants. Comma-stage embryos were examined for atg-9 and atg-9;epg-7 mutants. ∼200-cell stage embryos were examined for other autophagy mutants.


( A ) EPG - 7 coimmunoprecipitates with ATG - 9 : : HA .

(A) EPG-7 coimmunoprecipitates with ATG-9::HA.


( B ) EPG - 7 directly interacts with ATG - 9 in a GST pull - down .

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

(B) EPG-7 directly interacts with ATG-9 in a GST pull-down assay.


( C D ) Coimmunoprecipitation reveals that EPG - 7 associates with LGG - 3 ( C )

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

(C and D) Coimmunoprecipitation reveals that EPG-7 associates with LGG-3 (C)


( C D ) Coimmunoprecipitation reveals that EPG - 7 associates with LGG - 1 ( D ) .

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

(C and D) Coimmunoprecipitation reveals that EPG-7 associates with LGG-1 (D).


( E ) EPG - 7 directly interacts with LGG - 3 .

(E) EPG-7 directly interacts with LGG-3.


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

(F) EPG-7 fragments containing amino acids 852-1003 or 1004-1142, but not 762-851, interact with LGG-1.


( G ) Fragments of EPG - 7 containing 762 - 851 or 852 - 1003 directly interact with ATG - 18 .

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

(G) Fragments of EPG-7 containing amino acids 762-851 or 852-1003 directly interact with ATG-18.


( H ) Fragments of EPG - 7 containing 1 - 704 or 1143 - 1330 directly interact with UNC - 51 .

(H) Fragments of EPG-7 containing amino acids 1-704 or 1143-1330 directly interact with UNC-51.


( I ) SQST - 1 associates with LGG - 1 in vivo . Embryonic extracts from epg - 7 mutants show that interaction of SQST - 1 with LGG - 1 is EPG - 7 independent .

(I) SQST-1 associates with LGG-1 in vivo. Embryonic extracts from epg-7 mutants show that interaction of SQST-1 with LGG-1 is EPG-7 independent.


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

(J) Fragments of SQST-1 containing amino acids 351-499 and 604-693, but not 500-603, interact with LGG-1.


( K ) Fragments of SQST - 1 - containing 418 - 499 604 - 630 bind to LGG - 1 . Mutating putative LIR in 604 - 630 fragment ( 605YPDL608 to APDA ) does not affect binding .

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

(K) Fragments of SQST-1-containing amino acids 418-499 and 604-630 bind to LGG-1. Mutating the putative LIR in the 604-630 fragment (605YPDL608 to APDA) does not affect the binding.


( L M ) SQST - 1 ( del LGG - 1 - 1 + 2 ) : : GFP , in which two LGG - 1 - binding fragments ( 418 - 499 604 - 630 ) are deleted , is weakly expressed diffusely localized in cytoplasm in sqst - 1 ( ok2892 ) mutants . ( N O ) Loss of function of lgg - 1 dramatically elevates expression level of SQST - 1 ( del LGG - 1 - 1 + 2 ) : : GFP , which forms a large number of . ( L N ) DIC of shown in M O ,

O O O O B-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 O O O O O O O O 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 B-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 O O O O O O

(L and M) SQST-1(del LGG-1-1+2)::GFP, in which the two LGG-1-binding fragments (amino acids 418-499 and 604-630) are deleted, is weakly expressed and diffusely localized in the cytoplasm in sqst-1(ok2892) mutants. (N and O) Loss of function of lgg-1 dramatically elevates the expression level of SQST-1(del LGG-1-1+2)::GFP, which forms a large number of aggregates. (L and N) DIC images of the embryos shown in M and O, respectively


( P ) SQST - 1 ( del LGG - 1 - 1 + 2 ) : : GFP colocalize with EPG - 7 in lgg - 1 ; sqst - 1 mutants . Insets show a magnified view .

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

(P) SQST-1(del LGG-1-1+2)::GFP aggregates colocalize with EPG-7 in lgg-1;sqst-1 mutants. Insets show a magnified view.


Formation of ATG - 9 in various autophagy mutants . ( A B ) In wild - type , ATG - 9 : : GFP is diffusely localized in cytoplasm . ( A ) DIC image of shown in B . Insets show a magnified view . ( C ) ATG - 9 : : GFP forms a large number of small , intense in epg - 1 mutants . ( D E ) Accumulation of ATG - 9 : : GFP in unc - 51 ( D ) epg - 1 ( E ) mutants is independent of lgg - 1 . ( F ) ATG - 9 : : GFP largely disappear in epg - 1 ; epg - 7 mutants . ( G ) ATG - 9 : : GFP accumulates into large punctate structures in epg - 6 mutants . ( H ) Loss of function of lgg - 1 suppresses accumulation of ATG - 9 : : GFP in epg - 6 mutants . ( I ) Loss of function of epg - 7 has no effect on formation of ATG - 9 : : GFP in epg - 6 mutants . ATG - 9 : : GFP are smaller in epg - 6 ; epg - 7 mutants than those in epg - 6 single mutants . ( J - L ) ATG - 9 : : GFP accumulates into large punctate structures in epg - 4 mutants ( J ) accumulation is suppressed by simultaneously depleting activity of lgg - 1 ( K ) or epg - 7 ( L ) . ( M N ) In epg - 8 mutants , ATG - 9 accumulates into ( M ) that are suppressed by loss of lgg - 1 activity ( N ) . ( O ) epg - 7 mutants exhibit same distribution pattern of ATG - 9 : : GFP as wild - type . ( P ) of ATG - 9 : : GFP colocalized with EPG - 7 SQST - 1 in indicated autophagy mutants .

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

Formation of ATG-9 puncta in various autophagy mutants. (A and B) In wild-type embryos, ATG-9::GFP is diffusely localized in the cytoplasm. (A) DIC image of the embryo shown in B. Insets show a magnified view. (C) ATG-9::GFP forms a large number of small, intense puncta in epg-1 mutants. (D and E) Accumulation of ATG-9::GFP puncta in unc-51(D) and epg-1 (E) mutants is independent of lgg-1. (F) The ATG-9::GFP puncta largely disappear in epg-1;epg-7 mutants. (G) ATG-9::GFP accumulates into large punctate structures in epg-6 mutants. (H) Loss of function of lgg-1 suppresses the accumulation of ATG-9::GFP puncta in epg-6 mutants. (I) Loss of function of epg-7 has no effect on the formation of ATG-9::GFP puncta in epg-6 mutants. The ATG-9::GFP puncta are smaller in epg-6;epg-7 mutants than those in epg-6 single mutants. (J-L) ATG-9::GFP accumulates into large punctate structures in epg-4 mutants (J) and the accumulation is suppressed by simultaneously depleting the activity of lgg-1 (K) or epg-7 (L). (M and N) In epg-8 mutants, ATG-9 accumulates into aggregates (M) that are suppressed by loss of lgg-1 activity (N). (O) epg-7 mutants exhibit the same distribution pattern of ATG-9::GFP as wild-type embryos. (P) Percentage of ATG-9::GFP puncta colocalized with EPG-7 and SQST-1 aggregates in indicated autophagy mutants.


( Q - T ) ATG - 9 : : GFP in epg - 6 ( Q ) , epg - 8 ( R ) , unc - 51 ( S ) , atg - 18 ( T ) mutants colocalize with EPG - 7 .

(Q-T) The ATG-9::GFP puncta in epg-6 (Q), epg-8 (R), unc-51 (S), and atg-18 (T) mutants colocalize with EPG-7 aggregates.


( U V ) ATG - 9 : : GFP in epg - 6 ( U ) epg - 8 ( V ) mutants colocalize with SQST - 1 . ( W X ) In unc - 51 ( W ) atg - 18 ( X ) mutants , ATG - 9 : : GFP partially colocalizes with SQST - 1 .

(U and V) ATG-9::GFP puncta in epg-6 (U) and epg-8 (V) mutants colocalize with SQST-1 aggregates. (W and X) In unc-51 (W) and atg-18 (X) mutants, ATG-9::GFP partially colocalizes with SQST-1 aggregates.


( A2 ) of W07G4 . 5 : : GFP in wild type , epg - 7 mutants , atg - 3 L1 larvae before after 6 h starvation treatment . ( B2 ) Model for role of epg - 7 in degradation of SQST - 1 . SQST - 1 directly interacts with EPG - 7 is recruited into EPG - 7 . EPG - 7 also associates with multiple Atg , which may be recruited to SQST - 1 - EPG - 7 in a hierarchical order . IM , isolation . ( C2 ) Hierarchical relationship of scaffold EPG - 2 EPG - 7 other essential ATG EPG in aggrephagy

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

(A2) Levels of W07G4.5::GFP in wild type, epg-7 mutants, and atg-3 L1 larvae before and after 6 h starvation treatment. (B2) Model for the role of epg-7 in degradation of SQST-1 aggregates. SQST-1 directly interacts with EPG-7 and is recruited into EPG-7 aggregates. EPG-7 also associates with multiple Atg proteins, which may be recruited to the SQST-1-EPG-7 complex in a hierarchical order. IM, isolation membrane. (C2) Hierarchical relationship of the scaffold proteins EPG-2 and EPG-7 and other essential ATG and EPG proteins in the aggrephagy pathway


( A B ) C17E4 . 2 : : GFP is weakly expressed diffusely localized in a wild - type . ( A ) DIC image of shown in B . ( C D ) C17E4 . 2 : : GFP is expressed at dramatically elevated accumulates into many in lgg - 1 ( C ) epg - 7 ( D ) mutant .

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

(A and B) C17E4.2::GFP is weakly expressed and diffusely localized in a wild-type embryo. (A) DIC image of the embryo shown in B. (C and D) C17E4.2::GFP is expressed at dramatically elevated levels and accumulates into many aggregates in lgg-1 (C) and epg-7 (D) mutant embryos.


( E F ) W07G4 . 5 : : GFP is expressed in intestine a few are formed in a wild - type at fourfold stage . ( E ) DIC image of shown in F . ( G H ) W07G4 . 5 : : GFP is expressed at dramatically elevated accumulates into a large number of in atg - 3 ( G ) epg - 7 ( H ) mutants .

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

(E and F) W07G4.5::GFP is expressed in the intestine and a few aggregates are formed in a wild-type embryo at the fourfold stage. (E) DIC image of the embryo shown in F. (G and H) W07G4.5::GFP is expressed at dramatically elevated levels and accumulates into a large number of aggregates in atg-3 (G) and epg-7 (H) mutants.


( I ) of C33D9 . 6 : : GFP W07G4 . 5 : : GFP are dramatically elevated in atg - 3 epg - 7 mutant .

(I) Levels of C33D9.6::GFP and W07G4.5::GFP are dramatically elevated in atg-3 and epg-7 mutant embryos.


( J K ) C17E4 . 2 in atg - 3 mutants overlap with EPG - 7 ( J ) , but are separable from SQST - 1 ( K ) . Insets show a magnified view . ( L ) Colocalization frequency of C17E4 . 2 , C33D9 . 6 , W07G4 . 5 with EPG - 7 SQST - 1 in atg - 3 mutants .

(J and K) C17E4.2 aggregates in atg-3 mutants overlap with EPG-7 aggregates (J), but are separable from SQST-1 aggregates (K). Insets show a magnified view. (L) Colocalization frequency of C17E4.2, C33D9.6, and W07G4.5 aggregates with EPG-7 and SQST-1 aggregates in atg-3 mutants.


( M ) SQST - 1 : : GFP accumulates in hypodermal in epg - 7 mutants at larval stage . ( N ) SQST - 1 : : GFP disappear in rheb - 1 ( RNAi ) ; epg - 7 mutant larvae . ( O ) Dramatic accumulation of SQST - 1 : : GFP in hypodermal in atg - 3 mutant larvae . ( P ) SQST - 1 : : GFP persist in rheb - 1 ( RNAi ) ; atg - 3 mutant larvae .

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

(M) SQST-1::GFP accumulates in hypodermal cells in epg-7 mutants at the larval stage. (N) SQST-1::GFP aggregates disappear in rheb-1(RNAi);epg-7 mutant larvae. (O) Dramatic accumulation of SQST-1::GFP aggregates in hypodermal cells in atg-3 mutant larvae. (P) SQST-1::GFP aggregates persist in rheb-1(RNAi);atg-3 mutant larvae.


( Q - T ) At larval stages , W07G4 . 5 : : GFP forms in intestinal ( Q R ) . These disappear upon RNAi knockdown of let - 363 ( S T ) . ( U - X ) W07G4 . 5 : : GFP accumulate in intestinal in epg - 7 mutant larvae ( U V ) . These disappear when let - 363 is simultaneously depleted ( W X ) . ( Y Z ) In atg - 3 mutants , W07G4 . 5 : : GFP accumulate in intestinal ( Y ) persist after RNAi knockdown of let - 363 ( Z ) .

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

(Q-T) At larval stages, W07G4.5::GFP forms aggregates in intestinal cells (Q and R). These aggregates disappear upon RNAi knockdown of let-363 (S and T). (U-X) W07G4.5::GFP aggregates accumulate in intestinal cells in epg-7 mutant larvae (U and V). These aggregates disappear when let-363 is simultaneously depleted (W and X). (Y and Z) In atg-3 mutants, W07G4.5::GFP aggregates accumulate in intestinal cells (Y) and persist after RNAi knockdown of let-363 (Z).


( a ) Plasma ornithine concentrations in Oatrhg mice following intravenous injections of AAV - OAT at doses of 1x1013 ( n = 9 ) 3x1013 genome copies ( gc ) / Kg ( n = 6 ) , or AAV - GFP at dose of 1x1013 gc / Kg ( n = 9 ) . Normal range was established on wild - type mice ( n = 7 ) average ± 2SD was calculated . Data are shown as averages ± . * * * p < 0 . 001 , * * * * p < 0 . 0001 ( Two - way ANOVA ) .

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

(a) Plasma ornithine concentrations in Oatrhg mice following intravenous injections of AAV-OAT at the doses of 1x1013 (n=9) and 3x1013 genome copies (gc)/Kg (n=6), or AAV-GFP at the dose of 1x1013 gc/Kg (n=9). Normal range was established on wild-type mice (n=7) and average ± 2SD was calculated. Data are shown as averages ± SEM. ***p<0.001, ****p<0.0001 (Two-way ANOVA test).


( c ) Plasma ornithine lysine concentrations at 12 months of ( 10 - months post - injection ) in Oatrhg mice injected with AAV - OAT ( n = 3 ) or AAV - GFP ( n = 3 ) ; WT mouse controls ( n = 5 ) are also shown ; each sample was analyzed as triplicates , data are shown as averages ± . One - way ANOVA with Tukey correction ; * p < 0 . 05 ; * * * * p < 0 . 0001 .

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

(c) Plasma ornithine and lysine concentrations at 12 months of age (10-months post-injection) in Oatrhg mice injected with AAV-OAT (n=3) or AAV-GFP (n=3); WT mouse controls (n=5) are also shown; each sample was analyzed as triplicates, data are shown as averages ± SEM. One-way ANOVA test with Tukey correction; *p <0.05; ****p<0.0001.


( e ) Western blot for OAT GFP in livers of Oatrhg mice injected with AAV - GFP or AAV - OAT . A WT mouse liver was included as control . Vinculin was used as loading control .

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

(e) Western blot for OAT and GFP in livers of Oatrhg mice injected with AAV-GFP or AAV-OAT. A WT mouse liver was included as control. Vinculin was used as loading control.


( g ) Liver immunohistochemistry showing approximately 10 % of OAT - positive in Oatrhg mice injected with AAV - OAT . Livers of WT Oatrhg mice injected with AAV - GFP are shown as controls . 5x ( on left side ) 20x ( on right side ) fields of view are shown .

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

(g) Liver immunohistochemistry showing approximately 10% of OAT-positive cells in Oatrhg mice injected with AAV-OAT. Livers of WT and Oatrhg mice injected with AAV-GFP are shown as controls. 5x (images on the left side) and 20x (images on the right side) fields of view are shown.


( h ) Representative H & E of retinas of 11 - month - old Oatrhg mice injected with AAV - GFP or AVV - OAT at doses of 1x1013 or 3x1013 gc / Kg ; - matched WT mice uninjected Oatrhg mice were included as controls ( 40X ) . Abbreviations : OS , outer segment ; IS , inner segment ; RPE , retinal pigment epithelium ; ONL , outer layer ; INL , inner layer .

O O 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 B-CONTROLLED_VAR O O 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

(h) Representative H&E staining of the retinas of 11-month-old Oatrhg mice injected with AAV-GFP or AVV-OAT at the doses of 1x1013 or 3x1013 gc/Kg; age-matched WT mice and uninjected Oatrhg mice were included as controls (40X). Abbreviations: OS, outer segment; IS, inner segment; RPE, retinal pigment epithelium; ONL, outer nuclear layer; INL, inner nuclear layer.


( a ) Representative electron of mouse retina of an AAV - GFP injected Oatrhg mouse showing retinal pigmented epithelium ( RPE ) adjacent abnormal outer segment ( OS ) disk membranes ( black arrows ) . Phagolysosomes containing undigested OS disc membranes are present ( asterisks ) . ( b ) Retina of AAV - OAT injected Oatrhg mice showing photoreceptor disk membranes ( arrows ) RPE ultrastructure comparable to matched WT mice ( c , arrows ) . ( c ) Control retina of WT mouse is shown as comparison .

O 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 B-CONTROLLED_VAR 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

(a) Representative electron micrographs of the mouse retina of an AAV-GFP injected Oatrhg mouse showing retinal pigmented epithelium (RPE) and adjacent abnormal outer segment (OS) disk membranes (black arrows). Phagolysosomes containing undigested OS disc membranes are present (asterisks). (b) Retina of AAV-OAT injected Oatrhg mice showing photoreceptor disk membranes (white arrows) and RPE ultrastructure comparable to age matched WT mice (c, white arrows). (c) Control retina of WT mouse is shown as comparison.


( a ) Plasma ornithine concentrations in AAV - GFP - ( n = 3 ) AAV - OAT - injected ( n = 3 ) OatΔ mice . Normal range of wild - type mice is also shown . Each value plotted represents mean ± ; Two - way ANOVA , * p < 0 . 05 , * * p < 0 . 01 .

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

(a) Plasma ornithine concentrations in AAV-GFP- (n=3) and AAV-OAT-injected (n=3) OatΔ mice. Normal range of wild-type mice is also shown. Each value plotted represents mean ± SEM; Two-way ANOVA test, *p <0.05, **p<0.01.


( c ) Retinal H & E ( 40X ) of OatΔ mice injected with either AAV - GFP or AAV - OAT . Representative of three mice per groups are shown ; WT mice were included as control . Abbreviations : OS , outer segment ; IS , inner segment ; RPE , retinal pigment epithelium ; ONL , outer layer ; INL , inner layer .

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

(c) Retinal H&E (40X) of OatΔ mice injected with either AAV-GFP or AAV-OAT. Representative of three mice per groups are shown; WT mice were included as control. Abbreviations: OS, outer segment; IS, inner segment; RPE, retinal pigment epithelium; ONL, outer nuclear layer; INL, inner nuclear layer.


A - D Progression of microglial infiltration in ONL . Early in course of rod degeneration at P18 , few isolated apoptotic TUNEL - positive nuclei ( red , inset ) were present in ONL , with Iba1 + microglialsomata processes ( green ) confined to inner retina ( A ) . At P21 , microglia in outer plexiform layer ( OPL ) extended radial processes into ONL ( inset ) , with a concurrent increase in number of TUNEL + nuclei ( B ) . At P30 , retinalmicroglia infiltrated entire depth of markedly thinned ONL subretinal space ( SRS ) ( inset ) ( C ) . At P46 , microglia became less dense in ONL reverted to a more ramified ( D ) . Scale bar , 50 μm . E - G Quantitative analyses demonstrate that ONL atrophy progression , characterized by ONL thickness ( empty symbols ) number of rows of ONLnuclei ( filled symbols ) ( E ) , occurred concurrently with progression of ONL apoptosis , followed as density of TUNEL + nuclei in ONL ( F ) , microglia infiltration , followed as microglial density in ONL ( G ) ( n = 3 at each time point ) . Data points error bars indicate mean ± . H - K Higher magnification showing microglial process infiltration ( H ) , phagosome formation in infiltrating processes ( I ) , microglial transformation into amoeboid morphologies containing multiple phagosomes ( J , K ) .

O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-D Progression of microglial infiltration in the ONL. Early in the course of rod degeneration at P18, few isolated apoptotic TUNEL-positive nuclei (red, inset) were present in the ONL, with Iba1+microglialsomata and processes (green) confined to the inner retina (A). At P21, microglia in the outer plexiform layer (OPL) extended radial processes into the ONL (inset), with a concurrent increase in the number of TUNEL+nuclei (B). At P30, retinalmicroglia infiltrated the entire depth of the markedly thinned ONL and the subretinal space (SRS) (inset) (C). At P46, microglia became less dense in the ONL and reverted to a more ramified morphology (D). Scale bar, 50 μm.E-G Quantitative analyses demonstrate that ONL atrophy progression, characterized by ONL thickness (empty symbols) and number of rows of ONLnuclei (filled symbols) (E), occurred concurrently with the progression of ONL apoptosis, followed as the density of TUNEL+nuclei in the ONL (F), and microglia infiltration, followed as microglial density in the ONL (G) (n = 3 animals at each time point). Data points and error bars indicate mean ± SEM.H-K Higher magnification images showing microglial process infiltration (H), phagosome formation in infiltrating processes (I), and microglial transformation into amoeboid morphologies containing multiple phagosomes (J,K).


A - C Microglia infiltrating ONL demonstrate upregulation of phagocytic molecule , CD68 . Top in ( A ) show composites of CD68 ( red ) , Iba1 ( green ) , DAPI ( blue ) ; bottom show same field with CD68 only . At P18 , non - infiltrating microglia expressed low or undetectable of CD68 , a lysosome - associated . At P21 - 23 , microglia infiltrating ONL strongly upregulated CD68 ( arrowheads ) . At P30 , CD68 immunopositivity in ONL microglia decreased was mostly confined to amoeboid in subretinal space . ( B ) Magnified view of inset in ( A ) demonstrates localization of CD68 to microglia phagosomes . ( C ) CD68 expression ( of immunopositivity as a fraction of ONL ) demonstrated a peaked at P22 decreased by P30 . Scale bar , 20 μm .

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A-C Microglia infiltrating the ONL demonstrate upregulation of the phagocytic molecule, CD68. Top images in (A) show composites of CD68 (red), Iba1 (green), and DAPI (blue) staining; bottom images show the same field with CD68 staining only. At P18, non-infiltrating microglia expressed low or undetectable levels of CD68, a lysosome-associated membrane protein. At P21-23, microglia infiltrating the ONL strongly upregulated CD68 (arrowheads). At P30, CD68 immunopositivity in ONL microglia decreased and was mostly confined to amoeboid cells in the subretinal space. (B) Magnified view of inset in (A) demonstrates localization of CD68 to microglia phagosomes. (C) CD68 expression (area of immunopositivity as a fraction of the ONL) demonstrated a peaked at P22 and decreased by P30. Scale bar, 20 μm.


D - F Upregulation of MFG - E8 , a secreted glycoprotein that serves as a bridging molecule for phagocytosis receptors , in ONL . ( D ) MFG - E8 was low or absent in ONL at P18 , but emerged at P21 - 23 , localizing to photoreceptor bodies in a column - like pattern ( arrow ) as a punctate signal within infiltrating microglia ( arrowhead ) , before decreasing throughout ONL at P30 . ( E ) A magnified orthogonal view of inset from ( D ) demonstrating punctate MFG - E8 immunopositivity within ONL microglia . ( F ) of MFG - E8 expression demonstrated a prominent emergence at P22 a subsequent decrement by P30 . Scale bar , 20 μm .

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D-F Upregulation of MFG-E8, a secreted glycoprotein that serves as a bridging molecule for phagocytosis receptors, in the ONL. (D) MFG-E8 was low or absent in the ONL at P18, but emerged at P21-23, localizing to photoreceptor cell bodies in a column-like pattern (arrow) and as a punctate signal within infiltrating microglia (arrowhead), before decreasing throughout the ONL at P30. (E) A magnified orthogonal view of the inset from (D) demonstrating punctate MFG-E8 immunopositivity within ONL microglia. (F) Quantification of MFG-E8 expression demonstrated a prominent emergence at P22 and a subsequent decrement by P30. Scale bar, 20 μm.


G - K Increase in phosphatidylserine ( PS ) exposure in ONL during photoreceptor degeneration . ( G ) At P18 , PS immunopositivity is near absent in ONL , but increased significantly in ONLsomata at P21 - 23 , before decreasing at P30 . ( H ) Quantitation of PS exposure by image ( by fractional of PS immunopositivity within ONL ( top ) , mean of PS in ONL ( bottom ) ) demonstrated a transient increase at P22 . ( I ) Co - immunolabeling of rods with rhodopsin at P22 demonstrates that PS exposure was present in a majority of rods ( inset shows at high magnification co - labeling of PS rhodopsin in multiple rodsomata ) . ( J ) Conversely , immunolabeling of cones with conearrestin demonstrates sparse co - localization of PS in cones ( inset shows close juxtaposition but no colocalization of PS arrestin labeling ) . ( K ) Scoring of rhodopsin + rods arrestin + cones for PS co - labeling demonstrates that a large majority of rods , but only a small minority of cones , showed PS exposure ( two - sided unpaired t - , n = 3 at P22 ) . Scale bars = 20 μm .

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G-K Increase in phosphatidylserine (PS) exposure in the ONL during photoreceptor degeneration. (G) At P18, PS immunopositivity is near absent in the ONL, but increased significantly in ONLsomata at P21-23, before decreasing at P30. (H) Quantitation of PS exposure by image analysis (by fractional area of PS immunopositivity within the ONL (top), and the mean intensity of PS staining in the ONL (bottom)) demonstrated a transient increase at P22. (I) Co-immunolabeling of rods with rhodopsin at P22 demonstrates that PS exposure was present in a majority of rods (inset shows at high magnification the co-labeling of PS and rhodopsin in multiple rodsomata). (J) Conversely, immunolabeling of cones with conearrestin demonstrates the sparse co-localization of PS in cones (inset shows close juxtaposition but no colocalization of PS and arrestin labeling). (K) Scoring of rhodopsin+rods and arrestin+cones for PS co-labeling demonstrates that a large majority of rods, but only a small minority of cones, showed PS exposure (two-sided unpaired t-test, n = 3 animals at P22). Scale bars = 20 μm.


A - C Phagocytosis of rods by infiltrating microglia . ( A ) Representative example of a Iba1 + microglial process extending into ONL with a phagosome at its terminal end . Each phagosome contained a photoreceptornucleus ( labeled with DAPI , arrow ) that was identified as a rodphotoreceptor by rhodopsin immunopositivity ( superposition of Iba1 + phagosome with rhodopsin + soma in orthogonal views ) . ( B ) Example of an amoeboid microglia in ONL with multiple phagosomes containing both rhodopsin - positive ( arrowhead ) rhodopsin - negative ( arrow ) nuclei . ( C ) Rhodopsin + nuclei can be localized within CD68 - positive phagosomes in infiltrating microglia , indicating phagocytic engulfment of rods . Scale bar , 10 μm . D , E peak of microglial phagocytic activity occurred around P21 declined subsequently , as measured by number of rods phagocytosed ( D ) number of photoreceptors phagocytosed per microglial ( E ) ( n = 3 at each time point ) .

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A-C Phagocytosis of rods by infiltrating microglia. (A) Representative example of a Iba1+microglial process extending into the ONL with a phagosome at its terminal end. Each phagosome contained a photoreceptornucleus (labeled with DAPI, arrow) that was identified as a rodphotoreceptor by rhodopsin immunopositivity (superposition of Iba1+phagosome with rhodopsin+soma in orthogonal views). (B) Example of an amoeboid microglia in the ONL with multiple phagosomes containing both rhodopsin-positive (arrowhead) and rhodopsin-negative (arrow) nuclei. (C) Rhodopsin+nuclei can be localized within CD68-positive phagosomes in infiltrating microglia, indicating phagocytic engulfment of rods. Scale bar, 10 μm.D, E The peak of microglial phagocytic activity occurred around P21 and declined subsequently, as measured by the number of rods phagocytosed (D) and the number of photoreceptors phagocytosed per microglial cell (E) (n = 3 animals at each time point).