Figure 1A , B . MDCK - SIAT1 cells co - incubated with Luc - WSN / 33 and MH - S CM fractions . Replication was quantified by luminescence . ( A ) Replication curves display average luminescence reads from 5 wells per condition ( measured at 7 . 5 min intervals - displayed here at 30 min intervals ) from 1 experiment representative of 7 independent experiments . ( B ) Mean calculated area under the luminescence curves ( AUC ) for individual wells normalized to the corresponding mean of the control condition from these 7 experiments . C . Luc - WSN / 33 replication in MDCK - SIAT1 cells incubated with flow cytometry - quantified MH - S AM - EVs at specified EV : cell ratios . D . Luc - WSN / 33 replication in MDCK - SIAT1 cells incubated with CM fractions of J2 retrovirus - immortalized primary mouse AMs . E . Luc - WSN / 33 replication in MDCK - SIAT1 cells incubated with primary rat AM - derived EVs . F . Luc - WSN / 33 replication in MDCK - SIAT1 cells incubated with human THP - 1 macrophage - derived EVs .

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Figure 1A, B. MDCK-SIAT1 cells co-incubated with Luc-WSN/33 and MH-S CM fractions. Replication was quantified by luminescence. (A) Replication curves display average luminescence reads from 5 wells per condition (measured at 7.5 min intervals - displayed here at 30 min intervals) from 1 experiment representative of 7 independent experiments. (B) Mean calculated area under the luminescence curves (AUC) for individual wells normalized to the corresponding mean of the control condition from these 7 experiments.C. Luc-WSN/33 replication in MDCK-SIAT1 cells incubated with flow cytometry-quantified MH-S AM-EVs at specified EV:cell ratios.D. Luc-WSN/33 replication in MDCK-SIAT1 cells incubated with CM fractions of J2 retrovirus-immortalized primary mouse AMs.E. Luc-WSN/33 replication in MDCK-SIAT1 cells incubated with primary rat AM-derived EVs.F. Luc-WSN/33 replication in MDCK-SIAT1 cells incubated with human THP-1 macrophage-derived EVs.


Figure 2A , B . Luc - WSN / 33 replication co - incubated with MH - S EVs was assessed in ( A ) human A549 and ( B ) mouse MLE - 12 AECs . Data represent mean luminescence AUC from individual wells normalized to the mean of the corresponding control condition from 3 independent experiments . C . MLE - 12 AECs incubated with MH - S EVs infected with PR / 8 influenza . Replication was quantified by RT - qPCR at 12 h post infection . Data represent mean M gene transcripts normalized to β actin from 3 independent experiments . E Total lung RNA was isolated from mice on day 1 post infection and virus was quantified by RT - qPCR . Data represent mean M gene transcripts from individual mice normalized to β actin from 3 independent experiments ( 4 - 5 mice per group ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A, B. Luc-WSN/33 replication co-incubated with MH-S EVs was assessed in (A) human A549 and (B) mouse MLE-12 AECs. Data represent mean luminescence AUC from individual wells normalized to the mean of the corresponding control condition from 3 independent experiments.C. MLE-12 AECs incubated with MH-S EVs infected with PR/8 influenza. Replication was quantified by RT-qPCR at 12 h post infection. Data represent mean M gene transcripts normalized to β actin from 3 independent experiments.E Total lung RNA was isolated from mice on day 1 post infection and virus was quantified by RT-qPCR. Data represent mean M gene transcripts from individual mice normalized to β actin from 3 independent experiments (4-5 mice per group).


Figure 3A . EVs isolated from MH - S AMs and A549 AECs were co - incubated in parallel with Luc - WSN / 33 ( MOI = 0 . 5 ) in MDCK - SIAT1 cells . Data represent mean luminescence AUC from individual wells normalized to the mean of the corresponding control condition from 3 independent experiments . B . EVs isolated from MH - S cells treated with increasing concentrations of CSE were co - incubated with Luc - WSN / 33 in MLE - 12 cells . Data represent mean luminescence at 12 h post infection from individual wells normalized to the mean of untreated AM - EVs from 3 independent experiments per condition .

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Figure 3A. EVs isolated from MH-S AMs and A549 AECs were co-incubated in parallel with Luc-WSN/33 (MOI = 0.5) in MDCK-SIAT1 cells. Data represent mean luminescence AUC from individual wells normalized to the mean of the corresponding control condition from 3 independent experiments.B. EVs isolated from MH-S cells treated with increasing concentrations of CSE were co-incubated with Luc-WSN/33 in MLE-12 cells. Data represent mean luminescence at 12 h post infection from individual wells normalized to the mean of untreated AM-EVs from 3 independent experiments per condition.


Figure 4A . MLE - 12 AECs co - incubated with the listed WT strains ( MOI = 0 . 4 ) plus AM - EVs ( grey bars , EV : cell = 1 ) . Data represent mean M gene transcripts at 12 h post infection relative to the mean of the corresponding untreated strain ( white bars ) , each normalized to β actin from 2 - 4 independent experiments per strain . C . MHS EVs were co - incubated with the listed Luc - expressing strains plus AM - EVs ( grey bars , EV : cell = 2 ) in MDCK - SIAT1 cells . Data represent mean luminescence AUC from individual wells normalized to the means of the corresponding untreated strain ( white bars ) from 3 independent experiments per strain .

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Figure 4A. MLE-12 AECs co-incubated with the listed WT strains (MOI = 0.4) plus AM-EVs (grey bars, EV:cell = 1). Data represent mean M gene transcripts at 12 h post infection relative to the mean of the corresponding untreated strain (white bars), each normalized to β actin from 2-4 independent experiments per strain.C. MHS EVs were co-incubated with the listed Luc-expressing strains plus AM-EVs (grey bars, EV:cell = 2) in MDCK-SIAT1 cells. Data represent mean luminescence AUC from individual wells normalized to the means of the corresponding untreated strain (white bars) from 3 independent experiments per strain.


Figure 5A . MDCK - SIAT1 cells were pretreated with MH - S EVs ( EV : cell = 2 , ' EV pretreat ' ) or control for 8 h , washed , and subsequently incubated with Luc - WSN / 33 ( MOI = 1 ) . Data represent mean luminescence AUC from individual wells normalized to the means of the control condition from 3 independent experiments . MLE - 12 cells were incubated with WT PR / 8 + AM - EVs at 4 ° C ( to permit surface binding but not entry ) for 1 h , washed , and processed for ( B ) RNA isolation ( B ) Data represent mean M gene transcripts relative to the means of the corresponding untreated condition , each normalized to β actin from 3 independent experiments . C . MLE - 12 cells were incubated with WT PR / 8 + AM - EVs at 4 ° C ( to permit surface binding but not entry ) for 1 h , washed , and processed for ( C ) immunofluorescence microscopy . ( C ) Cells in both conditions were fixed and stained with anti - NP or isotype control Ab . Images are representative of two independent experiments . Scale bars = 50 μmD . A549 cells were incubated with WSN / 33 or HK / 14 + AM - EVs for 40 min , washed , fixed , and permeabilized . Cells were subsequently stained with Anti - NP ( green ) , Anti - Rab - 7 ( purple ) Abs , and DAPI ( blue ) . Images shown are representative of 8 independent experiments . Scale bars = 5 μmE , F . Co - localization analysis of ( E ) NP : DAPI and ( F ) NP : Rab7 for each condition was performed on Z - stack confocal images images of WSN / 33 ( black ) or HK / 14 ( red ) infected A549s . Data represent the mean NP : DAPI co - localization ratios and the mean NP : Rab7 Mander ' s co - localization coefficient for individual cells from these 8 experiments .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A. MDCK-SIAT1 cells were pretreated with MH-S EVs (EV:cell = 2, 'EV pretreat') or control for 8 h, washed, and subsequently incubated with Luc-WSN/33 (MOI = 1). Data represent mean luminescence AUC from individual wells normalized to the means of the control condition from 3 independent experiments.MLE-12 cells were incubated with WT PR/8 + AM-EVs at 4 °C (to permit surface binding but not entry) for 1 h, washed, and processed for (B) RNA isolation (B) Data represent mean M gene transcripts relative to the means of the corresponding untreated condition, each normalized to β actin from 3 independent experiments.C. MLE-12 cells were incubated with WT PR/8 + AM-EVs at 4 °C (to permit surface binding but not entry) for 1 h, washed, and processed for (C) immunofluorescence microscopy. (C) Cells in both conditions were fixed and stained with anti-NP or isotype control Ab. Images are representative of two independent experiments. Scale bars = 50 μmD. A549 cells were incubated with WSN/33 or HK/14 + AM-EVs for 40 min, washed, fixed, and permeabilized. Cells were subsequently stained with Anti-NP (green), Anti-Rab-7 (purple) Abs, and DAPI (blue). Images shown are representative of 8 independent experiments. Scale bars = 5 μmE, F. Co-localization analysis of (E) NP:DAPI and (F) NP:Rab7 for each condition was performed on Z-stack confocal images images of WSN/33 (black) or HK/14 (red) infected A549s. Data represent the mean NP:DAPI co-localization ratios and the mean NP:Rab7 Mander's co-localization coefficient for individual cells from these 8 experiments.


Figure 6A - D . Replication assessed by luminescence following 10 nM Baf - A1 addition at the indicated times following synchronized uptake of bound Luc - expressing virions . ( A , B ) Shown are examples from 1 experiment representative of a total of 3 - 11 independent experiments per strain . Data represent mean luminescence AUC normalized to the mean of the corresponding strain incubated in 0 . 1 % DMSO ( dashed line ) . ( C , D ) The kinetics of endosomal escape by Luc - WSN / 33 and HK / 14 strains are depicted by the loss of inhibition by high dose Baf - A1 between ( C ) 0 - 20 min and ( D ) 60 - 80 min . Data represent mean slope ( change in % inhibition / time interval ) from 5 wells per plate from 3 - 11 independent experiments . Shown are dose response curves ( left ) and corresponding IC50 values ( right ) for effects of the endosomal acidification inhibitors Baf - A1 on replication of Luc - expressing strains . Curves display mean luminescence AUC expressed as a percentage of replication of corresponding strains in control conditions from 3 - 5 independent experiments ( mean of 3 wells per condition ) for each strain and inhibitor concentration . Bar graphs depict mean IC50 values from these experiments . F . Shown are dose response curves ( left ) and corresponding IC50 values ( right ) for effects of the endosomal acidification inhibitors Chl on replication of Luc - expressing strains . Curves display mean luminescence AUC expressed as a percentage of replication of corresponding strains in control conditions from 3 - 5 independent experiments ( mean of 3 wells per condition ) for each strain and inhibitor concentration . Bar graphs depict mean IC50 values from these experiments .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6A-D. Replication assessed by luminescence following 10 nM Baf-A1 addition at the indicated times following synchronized uptake of bound Luc-expressing virions. (A, B) Shown are examples from 1 experiment representative of a total of 3-11 independent experiments per strain. Data represent mean luminescence AUC normalized to the mean of the corresponding strain incubated in 0.1% DMSO (dashed line). (C, D) The kinetics of endosomal escape by Luc-WSN/33 and HK/14 strains are depicted by the loss of inhibition by high dose Baf-A1 between (C) 0-20 min and (D) 60-80 min. Data represent mean slope (change in % inhibition/time interval) from 5 wells per plate from 3-11 independent experiments.Shown are dose response curves (left) and corresponding IC50 values (right) for effects of the endosomal acidification inhibitors Baf-A1 on replication of Luc-expressing strains. Curves display mean luminescence AUC expressed as a percentage of replication of corresponding strains in control conditions from 3-5 independent experiments (mean of 3 wells per condition) for each strain and inhibitor concentration. Bar graphs depict mean IC50 values from these experiments.F. Shown are dose response curves (left) and corresponding IC50 values (right) for effects of the endosomal acidification inhibitors Chl on replication of Luc-expressing strains. Curves display mean luminescence AUC expressed as a percentage of replication of corresponding strains in control conditions from 3-5 independent experiments (mean of 3 wells per condition) for each strain and inhibitor concentration. Bar graphs depict mean IC50 values from these experiments.


Figure 7A . Data are mean 525em ratio from 488 / 445 nm excitation intensities of internalized FDx particles vs . pH of clamping buffer fit to a standard curve from two independent experiments . B . Representative images of PBS - and DR - EV - treated MDCK - SIAT1 cells . Scale bars for full size images = 50 μm . Scale bars for cropped images = 10 μm . FDx particles are shown green and DR - EV particles are shown purple . White arrows are FDx endosomes containing DR - EVs . C . Measured pH of FDx endosomes in PBS - ( left bar ) and DR - EV - treated ( right bar ) MDCK - SIAT1 cells . The pH of FDx endosomes containing DR - EVs in DR - EV - treated cells ( middle bar ) ( corresponding to white arrows in Fig 7B ) . Data are the means of individual data points representing the average calculated pH of each endosome population within 1 hpf in each of 5 independent experiments .

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Figure 7A. Data are mean 525em ratio from 488/445 nm excitation intensities of internalized FDx particles vs. pH of clamping buffer fit to a standard curve from two independent experiments.B. Representative images of PBS- and DR-EV-treated MDCK-SIAT1 cells. Scale bars for full size images = 50 μm. Scale bars for cropped images = 10 μm. FDx particles are shown green and DR-EV particles are shown purple. White arrows are FDx endosomes containing DR-EVs.C. Measured pH of FDx endosomes in PBS- (left bar) and DR-EV-treated (right bar) MDCK-SIAT1 cells. The pH of FDx endosomes containing DR-EVs in DR-EV-treated cells (middle bar) (corresponding to white arrows in Fig 7B). Data are the means of individual data points representing the average calculated pH of each endosome population within 1 hpf in each of 5 independent experiments.


Figure 1 ( A ) . Human neutrophils stained for CD66b after incubation with RBCs infected with GFP + P . falciparum parasites ( white arrows ) . Nuclei were stained with DAPI ( blue ) , neutrophils were stained with CD66b ( red ) , GFP expressing parasites ( green ) . Scale bar , 5 µm . The upper and lower panel show two different cells . ( B ) . Giemsa staining of freshly isolated human neutrophils from a healthy donor incubated with iRBCs harboring late stages P . falciparum parasites ( black arrows ) . ( C ) . Flow cytometry analysis of human neutrophils incubated with opsonized or non - opsonized GFP + - late - stage iRBCs . Free neutrophils were used as control . ( D ) . Quantification of GFP + iRBCs phagocytosed by neutrophils . The phagocytosis was analysed by flow cytometry ( FACS ) . Data information :

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O 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-PANEL_START O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(A). Human neutrophils stained for CD66b after incubation with RBCs infected with GFP+ P. falciparum parasites (white arrows). Nuclei were stained with DAPI (blue), neutrophils were stained with CD66b (red), GFP expressing parasites (green). Scale bar, 5 µm. The upper and lower panel show two different cells.(B). Giemsa staining of freshly isolated human neutrophils from a healthy donor incubated with iRBCs harboring late stages P. falciparum parasites (black arrows).(C). Flow cytometry analysis of human neutrophils incubated with opsonized or non-opsonized GFP+-late-stage iRBCs. Free neutrophils were used as control.(D). Quantification of GFP+ iRBCs phagocytosed by neutrophils. The phagocytosis was analysed by flow cytometry (FACS). Data information:


Figure 2 ( A ) . Interaction between neutrophils and opsonized ( red ) or non - opsonized ( blue ) GFP + iRBCs by time in a " pulse - chase " experiment . The percentage of GFP + - neutrophils following 5 min of co - culture was set as 100 % . ( C ) . Parasitemia ( percentage % ) of NF54 iRBCs cultures with or without daily addition of freshly isolated neutrophils . ( D ) . Parasitemia ( percentage % ) of cultures of RBC infected by different P . falciparum lines ( NF54 , Dd2 , SL , PfEMP1 - KO ) . The parasites were kept in culture for six days in the presence ( Neut . ) or absence ( Cont . ) of neutrophils . ( E ) . Percentage change in parasitemia following neutrophil challenge of RBCs infected by different parasite lines ( NF54 , Dd2 , SL , PfEMP1 - KO ) . ( F ) . Flow cytometric analysis of the effect of neutrophil challenge on parasite cell cycle progression . Neutrophils were stained with an anti - CD11b antibody , while iRBCs were labeled with SYBR green . Gating for iRBCs were performed by gating for the SYBR green and CD11b negative population , excluding the neutrophils that were SYBR green negative , but CD11b positive . ( G ) . Short term neutrophil killing of late - stage NF54 - luciferase positive RBCs in the presence ( red ) or absence ( blue ) of catalase . ( H ) . Parasitemia of cultures of iRBCs challenged with neutrophils in the presence or absence of catalase . ( I ) . Short - term luciferase - based killing assay using neutrophils from three different donors in the presence or absence of 5 uM cytochalasin D .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(A). Interaction between neutrophils and opsonized (red) or non-opsonized (blue) GFP+ iRBCs by time in a "pulse-chase" experiment. The percentage of GFP+-neutrophils following 5 min of co-culture was set as 100%.(C). Parasitemia (percentage %) of NF54 iRBCs cultures with or without daily addition of freshly isolated neutrophils.(D). Parasitemia (percentage %) of cultures of RBC infected by different P. falciparum lines (NF54, Dd2, SL, PfEMP1-KO). The parasites were kept in culture for six days in the presence (Neut.) or absence (Cont.) of neutrophils.(E). Percentage change in parasitemia following neutrophil challenge of RBCs infected by different parasite lines (NF54, Dd2, SL, PfEMP1-KO).(F). Flow cytometric analysis of the effect of neutrophil challenge on parasite cell cycle progression. Neutrophils were stained with an anti-CD11b antibody, while iRBCs were labeled with SYBR green. Gating for iRBCs were performed by gating for the SYBR green and CD11b negative population, excluding the neutrophils that were SYBR green negative, but CD11b positive.(G). Short term neutrophil killing of late-stage NF54-luciferase positive RBCs in the presence (red) or absence (blue) of catalase.(H). Parasitemia of cultures of iRBCs challenged with neutrophils in the presence or absence of catalase.(I). Short-term luciferase-based killing assay using neutrophils from three different donors in the presence or absence of 5 uM cytochalasin D.


Figure 3 ( A ) . Representative flow cytometric analysis of human neutrophils cultured alone ( Control ) , incubated with late - stage NF54 parasites ( wild type ) or PfEMP1 Knock out ( PfEMP1 KO ) GFP + iRBC . ( B ) . Flow cytometric quantification of the effect of trypsin treatment on neutrophil interaction with wild type and PfEMP1 KO GFP + iRBC . ( C ) . Luciferase - based killing assay and neutrophil elimination of late - stage wild type ( wt ) and PfEMP1ko iRBC .

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Figure 3(A). Representative flow cytometric analysis of human neutrophils cultured alone (Control), incubated with late-stage NF54 parasites (wild type) or PfEMP1 Knock out (PfEMP1 KO) GFP+ iRBC.(B). Flow cytometric quantification of the effect of trypsin treatment on neutrophil interaction with wild type and PfEMP1 KO GFP+ iRBC.(C). Luciferase-based killing assay and neutrophil elimination of late-stage wild type (wt) and PfEMP1ko iRBC.


Figure 5 ( B ) . Short - term ( 6h ) neutrophil killing of unselected NF54 and antibody - selected PFD1235w expressing parasite lines . ( C ) . Flow cytometric quantification of neutrophil interaction with MitoTracker ( APC + ) stained NF54 iRBCs and antibody - selected PFD1235w expressing iRBC . ( D ) . Percent reduction in parasitemia of NF54 and antibody - selected PFD1235w expressing parasites after 5 days of co - culture with neutrophils , compared to unchallenged parasites ( E ) . var gene transcription profiles measured by qRT - PCR of antibody - selected PFD1235w expressing parasite line cultured in the absence ( upper panel ) or presence ( lower panel ) of neutrophils . Steady state mRNA levels of each individual var gene was calculated as in A . I .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O 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-PANEL_START O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(B). Short-term (6h) neutrophil killing of unselected NF54 and antibody-selected PFD1235w expressing parasite lines.(C). Flow cytometric quantification of neutrophil interaction with MitoTracker (APC+) stained NF54 iRBCs and antibody-selected PFD1235w expressing iRBC.(D). Percent reduction in parasitemia of NF54 and antibody-selected PFD1235w expressing parasites after 5 days of co-culture with neutrophils, compared to unchallenged parasites(E). var gene transcription profiles measured by qRT-PCR of antibody-selected PFD1235w expressing parasite line cultured in the absence (upper panel) or presence (lower panel) of neutrophils. Steady state mRNA levels of each individual var gene was calculated as in A. I.


Figure 1SDS - PAGE analysis of the binding of Strep - tagged REC114 variants ( using a single Strep - tag ( WSHPQFEK ) ) to co - expressed His - MEI41 - 127 after purification on Strep - Tactin resin . SDS - PAGE analysis of the binding of His - MBP - REC114226 - 254 to co - expressed His - MEI4 variants after purification on Amylose resin . Native MS spectrum of co - purified Strep - REC114 and MEI41 - 127 . The main complexes are Strep - REC114 : MEI41 - 127 in ratios 2 : 1 and 4 : 2 . There are also free Strep - REC114 and 1 : 1 Strep - REC114 - MEI4 complex . Sedimentation velocity profiles of the REC114226 - 254 - MEI41 - 43 complex obtained at 280 nm at three concentrations ( 0 . 2 mg / ml , 1 mg / ml and 4 . 5 mg / ml ) in a solution containing 20 mM Tris pH 8 . 0 and 200 mM NaCl . Expected sedimentation coefficient for the 2 : 1 complex is 1 . 13S and for 4 : 2 is 1 . 58S . The sedimentation coefficient of the main peak increases with increasing protein concentration : s = 1 . 2S at 0 . 2 mg / ml , s = 1 . 44S at 1 mg / ml and s = 1 . 64S at 4 mg / ml . These results suggest a fast exchange between the two forms .

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Figure 1SDS-PAGE analysis of the binding of Strep-tagged REC114 variants (using a single Strep-tag (WSHPQFEK)) to co-expressed His-MEI41-127 after purification on Strep-Tactin resin. SDS-PAGE analysis of the binding of His-MBP-REC114226-254 to co-expressed His-MEI4 variants after purification on Amylose resin.Native MS spectrum of co-purified Strep-REC114 and MEI41-127. The main complexes are Strep-REC114:MEI41-127 in ratios 2:1 and 4:2. There are also free Strep-REC114 and 1:1 Strep-REC114-MEI4 complex.Sedimentation velocity profiles of the REC114226-254-MEI41-43 complex obtained at 280 nm at three concentrations (0.2 mg/ml, 1 mg/ml and 4.5 mg/ml) in a solution containing 20 mM Tris pH 8.0 and 200 mM NaCl. Expected sedimentation coefficient for the 2:1 complex is 1.13S and for 4:2 is 1.58S. The sedimentation coefficient of the main peak increases with increasing protein concentration: s=1.2S at 0.2 mg/ml, s=1.44S at 1 mg/ml and s=1.64S at 4 mg/ml. These results suggest a fast exchange between the two forms.


Figure 2Native MS spectrum of His - MBP - REC114226 - 254 . This protein is a dimer .

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Figure 2Native MS spectrum of His-MBP-REC114226-254. This protein is a dimer.


Figure 3E . Pull - down experiments of full - length Strep - tagged REC114 mutants indicated above the lanes with His - MBP - MEI41 - 43 . All proteins were first purified by affinity chromatography and gel filtration . A total of 0 . 9 % of the input ( lanes 1 - 6 ) and 1 % of the eluates ( lanes 7 - 12 ) were analyzed on 15 % SDS - PAGE gels stained with coomassie brilliant blue . Control lanes 1 - 2 and 7 - 8 show inputs and elutions of REC114 and MEI4 alone , respectively . The MEI4 sample contains also free MBP tag . The REC114 sample also contains a degradation product , likely corresponding to its PH domain . F . Pull - down experiments of Strep - tagged REC114 with His - MBP - MEI41 - 43 mutants indicated above the lanes . All proteins were first purified by affinity chromatography and gel filtration . A total of 0 . 9 % of the input ( lanes 1 - 4 ) and 1 % of the eluates ( lanes 5 - 8 ) were analyzed on 15 % SDS - PAGE gels stained with coomassie brilliant blue . The MEI4 sample contains also free MBP tag . The REC114 sample also contains a degradation product , likely corresponding to its PH domain .

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Figure 3E. Pull-down experiments of full-length Strep-tagged REC114 mutants indicated above the lanes with His-MBP-MEI41-43. All proteins were first purified by affinity chromatography and gel filtration. A total of 0.9% of the input (lanes 1-6) and 1% of the eluates (lanes 7-12) were analyzed on 15% SDS-PAGE gels stained with coomassie brilliant blue. Control lanes 1-2 and 7-8 show inputs and elutions of REC114 and MEI4 alone, respectively. The MEI4 sample contains also free MBP tag. The REC114 sample also contains a degradation product, likely corresponding to its PH domain.F. Pull-down experiments of Strep-tagged REC114 with His-MBP-MEI41-43 mutants indicated above the lanes. All proteins were first purified by affinity chromatography and gel filtration. A total of 0.9% of the input (lanes 1-4) and 1% of the eluates (lanes 5-8) were analyzed on 15% SDS-PAGE gels stained with coomassie brilliant blue. The MEI4 sample contains also free MBP tag. The REC114 sample also contains a degradation product, likely corresponding to its PH domain.


Figure 4Molecular mass determination of IHO11 - 281 , IHO1125 - 281 and IHO1196 - 245 by MALLS using Superose 6 column . The measured molecular mass of the main peak of each construct is 120 kDa , 67 kDa and 24 kDa , respectively . These values correspond to calculated molecular weights of IHO1 tetramers ( 125 kDa , 72 kDa and 24 kDa , respectively ) . The samples were injected at 5 - 10mg / ml .

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Figure 4Molecular mass determination of IHO11-281, IHO1125-281 and IHO1196-245 by MALLS using Superose 6 column. The measured molecular mass of the main peak of each construct is 120 kDa, 67 kDa and 24 kDa, respectively. These values correspond to calculated molecular weights of IHO1 tetramers (125 kDa, 72 kDa and 24 kDa, respectively). The samples were injected at 5-10mg/ml.


Figure 5Superose 6 gel filtration elution profile of His - MBP - IHO11 - 281 mixed with Strep - REC114 - His - MEI41 - 127 . Overlay with individual elution profiles of His - MBP - IHO11 - 281 and the Strep - REC114 - His - MEI41 - 127 complexSDS - PAGE analysis of fraction 1 - 13 of the Superose 6 gel filtration elution profile shown in A . L indicates input sample loaded onto the column . Superose 6 gel filtration elution profile of His - MBP - IHO124 - 281 mixed with Strep - REC114 - His - MEI41 - 127 , showing lack of binding . Overlay with individual elution profiles of His - MBP - IHO124 - 281 and the Strep - REC114 - His - MEI41 - 127 complexSDS - PAGE analysis of fraction 1 - 13 of the Superose 6 gel filtration elution profile shown in C . L indicates input sample loaded onto the column . ITC measurement of the interaction affinity between of His - MBP - IHO11 - 281 and Strep - REC114 - His - MEI41 - 127 . ITC measurement of the interaction affinity between of His - MBP - IHO124 - 281 and Strep - REC114 - His - MEI41 - 127 ITC measurement of the interaction affinity between of His - MBP - IHO11 - 23 and Strep - REC114 - His - MEI41 - 127 .

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Figure 5Superose 6 gel filtration elution profile of His-MBP-IHO11-281 mixed with Strep-REC114-His-MEI41-127. Overlay with individual elution profiles of His-MBP-IHO11-281 and the Strep-REC114-His-MEI41-127 complexSDS-PAGE analysis of fraction 1-13 of the Superose 6 gel filtration elution profile shown in A. L indicates input sample loaded onto the column.Superose 6 gel filtration elution profile of His-MBP-IHO124-281 mixed with Strep-REC114-His-MEI41-127, showing lack of binding. Overlay with individual elution profiles of His-MBP-IHO124-281 and the Strep-REC114-His-MEI41-127 complexSDS-PAGE analysis of fraction 1-13 of the Superose 6 gel filtration elution profile shown in C. L indicates input sample loaded onto the column.ITC measurement of the interaction affinity between of His-MBP-IHO11-281 and Strep-REC114-His-MEI41-127. ITC measurement of the interaction affinity between of His-MBP-IHO124-281 and Strep-REC114-His-MEI41-127 ITC measurement of the interaction affinity between of His-MBP-IHO11-23 and Strep-REC114-His-MEI41-127.


Figure 6ITC measurement of the interaction affinity between of His - MBP - IHO11 - 23 and the L104D mutant of Strep - REC1141 - 159 . SDS - PAGE analysis of fractions 1 - 13 of Superose 6 gel filtration elution profiles of His - MBP - IHO11 - 281 mixed with His - MBP - ANKRD311808 - 1857 , Strep - REC114 and His - MEI41 - 127 ( upper panel ) ; mixture of His - MBP - ANKRD311808 - 1857 with Strep - REC114 - His - MEI41 - 127 ( middle panel ) and His - MBP - IHO11 - 281 mixed with Strep - REC114 and His - MEI41 - 127 ( lower panel ) . L indicates input sample loaded onto the column . The red frame highlights the complex of IHO1 bound to REC114 and MEI4 ( lower panel ) and the lack of this complex in presence of ANKRD31 ( upper panel ) . Orange square shows equivalent elution of ANKRD31 , REC114 and MEI4 in presence or absence of IHO1 . Superose 6 gel filtration elution profiles of His - MBP - IHO11 - 281 mixed with His - MBP - ANKRD311808 - 1857 , Strep - REC114 and His - MEI41 - 127 ( orange ) ; His - MBP - ANKRD311808 - 1857 , Strep - REC114 and His - MEI41 - 127 ( blue ) and His - MBP - IHO11 - 281 mixed with Strep - REC114 - His - MEI41 - 127 ( green ) . Superose 6 gel filtration elution profiles of His - MBP - IHO11 - 281 mixed with His - MBP - ANKRD311808 - 1857 , Strep - REC114 and His - MEI41 - 127 ( orange ) ; His - MBP - ANKRD311808 - 1857 , Strep - REC114 and His - MEI41 - 127 ( blue ) and His - MBP - IHO11 - 281 ( green ) .

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Figure 6ITC measurement of the interaction affinity between of His-MBP-IHO11-23 and the L104D mutant of Strep-REC1141-159.SDS-PAGE analysis of fractions 1-13 of Superose 6 gel filtration elution profiles of His-MBP-IHO11-281 mixed with His-MBP-ANKRD311808-1857, Strep-REC114 and His-MEI41-127 (upper panel); mixture of His-MBP-ANKRD311808-1857 with Strep-REC114-His-MEI41-127 (middle panel) and His-MBP-IHO11-281 mixed with Strep-REC114 and His-MEI41-127 (lower panel). L indicates input sample loaded onto the column. The red frame highlights the complex of IHO1 bound to REC114 and MEI4 (lower panel) and the lack of this complex in presence of ANKRD31 (upper panel). Orange square shows equivalent elution of ANKRD31, REC114 and MEI4 in presence or absence of IHO1.Superose 6 gel filtration elution profiles of His-MBP-IHO11-281 mixed with His-MBP-ANKRD311808-1857, Strep-REC114 and His-MEI41-127 (orange); His-MBP-ANKRD311808-1857, Strep-REC114 and His-MEI41-127 (blue) and His-MBP-IHO11-281 mixed with Strep-REC114 -His-MEI41-127 (green). Superose 6 gel filtration elution profiles of His-MBP-IHO11-281 mixed with His-MBP-ANKRD311808-1857, Strep-REC114 and His-MEI41-127 (orange); His-MBP-ANKRD311808-1857, Strep-REC114 and His-MEI41-127 (blue) and His-MBP-IHO11-281 (green).


Figure 3B . Validation of the TNF - RSC interacting proteins . A549 cells were stimulated with FLAG - TNF - α for the indicated time and the TNF - RSCs were isolated as described in Figure 3A . TNF - α - stimulation - dependent interaction of the indicated proteins was confirmed by isolating the TNF - RSC and blotting with the indicated antibodies . The amount of the analyzed proteins in the input material used for the TNF - RSC isolation is also shown . C . Quantification of ubiquitin linkage peptides in the TNF - RSC . The box plot shows the relative abundance of K48 , K63 and linear ubiquitin linkage - specific peptides in the TNF - RSCs isolated from TNF - α - stimulated ( 15 min ) and control cells quantified by SILAC - based MS . E . The list of proteins enriched in SPATA2pull - down . The table shows proteins that were enriched in GFP - SPATA2pull - down . The columns under " Unstimulated " shows SILAC ratios of the indicated proteins in SPATA2 pull - downs from unstimulated cells , whereas the columns under " Stimulated ( TNF - α 15 min ) " shows SILAC ratios of the indicated proteins in SPATA2 pull - downs from cells stimulated with TNF - α for 15 minutes . F . Validation of TNF - α - stimulation - independent association of SPATA2 with CYLD . GFP - SPATA2 was immunoprecipitated from unstimulated and TNF - α - stimulated cells , and probed with CYLD antibody . G . HOIP interacts with SPATA2 . FLAG - HOIP expressing A20 . 2J cells were SILAC labeled and FLAG - HOIP was immunoprecipitated from unstimulated cells and associated proteins were quantified using LC - MS / MS . The table contains proteins that were enriched in HOIPpull - down compared to control pull - down . H . Validation of SPATA2 interaction with HOIP . GFP - tagged RNF31 was purified from unstimulated or TNF - a - stimulated ( 15 min ) HCT116 cells and the immunoprecipitates were blotted with SPATA2 , CYLD and GFP ( HOIP ) antibodies . The lower panels show expression of the indicated proteins in the input . The asterisk indicates unspecific band .

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Figure 3B. Validation of the TNF-RSC interacting proteins. A549 cells were stimulated with FLAG-TNF-α for the indicated time and the TNF-RSCs were isolated as described in Figure 3A. TNF-α-stimulation-dependent interaction of the indicated proteins was confirmed by isolating the TNF-RSC and blotting with the indicated antibodies. The amount of the analyzed proteins in the input material used for the TNF-RSC isolation is also shown.C. Quantification of ubiquitin linkage peptides in the TNF-RSC. The box plot shows the relative abundance of K48, K63 and linear ubiquitin linkage-specific peptides in the TNF-RSCs isolated from TNF-α-stimulated (15 min) and control cells quantified by SILAC-based MS.E. The list of proteins enriched in SPATA2pull-down. The table shows proteins that were enriched in GFP-SPATA2pull-down. The columns under "Unstimulated" shows SILAC ratios of the indicated proteins in SPATA2 pull-downs from unstimulated cells, whereas the columns under "Stimulated (TNF-α 15 min)" shows SILAC ratios of the indicated proteins in SPATA2 pull-downs from cells stimulated with TNF-α for 15 minutes.F. Validation of TNF-α-stimulation-independent association of SPATA2 with CYLD. GFP-SPATA2 was immunoprecipitated from unstimulated and TNF-α-stimulated cells, and probed with CYLD antibody.G. HOIP interacts with SPATA2. FLAG-HOIP expressing A20.2J cells were SILAC labeled and FLAG-HOIP was immunoprecipitated from unstimulated cells and associated proteins were quantified using LC-MS/MS. The table contains proteins that were enriched in HOIPpull-down compared to control pull-down.H. Validation of SPATA2 interaction with HOIP. GFP-tagged RNF31 was purified from unstimulated or TNF-a-stimulated (15 min) HCT116 cells and the immunoprecipitates were blotted with SPATA2, CYLD and GFP (HOIP) antibodies. The lower panels show expression of the indicated proteins in the input. The asterisk indicates unspecific band.


Figure 4B . The predicted PUB domain in the N - terminus of SPATA2 interacts with CYLD , but not with LUBAC or other TNF - RSC components . GFP - SPATA2 and the indicated deletion mutants were purified from A549 cells that were stimulated with TNF - α for 15 minutes , and the enriched proteins were quantified using SILAC - based MS . C . Validation of SPATA2 N - terminus interaction with CYLD . GFP - SPATA2 and the indicated deletion mutants were purified from A549 cells as described in Figure 5A , and association of CYLD was analyzed by immunoblotting . The lower panels show controls . D . The USP domain of CYLD interacts with SPATA2 . STREP - FLAG - CYLD and the indicated deletion mutants were purified from A549 cells using Step - Tactin sepharose and blotted with SPATA2 antibody . The lower panels show controls . E . The C - terminus of SPATA2 interacts with HOIP . A549 cells were transiently transfected with GFP - tagged SPATA2 , SPATA2 D200 and SPATA2 200 * , and SPATA2 and the mutants were immunoprecipitated using GFP - trap beads . The immunoprecipitates were immunoblotted with HOIP , CYLD and GFP ( HOIP ) antibodies . The lower panels show input control , and the * shows unspecific band .

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Figure 4B. The predicted PUB domain in the N-terminus of SPATA2 interacts with CYLD, but not with LUBAC or other TNF-RSC components. GFP-SPATA2 and the indicated deletion mutants were purified from A549 cells that were stimulated with TNF-α for 15 minutes, and the enriched proteins were quantified using SILAC-based MS.C. Validation of SPATA2 N-terminus interaction with CYLD. GFP-SPATA2 and the indicated deletion mutants were purified from A549 cells as described in Figure 5A, and association of CYLD was analyzed by immunoblotting. The lower panels show controls.D. The USP domain of CYLD interacts with SPATA2. STREP-FLAG-CYLD and the indicated deletion mutants were purified from A549 cells using Step-Tactin sepharose and blotted with SPATA2 antibody. The lower panels show controls.E. The C-terminus of SPATA2 interacts with HOIP. A549 cells were transiently transfected with GFP-tagged SPATA2, SPATA2 D200 and SPATA2 200*, and SPATA2 and the mutants were immunoprecipitated using GFP-trap beads. The immunoprecipitates were immunoblotted with HOIP, CYLD and GFP (HOIP) antibodies. The lower panels show input control, and the * shows unspecific band.


Figure 5B . Effect of SPATA2 knockdown on association of proteins with TNF - RSC . The list shows SILAC ratios of TNF - RSC - associated proteins in control cells and SPATA2 knockdown cells . The proteins that showed reduced association with the TNF - RSC after SPATA2 knockdown are highlighted red . C . Validation of SPATA2 - dependent association of CYLD with the TNF - RSC . The TNF - RSC was purified under the indicated conditions and association of SPATA2 , CYLD , RIPK1 and HOIP with the TNF - RSC was analyzed by immunoblotting . The input controls show expression of the analyzed proteins in cell lysates . The asterisk denotes unspecific bands .

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Figure 5B. Effect of SPATA2 knockdown on association of proteins with TNF-RSC. The list shows SILAC ratios of TNF-RSC-associated proteins in control cells and SPATA2 knockdown cells. The proteins that showed reduced association with the TNF-RSC after SPATA2 knockdown are highlighted red.C. Validation of SPATA2-dependent association of CYLD with the TNF-RSC. The TNF-RSC was purified under the indicated conditions and association of SPATA2, CYLD, RIPK1 and HOIP with the TNF-RSC was analyzed by immunoblotting. The input controls show expression of the analyzed proteins in cell lysates. The asterisk denotes unspecific bands.


Figure 6A . SPATA2 regulates TNF - a - induced NF - κB activation . HEK293T cells were transfected with the SPATA2 siRNA ( SMARTpool or with to individual siRNA ) or a non - targeting control siRNA , and NF - kB activation was monitored with luciferase - based NF - kB reporter assays . Error bars specify the standard error of the mean ( SEM ) of three independent experiments . p * * < 0 . 05 ( two - tailed , unpaired Student ´ s t - test ) . B . Knockdown of SPATA2 enhances expression of the NF - κB target genes . HEK293T cells were transfected with SPATA2 siRNA or a non - targeting control siRNA and the expression of the indicated mRNAs was measured in cells stimulated with TNF - a for 0 , 3 and 6 hours with qPCR . C . SPATA2 is involved in TNF - α - induced necroptosis . SPATA2 was knocked down in L929 cells using RNAi , and cells were pretreated with DMSO , Z - VAD , or Z - VAD + NEC1 for 1 hour followed by treatment with TNF - α for 2 . 5 hours . Cell viability was assessed with propidium iodide staining and flow cytometry . Knockdown of SPATA2 mRNA was verified using real time PCR . p * * < 0 . 05 ( two - tailed , unpaired Student ´ s t - test ) . D . Knockdown of SPATA2 reduces phosphorylation of MLKL in TNF - α - induced necroptosis . TNF - α - dependent necroptosis was induced in L929 as described in Figure 6C . Phosphorylation of MLKL was analyzed using S358 phospho - specific antibody .

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Figure 6A. SPATA2 regulates TNF-a-induced NF-κB activation. HEK293T cells were transfected with the SPATA2 siRNA (SMARTpool or with to individual siRNA) or a non-targeting control siRNA, and NF-kB activation was monitored with luciferase-based NF-kB reporter assays. Error bars specify the standard error of the mean (SEM) of three independent experiments. p** < 0.05 (two-tailed, unpaired Student´s t-test).B. Knockdown of SPATA2 enhances expression of the NF-κB target genes. HEK293T cells were transfected with SPATA2 siRNA or a non-targeting control siRNA and the expression of the indicated mRNAs was measured in cells stimulated with TNF-a for 0, 3 and 6 hours with qPCR.C. SPATA2 is involved in TNF-α-induced necroptosis. SPATA2 was knocked down in L929 cells using RNAi, and cells were pretreated with DMSO, Z-VAD, or Z-VAD+NEC1 for 1 hour followed by treatment with TNF-α for 2.5 hours. Cell viability was assessed with propidium iodide staining and flow cytometry. Knockdown of SPATA2 mRNA was verified using real time PCR. p** < 0.05 (two-tailed, unpaired Student´s t-test).D. Knockdown of SPATA2 reduces phosphorylation of MLKL in TNF-α-induced necroptosis. TNF-α-dependent necroptosis was induced in L929 as described in Figure 6C. Phosphorylation of MLKL was analyzed using S358 phospho-specific antibody.


Figure 1A ) Purified His - α - synuclein protein was agitated for 0 - 2 weeks ( lane 1 , 0weeks ; lanes 2 and 3 , 1week ; lanes 4 and 5 , 2weeks ) . Aliquots from each sample were separated into supernatant ( lanes 2 and 4 ) and pellet ( lanes3 and 5 ) fractions . These fractions were subjected to immunoblotting with anti - α - synuclein antibody . Numbers at left are molecular weight markers ( MW ) in kilodaltons . ( B ) Fibril formation of α - synuclein was measured with ProteoStat , an aggregation detection dye . a . u . , arbitrary units . ( C ) AFM images of α - synuclein agitated for 0 or 2 weeks were acquired in air using the dynamic mode . A high - magnification view of the boxed area is shown in the lower panel . The scale on the right represents the height of pixels in the image . Scale bars , 100 nm . ( D ) α - Synuclein inclusions in fibril - introduced HEK293 cells were detected immunocytochemically with anti - phosphorylated α - synuclein ( P - αSyn ) , anti - ubiquitin ( Ub ) , and anti - p62 ( p62 ) antibodies . Confocal images reconstructed in the z - axis along the white lines are shown in the right panel ( Z - axis ) . Blue , DAPI . Scale bar , 10 µm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A) Purified His-α-synuclein protein was agitated for 0-2 weeks (lane 1, 0weeks; lanes 2 and 3, 1week; lanes 4 and 5, 2weeks). Aliquots from each sample were separated into supernatant (lanes 2 and 4) and pellet (lanes3 and 5) fractions. These fractions were subjected to immunoblotting with anti-α-synuclein antibody. Numbers at left are molecular weight markers (MW) in kilodaltons.(B) Fibril formation of α-synuclein was measured with ProteoStat, an aggregation detection dye. a.u., arbitrary units.(C) AFM images of α-synuclein agitated for 0 or 2 weeks were acquired in air using the dynamic mode. A high-magnification view of the boxed area is shown in the lower panel. The scale on the right represents the height of pixels in the image. Scale bars, 100 nm.(D) α-Synuclein inclusions in fibril-introduced HEK293 cells were detected immunocytochemically with anti-phosphorylated α-synuclein (P-αSyn), anti-ubiquitin (Ub), and anti-p62 (p62) antibodies. Confocal images reconstructed in the z-axis along the white lines are shown in the right panel (Z-axis). Blue, DAPI. Scale bar, 10 µm.


Figure 2 ( A ) Autophagic clearance of α - synuclein inclusions was verified by immunocytochemical analysis with anti - phosphorylated α - synuclein ( P - αSyn ) and anti - LC3 ( LC3 ) antibodies . Confocal images reconstructed in the z - axis along the white lines are shown in the right panel ( Z - axis ) . Blue , DAPI . Scale bar , 10 µm . Phosphorylated α - synuclein - positive inclusions were sequestrated into LC3 - positive autophagosomes . ( B ) Similarly , this sequestration was confirmed in HEK293 cells stably expressing DsRed - LC3 . The upper and lower panels show α - synuclein fibrils - and mock - introduced cells , respectively . Blue , DAPI . Scale bar , 10 µm .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(A) Autophagic clearance of α-synuclein inclusions was verified by immunocytochemical analysis with anti-phosphorylated α-synuclein (P-αSyn) and anti-LC3 (LC3) antibodies. Confocal images reconstructed in the z-axis along the white lines are shown in the right panel (Z-axis). Blue, DAPI. Scale bar, 10 µm. Phosphorylated α-synuclein-positive inclusions were sequestrated into LC3-positive autophagosomes.(B) Similarly, this sequestration was confirmed in HEK293 cells stably expressing DsRed-LC3. The upper and lower panels show α-synuclein fibrils-and mock-introduced cells, respectively. Blue, DAPI. Scale bar, 10 µm.


Figure 3 ( A ) HEK293 cells stably expressing GFP - LC3 were transfected with control ( upper ) or Atg - 5 siRNA ( lower ) . After 36 h , α - synuclein fibrils were introduced into them for 4 h , followed by immunostaining with anti - phospho - α - synuclein antibody ( P - αSyn ) . Blue , DAPI . Scale bar , 10 µm . ( B ) The number of GFP - LC3 - positive α - synuclein inclusions ( % ) were assessed in randomly chosen fields ( n = 5 ) with 48 - 141 inclusions . Statistical analysis was performed with t - test ( * p < 0 . 01 ) . ( C ) Clearance of α - synuclein inclusions in control ( solid bar ) or Atg - 5 ( open bar ) knockdown cells was measured at 4 h and 24 h after α - synuclein fibrils . The number of phosphorylated α - synuclein - positive cells was assessed in randomly chosen fields ( n = 5 ) . Data from each experiment are were normalized to Cont - siRNA - 4 h ( 100 % ) and represented as relative value of α - synuclein inclusions ± s . e . m . Statistical analysis was performed with one - way ANOVA ( post - hoc Tukey ' s test ) . * p < 0 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(A) HEK293 cells stably expressing GFP-LC3 were transfected with control (upper) or Atg-5 siRNA (lower). After 36 h, α-synuclein fibrils were introduced into them for 4 h, followed by immunostaining with anti-phospho-α-synuclein antibody (P-αSyn). Blue, DAPI. Scale bar, 10 µm.(B) The number of GFP-LC3-positive α-synuclein inclusions (%) were assessed in randomly chosen fields (n = 5) with 48-141 inclusions. Statistical analysis was performed with t-test (*p<0.01).(C) Clearance of α-synuclein inclusions in control (solid bar) or Atg-5 (open bar) knockdown cells was measured at 4 h and 24 h after α-synuclein fibrils. The number of phosphorylated α-synuclein-positive cells was assessed in randomly chosen fields (n = 5). Data from each experiment are were normalized to Cont-siRNA-4 h (100%) and represented as relative value of α-synuclein inclusions ± s.e.m. Statistical analysis was performed with one-way ANOVA (post-hoc Tukey's test). *p<0.01


Figure 4 ( A ) Autophagy flux was analyzed by LC3 protein monitoring . α - Synuclein fibrils - introduced HEK293 cells were treated with ( + ) / without ( − ) bafilomycin A1 ( BafA1 ) for 2 h . Cell lysates were subjected to immunoblotting analysis using anti - LC3 ( upper panel ) or anti - actin ( lower panel ) antibodies . Quantification of the relative levels of LC3 - II / Actin or autophagic flux ( Baf + / Baf - ) is shown with the ratio . Relative level of LC3 - II / Actin was represented by mean ± s . d . as a graph . Statistical analysis was performed with one - way ANOVA ( post - hoc Tukey ' s test ) . * p < 0 . 05 . This experiment was repeated three times . ( B ) α - Synuclein fibrils were introduced into HEK293 cells , followed by addition of DMSO , 100 nM bafilomycin ( BafA1 ) , or 200 nM rapamycin ( Rapa ) . Cells were stained by anti - phosphorylated α - synuclein antibody ( green ) and DAPI ( blue ) 1 h ( left panels ) or 24 h ( right panels ) after introduction . ( C ) The number of phosphorylated α - synuclein - positive cells per unit area ( 0 . 6 mm2 ) was assessed in randomly chosen fields ( n = 6 ) . Data represent mean ± s . e . m . Statistical analysis was performed with one - way ANOVA ( post - hoc Tukey ' s test ) . * p < 0 . 05 , * * p < 0 . 001 . ( D ) Colocalization of α - synuclein inclusions and lysosomes . α - Synuclein inclusions ( P - αSyn ) and lysosomes ( LAMP1 ) were immunostained with anti - phospho - α - synuclein polyclonal antibody ( green ) and anti - LAMP1 antibody ( red ) 1 h ( left panel ) or 4 h ( middle panel ) after fibril introduction . Fibril - unintroduced cells were used as mock control ( right panel ) . Arrowheads and arrows indicate localized or unlocalized α - synuclein inclusions to lysosomes , respectively . Blue , DAPI . Scale bars , 10 µm . ( E ) Colocalization of α - synuclein inclusions ( n = 41 - 73 ) to lysosomes were measured in randomly chosen fields . The experiment was repeated three times . Data represent mean ± s . e . m . Statistical analysis was performed with one - way ANOVA ( post - hoc Tukey ' s test ) . * p < 0 . 01 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(A) Autophagy flux was analyzed by LC3 protein monitoring. α-Synuclein fibrils-introduced HEK293 cells were treated with (+)/without (−) bafilomycin A1 (BafA1) for 2 h. Cell lysates were subjected to immunoblotting analysis using anti-LC3 (upper panel) or anti-actin (lower panel) antibodies. Quantification of the relative levels of LC3-II/Actin or autophagic flux (Baf+/Baf-) is shown with the ratio. Relative level of LC3-II/Actin was represented by mean ± s.d. as a graph. Statistical analysis was performed with one-way ANOVA (post-hoc Tukey's test). *p<0.05. This experiment was repeated three times.(B) α-Synuclein fibrils were introduced into HEK293 cells, followed by addition of DMSO, 100 nM bafilomycin (BafA1), or 200 nM rapamycin (Rapa). Cells were stained by anti-phosphorylated α-synuclein antibody (green) and DAPI (blue) 1 h (left panels) or 24 h (right panels) after introduction.(C) The number of phosphorylated α-synuclein-positive cells per unit area (0.6 mm2) was assessed in randomly chosen fields (n = 6). Data represent mean ± s.e.m. Statistical analysis was performed with one-way ANOVA (post-hoc Tukey's test). *p<0.05, **p<0.001.(D) Colocalization of α-synuclein inclusions and lysosomes. α-Synuclein inclusions (P-αSyn) and lysosomes (LAMP1) were immunostained with anti-phospho-α-synuclein polyclonal antibody (green) and anti-LAMP1 antibody (red) 1 h (left panel) or 4 h (middle panel) after fibril introduction. Fibril-unintroduced cells were used as mock control (right panel). Arrowheads and arrows indicate localized or unlocalized α-synuclein inclusions to lysosomes, respectively. Blue, DAPI. Scale bars, 10 µm.(E) Colocalization of α-synuclein inclusions (n = 41-73) to lysosomes were measured in randomly chosen fields. The experiment was repeated three times. Data represent mean ± s.e.m. Statistical analysis was performed with one-way ANOVA (post-hoc Tukey's test). *p<0.01.


Figure 5 ( A ) HEK293 cells stably expressing GFP - LC3 were transfected with p62 - siRNA ( right panels ) or control - siRNA ( left panels ) . Phosphorylated α - synuclein - positive inclusions ( P - αSyn ) are indicated by arrowheads . In p62 - knockdown cells , the expression and accumulation of p62 were not detected , and inclusions were never sequestrated by GFP - LC3 autophagosomes . Scale bars , 20 µm . ( B ) The number of GFP - LC3 - positive α - synuclein inclusions ( % ) in Fig . 5A were assessed in randomly chosen fields with 32 - 44 inclusions ( n = 3 ) . Data represent mean ± s . e . m . Statistical analysis was performed with t - test ( * p < 0 . 05 ) . ( C ) α - Synuclein fibrils were introduced into control ( Cont ) or p62 - knockdown ( p62 ) cells . Four hours after introduction , cell lysates were subjected to immunoblotting analysis using anti - LC3 ( upper panel ) or anti - actin ( lower panel ) antibodies . Quantification of the relative levels of LC3 - II / Actin is shown with the ratio . Relative level of LC3 - II / Actin was represented by mean ± s . d . as a graph . Statistical analysis was performed with one - way ANOVA ( post - hoc Tukey ' s test ) . * p < 0 . 01 . This experiment was repeated three times . ( D ) Clearance of α - synuclein inclusions in control ( solid bar ) or p62 ( open bar ) knockdown cells was measured at 4 h and 24 h after α - synuclein fibrils . The number of phosphorylated α - synuclein - positive cells was assessed in randomly chosen fields ( n = 5 ) . Data from each experiment were normalized to Cont - siRNA - 4 h ( 100 % ) and represented as relative value of α - synuclein inclusions ± s . e . m . Statistical analysis was performed with one - way ANOVA ( post - hoc Tukey ' s test ) . * p < 0 . 01 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(A) HEK293 cells stably expressing GFP-LC3 were transfected with p62-siRNA (right panels) or control-siRNA (left panels). Phosphorylated α-synuclein-positive inclusions (P-αSyn) are indicated by arrowheads. In p62-knockdown cells, the expression and accumulation of p62 were not detected, and inclusions were never sequestrated by GFP-LC3 autophagosomes. Scale bars, 20 µm.(B) The number of GFP-LC3-positive α-synuclein inclusions (%) in Fig. 5A were assessed in randomly chosen fields with 32-44 inclusions (n = 3). Data represent mean ± s.e.m. Statistical analysis was performed with t-test (*p<0.05).(C) α-Synuclein fibrils were introduced into control (Cont) or p62-knockdown (p62) cells. Four hours after introduction, cell lysates were subjected to immunoblotting analysis using anti-LC3 (upper panel) or anti-actin (lower panel) antibodies. Quantification of the relative levels of LC3-II/Actin is shown with the ratio. Relative level of LC3-II/Actin was represented by mean ± s.d. as a graph. Statistical analysis was performed with one-way ANOVA (post-hoc Tukey's test). *p<0.01. This experiment was repeated three times.(D) Clearance of α-synuclein inclusions in control (solid bar) or p62 (open bar) knockdown cells was measured at 4 h and 24 h after α-synuclein fibrils. The number of phosphorylated α-synuclein-positive cells was assessed in randomly chosen fields (n = 5). Data from each experiment were normalized to Cont-siRNA-4 h (100%) and represented as relative value of α-synuclein inclusions ± s.e.m. Statistical analysis was performed with one-way ANOVA (post-hoc Tukey's test). *p<0.01.


Figure 6 ( A ) α - Synuclein fibrils were introduced into HEK293 cells stably expressing both DsRed - LC3 and EGFP - parkin . After 24 h , the cells were immunostained with anti - phosphorylated α - synuclein ( P - αSyn ) . There was no alteration in the localization of EGFP - parkin . The arrows indicate α - synuclein inclusion . Scale bar , 10 µm . ( B ) Mitochondrial clearance was confirmed in mock - ( upper panels ) or α - synuclein fibrils - introduced cells ( lower panels ) . After introduction , EGFP - parkin cells were treated with DMSO ( left ) or 10 µM CCCP for 4 h ( middle ) or 16 h ( right ) . Cells were examined immunocytochemically with anti - Tom20 ( red ) and anti - phosphorylated α - synuclein antibody ( P - αSyn ; aqua ) . Figures were presented as merged images . Individual images were provided in supplemental figure ( Fig . S8 ) . Scale bar , 10 µm .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(A) α-Synuclein fibrils were introduced into HEK293 cells stably expressing both DsRed-LC3 and EGFP-parkin. After 24 h, the cells were immunostained with anti-phosphorylated α-synuclein (P-αSyn). There was no alteration in the localization of EGFP-parkin. The arrows indicate α-synuclein inclusion. Scale bar, 10 µm.(B) Mitochondrial clearance was confirmed in mock- (upper panels) or α-synuclein fibrils-introduced cells (lower panels). After introduction, EGFP-parkin cells were treated with DMSO (left) or 10 µM CCCP for 4 h (middle) or 16 h (right). Cells were examined immunocytochemically with anti-Tom20 (red) and anti-phosphorylated α-synuclein antibody (P-αSyn; aqua). Figures were presented as merged images. Individual images were provided in supplemental figure (Fig. S8). Scale bar, 10 µm.


Figure 1Western blot for p53 , phosphorylated p53 at Ser15 ( p53 pS15 ) , and vinculin ( vinc ) in HCT116 treated with 300 μM 5 - FU for 8 h and silenced for PHD1 , PHD2 , or PHD3 . qRT - PCR for PHD1 , PHD2 , and PHD3 in HCT116 silenced for PHD1 ( * P = 0 . 0006 toward Scr ) , PHD2 ( * P = 0 . 0004 toward Scr ) , or PHD3 ( * P = 0 . 0075 toward Scr ) . A two - tailed unpaired t - test was performed with n = 3 / group . Western blot of p53 pS15 , p53 , PHD1 , and vinculin in HCT116 treated with 300 μM 5 - FU for 8 h and silenced for PHD1 or a scrambled ( Scr ) control . qRT - PCR for PHD1 showing silencing efficacy with both siRNAs 1 ( * P = 0 . 0017 toward Scr ) and 2 ( * P = 0 . 0023 toward Scr ) compared to the scrambled control in HCT116 . A two - tailed unpaired t - test was performed with n = 3 / group . Western blot for p53 , p53 pS15 , and vinc in HCT116 silenced for PHD1 with two different constructs ( constructs 1 and 2 ) upon exposure to 300 μM 5 - FU for 8 h . Western blot for p53 pS15 , p53 , and vinculin in HCT116 silenced for PHD1 and treated with either 200 nM SN - 38 or 20 μM oxaliplatin for 8 h . RNA levels of PHD1 in LIM1215 silenced for PHD1 . ( * P = 0 . 0099 toward Scr in a two - tailed unpaired t - test with n = 3 / group . ) Western blot for p53 pS15 , p53 , and vinc in LIM1215 upon silencing of PHD1 and treatment for 8 h with 200 μM 5 - FU .

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Figure 1Western blot for p53, phosphorylated p53 at Ser15 (p53 pS15), and vinculin (vinc) in HCT116 treated with 300 μM 5-FU for 8 h and silenced for PHD1, PHD2, or PHD3.qRT-PCR for PHD1, PHD2, and PHD3 in HCT116 silenced for PHD1 (*P = 0.0006 toward Scr), PHD2 (*P = 0.0004 toward Scr), or PHD3 (*P = 0.0075 toward Scr). A two-tailed unpaired t-test was performed with n = 3/group.Western blot of p53 pS15, p53, PHD1, and vinculin in HCT116 treated with 300 μM 5-FU for 8 h and silenced for PHD1 or a scrambled (Scr) control.qRT-PCR for PHD1 showing silencing efficacy with both siRNAs 1 (*P = 0.0017 toward Scr) and 2 (*P = 0.0023 toward Scr) compared to the scrambled control in HCT116. A two-tailed unpaired t-test was performed with n = 3/group.Western blot for p53, p53 pS15, and vinc in HCT116 silenced for PHD1 with two different constructs (constructs 1 and 2) upon exposure to 300 μM 5-FU for 8 h.Western blot for p53 pS15, p53, and vinculin in HCT116 silenced for PHD1 and treated with either 200 nM SN-38 or 20 μM oxaliplatin for 8 h.RNA levels of PHD1 in LIM1215 silenced for PHD1. (*P = 0.0099 toward Scr in a two-tailed unpaired t-test with n = 3/group.)Western blot for p53 pS15, p53, and vinc in LIM1215 upon silencing of PHD1 and treatment for 8 h with 200 μM 5-FU.


Figure 2A Western blot for p53 , cleaved caspase - 3 ( cleaved casp3 ) , cleaved parp , and vinculin ( vinc ) in p53wt / wt and p53 − / − HCT116 silenced for PHD1 upon exposure to 300 μM 5 - FU for 24 h , showing an increased apoptotic response to 5 - FU treatment in PHD1 - silenced cells compared to the scrambled control . B Confirmation of these results by FACS analysis of propidium iodide - stained PHD1 - silenced p53wt / wt and p53 − / − HCT116 cells exposed for 26 h to 300 - μM 5 - FU treatment . * P = 0 . 002 toward p53wt / wt HCT116 shScr 5 - FU - treated ( two - tailed unpaired t - test ) with n = 6 for non - treated p53wt / wt HCT116 and n = 3 in all other groups . C Detection of cleaved casp3 , cleaved parp , and vinc in HCT116 silenced with a second siRNA for PHD1 . D , E Apoptosis as detected by Western blot for cleaved casp3 and parp in PHD1 - silenced HCT116 treated for 20 h with 200 nM SN - 38 ( D ) or for 24 h with 20 μM oxaliplatin ( E ) . F Western blot for cleaved casp3 , parp , and vinc in LIM1215 silenced for PHD1 and treated for 24 h with 200 μM 5 - FU . G Similar apoptosis levels were detected by Western blot for cleaved casp3 and parp with vinc as a loading control in p53S15A → p53 − / − HCT116 silenced for PHD1 or scrambled ( Scr ) control and treated for 20 h with 300 μM 5 - FU .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A Western blot for p53, cleaved caspase-3 (cleaved casp3), cleaved parp, and vinculin (vinc) in p53wt/wt and p53−/− HCT116 silenced for PHD1 upon exposure to 300 μM 5-FU for 24 h, showing an increased apoptotic response to 5-FU treatment in PHD1-silenced cells compared to the scrambled control.B Confirmation of these results by FACS analysis of propidium iodide-stained PHD1-silenced p53wt/wt and p53−/− HCT116 cells exposed for 26 h to 300-μM 5-FU treatment. *P = 0.002 toward p53wt/wt HCT116 shScr 5-FU-treated (two-tailed unpaired t-test) with n = 6 for non-treated p53wt/wt HCT116 and n = 3 in all other groups.C Detection of cleaved casp3, cleaved parp, and vinc in HCT116 silenced with a second siRNA for PHD1.D, E Apoptosis as detected by Western blot for cleaved casp3 and parp in PHD1-silenced HCT116 treated for 20 h with 200 nM SN-38 (D) or for 24 h with 20 μM oxaliplatin (E).F Western blot for cleaved casp3, parp, and vinc in LIM1215 silenced for PHD1 and treated for 24 h with 200 μM 5-FU.G Similar apoptosis levels were detected by Western blot for cleaved casp3 and parp with vinc as a loading control in p53S15A→p53−/− HCT116 silenced for PHD1 or scrambled (Scr) control and treated for 20 h with 300 μM 5-FU.


Figure 3Representative images and quantification of the colony formation capacity of p53wt / wt HCT116 cells transduced with a doxycycline - inducible shScr or shPHD1 silencing construct , treated for 24 h with doxycycline with or without additional treatment for 8 h with 300 μM 5 - FU . * P = 0 . 008 toward shScr , two - tailed unpaired t - test and n = 3 / group . Tumor volume of p53wt / wtHCT116 cells transduced with a doxycycline - inducible shScr or shPHD1 silencing construct injected subcutaneously in nude mice and treated with 5 - FU . * P = 0 . 045 toward shScr 5 - FU - treated mice by two - way ANOVA with n = 6 for p53wt / wtHCT116 shScr , n = 7 for p53wt / wtHCT116 shPHD1 and p53wt / wtHCT116 shScr 5 - FU - treated , and n = 8 for p53wt / wtHCT116 shPHD15 - FU - treated . Tumor volume of p53 − / − HCT116 cells transduced with a doxycycline - inducible shScr or shPHD1 silencing construct injected subcutaneously in nude mice and treated with 5 - FU . n = 6 for p53 − / − HCT116 shScr , p53 − / − HCT116 shPHD1 , and p53 − / − HCT116 shScr 5 - FU - treated and n = 8 for p53 − / − HCT116 shPHD15 - FU - treated .

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Figure 3Representative images and quantification of the colony formation capacity of p53wt/wt HCT116 cells transduced with a doxycycline-inducible shScr or shPHD1 silencing construct, treated for 24 h with doxycycline with or without additional treatment for 8 h with 300 μM 5-FU. *P = 0.008 toward shScr, two-tailed unpaired t-test and n = 3/group.Tumor volume of p53wt/wtHCT116 cells transduced with a doxycycline-inducible shScr or shPHD1 silencing construct injected subcutaneously in nude mice and treated with 5-FU. *P = 0.045 toward shScr 5-FU-treated mice by two-way ANOVA with n = 6 for p53wt/wtHCT116 shScr, n = 7 for p53wt/wtHCT116 shPHD1 and p53wt/wtHCT116 shScr 5-FU-treated, and n = 8 for p53wt/wtHCT116 shPHD15-FU-treated.Tumor volume of p53−/−HCT116 cells transduced with a doxycycline-inducible shScr or shPHD1 silencing construct injected subcutaneously in nude mice and treated with 5-FU. n = 6 for p53−/−HCT116 shScr, p53−/−HCT116 shPHD1, and p53−/−HCT116 shScr 5-FU-treated and n = 8 for p53−/−HCT116 shPHD15-FU-treated.


Figure 4Western blot for p53 pS15 , p53 , and vinculin ( vinc ) in PHD1 - silenced HCT116 upon treatment with 300 μM 5 - FU for 8 h with or without 0 . 5 mM DMOG for 26 h . Vinculin ( vinc ) is used as a loading control . Western blot detection of PHD1 - Flag and p53 in whole cell extracts ( WCE ) or after immunoprecipitation against the Flag - tag of overexpressed PHD1 - Flag in HEK293T cells . Western blot detection of PHD1 - Flag and p53 after immunoprecipitation against the Flag - tag of recombinant PHD1 - Flag incubated with recombinant p53 . Western blot for p53 and PHD1 in WCE or after immunoprecipitation of endogenous p53 in p53wt / wt HCT116 cells , silenced with a Scr control , siPHD1 , or sip53 , or after immunoprecipitation of p53wt / wt HCT116 cell lysates with an IgG control .

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Figure 4Western blot for p53 pS15, p53, and vinculin (vinc) in PHD1-silenced HCT116 upon treatment with 300 μM 5-FU for 8 h with or without 0.5 mM DMOG for 26 h. Vinculin (vinc) is used as a loading control.Western blot detection of PHD1-Flag and p53 in whole cell extracts (WCE) or after immunoprecipitation against the Flag-tag of overexpressed PHD1-Flag in HEK293T cells.Western blot detection of PHD1-Flag and p53 after immunoprecipitation against the Flag-tag of recombinant PHD1-Flag incubated with recombinant p53.Western blot for p53 and PHD1 in WCE or after immunoprecipitation of endogenous p53 in p53wt/wt HCT116 cells, silenced with a Scr control, siPHD1, or sip53, or after immunoprecipitation of p53wt/wt HCT116 cell lysates with an IgG control.


Figure 5Western blot for p53 pS15 , p53 , and vinculin ( vinc ) in HCT116 treated with 300 μM 5 - FU upon silencing of PHD1 and p38α , alone or in combination . Vinculin ( vinc ) is used as a loading control . Detection of p53 pS15 and p53 after in vitro phosphorylation by p38α , p38β , or p38γ of p53immunoprecipitated from cells silenced for a control ( SIMA ) or PHD1 . Detection by Western blot of p53 and p38 from whole cell extracts ( WCE ) or after immunoprecipitation of p53 from cell silenced for a Scr control or siPHD1 and treated for 1 h with 300 μM 5 - FU .

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Figure 5Western blot for p53 pS15, p53, and vinculin (vinc) in HCT116 treated with 300 μM 5-FU upon silencing of PHD1 and p38α, alone or in combination. Vinculin (vinc) is used as a loading control.Detection of p53 pS15 and p53 after in vitro phosphorylation by p38α, p38β, or p38γ of p53immunoprecipitated from cells silenced for a control (SIMA) or PHD1.Detection by Western blot of p53 and p38 from whole cell extracts (WCE) or after immunoprecipitation of p53 from cell silenced for a Scr control or siPHD1 and treated for 1 h with 300 μM 5-FU.


Figure 6Transcription analysis of CDKN1A , GADD45 , MDM2 , BAX , PUMA in PHD1 - silenced p53wt / wt HCT116 either untreated or treated for 8 h with 300 μM 5 - FU . Western blot for cleaved parp and vinculin ( vinc ) in PHD1 - silenced HCT116 treated for 20 h with 300 μM 5 - FU alone or in combination with 3 μg / ml α - amanitin . PHD1 mRNA levels in p53R248 / − HCT116 silenced for PHD1 . * P = 0 . 0005 toward scrambled control with a two - tailed unpaired t - test and n = 3 / group . Western blot for p53 pS15 , p53 , and vinculin in PHD1 - silenced p53R248 / − HCT116 upon 8 - h treatment with 300 μM 5 - FU . Western blot for cleaved casp3 and vinculin in p53R248 / − HCT116 silenced for PHD1 and treated with 300 μM 5 - FU for 24 h .

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Figure 6Transcription analysis of CDKN1A, GADD45, MDM2, BAX, PUMA in PHD1-silenced p53wt/wt HCT116 either untreated or treated for 8 h with 300 μM 5-FU.Western blot for cleaved parp and vinculin (vinc) in PHD1-silenced HCT116 treated for 20 h with 300 μM 5-FU alone or in combination with 3 μg/ml α-amanitin.PHD1 mRNA levels in p53R248/− HCT116 silenced for PHD1. *P = 0.0005 toward scrambled control with a two-tailed unpaired t-test and n = 3/group.Western blot for p53 pS15, p53, and vinculin in PHD1-silenced p53R248/− HCT116 upon 8-h treatment with 300 μM 5-FU.Western blot for cleaved casp3 and vinculin in p53R248/− HCT116 silenced for PHD1 and treated with 300 μM 5-FU for 24 h.


Figure 7Western blot for pH2AX and vinculin ( vinc ) in p53wt / wt and p53 − / − HCT116 cells after 24 - h treatment with 300 μM 5 - FU . Detection of pH2AX and vinc in p53S15A → p53 − / − HCT116 cells silenced for PHD1 and treated with 300 μM 5 - FU for 24 h . Detection of pH2AX and vinc in HCT116 silenced with a second silencing construct of PHD1 and treated for 24 h with 300 μM 5 - FU . Western blot for pH2AX and vinc in HCT116 treated for 20 h with 200 nM SN - 38 or 24 h with 20 μM oxaliplatin . Western blot for pH2AX and vinc in p53R248 / − HCT116 cells silenced for PHD1 upon treatment with 300 μM 5 - FU for 24 h . Detection by Western blot of p53 and XPB from whole cell extracts ( WCE ) or after immunoprecipitation of p53 from cells silenced for Scr control or siPHD1 and treated for 4 or 8 h with 300 μM 5 - FU . Western blot for pH2AX and vinc in HCT116 silenced for PHD1 alone or in combination with silencing for XPB upon treatment with 300 μM 5 - FU for 20 h .

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Figure 7Western blot for pH2AX and vinculin (vinc) in p53wt/wt and p53−/− HCT116 cells after 24-h treatment with 300 μM 5-FU.Detection of pH2AX and vinc in p53S15A→p53−/− HCT116 cells silenced for PHD1 and treated with 300 μM 5-FU for 24 h.Detection of pH2AX and vinc in HCT116 silenced with a second silencing construct of PHD1 and treated for 24 h with 300 μM 5-FU.Western blot for pH2AX and vinc in HCT116 treated for 20 h with 200 nM SN-38 or 24 h with 20 μM oxaliplatin.Western blot for pH2AX and vinc in p53R248/− HCT116 cells silenced for PHD1 upon treatment with 300 μM 5-FU for 24 h.Detection by Western blot of p53 and XPB from whole cell extracts (WCE) or after immunoprecipitation of p53 from cells silenced for Scr control or siPHD1 and treated for 4 or 8 h with 300 μM 5-FU.Western blot for pH2AX and vinc in HCT116 silenced for PHD1 alone or in combination with silencing for XPB upon treatment with 300 μM 5-FU for 20 h.


Figure 1 ( C ) miR - 574 - 5p and miR - 574 - 3p were highly expressed in the explanted failing hearts from chronic HF patients ( n = 17 ) versus donor non - failing hearts ( n = 6 ) . ( D ) miR - 574 - 5p and miR - 574 - 3p were induced in the hearts of mice with isoproterenol ( ISO ) infusion ( 4 weeks ; n = 10 - 12 ) . ( E ) miR - 574 - 5p and miR - 574 - 3p were induced in the hearts of mice under transverse aortic constriction ( TAC ) surgery ( 4 weeks ; n = 8 - 10 ) .

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Figure 1(C) miR-574-5p and miR-574-3p were highly expressed in the explanted failing hearts from chronic HF patients (n=17) versus donor non-failing hearts (n=6).(D) miR-574-5p and miR-574-3p were induced in the hearts of mice with isoproterenol (ISO) infusion (4 weeks; n=10-12).(E) miR-574-5p and miR-574-3p were induced in the hearts of mice under transverse aortic constriction (TAC) surgery (4 weeks; n=8-10).


Figure 2 ( A ) H & E of hearts from WT and miR - 574 - / - mice with or without isoproterenol ( ISO ) treatment for 4 weeks . Mice were between 8 - 10 weeks old females . ( B ) Ratio of HW / TL ( heart weight / tibia length ) in WT and miR - 574 - / - mice ( n = 8 / 7 / 8 / 6 for 4 groups of WT , Veh . ; WT , ISO ; KO , Veh . ; KO , ISO ) . ( C ) WGA ( wheat germ agglutinin ) staining in WT and miR - 574 - / - mice . Cross - sectional area ( CSA ) of CMs was measured and quantified ( n ≥ 500 cells ) . Scale bar : 50 μm . In the violin plot , black line shows median value for the group and peach dashed lines represent two quartile lines in each group . ( D ) Picrosirius red staining in WT and miR - 574 - / - mice ( n = 6 per group ) . Scale bar : 1 mm . ( E ) RT - qPCR of fetal cardiac genes in WT and miR - 574 - / - mice with ISO treatment ( n = 3 per group ) . ANF , atrial natriuretic factor ; BNP , B - type natriuretic peptide ; Myh6 / Myh7 , myosin heavy polypeptide 6 / 7 ; Col1a2 / Col3a1 , procollagen , type I , α2 ; type III , α1 . ( F ) TUNEL assay for heart tissue sections from WT and miR - 574 - / - mice under ISO versus vehicle treatment ( n = 6 per group ) . Scale bar : 10 μm .

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Figure 2(A) H&E of hearts from WT and miR-574-/- mice with or without isoproterenol (ISO) treatment for 4 weeks. Mice were between 8-10 weeks old females.(B) Ratio of HW/TL (heart weight/tibia length) in WT and miR-574-/- mice (n=8/7/8/6 for 4 groups of WT, Veh.; WT, ISO; KO, Veh.; KO, ISO).(C) WGA (wheat germ agglutinin) staining in WT and miR-574-/- mice. Cross-sectional area (CSA) of CMs was measured and quantified (n≥500 cells). Scale bar: 50 μm. In the violin plot, black line shows median value for the group and peach dashed lines represent two quartile lines in each group.(D) Picrosirius red staining in WT and miR-574-/- mice (n=6 per group). Scale bar: 1 mm.(E) RT-qPCR of fetal cardiac genes in WT and miR-574-/- mice with ISO treatment (n=3 per group). ANF, atrial natriuretic factor; BNP, B-type natriuretic peptide; Myh6/Myh7, myosin heavy polypeptide 6/7; Col1a2/Col3a1, procollagen, type I, α2; type III, α1.(F) TUNEL assay for heart tissue sections from WT and miR-574-/- mice under ISO versus vehicle treatment (n=6 per group). Scale bar: 10 μm.


Figure 3 ( A ) H & E staining of hearts from WT and miR - 574 - / - mice 4 weeks after transverse aortic constriction ( TAC ) surgery . ( B ) Ratio of HW / TL in WT and miR - 574 - / - mice ( n = 8 per group ) . ( C ) WGA staining of hearts from WT and miR - 574 - / - mice 4 weeks after TAC surgery ( n ≥ 500 cells ) . Scale bar : 20 μm . In the violin plot , black line shows median value for the group and peach dashed lines represent two quartile lines in each group . ( D ) Picrosirius red staining of hearts from WT and miR - 574 - / - mice under TAC surgery ( n = 8 per group ) . Scale bar : 1 mm . ( E ) RT - qPCR of fetal cardiac genes in WT and miR - 574 - / - mice with TAC surgery ( n = 4 per group ) . ( F ) Echocardiography measurement of cardiac functions of hearts from WT and miR - 574 - / - mice 4 weeks post TAC ( n = 8 per group ) . FS , fractional shortening .

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Figure 3(A) H&E staining of hearts from WT and miR-574-/- mice 4 weeks after transverse aortic constriction (TAC) surgery.(B) Ratio of HW/TL in WT and miR-574-/- mice (n=8 per group).(C) WGA staining of hearts from WT and miR-574-/- mice 4 weeks after TAC surgery (n≥500 cells). Scale bar: 20 μm. In the violin plot, black line shows median value for the group and peach dashed lines represent two quartile lines in each group.(D) Picrosirius red staining of hearts from WT and miR-574-/- mice under TAC surgery (n=8 per group). Scale bar: 1 mm.(E) RT-qPCR of fetal cardiac genes in WT and miR-574-/- mice with TAC surgery (n=4 per group).(F) Echocardiography measurement of cardiac functions of hearts from WT and miR-574-/- mice 4 weeks post TAC (n=8 per group). FS, fractional shortening.


Figure 4The schematic of therapeutic treatment of the TAC model using miRNA mimics followed by phenotypic characterizations . HW / TL ratio is measured ( n = 4 - 10 per group ) . Ctrl - miR : negative control miRNA mimics . ( B ) H & E and WGA staining of murine hearts in the therapeutic models . Scale bar : 1 mm for H & E and 50 μm for WGA . Cardiomyocyte surface area was quantified from WGA staining in the right panel . > 120 CMs are quantified in each group . The black line shows medium value for the group and the dotted lines represent two quartile lines in each group . ( C ) Picrosirius red staining of murine hearts in the therapeutic models . Scale bar : 1 mm . The fibrotic area was quantified in the right panel ( n = 4 - 10 per group ) . ( D ) Echocardiography analysis of the cardiac function of mice in the therapeutic models at the end - point of 6 weeks post - surgery ( n = 4 - 10 per group ) .

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Figure 4The schematic of therapeutic treatment of the TAC model using miRNA mimics followed by phenotypic characterizations. HW/TL ratio is measured (n=4-10 per group). Ctrl-miR: negative control miRNA mimics.(B) H&E and WGA staining of murine hearts in the therapeutic models. Scale bar: 1 mm for H&E and 50 μm for WGA. Cardiomyocyte surface area was quantified from WGA staining in the right panel. >120 CMs are quantified in each group. The black line shows medium value for the group and the dotted lines represent two quartile lines in each group.(C) Picrosirius red staining of murine hearts in the therapeutic models. Scale bar: 1 mm. The fibrotic area was quantified in the right panel (n=4-10 per group).(D) Echocardiography analysis of the cardiac function of mice in the therapeutic models at the end-point of 6 weeks post-surgery (n=4-10 per group).


Figure 5 ( A ) Volcano curve analysis of dysregulated genes in miR - 574 - / - versus WT heart . The Padj is the P - values adjusted for multiple testing with correction using the Benjamini - Hochberg procedure . The dots above the dashed line show Padj < 0 . 05 . ( B ) Heatmap of significantly upregulated genes in miR - 574 - / - mice at baseline analyzed by RNA - Seq . P60 male mice , n = 3 per group , Padj < 0 . 05 . The Padj is the P - values adjusted for multiple testing with correction using the Benjamini - Hochberg procedure . ( E ) Expression of Fam210a mRNA in the heart of miR - 574 - / - and WT mice from 3 biological replicates . ( F ) Expression of FAM210A protein in the heart of miR - 574 - / - and WT mice . Protein intensity was quantified in the right panel ( n = 6 per group ) . ( H ) Dual luciferase reporter assays with co - transfection of miR - 574 - 5p mimics and FLuc - Fam210a 3 ' UTR bearing wild type and mutant seed sequences . The assays were performed in three biological replicates . ( I ) Dual luciferase reporter assays with co - transfection of miR - 574 - 3p mimics and FLuc - Fam210a 3 ' UTR bearing wild type and mutant seed sequences . The assays were performed in three biological replicates .

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Figure 5(A) Volcano curve analysis of dysregulated genes in miR-574-/- versus WT heart. The Padj is the P-values adjusted for multiple testing with correction using the Benjamini-Hochberg procedure. The dots above the dashed line show Padj<0.05.(B) Heatmap of significantly upregulated genes in miR-574-/- mice at baseline analyzed by RNA-Seq. P60 male mice, n=3 per group, Padj<0.05. The Padj is the P-values adjusted for multiple testing with correction using the Benjamini-Hochberg procedure.(E) Expression of Fam210a mRNA in the heart of miR-574-/- and WT mice from 3 biological replicates.(F) Expression of FAM210A protein in the heart of miR-574-/- and WT mice. Protein intensity was quantified in the right panel (n=6 per group).(H) Dual luciferase reporter assays with co-transfection of miR-574-5p mimics and FLuc-Fam210a 3'UTR bearing wild type and mutant seed sequences. The assays were performed in three biological replicates.(I) Dual luciferase reporter assays with co-transfection of miR-574-3p mimics and FLuc-Fam210a 3'UTR bearing wild type and mutant seed sequences. The assays were performed in three biological replicates.