( E - G ) Autaptic neurons of munc18 - 1 null mutant mice rescued with lentiviruses expressing M18WT were superfused with WIN55 , 212 - 2 ( 1 M ) , WIN55 , 212 - 2 ( 1 M ) plus AM251 ( 1 M ) or vehicle only ( DMSO ) . ( E ) Amplitude of excitatory synapse response normalized to t = 0 s . CB1R agonist WIN55 , 212 - 2 ( 1 M ) or vehicle only ( DMSO ) were applied at t = 60 s for 30 s . ( WT + WIN n = 13 ; WT + DMSO n = 16 , * p < 0 . 05 ) . Note the broke Y - axis . ( F ) Paired pulse ratio ( 20 ms interval ) at t = 0 and t = 240 s in M18WT neurons superfused with CB1R agonist WIN55 , 212 - 2 ( 1 M ) at t = 60 s for 30 s . WIN55 , 212 - 2 application increases PP ratios indicative of a reduction of synaptic release probability ( t = 0 s : 0 . 94 ± 0 . 1 , n = 13 ; t = 240 s : 1 . 36 ± 0 . 2 , n = 16 , * p < 0 . 05 ) . ( G ) Amplitude of excitatory synapse response normalized to t = 0 s . CB1R agonist WIN55 , 212 - 2 ( 1 M ) was applied together with the CB1R antagonist AM251 ( 1 M ) at t = 60 s for 30 s . ( WT + WIN n = 13 , same trace as 6F ; WT + WIN + AM251 n = 15 , * p < 0 . 05 ) . Note the broke Y - axis .

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

(E-G) Autaptic neurons of munc18-1 null mutant mice rescued with lentiviruses expressing M18WT were superfused with WIN55,212-2 (1 M), WIN55,212-2 (1 M) plus AM251 (1 M) or vehicle only (DMSO).(E) Amplitude of excitatory synapse response normalized to t=0 s. CB1R agonist WIN55,212-2 (1 M) or vehicle only (DMSO) were applied at t=60 s for 30 s. (WT + WIN n = 13; WT + DMSO n = 16, * p < 0.05). Note the broke Y-axis.(F) Paired pulse ratio (20 ms interval) at t = 0 and t = 240 s in M18WT neurons superfused with CB1R agonist WIN55,212-2 (1 M) at t = 60 s for 30 s. WIN55,212-2 application increases PP ratios indicative of a reduction of synaptic release probability (t = 0 s: 0.94 ± 0.1, n = 13; t = 240 s: 1.36 ± 0.2, n = 16, * p < 0.05).(G) Amplitude of excitatory synapse response normalized to t = 0 s. CB1R agonist WIN55,212-2 (1 M) was applied together with the CB1R antagonist AM251 (1 M) at t = 60 s for 30 s. (WT + WIN n = 13, same trace as 6F; WT + WIN + AM251 n = 15, * p < 0.05). Note the broke Y-axis.


( H - J ) Hippocampal autaptic neurons of munc18 - 1 null mutant mice were rescued with M18WT or M18S241A . mGluR2 / 3 agonist LY379268 ( 1 M ) was applied by bath - perfusion for 4 min . ( H ) Ratio of amplitude of evoked release events after ( t = 240 s ) and before ( t = 0 s ) LY379268 application ( M18WT : 55 . 0 ± 4 . 7 % , n = 14 ; M18S241A : 81 . 1 ± 3 . 6 % , n = 16 , * p < 0 . 05 ) . ( I ) Ratio of frequency of spontaneous release events after ( t = 240 s ) and before ( t = 0 s ) LY379268 application ( M18WT : 63 . 0 ± 5 . 7 % , n = 14 ; M18S241A : 85 . 1 ± 3 . 4 % , n = 16 , * p < 0 . 05 ) . ( J ) Ratio of the amplitude of spontaneous release events after ( t = 240 s ) and before ( t = 0 s ) WIN55 , 212 - 2 application ( M18WT : 81 . 0 ± 1 . 3 % , n = 14 ; M18S241A : 82 . 1 ± 3 . 3 % , n = 16 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-J) Hippocampal autaptic neurons of munc18-1 null mutant mice were rescued with M18WT or M18S241A. mGluR2/3 agonist LY379268 (1 M) was applied by bath-perfusion for 4 min.(H) Ratio of amplitude of evoked release events after (t = 240 s) and before (t = 0 s) LY379268 application (M18WT: 55.0 ± 4.7 %, n = 14; M18S241A: 81.1 ± 3.6 %, n = 16, * p < 0.05).(I) Ratio of frequency of spontaneous release events after (t = 240 s) and before (t = 0 s) LY379268 application (M18WT: 63.0 ± 5.7 %, n = 14; M18S241A: 85.1 ± 3.4 %, n = 16, * p < 0.05).(J) Ratio of the amplitude of spontaneous release events after (t = 240 s) and before (t = 0 s) WIN55,212-2 application (M18WT: 81.0 ± 1.3 %, n = 14; M18S241A: 82.1 ± 3.3 %, n = 16).


( A - D ) DSE duration and amplitude is decreased in CA1 pyramidal neurons upon ERK pathway inhibition by a pre - treatment with PD98059 ( PD ; 10 µM ) compared to 0 . 1 % DMSO ( control ) . ( A ) Average EPSC amplitude during 25 s baseline recording prior to DSE induction shows no significant differences in EPSC amplitude between the DMSO and PD pretreated groups ( DMSO : 196 . 36 + / - 16 . 51 pA , n = 10 ; PD : 196 . 42 + / - 18 . 61 pA , n = 11 , p = 0 . 9 ) . ( B ) DSE in neurons of DMSO pre - treated slices , induced by a 10s depolarization to 0 mV ( arrow ) . Inset : example traces of 2 averaged EPSCs before ( black ) and after depolarization ( grey ) . ( C ) DSE in slices pre - treated with PD . Insets : example traces of 2 averaged EPSCs before ( red ) and after depolarization ( grey ) . ( D ) Comparison of DSE between control and PD . PD pre - treatment limits amplitude and duration of DSE . * * = p < 0 . 01 ; unpaired T - test with Welch ' s correction . Numbers in bars represent the number of neurons in each group .

O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) DSE duration and amplitude is decreased in CA1 pyramidal neurons upon ERK pathway inhibition by a pre-treatment with PD98059 (PD; 10 µM) compared to 0.1% DMSO (control).(A) Average EPSC amplitude during 25 s baseline recording prior to DSE induction shows no significant differences in EPSC amplitude between the DMSO and PD pretreated groups (DMSO: 196.36 +/- 16.51 pA, n = 10; PD: 196.42 +/- 18.61 pA, n = 11, p = 0.9).(B) DSE in neurons of DMSO pre-treated slices, induced by a 10s depolarization to 0 mV (arrow). Inset: example traces of 2 averaged EPSCs before (black) and after depolarization (grey).(C) DSE in slices pre-treated with PD. Insets: example traces of 2 averaged EPSCs before (red) and after depolarization (grey).(D) Comparison of DSE between control and PD. PD pre-treatment limits amplitude and duration of DSE. ** = p < 0.01; unpaired T-test with Welch's correction. Numbers in bars represent the number of neurons in each group.


( E - H ) ERK - pathway inhibition by pre - treatment with PD98059 ( PD ; 10 µM ) blocks the effect of WIN55 , 212 - 2 ( WIN ; 2 µM ) on the amplitude of EPSCs recorded in CA1 pyramidal neurons . ( E ) Average EPSC amplitude during 5 min baseline recording prior to DSE induction shows no significant differences in EPSC amplitude between the DMSO and PD pretreated groups ( DMSO : 295 . 69 + / - 28 . 73 pA , n = 6 ; PD : 270 . 86 + / - 32 . 14 pA , n = 6 , p = 0 . 6 ) . ( F ) Effect of WIN application ( grey bar , 30 min ) on EPSC - amplitude in CA1 pyramidal neurons of 0 . 1 % DMSO ( control ) pre - treated slices . Inset : example traces of averaged EPSCs of baseline ( black ) and the last 5 min ( grey ) . ( G ) Effect of WIN application ( grey bar , 30 min ) on EPSC - amplitude in CA1 pyramidal neurons of PD pre - treated slices . Inset : example traces of averaged EPSCs of baseline ( red ) and the last 5 minutes ( grey ) . ( H ) Comparison of the effect size of the last 5 minutes in both groups shows a significant difference . * = p < 0 . 05 ; unpaired T - test . Numbers in bars represent number of slices in each group .

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

(E-H) ERK-pathway inhibition by pre-treatment with PD98059 (PD; 10 µM) blocks the effect of WIN55,212-2 (WIN; 2 µM) on the amplitude of EPSCs recorded in CA1 pyramidal neurons.(E) Average EPSC amplitude during 5 min baseline recording prior to DSE induction shows no significant differences in EPSC amplitude between the DMSO and PD pretreated groups (DMSO: 295.69 +/- 28.73 pA, n = 6; PD: 270.86 +/- 32.14 pA, n = 6, p= 0.6).(F) Effect of WIN application (grey bar, 30 min) on EPSC-amplitude in CA1 pyramidal neurons of 0.1% DMSO (control) pre-treated slices. Inset: example traces of averaged EPSCs of baseline (black) and the last 5 min (grey).(G) Effect of WIN application (grey bar, 30 min) on EPSC-amplitude in CA1 pyramidal neurons of PD pre-treated slices. Inset: example traces of averaged EPSCs of baseline (red) and the last 5 minutes (grey).(H) Comparison of the effect size of the last 5 minutes in both groups shows a significant difference. * = p < 0.05; unpaired T-test. Numbers in bars represent number of slices in each group.


( A ) Western blot analysis of the immunoproteasome subunits LMP2 , LMP7 and β - actin ( loading control ) in three PolgA + / + and three PolgAmut / mut cell lines .

O O O O O O O O O O O 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) Western blot analysis of the immunoproteasome subunits LMP2, LMP7 and β-actin (loading control) in three PolgA+/+ and three PolgAmut/mut cell lines.


( B ) RT - qPCR analysis of immunoproteasome subunits Psmb8 , Psmb9 , Psmb10 in three different PolgA + / + and three PolgAmut / mut cell lines .

O O O O O O 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) RT-qPCR analysis of immunoproteasome subunits Psmb8, Psmb9, Psmb10 in three different PolgA+/+ and three PolgAmut/mut cell lines.


( C ) Activity assay for the catalytic subunits of the proteasome using the pan activity - based probe ( ABP ) MV151 , the β1 / LMP2 specific LW124 , and the β5 / LMP7 specific MVB127 ABPs in three PolgA + / + and three PolgAmut / mut cell lines . Resolution of β5 / LMP7 in the SDS gel is hampered as both subunits are of similar molecular weight and thus do not separate in our gels . Total protein staining was used as a loading control .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Activity assay for the catalytic subunits of the proteasome using the pan activity-based probe (ABP) MV151, the β1/LMP2 specific LW124, and the β5/LMP7 specific MVB127 ABPs in three PolgA+/+ and three PolgAmut/mut cell lines. Resolution of β5/LMP7 in the SDS gel is hampered as both subunits are of similar molecular weight and thus do not separate in our gels. Total protein staining was used as a loading control.


( D ) representative Western blots for the analysis of the cGAS / STING signaling pathway in PolgA + / + and PolgAmut / mut cells with β - actin serving as loading control .

(D) representative Western blots for the analysis of the cGAS/STING signaling pathway in PolgA+/+ and PolgAmut/mut cells with β-actin serving as loading control.


( E ) Secreted IFNβ in supernatants from three distinct PolgA + / + and PolgAmut / mut cell lines . The 15 . 81 pg / ml detected by ELISA in PolgAmut / mut cell supernatants corresponds to 18 . 97 IU / ml recombinant mouse ( rm ) IFN - β according to the R & D Systems cytokine conversion

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

(E) Secreted IFNβ in supernatants from three distinct PolgA+/+ and PolgAmut/mut cell lines. The 15.81 pg/ml detected by ELISA in PolgAmut/mut cell supernatants corresponds to 18.97 IU/ml recombinant mouse (rm) IFN-β according to the R&D Systems cytokine conversion table


( F ) RT - qPCR analysis of Stat1 in three different PolgA + / + and three PolgAmut / mut cell lines .

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) RT-qPCR analysis of Stat1 in three different PolgA+/+ and three PolgAmut/mut cell lines.


( A ) Western blot analysis of LMP2 , LMP7 , STAT1 and β - actin ( loading control ) in PolgA + / + cells after incubation with either PolgA + / + or PolgAmut / mut cell - conditioned medium for 48 h . Densitometric analysis of LMP2 , LMP7 and STAT1 expression normalized to β - actin loading control set to 1 in PolgA + / + incubated with either PolgA + / + or PolgAmut / mut cell - conditioned medium from three different cell lines .

(A) Western blot analysis of LMP2, LMP7, STAT1 and β-actin (loading control) in PolgA+/+ cells after incubation with either PolgA+/+ or PolgAmut/mut cell-conditioned medium for 48 h. Densitometric analysis of LMP2, LMP7 and STAT1 expression normalized to β-actin loading control set to 1 in PolgA+/+ incubated with either PolgA+/+ or PolgAmut/mut cell-conditioned medium from three different cell lines.


( B ) Western blot analysis of LMP2 , LMP7 , STAT1 , and β - actin ( loading control ) upon siRNA - mediated silencing of cGAS or STING in PolgAmut / mut cells compared to untransfected ( Ctrl ) and non - sense scrambled transfection control ( NC ) in three independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Western blot analysis of LMP2, LMP7, STAT1, and β-actin (loading control) upon siRNA-mediated silencing of cGAS or STING in PolgAmut/mut cells compared to untransfected (Ctrl) and non-sense scrambled transfection control (NC) in three independent experiments.


( E ) Western blot analysis of LMP2 , LMP7 , STAT1 , and β - actin ( loading control ) in PolgA + / + cells transfected with mouse wildtype mtDNA , mtDNA pretreated with DNase I or only DNase I for 48 h . Densitometric analysis of LMP2 , LMP7 and STAT1 expression normalized to β - actin loading control in PolgA + / + cells . Values are given as fold over PolgA + / + control with the mean of normalized PolgA + / + signal normalized to 1 . ( F ) Western blot analysis of LMP2 , LMP7 , STAT1 and β - actin ( loading control ) in PolgA + / + cells transfected with cGAMP for 24 h . Densitometric analysis of LMP2 , LMP7 and STAT1 expression normalized to β - actin loading control ( Ctrl ) set to 1 .

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

(E) Western blot analysis of LMP2, LMP7, STAT1, and β-actin (loading control) in PolgA+/+ cells transfected with mouse wildtype mtDNA, mtDNA pretreated with DNase I or only DNase I for 48 h. Densitometric analysis of LMP2, LMP7 and STAT1 expression normalized to β-actin loading control in PolgA+/+ cells. Values are given as fold over PolgA+/+ control with the mean of normalized PolgA+/+ signal normalized to 1. (F) Western blot analysis of LMP2, LMP7, STAT1 and β-actin (loading control) in PolgA+/+ cells transfected with cGAMP for 24 h. Densitometric analysis of LMP2, LMP7 and STAT1 expression normalized to β-actin loading control (Ctrl) set to 1.


( G ) Protein expression of LMP2 , LMP7 and β - actin ( loading control ) in wildtype ( WT ) , cGAS KO or STINGgt / gt losss of function MEFs that had been transfected with 200 ng / ml mtDNA for 48 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 O O O O O O O O O O O O O O O O O O O O O O O

(G) Protein expression of LMP2, LMP7 and β-actin (loading control) in wildtype (WT), cGAS KO or STINGgt/gt losss of function MEFs that had been transfected with 200 ng/ml mtDNA for 48 h.


( B ) UTY - assay of CD8 + reporter T cells upon co - culture with different ratios of PolgA + / + or PolgAmut / mut cells . Data are combined results of three independent experiments and normalized to the signal of maximum induction of PolgA + / + cells in co - culture with CD8 + reporter T cells .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) UTY-assay of CD8+ reporter T cells upon co-culture with different ratios of PolgA+/+ or PolgAmut/mut cells. Data are combined results of three independent experiments and normalized to the signal of maximum induction of PolgA+/+ cells in co-culture with CD8+ reporter T cells.


( E ) UTY - assay of CD8 + reporter T cells upon 1 : 2 co - culture ( splenocytes : T cells ) with splenocytes isolated from either WT or LMP7 KO mice and transfected with mtDNA . Splenocytes were isolated from 4 mice each in two different preparations .

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

(E) UTY-assay of CD8+ reporter T cells upon 1:2 co-culture (splenocytes:T cells) with splenocytes isolated from either WT or LMP7 KO mice and transfected with mtDNA. Splenocytes were isolated from 4 mice each in two different preparations.


( A ) Western blot analysis of immunoproteasome subunits LMP2 , LMP7 , and of STAT1 in pmAT2 cells transfected with mtDNA or mtDNA pretreated with DNase I for 48 h or treated with IFNβ for 24 h . β - actin was used as a loading control . Densitometric analysis of LMP2 , LMP7 and STAT1 expression normalized to β - actin loading control in pmAT2 from three independent pmAT2 preparations .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Western blot analysis of immunoproteasome subunits LMP2, LMP7, and of STAT1 in pmAT2 cells transfected with mtDNA or mtDNA pretreated with DNase I for 48 h or treated with IFNβ for 24 h. β-actin was used as a loading control. Densitometric analysis of LMP2, LMP7 and STAT1 expression normalized to β-actin loading control in pmAT2 from three independent pmAT2 preparations.


( C ) Representative Western blot analysis of phLF for LMP2 , LMP7 , STAT1 , and β - actin ( loading control ) upon transfection of mtDNA or mtDNA pretreated with DNase I for 24 h , 48 h or 72 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 O O O O O O O O O O O O O O O O O O O O

(C) Representative Western blot analysis of phLF for LMP2, LMP7, STAT1, and β-actin (loading control) upon transfection of mtDNA or mtDNA pretreated with DNase I for 24 h, 48 h or 72 h.


( G ) Western blot analysis of STING , pTBK1 , TBK1 , pIRF3 , IRF3 , pIκBα , IκBα , pP65 , P65 and β - actin ( loading control ) in phLFs transfected with mtDNA for 2 h , 6 h , or 24 h , respectively .

O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Western blot analysis of STING, pTBK1, TBK1, pIRF3, IRF3, pIκBα, IκBα, pP65, P65 and β-actin (loading control) in phLFs transfected with mtDNA for 2 h, 6 h, or 24 h, respectively.


( B ) Flow cytometric detection of Influenza - M1 / HLA - A2 complex in phLF upon transfection with mtDNA , mtDNA pretreated with DNase I , or treatment with IFNβ ( n = 3 independent experiments ) .

O O O O O O O O O O O O O O O O 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) Flow cytometric detection of Influenza-M1/HLA-A2 complex in phLF upon transfection with mtDNA, mtDNA pretreated with DNase I, or treatment with IFNβ (n=3 independent experiments).


( D ) CD8 + T cell activation assay of Influenza - M1 / HLA - A2 restricted CD8 + T cells upon co - culture with mtDNA - transfected phLFs loaded with M1 peptide or upon treatment with IFNβ or IFNγ displaying secretion of IL - 2 from three different biological experiments .

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

(D) CD8+ T cell activation assay of Influenza-M1/HLA-A2 restricted CD8+ T cells upon co-culture with mtDNA-transfected phLFs loaded with M1 peptide or upon treatment with IFNβ or IFNγ displaying secretion of IL-2 from three different biological experiments.


( A ) Western blot analysis of the immunoproteasome subunits LMP2 and LMP7 in wild type ( WT ) , cGAS KO and STINGgt / gt loss of function MEFs transfected with 1 μg / ml HT - DNA or plasmid - DNA for 48 h . β - actin was used as a loading control . Densitometric analysis of LMP2 and LMP7 expression normalized to β - actin loading control in wild type ( WT ) controls ( ctrl ) from three independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Western blot analysis of the immunoproteasome subunits LMP2 and LMP7 in wild type (WT), cGAS KO and STINGgt/gt loss of function MEFs transfected with 1 μg/ml HT-DNA or plasmid-DNA for 48 h. β-actin was used as a loading control. Densitometric analysis of LMP2 and LMP7 expression normalized to β-actin loading control in wild type (WT) controls (ctrl) from three independent experiments.


( B ) UTY - assay of CD8 + reporter T cells upon 1 : 2 co - culture ( splenocytes : T cells ) with splenocytes isolated from either wildtype ( WT ) or LMP7 KO mice and transfected with htDNA . Splenocytes were isolated from 4 mice each in two different preparations .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) UTY-assay of CD8+ reporter T cells upon 1:2 co-culture (splenocytes:T cells) with splenocytes isolated from either wildtype (WT) or LMP7 KO mice and transfected with htDNA. Splenocytes were isolated from 4 mice each in two different preparations.


( C ) Western blot analysis of immunoproteasome subunits LMP2 and LMP7 in wild type ( WT ) , heterozygous STAT1 mutated ( STAT1 * + / - ) and homozygous STAT1 mutated ( STAT1 * - / - ) skin fibroblasts transfected with 1 μg / ml HT - DNA or pretreated HT - DNA with DNase I for 48 h .

(C) Western blot analysis of immunoproteasome subunits LMP2 and LMP7 in wild type (WT), heterozygous STAT1 mutated (STAT1*+/-) and homozygous STAT1 mutated (STAT1*-/-) skin fibroblasts transfected with 1 μg/ml HT-DNA or pretreated HT-DNA with DNase I for 48 h.


( D - E ) Representative immunofluorescence detection of LMP2 ( red ) , CD8 + ( green ) and PD - 1 ( red ) in control and IPF lungs , arrows indicate double positive CD8 + PD - 1 + cells , magnifications 10X , 20X and 40X correspond to a scale bar of 100 µm . Arrows indicate epithelial cells ( E ) or macrophages ( M ) and cells stained positive for CD8 .

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

(D-E) Representative immunofluorescence detection of LMP2 (red), CD8+ (green) and PD-1(red) in control and IPF lungs, arrows indicate double positive CD8+ PD-1+ cells, magnifications 10X, 20X and 40X correspond to a scale bar of 100 µm. Arrows indicate epithelial cells (E) or macrophages (M) and cells stained positive for CD8.


( F ) Flow cytometric analysis of CD3 , CD8 , and CCR7 expression on peripheral blood mononuclear cells ( PBMCs ) of control ( n = 21 ) and IPF ( n = 66 ) patients . Data are presented as gated % of parent . Data were analyzed with two - tailed unpaired Student ' s t - test . Asterisks indicate significance as * P < 0 . 05 .

(F) Flow cytometric analysis of CD3, CD8, and CCR7 expression on peripheral blood mononuclear cells (PBMCs) of control (n=21) and IPF (n=66) patients. Data are presented as gated % of parent. Data were analyzed with two-tailed unpaired Student's t-test. Asterisks indicate significance as *P<0.05.


( C , D ) Quantification of SCOT and BDH1 expression levels relative to VCP in various murine organs . Data are presented as mean ± SD . n = 3 ; * , p < 0 . 05 ; * * * , p < 0 . 001 unpaired Student ' s t - test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, D) Quantification of SCOT and BDH1 expression levels relative to VCP in various murine organs. Data are presented as mean ± SD. n=3; *, p<0.05; ***, p<0.001 unpaired Student's t-test.


( G ) Immunoblot of SCOT , BDH1 , CD31 and VCP in cardiac endothelial cells ( EC ) and non - endothelial cell fraction ( non - EC ) isolated from adult C57Bl / 6J mice .

O O O O O O O O O O O O O O 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) Immunoblot of SCOT, BDH1, CD31 and VCP in cardiac endothelial cells (EC) and non-endothelial cell fraction (non-EC) isolated from adult C57Bl/6J mice.


( A , B ) Murine cardiac endothelial cells ( MCEC ) were treated with 2 mM D - β - hydroxybutyrate ( β - OHB ) or 2 mM acetoacetate ( AcAc ) for 24 hours . Quantification of concentrations of TCA cycle intermediates compared to control treatment . Data are presented as mean ± SD . n ≥ 4 ; ns , not significant ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 unpaired Student ' s t - test .

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 I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR 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 O O O O O O O O O O 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, B) Murine cardiac endothelial cells (MCEC) were treated with 2 mM D-β-hydroxybutyrate (β-OHB) or 2 mM acetoacetate (AcAc) for 24 hours. Quantification of concentrations of TCA cycle intermediates compared to control treatment. Data are presented as mean ± SD. n≥4; ns, not significant; *, p<0.05; **, p<0.01; ***, p<0.001 unpaired Student's t-test.


( D ) Quantifications of [ 13C ] - labeled fractions of amino acids ( asparagine , L - aspartate , L - glutamate , L - proline ) , putative lipid species ( nervonic acid , 1 - palmitoylglycerol , ceramide ( d18 : 1 / 16 : 0 ) ) and uridine diphosphate ( UDP ) species ( uridine monophosphate , uridine 5 ' - diphosphate , UDP - N - acetylglucosamine ) in MCEC treated with 1 mM 13C4 - β - hydroxybutyrate or 1 mM 13C4 - acetoacetate for 24 hours or the same concentration of unlabeled ketone bodies ( [ 12C ] isotope ) . Data are presented as mean ± SD . n ≥ 4 ; ns , not significant ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 unpaired Student ' s t - test .

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

(D) Quantifications of [13C]-labeled fractions of amino acids (asparagine, L-aspartate, L-glutamate, L-proline), putative lipid species (nervonic acid, 1-palmitoylglycerol, ceramide (d18:1/16:0)) and uridine diphosphate (UDP) species (uridine monophosphate, uridine 5'-diphosphate, UDP-N-acetylglucosamine) in MCEC treated with 1 mM 13C4-β-hydroxybutyrate or 1 mM 13C4-acetoacetate for 24 hours or the same concentration of unlabeled ketone bodies ([12C] isotope). Data are presented as mean ± SD. n≥4; ns, not significant; *, p<0.05; **, p<0.01; ***, p<0.001 unpaired Student's t-test.


( A , B ) Murine cardiac endothelial cells ( MCEC ) were treated with different concentrations of D - β - hydroxybutyrate ( βOHB ) or acetoacetate ( AcAc ) for 24 hours . Relative BrdU absorbance was quantified compared to control treatment ( H2O for βOHB and EtOH for AcAc ) . Data are presented as mean ± SD . n ≥ 3 ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 unpaired Student ' s t - test .

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

(A, B) Murine cardiac endothelial cells (MCEC) were treated with different concentrations of D-β-hydroxybutyrate (βOHB) or acetoacetate (AcAc) for 24 hours. Relative BrdU absorbance was quantified compared to control treatment (H2O for βOHB and EtOH for AcAc). Data are presented as mean ± SD. n≥3; *, p<0.05; **, p<0.01; ***, p<0.001 unpaired Student's t-test.


( I - M ) Angiogenic capacity of MCEC in response to ketone body treatment was analyzed using a spheroid - based sprouting assay . Spheroids were treated with media containing 30 mM D - β - hydroxybutyrate ( βOHB ) , 30 mM acetoacetate ( AcAc ) , 10 mM acetate , 1 mM octanoate , 1 mM butyrate , 30 mM L - β - hydroxybutyrate ( L - βOHB ) , 1 mM niacin or the respective controls ( final concentration of reagents is diluted to approximately 10 % ) and analyzed after 48 hours . The ( I , L ) average number of sprouts per spheroid , the ( J , M ) accumulated total sprout length and ( K ) average sprout length were quantified . Data are presented as mean ± SD . n ≥ 3 ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 unpaired Student ' s t - test .

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

(I-M) Angiogenic capacity of MCEC in response to ketone body treatment was analyzed using a spheroid-based sprouting assay. Spheroids were treated with media containing 30 mM D-β-hydroxybutyrate (βOHB), 30 mM acetoacetate (AcAc), 10 mM acetate, 1 mM octanoate, 1 mM butyrate, 30 mM L-β-hydroxybutyrate (L- βOHB), 1 mM niacin or the respective controls (final concentration of reagents is diluted to approximately 10 %) and analyzed after 48 hours. The (I, L) average number of sprouts per spheroid, the (J, M) accumulated total sprout length and (K) average sprout length were quantified. Data are presented as mean ± SD. n≥3; *, p<0.05; **, p<0.01; ***, p<0.001 unpaired Student's t-test.


( B , Angiogenic capacity of Oxct1 knockout cells in response to ketone body treatment was analyzed using a spheroid - based sprouting assay . Spheroids were treated with media containing 30 mM D - β - hydroxybutyrate ( βOHB ) , 30 mM acetoacetate ( AcAc ) or the respective controls ( final concentration of reagents is diluted to approximately 10 % ) for 48 hours . The average number of sprouts per spheroid was quantified . Data are presented as mean ± SD . n = 4 ; * , p < 0 . 05 ; * * , p < 0 . 01 ; unpaired Student ' s t - test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Angiogenic capacity of Oxct1 knockout cells in response to ketone body treatment was analyzed using a spheroid-based sprouting assay. Spheroids were treated with media containing 30 mM D-β-hydroxybutyrate (βOHB), 30 mM acetoacetate (AcAc) or the respective controls (final concentration of reagents is diluted to approximately 10 %) for 48 hours. The average number of sprouts per spheroid was quantified. Data are presented as mean ± SD. n=4; *, p<0.05; **, p<0.01; unpaired Student's t-test.


( C , D ) Heatmaps showing expression levels of genes involved in KEGG pathways ( C ) fatty acid degradation and ( D ) ketone body metabolism in cardiac endothelial cells isolated from male C57Bl / 6J mice that were fed a ketogenic diet or control diet for two weeks .

(C, D) Heatmaps showing expression levels of genes involved in KEGG pathways (C) fatty acid degradation and (D) ketone body metabolism in cardiac endothelial cells isolated from male C57Bl/6J mice that were fed a ketogenic diet or control diet for two weeks.


( A , B ) Representative images of heart sections of animals kept on a ketogenic diet or control diet for two weeks stained against ERG and Ki67 or EdU , double positive cells are indicated by arrowheads , and quantification of double positive cells in heart sections per high power field ( HPF ) . Scale bar : 50 μm . Data are presented as mean ± SD . n ≥ 3 ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 unpaired Student ' s t - test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, B) Representative images of heart sections of animals kept on a ketogenic diet or control diet for two weeks stained against ERG and Ki67 or EdU, double positive cells are indicated by arrowheads, and quantification of double positive cells in heart sections per high power field (HPF). Scale bar: 50 μm. Data are presented as mean ± SD. n≥3; *, p<0.05; **, p<0.01; ***, p<0.001 unpaired Student's t-test.


( F ) Quantification of Ki67 + endothelial cells in several organs of mice kept on a ketogenic diet or control diet for four weeks ( gastrocnemius and soleus muscle ) or two weeks ( brain , lungs , subcutaneous adipose tissue , liver ) . Data are presented as mean ± SD . n ≥ 3 ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 unpaired Student ' s t - test .

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

(F) Quantification of Ki67+ endothelial cells in several organs of mice kept on a ketogenic diet or control diet for four weeks (gastrocnemius and soleus muscle) or two weeks (brain, lungs, subcutaneous adipose tissue, liver). Data are presented as mean ± SD. n≥3; *, p<0.05; **, p<0.01; ***, p<0.001 unpaired Student's t-test.


( B ) Representative images of heart sections of mice kept on a control or ketogenic diet eight weeks after TAC surgery stained against Ki67 and ERG . Scale bar : 50 µm .

O O O O O O O O O O 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) Representative images of heart sections of mice kept on a control or ketogenic diet eight weeks after TAC surgery stained against Ki67 and ERG. Scale bar: 50 µm.


B . Normalized number of filaments nucleated over time , from SPIN90 - Arp2 / 3 exposed to 2 μΜ G - actin ( 15 % labeled with Alexa488 ) and 1 μΜ profilin , with 0 or 0 . 5 µM of GST - VCA from different NPFs . Solid lines are exponential fits , yielding nucleation rates knuc = ( 1 . 06 ± 0 . 03 ) х10 - 3 s - 1 without VCA , and knuc = ( 3 . 23 ± 0 . 08 ) х10 - 3 , ( 2 . 58 ± 0 . 05 ) х10 - 3 , and ( 2 . 28 ± 0 . 03 ) х10 - 3 s - 1 with VCA from N - WASP , WASP and WASH , respectively .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Normalized number of filaments nucleated over time, from SPIN90-Arp2/3 exposed to 2 μΜ G-actin (15% labeled with Alexa488) and 1 μΜ profilin, with 0 or 0.5 µM of GST-VCA from different NPFs. Solid lines are exponential fits, yielding nucleation rates knuc=(1.06±0.03)х10-3 s-1 without VCA, and knuc=(3.23±0.08)х10-3, (2.58±0.05)х10-3, and (2.28±0.03)х10-3 s-1 with VCA from N-WASP, WASP and WASH, respectively.


Dissociation of SPIN90 - Arp2 / 3 - nucleated filaments . ( B ) The fraction of filaments still attached to the surface , versus time , for different concentrations of GST - NWASP - VCA . Black lines are exponential fits .

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Dissociation of SPIN90-Arp2/3-nucleated filaments. (B) The fraction of filaments still attached to the surface, versus time, for different concentrations of GST-NWASP-VCA. Black lines are exponential fits.


Dissociation of SPIN90 - Arp2 / 3 - nucleated filaments . ( C ) Detachment rates , determined by exponential fits of survival curves ( as in B ) as a function of the concentration of VCA motifs from different NPFs . The data are fitted by a Michaelis - Menten equation ( resulting in an apparent KD of 0 . 10 ± 0 . 01 , 0 . 65 ± 0 . 36 and 0 . 82 ± 0 . 14 µM , for N - WASP , WASH and WASP , respectively ; and a maximum detachment rate of 0 . 033 ± 0 . 001 , 0 . 031 ± 0 . 006 , 0 . 010 ± 0 . 001 s - 1 for WASH , N - WASP and WASP , respectively ) .

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

Dissociation of SPIN90-Arp2/3-nucleated filaments. (C) Detachment rates, determined by exponential fits of survival curves (as in B) as a function of the concentration of VCA motifs from different NPFs. The data are fitted by a Michaelis-Menten equation (resulting in an apparent KD of 0.10±0.01, 0.65±0.36 and 0.82±0.14 µM, for N-WASP, WASH and WASP, respectively; and a maximum detachment rate of 0.033± 0.001, 0.031± 0.006, 0.010± 0.001 s-1 for WASH, N-WASP and WASP, respectively).


Dissociation of Arp2 / 3 - mediated branches . ( E ) The fraction of remaining branches , versus time , for different concentrations of GST - NWASP - VCA . Black lines are exponential fits .

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

Dissociation of Arp2/3-mediated branches. (E) The fraction of remaining branches, versus time, for different concentrations of GST-NWASP-VCA. Black lines are exponential fits.


Dissociation of SPIN90 - Arp2 / 3 - nucleated filaments . ( A ) Schematic of the detachment experiment . Actin filaments were first nucleated from a SPIN90 - Arp2 / 3 - decorated surface , and elongated . Buffer with different concentrations of VCA was flowed in , and filament detachment events were monitored over time . Dissociation of Arp2 / 3 - mediated branches . ( F ) Debranching rates , determined by exponential fits of survival curves ( as in E ) as a function of the concentration of VCA motifs from different NPFs . The data are fitted by a Michaelis - Menten equation ( resulting in an apparent KD of 0 . 57 ± 0 . 35 , 0 . 81 ± 0 . 36 and 0 . 53 ± 0 . 23 µM , for WASH , N - WASP and WASP , respectively ; and a maximum detachment rate of 0 . 020 ± 0 . 004 , 0 . 012 ± 0 . 002 , 0 . 005 ± 0 . 001 s - 1 for WASH , N - WASP and WASP , respectively ) .

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

Dissociation of SPIN90-Arp2/3-nucleated filaments. (A) Schematic of the detachment experiment. Actin filaments were first nucleated from a SPIN90-Arp2/3-decorated surface, and elongated. Buffer with different concentrations of VCA was flowed in, and filament detachment events were monitored over time. Dissociation of Arp2/3-mediated branches. (F) Debranching rates, determined by exponential fits of survival curves (as in E) as a function of the concentration of VCA motifs from different NPFs. The data are fitted by a Michaelis-Menten equation (resulting in an apparent KD of 0.57±0.35, 0.81±0.36 and 0.53±0.23 µM, for WASH, N-WASP and WASP, respectively; and a maximum detachment rate of 0.020± 0.004, 0.012± 0.002, 0.005± 0.001 s-1 for WASH, N-WASP and WASP, respectively).


A . The fraction of filaments still attached to the surface by SPIN90 - Arp2 / 3 , over time , when exposed to different concentrations of GMF . For each experiment , n = 36 - 43 filaments . Black lines are exponential fits . Each experiment was repeated 3 times ( technical replicates ) , yielding similar results .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. The fraction of filaments still attached to the surface by SPIN90-Arp2/3, over time, when exposed to different concentrations of GMF. For each experiment, n=36-43 filaments. Black lines are exponential fits. Each experiment was repeated 3 times (technical replicates), yielding similar results.


B . Detachment rates , determined by exponential fits of survival curves shown in A , as a function of the concentration of GMF . The error bars are determined by fitting the edges of the 95 % confidence intervals ( not shown , for readability ) of the survival curves . The data are fitted by a Michaelis - Menten equation , yielding an apparent dissociation constant KD = 70 ± 23 nM and a maximum detachment rate of 0 . 017 ± 0 . 001s - 1 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Detachment rates, determined by exponential fits of survival curves shown in A, as a function of the concentration of GMF. The error bars are determined by fitting the edges of the 95% confidence intervals (not shown, for readability) of the survival curves. The data are fitted by a Michaelis-Menten equation, yielding an apparent dissociation constant KD=70±23 nM and a maximum detachment rate of 0.017±0.001s-1.


Assessing the presence of Arp2 / 3 on SPIN90 after filament departure . ( B - C ) The fraction of SPIN90 that re - nucleated a filament 500 seconds after the detachment of the initial filament , was quantified for different conditions .

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

Assessing the presence of Arp2/3 on SPIN90 after filament departure. (B-C) The fraction of SPIN90 that re-nucleated a filament 500 seconds after the detachment of the initial filament, was quantified for different conditions.


GMF and VCA interfere with the SPIN90 - Arp2 / 3 interaction , in the absence of filament . ( G ) Immuno - blots from pull - down assays where GST beads , decorated with GST or GST - SPIN90 - Cter ( the functional domain of SPIN90 ) , were incubated with 0 . 4 μΜ Arp2 / 3 and gradient concentrations of GMF or VCA for 1 hour at room temperature . After the unbound protein was washed out , the amount of Arp2 / 3 attached to the beads was detected by Anti - ArpC2 antibody . Ponceau red straining of the membrane verified that the beads were loaded with equal amounts of GST or GST - SPIN90 ( Appendix Figure S2 ) .

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

GMF and VCA interfere with the SPIN90-Arp2/3 interaction, in the absence of filament. (G) Immuno-blots from pull-down assays where GST beads, decorated with GST or GST-SPIN90-Cter (the functional domain of SPIN90), were incubated with 0.4 μΜ Arp2/3 and gradient concentrations of GMF or VCA for 1 hour at room temperature. After the unbound protein was washed out, the amount of Arp2/3 attached to the beads was detected by Anti-ArpC2 antibody. Ponceau red straining of the membrane verified that the beads were loaded with equal amounts of GST or GST-SPIN90 (Appendix Figure S2).


GMF and VCA interfere with the SPIN90 - Arp2 / 3 interaction , in the absence of filament . ( H - I ) Quantification of the amount of ArpC2 remaining on the beads , in different conditions .

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

GMF and VCA interfere with the SPIN90-Arp2/3 interaction, in the absence of filament. (H-I) Quantification of the amount of ArpC2 remaining on the beads, in different conditions.


A - C . Survival fractions for branches exposed to different forces in the microfluidics chamber , while being exposed to 0 . 15µM G - actin , alone ( gray curves , A ) , with 1 µM VCA ( blue curves , B ) , or with 1 µM GMF ( red curves , C ) . The shaded areas represent 95 % confidence intervals . Black lines are exponential fits .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-C. Survival fractions for branches exposed to different forces in the microfluidics chamber, while being exposed to 0.15µM G-actin, alone (gray curves, A), with 1 µM VCA (blue curves, B), or with 1 µM GMF (red curves, C). The shaded areas represent 95% confidence intervals. Black lines are exponential fits.


D . Summary of the debranching rates , resulting from the exponential fits in ( A - C ) .

D. Summary of the debranching rates, resulting from the exponential fits in (A-C).


E - G . Survival fractions for SPIN90 - Arp2 / 3 - nucleated filaments exposed to different forces in the microfluidics chamber , while being exposed to 0 . 15µM G - actin , alone ( gray curves , E ) , with 0 . 1 µM VCA ( blue curves , F ) , or with 0 . 1 µM GMF ( red curves , G ) . The shaded areas represent 95 % confidence intervals . Black lines are exponential fits .

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

E-G. Survival fractions for SPIN90-Arp2/3-nucleated filaments exposed to different forces in the microfluidics chamber, while being exposed to 0.15µM G-actin, alone (gray curves, E), with 0.1 µM VCA (blue curves, F), or with 0.1 µM GMF (red curves, G). The shaded areas represent 95% confidence intervals. Black lines are exponential fits.


H . Summary of the detachment rates for SPIN90 - Arp2 / 3 - nucleated filaments , resulting from the exponential fits in ( E - G ) .

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H. Summary of the detachment rates for SPIN90-Arp2/3-nucleated filaments, resulting from the exponential fits in (E-G).


A . Fraction of remaining branches , over time , starting at an average of 5 and 25 minutes after branch nucleation , in the presence ( blue ) or absence ( gray ) of VCA . Lines are exponential fits , yielding debranching rates of ( 3 . 7 ± 0 . 6 ) x10 - 4 s - 1 and ( 20 ± 8 ) x10 - 4 s - 1 without VCA for 5 - and 25 - minute - old branches , respectively ; and debranching rates of ( 3 . 8 ± 1 . 3 ) x10 - 3 s - 1 and ( 8 . 2 ± 2 . 8 ) x10 - 3 s - 1 with 0 . 5 µM VCA for 5 - and 25 - minute - old branches , respectively .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Fraction of remaining branches, over time, starting at an average of 5 and 25 minutes after branch nucleation, in the presence (blue) or absence (gray) of VCA. Lines are exponential fits, yielding debranching rates of (3.7±0.6)x10-4 s-1 and (20±8)x10-4 s-1 without VCA for 5- and 25-minute-old branches, respectively; and debranching rates of (3.8±1.3)x10-3 s-1 and (8.2±2.8)x10-3 s-1 with 0.5 µM VCA for 5- and 25-minute-old branches, respectively.


B . Fraction of attached SPIN90 - nucleated filaments , over time , starting at an average of 5 and 25 minutes after branch nucleation , in the presence ( blue ) or absence ( gray ) of VCA . Lines are exponential fits , yielding detachment rates of ( 12 ± 7 ) x10 - 4 s - 1 and ( 7 ± 3 ) x10 - 4 s - 1 without VCA for 5 - and 25 - minute - old filaments , respectively ( p = 0 . 2 , logrank test ) ; and detachment rates of ( 2 . 5 ± 1 . 1 ) x10 - 2 s - 1 and ( 3 . 1 ± 1 . 7 ) x10 - 2 s - 1 with 1 µM VCA for 5 - and 25 - minute - old filaments , respectively ( p = 0 . 4 , logrank test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Fraction of attached SPIN90-nucleated filaments, over time, starting at an average of 5 and 25 minutes after branch nucleation, in the presence (blue) or absence (gray) of VCA. Lines are exponential fits, yielding detachment rates of (12±7)x10-4 s-1 and (7±3)x10-4 s-1 without VCA for 5- and 25-minute-old filaments, respectively (p=0.2, logrank test); and detachment rates of (2.5±1.1)x10-2 s-1 and (3.1±1.7)x10-2 s-1 with 1 µM VCA for 5- and 25-minute-old filaments, respectively (p=0.4, logrank test).


A . The fraction of remaining branches , versus time , exposed to 0 . 16 µM G - actin ( black ) supplemented with 50 nM cortactin ( red ) . The branch junctions were exposed to an average force of 1 . 4 pN .

O O O O O O O O O O O O O O O O O O O O O O O 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. The fraction of remaining branches, versus time, exposed to 0.16 µM G-actin (black) supplemented with 50 nM cortactin (red). The branch junctions were exposed to an average force of 1.4 pN.


B . The fraction of remaining SPIN90 - Arp2 / 3 - nucleated filaments , versus time , exposed to 0 . 15 µM G - actin ( black ) supplemented with 10 nM cortactin ( red ) .

B. The fraction of remaining SPIN90-Arp2/3-nucleated filaments, versus time, exposed to 0.15 µM G-actin (black) supplemented with 10 nM cortactin (red).


C . The fraction of remaining SPIN90 - Arp2 / 3 - nucleated filaments , versus time , exposed to 0 . 15 µM G - actin , with 0 . 5 µM VCA ( black ) supplemented with 10 nM cortactin ( red ) .

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

C. The fraction of remaining SPIN90-Arp2/3-nucleated filaments, versus time, exposed to 0.15 µM G-actin, with 0.5 µM VCA (black) supplemented with 10 nM cortactin (red).


( a ) p62 localizes to the MR during cytokinesis and abscission , and is taken up together with the MR by one daughter cell . U2OS cells were fixed and stained with anti - p62 ( red ) and anti - MKLP1 ( green ) antibodies . Scale bars are 10 μm . Colocalization of p62 with MKLP1 of the MR was regularly observed in cells at this stage of cell division . The arrowheads point to MRs , which are shown enlarged in the insets . The quantification is presented in Supplementary Information Fig . S1a .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) p62 localizes to the MR during cytokinesis and abscission, and is taken up together with the MR by one daughter cell. U2OS cells were fixed and stained with anti-p62 (red) and anti-MKLP1 (green) antibodies. Scale bars are 10 μm. Colocalization of p62 with MKLP1 of the MR was regularly observed in cells at this stage of cell division. The arrowheads point to MRs, which are shown enlarged in the insets. The quantification is presented in Supplementary Information Fig. S1a.


( b ) siRNA - mediated depletion of p62 from U2OS cells results in moderate MR accumulation , compared with a control depletion ( lamin A / C ) . For quantification , MRs were counted in control cells and in p62 - depleted cells that were negative for p62 staining . Data are means ± s . d . of three independent experiments ( n , total number of cells counted ; P , probability of a two - tailed paired t - test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) siRNA-mediated depletion of p62 from U2OS cells results in moderate MR accumulation, compared with a control depletion (lamin A/C). For quantification, MRs were counted in control cells and in p62-depleted cells that were negative for p62 staining. Data are means ± s.d. of three independent experiments (n, total number of cells counted; P, probability of a two-tailed paired t-test).


( a ) Atg8 ( LC3 ) localizes to the MR during abscission . The upper panels show stills from time - lapse microscopy of GFP - Atg8 - expressing cells . The arrowhead points to the accumulation of Atg8 at the site of the MR during abscission . The enlarged clipping on the right shows the midbody area at time - point 68 min . Scale bar is 10 μm .

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(a) Atg8 (LC3) localizes to the MR during abscission. The upper panels show stills from time-lapse microscopy of GFP-Atg8-expressing cells. The arrowhead points to the accumulation of Atg8 at the site of the MR during abscission. The enlarged clipping on the right shows the midbody area at time-point 68 min. Scale bar is 10 μm.


( b ) Cytosolic MRs are found inside Atg8 - positive vesicles . U2OS cells stably expressing GFP - Atg8 were fixed and stained for MKLP1 ( red ) and α - tubulin ( blue ) . Scale bars are 10 μm and 1 μm for the enlarged boxed areas . Putative different stages of autophagosomes ( left ) and lysosomes ( right ) are shown .

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(b) Cytosolic MRs are found inside Atg8-positive vesicles. U2OS cells stably expressing GFP-Atg8 were fixed and stained for MKLP1 (red) and α-tubulin (blue). Scale bars are 10 μm and 1 μm for the enlarged boxed areas. Putative different stages of autophagosomes (left) and lysosomes (right) are shown.


( c ) Juxtanuclear MRs are found in Rab7 - and LAMP2 - positive lysosomes . U2OS cells stably expressing DsRed - Rab7 were fixed and stained for MKLP1 α - tubulin and DAPI ( left panels ) ; wild - type U2OS were stained for MKLP1 and LAMP2 ( right panel ) . Scale bars are 10 μm . The insets are enlargements of the engulfed MRs . Scale bars are 1 μm .

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(c) Juxtanuclear MRs are found in Rab7- and LAMP2-positive lysosomes. U2OS cells stably expressing DsRed-Rab7 were fixed and stained for MKLP1 α-tubulin and DAPI (left panels); wild-type U2OS were stained for MKLP1 and LAMP2 (right panel). Scale bars are 10 μm. The insets are enlargements of the engulfed MRs. Scale bars are 1 μm.


( a ) Accumulation of MRs in cells treated with the PI ( 3 ) K inhibitor 3 - MA . Left panels show HeLa cells treated with 3 - MA ( 4 mM ) for 3 days . Cells were fixed and stained . Images are colour - inverted for better visualization . The quantification of this experiment is shown on the upper right ( mean ± s . d . of three independent experiments ; n = total number of cells counted ) . The lower right panel shows an enlarged area of 3 - MA - treated cells in true colour . Scale bars are 20 μm .

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

(a) Accumulation of MRs in cells treated with the PI(3)K inhibitor 3-MA. Left panels show HeLa cells treated with 3-MA (4 mM) for 3 days. Cells were fixed and stained. Images are colour-inverted for better visualization. The quantification of this experiment is shown on the upper right (mean ± s.d. of three independent experiments; n = total number of cells counted). The lower right panel shows an enlarged area of 3-MA-treated cells in true colour. Scale bars are 20 μm.


( b ) siRNA - mediated depletion of Atg6 ( Beclin - 1 ) and Atg7 from HeLa cells causes MR accumulations . The right panel shows the quantification of MRs per cell ( mean ± s . d . of three independent experiments ) ; the left panel the efficiency of Atg6 and Atg7 depletion .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) siRNA-mediated depletion of Atg6 (Beclin-1) and Atg7 from HeLa cells causes MR accumulations. The right panel shows the quantification of MRs per cell (mean ± s.d. of three independent experiments); the left panel the efficiency of Atg6 and Atg7 depletion.


( c ) Inhibition of lysosomal acidification leads to MR accumulation . Panels on the left show HeLa cells left untreated or treated with chloroquine ( 20 μM ) or ammonium chloride ( 10 mM ) for 3 days . Scale bar are 20 μm . Cells were fixed and stained as indicated . A quantification of this experiment is shown on the left ( mean ± s . d . of three independent experiments ; n , total number of cells ; P , probability of a two - tailed paired t - test ) .

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 O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Inhibition of lysosomal acidification leads to MR accumulation. Panels on the left show HeLa cells left untreated or treated with chloroquine (20 μM) or ammonium chloride (10 mM) for 3 days. Scale bar are 20 μm. Cells were fixed and stained as indicated. A quantification of this experiment is shown on the left (mean ± s.d. of three independent experiments; n, total number of cells; P, probability of a two-tailed paired t-test).


( a ) Cell - cycle lengths of different cell lines correlate well with the average amount of MRs . Indicated cell lines were grown on coverslips , fixed and MRs were counted after staining with anti - MKLP1 antibodies . Cell - cycle lengths were assessed as described in the Methods . Data are mean ± s . d . of three independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Cell-cycle lengths of different cell lines correlate well with the average amount of MRs. Indicated cell lines were grown on coverslips, fixed and MRs were counted after staining with anti-MKLP1 antibodies. Cell-cycle lengths were assessed as described in the Methods. Data are mean ± s.d. of three independent experiments.


( b ) The degree of MR persistence depends on the time and not on cell - cycle duration . HeLa cells were either left untreated or were arrested in S - phase with hydroxyurea ( HU , 5 mM ) . Cells were fixed and stained as indicated . Scale bars are 20 μm . The right panel shows quantification of MR counting ( mean ± s . d . of three independent experiments ; n , total number of cells ; P , probability of a two - tailed paired t - test ) .

O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) The degree of MR persistence depends on the time and not on cell-cycle duration. HeLa cells were either left untreated or were arrested in S-phase with hydroxyurea (HU, 5 mM). Cells were fixed and stained as indicated. Scale bars are 20 μm. The right panel shows quantification of MR counting (mean ± s.d. of three independent experiments; n, total number of cells; P, probability of a two-tailed paired t-test).


( a ) Fibroblasts of individuals with mucolipidosis II ( I - cell disease ; upper right panel ) or Hurler syndrome ( lower right panel ) were compared with parental control cells ( left panels ) . The alleles are indicated . Cells were fixed and stained with the indicated antibodies and DAPI to visualize DNA . Arrowheads point to the accumulated MRs . Scale bars are 20 μm . ( b ) Quantification of a ( mean ± s . d . of three independent experiments ; n , total number of cells ; P , probability of a two - tailed paired t - test ) .

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

(a) Fibroblasts of individuals with mucolipidosis II (I-cell disease; upper right panel) or Hurler syndrome (lower right panel) were compared with parental control cells (left panels). The alleles are indicated. Cells were fixed and stained with the indicated antibodies and DAPI to visualize DNA. Arrowheads point to the accumulated MRs. Scale bars are 20 μm. (b) Quantification of a (mean ± s.d. of three independent experiments; n, total number of cells; P, probability of a two-tailed paired t-test).


A Expression patterns of pSEU : : SEU - GFP and pWOX5 : : GFP in the embryos of the indicated genotypes at dermatogen , early globular , and heart stages . White arrows indicate the QC precursor cell , and white dashed lines indicate embryos . Scale bars : 10 µm .

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A Expression patterns of pSEU::SEU-GFP and pWOX5::GFP in the embryos of the indicated genotypes at dermatogen, early globular, and heart stages. White arrows indicate the QC precursor cell, and white dashed lines indicate embryos. Scale bars: 10 µm.


B Quantification of pWOX5 : : GFP GFP fluorescence in wild - type ( WT ) and seu - 3 mutant embryos . Fluorescence intensity at the early globular stage of WT embryos was set to 1 . Data Information : In ( B ) data represent mean ± SD of three independent replicates . n denotes the total number of scored samples . Individual values ( black dots ) are shown . * * P < 0 . 01 , * P < 0 . 05 ( Student ' s t - test ) .

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B Quantification of pWOX5::GFP GFP fluorescence in wild-type (WT) and seu-3 mutant embryos. Fluorescence intensity at the early globular stage of WT embryos was set to 1. Data Information: In (B) data represent mean ± SD of three independent replicates. n denotes the total number of scored samples. Individual values (black dots) are shown. **P < 0.01, *P < 0.05 (Student's t-test).


C , D Expression pattern of pWOX5 : : GFP in WT ( C ) and seu - 3 ( D ) embryos at 5 days after germination ( DAG ) . Scale bars : 20 µm . E Quantification of GFP fluorescence in the QC of pWOX5 : : GFP transgenic plants , as shown in ( C ) and ( D ) . Fluorescence intensity was normalized to the WT . Data Information : ( E ) , data represent mean ± SD of three independent replicates . n denotes the total number of scored samples . Individual values ( black dots ) are shown . * * P < 0 . 01 , * P < 0 . 05 ( Student ' s t - test ) .

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C, D Expression pattern of pWOX5::GFP in WT (C) and seu-3 (D) embryos at 5 days after germination (DAG). Scale bars: 20 µm. E Quantification of GFP fluorescence in the QC of pWOX5::GFP transgenic plants, as shown in (C) and (D). Fluorescence intensity was normalized to the WT. Data Information: (E), data represent mean ± SD of three independent replicates. n denotes the total number of scored samples. Individual values (black dots) are shown. **P < 0.01, *P < 0.05 (Student's t-test).


F RT - qPCR analysis of the relative expression levels of WOX5 in WT and seu - 3 roots . Total RNA was extracted from 5 mm root tip sections of seedlings at 5 DAG . Data Information : ( F ) data represent mean ± SD of three independent replicates . n denotes the total number of scored samples . Individual values ( black dots ) are shown . * * P < 0 . 01 , * P < 0 . 05 ( Student ' s t - test ) .

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F RT-qPCR analysis of the relative expression levels of WOX5 in WT and seu-3 roots. Total RNA was extracted from 5 mm root tip sections of seedlings at 5 DAG. Data Information: (F) data represent mean ± SD of three independent replicates. n denotes the total number of scored samples. Individual values (black dots) are shown. **P < 0.01, *P < 0.05 (Student's t-test).


G - J Modified pseudo - Schiff propidium iodide ( mPS - PI ) staining of stem cell niche areas in the indicated genotypes at 5 DAG . Blue arrows indicate the QC , and red arrows indicate the columella stem cells ( CSCs ) . The numbers denote total number of scored samples , with similar phenotypes showing in ( G - J ) . Scale bars : 20 μm . K Quantification of the CSC layer in the indicated genotypes at 5 DAG . Data Information ( K ) , data represent mean ± SD of three independent replicates . n denotes the total number of scored samples . Individual values ( black dots ) are shown . * * P < 0 . 01 , * P < 0 . 05 ( Student ' s t - test ) .

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G-J Modified pseudo-Schiff propidium iodide (mPS-PI) staining of stem cell niche areas in the indicated genotypes at 5 DAG. Blue arrows indicate the QC, and red arrows indicate the columella stem cells (CSCs). The numbers denote total number of scored samples, with similar phenotypes showing in (G-J). Scale bars: 20 μm. K Quantification of the CSC layer in the indicated genotypes at 5 DAG. Data Information (K), data represent mean ± SD of three independent replicates. n denotes the total number of scored samples. Individual values (black dots) are shown. **P < 0.01, *P < 0.05 (Student's t-test).


L , M Double staining of the QC184 β - glucuronidase ( GUS ) marker ( light blue ) and starch granules ( dark brown ) in WT ( L ) and seu - 3 ( M ) seedlings at 5 DAG . In white arrows indicate the CSCs . Scale bars : 20 μm .

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L, M Double staining of the QC184 β-glucuronidase (GUS) marker (light blue) and starch granules (dark brown) in WT (L) and seu-3 (M) seedlings at 5 DAG. In white arrows indicate the CSCs. Scale bars: 20 μm.


N , O Expression pattern of J2341 in WT ( N ) and seu - 3 ( O ) seedlings at 5 DAG . P , Q Expression pattern of CS9227 in WT ( P ) and seu - 3 ( Q ) seedlings at 5 DAG . In white arrows indicate the CSCs . Scale bars : 20 μm .

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N, O Expression pattern of J2341 in WT (N) and seu-3 (O) seedlings at 5 DAG. P, Q Expression pattern of CS9227 in WT (P) and seu-3 (Q) seedlings at 5 DAG. In white arrows indicate the CSCs. Scale bars: 20 μm.


A - C Expression pattern of pWOX5 : : GFP in WT ( A ) , scr - 3 ( B ) and plt1 - 4 plt2 - 2 ( C ) at 5 DAG . D Quantification of GFP fluorescence in the QC of pWOX5 : : GFP transgenic seedlings , as shown in ( A - C ) . GFP signal intensity in each genotype was normalized relative to that in the WT . Data information : In ( D ) n denotes the total number of scored samples . Individual values ( black dots ) are shown . Data represent mean ± SD of three independent replicates . In ( D ) different lowercase letters indicate significant differences by one - way ANOVA followed by Tukey ' s multiple comparison test ( P < 0 . 01 ) .

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A-C Expression pattern of pWOX5::GFP in WT (A), scr-3 (B) and plt1-4 plt2-2 (C) at 5 DAG. D Quantification of GFP fluorescence in the QC of pWOX5::GFP transgenic seedlings, as shown in (A-C). GFP signal intensity in each genotype was normalized relative to that in the WT. Data information: In (D) n denotes the total number of scored samples. Individual values (black dots) are shown. Data represent mean ± SD of three independent replicates. In (D) different lowercase letters indicate significant differences by one-way ANOVA followed by Tukey's multiple comparison test (P < 0.01).


E RT - qPCR analysis of the relative expression levels of WOX5 in WT , scr - 3 , and plt1 - 4 plt2 - 2 roots . Total RNA was extracted from 5 mm root tip sections of seedlings at 5 DAG . Data information : In ( E ) , * * P < 0 . 01 ( Student ' s t - test ) . Scale bars : 20 µm .

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E RT-qPCR analysis of the relative expression levels of WOX5 in WT, scr-3, and plt1-4 plt2-2 roots. Total RNA was extracted from 5 mm root tip sections of seedlings at 5 DAG. Data information: In (E) , **P < 0.01 (Student's t-test). Scale bars: 20 µm.


F Primary root length of the indicated genotypes at 5 DAG . Data information : In ( F ) n denotes the total number of scored samples . Individual values ( black dots ) are shown . Data represent mean ± SD of three independent replicates . In ( F ) , different lowercase letters indicate significant differences by one - way ANOVA followed by Tukey ' s multiple comparison test ( P < 0 . 01 ) .

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F Primary root length of the indicated genotypes at 5 DAG. Data information: In (F) n denotes the total number of scored samples. Individual values (black dots) are shown. Data represent mean ± SD of three independent replicates. In (F), different lowercase letters indicate significant differences by one-way ANOVA followed by Tukey's multiple comparison test (P < 0.01).


G - J Representative confocal images of the indicated genotypes at 5 DAG . White arrows indicate the QC .

G-J Representative confocal images of the indicated genotypes at 5 DAG. White arrows indicate the QC.


K Quantification of GFP fluorescence in the QC of pSCR : : GFP and pSCR : : GFP - SCR transgenic seedlings , as shown GFP signal intensity of each genotype was normalized relative to that of the WT . Data information ( K ) , n denotes the total number of scored samples . Individual values ( black dots ) are shown . Data represent mean ± SD of three independent replicates . ( K ) , * * P < 0 . 01 ( Student ' s t - test ) . Scale bars : 20 µm .

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K Quantification of GFP fluorescence in the QC of pSCR::GFP and pSCR::GFP-SCR transgenic seedlings, as shown GFP signal intensity of each genotype was normalized relative to that of the WT. Data information (K), n denotes the total number of scored samples. Individual values (black dots) are shown. Data represent mean ± SD of three independent replicates. (K), **P < 0.01 (Student's t-test). Scale bars: 20 µm.


L - Q Root stem cell niche phenotypes of the indicated genotypes at 5 DAG . White dashed lines indicate the QC region . The numbers denote total number of scored samples , with similar phenotypes showing in ( L - Q ) .

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L-Q Root stem cell niche phenotypes of the indicated genotypes at 5 DAG. White dashed lines indicate the QC region. The numbers denote total number of scored samples, with similar phenotypes showing in (L-Q).


A Yeast two - hybrid ( Y2H ) assays showing the interaction between SEU and SCR . The yeast transformants were plated on synthetic defined ( SD ) media lacking Leu and Trp ( SD / - 2 ) or lacking Ade , His , Leu , and Trp ( SD / - 4 ) to assess protein - protein interactions . AD , GAL4 activation domain ; BD , GAL4 DNA - binding domain .

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A Yeast two-hybrid (Y2H) assays showing the interaction between SEU and SCR. The yeast transformants were plated on synthetic defined (SD) media lacking Leu and Trp (SD/-2) or lacking Ade, His, Leu, and Trp (SD/-4) to assess protein-protein interactions. AD, GAL4 activation domain; BD, GAL4 DNA-binding domain.


B In vitro pull - down assays showing that SEU directly interacts with SCR but not with SHR . SCR - FLAG was pulled down by SEU - MBP immobilized on amylose resin . Protein bound to the amylose resin was eluted and analyzed by immunoblotting using anti - FLAG antibody . The asterisk indicates the position of SEU - MBP .

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B In vitro pull-down assays showing that SEU directly interacts with SCR but not with SHR. SCR-FLAG was pulled down by SEU-MBP immobilized on amylose resin. Protein bound to the amylose resin was eluted and analyzed by immunoblotting using anti-FLAG antibody. The asterisk indicates the position of SEU-MBP.


C Verification of in vivo interactions between SCR and SEU in N . benthamiana leaves via Co - IP assays . SCR - GFP and SEU - myc were transiently coexpressed in N . benthamiana leaves . Protein samples were immunoprecipitated using anti - myc antibody .

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C Verification of in vivo interactions between SCR and SEU in N. benthamiana leaves via Co-IP assays. SCR-GFP and SEU-myc were transiently coexpressed in N. benthamiana leaves. Protein samples were immunoprecipitated using anti-myc antibody.


D Co - IP assays of SEU with SCR in Arabidopsis . Protein extracts from WT and SCR - GFP roots were isolated at 5 DAG and immunoprecipitated with anti - GFP antibody .

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D Co-IP assays of SEU with SCR in Arabidopsis. Protein extracts from WT and SCR-GFP roots were isolated at 5 DAG and immunoprecipitated with anti-GFP antibody.


F , G SCR and SEU physically bind to the WOX5 promoter , as shown by ChIP - qPCR analysis . Chromatin was isolated from 5 mm root tip sections of seedlings at 5 DAG , sonicated , and immunoprecipitated using anti - GFP antibody . The precipitated DNA was used as a template for qPCR analysis . A , B , and I indicated the PCR amplicons ACT7 , control . Data information : In ( F ) , ( G ) data represent mean ± SD of three independent replicates . * * P < 0 . 01 ( Student ' s t - test ) .

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F, G SCR and SEU physically bind to the WOX5 promoter, as shown by ChIP-qPCR analysis. Chromatin was isolated from 5 mm root tip sections of seedlings at 5 DAG, sonicated, and immunoprecipitated using anti-GFP antibody. The precipitated DNA was used as a template for qPCR analysis. A, B, and I indicated the PCR amplicons ACT7, control. Data information: In (F), (G) data represent mean ± SD of three independent replicates. **P < 0.01 (Student's t-test).


H Mutation of the SCR gene impairs the recruitment of SEU to the WOX5 promoter , as shown by ChIP - qPCR analysis . Chromatin was extracted from SEU - GFP and SEU - GFP scr - 3 seedlings at 5 DAG and precipitated with anti - GFP antibody . ChIP signals were quantified by qPCR as a percentage of total input DNA . Data information : In ( H ) data represent mean ± SD of three independent replicates . * * P < 0 . 01 ( Student ' s t - test ) .

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H Mutation of the SCR gene impairs the recruitment of SEU to the WOX5 promoter, as shown by ChIP-qPCR analysis. Chromatin was extracted from SEU-GFP and SEU-GFP scr-3 seedlings at 5 DAG and precipitated with anti-GFP antibody. ChIP signals were quantified by qPCR as a percentage of total input DNA. Data information: In (H) data represent mean ± SD of three independent replicates. **P < 0.01 (Student's t-test).


I SEU stimulated SCR - mediated WOX5 promoter activation in transient expression assays in N . benthamiana leaves . The pWOX5 : : LUC reporter was cotransformed with the indicated effector constructs . The pWOX5 : : LUC activity was normalized relative to the internal control ( LUC / Renilla luciferase [ REN ] ) . The schematic diagram shows the construct used in the transient expression assays . Arrows indicate promoter regions , and boxes indicate coding sequences . Different lowercase letters indicate significant differences by one - way ANOVA followed by Tukey ' s multiple comparison test ( P < 0 . 01 ) . Data information : In ( I ) data represent mean ± SD of three independent replicates . * * P < 0 . 01 ( Student ' s t - test ) .

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I SEU stimulated SCR-mediated WOX5 promoter activation in transient expression assays in N. benthamiana leaves. The pWOX5::LUC reporter was cotransformed with the indicated effector constructs. The pWOX5::LUC activity was normalized relative to the internal control (LUC/Renilla luciferase [REN]). The schematic diagram shows the construct used in the transient expression assays. Arrows indicate promoter regions, and boxes indicate coding sequences. Different lowercase letters indicate significant differences by one-way ANOVA followed by Tukey's multiple comparison test (P < 0.01). Data information: In (I) data represent mean ± SD of three independent replicates. **P < 0.01 (Student's t-test).


J - M Representative confocal images of the indicated genotypes at 5 DAG . Scale bars : 20 μm . Insets show the QC region in which the solid white lines indicate the QC of WT , and the dashed white lines indicate the QC of seu - 3 . ( Insets scale bars : 5 μm ) . N Quantification of GFP signal intensity in the QC of WT and seu - 3 seedlings expressing pWOX5 : : SEU - GFP and pWOX5 : : SCR - GFP . GFP signal intensity in seu - 3 seedlings was normalized relative to the WT . Individual values ( black dots ) are shown . n denotes the total number of scored samples . Data information : In ( N ) , data represent mean ± SD of three independent replicates . * * P < 0 . 01 ( Student ' s t - test ) .

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J-M Representative confocal images of the indicated genotypes at 5 DAG. Scale bars: 20 μm. Insets show the QC region in which the solid white lines indicate the QC of WT, and the dashed white lines indicate the QC of seu-3. (Insets scale bars: 5 μm). N Quantification of GFP signal intensity in the QC of WT and seu-3 seedlings expressing pWOX5::SEU-GFP and pWOX5::SCR-GFP. GFP signal intensity in seu-3 seedlings was normalized relative to the WT. Individual values (black dots) are shown. n denotes the total number of scored samples. Data information: In (N), data represent mean ± SD of three independent replicates. **P < 0.01 (Student's t-test).


A Y2H assays showing that SEU interacts with SDG4 and SDG25 . The yeast transformants were plated on SD / - 2 and SD / - 4 media to assess protein - protein interactions .

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A Y2H assays showing that SEU interacts with SDG4 and SDG25. The yeast transformants were plated on SD/-2 and SD/-4 media to assess protein-protein interactions.


C SDG4 physically bind to the WOX5 promoter , as shown by ChIP - qPCR analysis . ACT7 , control . Data information Chromatin was isolated from 5 mm root tip sections of seedlings at 5 DAG , sonicated , and immunoprecipitated using anti - GFP or anti - H3K4me3 antibodies . The precipitated DNA was used as a template for qPCR analysis . In ( C ) data represent mean ± SD of three independent replicates . * * P < 0 . 01 , * P < 0 . 05 ( Student ' s t - test ) .

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C SDG4 physically bind to the WOX5 promoter, as shown by ChIP-qPCR analysis. ACT7, control. Data information Chromatin was isolated from 5 mm root tip sections of seedlings at 5 DAG, sonicated, and immunoprecipitated using anti-GFP or anti-H3K4me3 antibodies. The precipitated DNA was used as a template for qPCR analysis. In (C) data represent mean ± SD of three independent replicates. **P < 0.01, *P < 0.05 (Student's t-test).


D , E ChIP - qPCR analysis indicating that mutations in SDG4 and SEU genes impair H3K4me3 deposition in the WOX5 promoter . ChIP signal was normalized to the WT . Individual values ( black dots ) are shown . Data information : Chromatin was isolated from 5 mm root tip sections of seedlings at 5 DAG , sonicated , and immunoprecipitated using anti - GFP or anti - H3K4me3 antibodies . The precipitated DNA was used as a template for qPCR analysis . In ( D ) , ( E ) data represent mean ± SD of three independent replicates . * * P < 0 . 01 , * P < 0 . 05 ( Student ' s t - test ) .

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D, E ChIP-qPCR analysis indicating that mutations in SDG4 and SEU genes impair H3K4me3 deposition in the WOX5 promoter. ChIP signal was normalized to the WT. Individual values (black dots) are shown. Data information: Chromatin was isolated from 5 mm root tip sections of seedlings at 5 DAG, sonicated, and immunoprecipitated using anti-GFP or anti-H3K4me3 antibodies. The precipitated DNA was used as a template for qPCR analysis. In (D), (E) data represent mean ± SD of three independent replicates. **P < 0.01, *P < 0.05 (Student's t-test).


F , G Expression pattern of pWOX5 : : GFP in the indicated genotypes at 5 DAG . Scale bars : 20 µm . H Quantification of GFP fluorescence in the indicated genotypes expressing pWOX5 : : GFP . GFP signal intensity in the WT was set to 1 . n denotes the total number of scored samples . Data information : In ( H ) data represent mean ± SD of three independent replicates . * * P < 0 . 01 , * P < 0 . 05 ( Student ' s t - test ) .

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F, G Expression pattern of pWOX5::GFP in the indicated genotypes at 5 DAG. Scale bars: 20 µm. H Quantification of GFP fluorescence in the indicated genotypes expressing pWOX5::GFP. GFP signal intensity in the WT was set to 1. n denotes the total number of scored samples. Data information: In (H) data represent mean ± SD of three independent replicates. **P < 0.01, *P < 0.05 (Student's t-test).


I RT - qPCR analysis of the relative expression levels of WOX5 in WT and ashr3 - 1 roots . Total RNA was extracted from 5 mm root tip sections of seedlings at 5 DAG . Individual values ( black dots ) are shown . Data information : In ( I ) data represent mean ± SD of three independent replicates . * * P < 0 . 01 , * P < 0 . 05 ( Student ' s t - test ) .

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I RT-qPCR analysis of the relative expression levels of WOX5 in WT and ashr3-1 roots. Total RNA was extracted from 5 mm root tip sections of seedlings at 5 DAG. Individual values (black dots) are shown. Data information: In (I) data represent mean ± SD of three independent replicates. **P < 0.01, *P < 0.05 (Student's t-test).


J - M mPS - PI staining of stem cell niche in the indicated genotypes at 5 DAG . Blue arrows indicate the QC , and red arrows indiate the CSCs . The numbers denote total number of scored samples , with same phenotypes showing in ( J - M ) . Scale bars : 10 μm . N Quantification of the CSC layer in the indicated genotypes at 5 DAG . Data information In ( N ) , data represent mean ± SD of three independent replicates . * * P < 0 . 01 , * P < 0 . 05 ( Student ' s t - test ) .

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J-M mPS-PI staining of stem cell niche in the indicated genotypes at 5 DAG. Blue arrows indicate the QC, and red arrows indiate the CSCs. The numbers denote total number of scored samples, with same phenotypes showing in (J-M). Scale bars: 10 μm. N Quantification of the CSC layer in the indicated genotypes at 5 DAG. Data information In (N), data represent mean ± SD of three independent replicates. **P < 0.01, *P < 0.05 (Student's t-test).


A - G Representative confocal images of the indicated genotypes at 5 DAG . The white dashed lines indicate the QC region . Scale bars : 20 μm .

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A-G Representative confocal images of the indicated genotypes at 5 DAG. The white dashed lines indicate the QC region. Scale bars: 20 μm.


H Domain mapping of SEU involved in interactions with SCR and SDG4 . The yeast transformants were plated on SD / - 2 and SD / - 4 media to assess protein - protein interactions .

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H Domain mapping of SEU involved in interactions with SCR and SDG4. The yeast transformants were plated on SD/-2 and SD/-4 media to assess protein-protein interactions.


I SEU associates with SCR and SDG4 in N . benthamiana leaves , as shown by Co - IP assays . SDG4 - myc , SCR - HA , and SEU - GFP were coinfiltrated into N . benthamiana leaves . Protein samples were immunoprecipitated with anti - GFP antibody and immunoblotted with anti - myc and anti - HA antibody . N . benthamiana leaves coinfiltrated with SDG4 - myc and SCR - HA were used as a negative control .

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I SEU associates with SCR and SDG4 in N. benthamiana leaves, as shown by Co-IP assays. SDG4-myc, SCR-HA, and SEU-GFP were coinfiltrated into N. benthamiana leaves. Protein samples were immunoprecipitated with anti-GFP antibody and immunoblotted with anti-myc and anti-HA antibody. N. benthamiana leaves coinfiltrated with SDG4-myc and SCR-HA were used as a negative control.


J Co - IP assays of SCR with SEU and SDG4 in Arabidopsis . Proteins extracted from SDG4 - myc and SDG4 - myc SCR - GFP plants were immunoprecipitated using anti - GFP antibody , and immunoblotted using anti - myc and anti - SEU antibodies .

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J Co-IP assays of SCR with SEU and SDG4 in Arabidopsis. Proteins extracted from SDG4-myc and SDG4-myc SCR-GFP plants were immunoprecipitated using anti-GFP antibody, and immunoblotted using anti-myc and anti-SEU antibodies.


( B ) Dot plot indicating fraction of tf - Reporters delivered to the lysosome by ATG7 - independent mechanisms . The median red : green ratio of each population was used to calculate fractional delivery according to the following formula : ( ratiosgATG7 - ratiosgATG9A ) / ( ratioNegativeCtl1 - ratiosgATG9A ) , with the assumption that sgATG9A yields a true autophagy - null phenotype . Mean values for each reporter are indicated by a black bar . n = 3 for each reporter .

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(B) Dot plot indicating fraction of tf-Reporters delivered to the lysosome by ATG7-independent mechanisms. The median red:green ratio of each population was used to calculate fractional delivery according to the following formula: (ratiosgATG7-ratiosgATG9A)/(ratioNegativeCtl1-ratiosgATG9A), with the assumption that sgATG9A yields a true autophagy-null phenotype. Mean values for each reporter are indicated by a black bar. n=3 for each reporter.


( C ) Wild - type ( WT ) K562 cells and indicated deletion isolates were treated with 100 nM Bafilomycin A1 ( BafA1 ) or DMSO for 18h and analyzed by flow cytometry for red : green ratio of tf - NBR1 . Median values for each sample are identified by a black line within each violin . The red dotted line across all samples corresponds to the red : green ratio in wild - type cells . The red solid line across all samples corresponds to the ratio observed under maximally inhibited conditions ( ATG9AKO cells ) . n = 10 , 000 cells .

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(C) Wild-type (WT) K562 cells and indicated deletion isolates were treated with 100 nM Bafilomycin A1 (BafA1) or DMSO for 18h and analyzed by flow cytometry for red:green ratio of tf-NBR1. Median values for each sample are identified by a black line within each violin. The red dotted line across all samples corresponds to the red:green ratio in wild-type cells. The red solid line across all samples corresponds to the ratio observed under maximally inhibited conditions (ATG9AKO cells). n = 10,000 cells.


( D ) Representative confocal micrographs ( as maximum intensity projections ) of wild - type K562 cells and indicated deletion isolates expressing tf - NBR1 . Selected regions ( white box ) of micrographs are shown as single and merged channels from fluorescence microscopy . Red fluorescent protein ( RFP ) , magenta ; Green fluorescent protein ( GFP ) , green ; Hoechst , blue . Scale bars : large panels , 5 µm ; small panels , 1 µm . All images scaled equally .

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(D) Representative confocal micrographs (as maximum intensity projections) of wild-type K562 cells and indicated deletion isolates expressing tf-NBR1. Selected regions (white box) of micrographs are shown as single and merged channels from fluorescence microscopy. Red fluorescent protein (RFP), magenta; Green fluorescent protein (GFP), green; Hoechst, blue. Scale bars: large panels, 5 µm; small panels, 1 µm. All images scaled equally.


( E ) Correlative light and electron microscopy ( CLEM ) of K562 ATG7KO cells expressing tf - NBR1 under basal conditions . Analysis workflow is indicated by green arrows . White arrows indicate representative structures of interest . White boxes demarcate zoomed areas in subsequent images . NBR1 , green ; Hoechst , blue . Scale bar ( small images ) , 2 . 5 µm . Scale bar ( large images ) , 250 nm .

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(E) Correlative light and electron microscopy (CLEM) of K562 ATG7KO cells expressing tf-NBR1 under basal conditions. Analysis workflow is indicated by green arrows. White arrows indicate representative structures of interest. White boxes demarcate zoomed areas in subsequent images. NBR1, green; Hoechst, blue. Scale bar (small images), 2.5 µm. Scale bar (large images), 250 nm.


( A ) Box plot of average beta scores ( similar to log - fold change ) for tf - NBR1 flux in indicated deletion cell lines . Median values ( central band ) , inner quartiles ( boxed regions ) , 1 . 5 interquartile ranges ( whiskers ) , and outliers ( dots ) are indicated . Representative hits across all three genetic backgrounds are indicated in blue .

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(A) Box plot of average beta scores (similar to log-fold change) for tf-NBR1 flux in indicated deletion cell lines. Median values (central band), inner quartiles (boxed regions), 1.5 interquartile ranges (whiskers), and outliers (dots) are indicated. Representative hits across all three genetic backgrounds are indicated in blue.


( B ) Gene correlation plot of average beta scores for lipidation dependent autophagy ( tf - NDP52 , from ( Shoemaker et al , 2019 ) ) and lipidation independent autophagy ( average of tf - NBR1 from ATG7KO , ATG10 KO and ATG3 KO cells Highlighted in red are genes with a beta score > 0 . 5 across all three lipidation - deficient cell lines . In black are lipidation components . Dashed lines , top 1 % of beta scores .

(B) Gene correlation plot of average beta scores for lipidation dependent autophagy (tf-NDP52, from (Shoemaker et al, 2019)) and lipidation independent autophagy (average of tf-NBR1 from ATG7KO, ATG10 KO and ATG3 KO cells Highlighted in red are genes with a beta score > 0.5 across all three lipidation-deficient cell lines. In black are lipidation components. Dashed lines, top 1% of beta scores.


( C ) Wild - type K562 cells and indicated deletion isolates expressing Cas9 and tf - NDP52 or tf - NBR1 were transduced with individual sgRNAs for indicated genes . The median red : green ratio of each population was used to calculate the fold - repression according to the following formula : ( ratiosgGene - ratiosgATG9A ) / ( ratioNegativeCtl - ratiosgATG9A ) , with the assumption that sgATG9A yields a true autophagy - null phenotype . Deeper shades of red indicate stronger suppressor phenotypes . Genes were clustered on the basis of their patterns of genetic interactions .

(C) Wild-type K562 cells and indicated deletion isolates expressing Cas9 and tf-NDP52 or tf-NBR1 were transduced with individual sgRNAs for indicated genes. The median red:green ratio of each population was used to calculate the fold-repression according to the following formula: (ratiosgGene-ratiosgATG9A)/(ratioNegativeCtl-ratiosgATG9A), with the assumption that sgATG9A yields a true autophagy-null phenotype. Deeper shades of red indicate stronger suppressor phenotypes. Genes were clustered on the basis of their patterns of genetic interactions.


( A ) Wild - type ( WT ) K562 cells and indicated deletion isolates were treated with 100nM Bafilomycin A1 ( BafA1 ) or DMSO for 18h and analyzed by flow cytometry for red : green ratio of tf - NBR1 . Median values for each sample are identified by a black line within each violin . The red dotted line across all samples corresponds to the red : green ratio in wild - type cells . The red solid line across all samples corresponds to the ratio observed under maximally inhibited conditions ( ATG9AKO cells ) . ( n > 10 , 000 cells ) .

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(A) Wild-type (WT) K562 cells and indicated deletion isolates were treated with 100nM Bafilomycin A1 (BafA1) or DMSO for 18h and analyzed by flow cytometry for red:green ratio of tf-NBR1. Median values for each sample are identified by a black line within each violin. The red dotted line across all samples corresponds to the red:green ratio in wild-type cells. The red solid line across all samples corresponds to the ratio observed under maximally inhibited conditions (ATG9AKO cells). (n > 10,000 cells).