C Fraction of expressed genes ( genes with non - zero counts in DESeq2 ) were calculated which exhibit individual or combinations of differential gene expression ( DGE ) , differential transcript usage ( DTU ) and / or alternative splicing ( AS ) events in HEK293 and HeLa WT cells overexpressing UPF3A using the respective computational analysis ( cutoffs are indicated ) . AS and DTU events were collapsed on the gene level . For DGE , p - values were calculated by DESeq2 using a two - sided Wald test and corrected for multiple testing using the Benjamini - Hochberg method . For DTU , p - values were calculated by IsoformSwitchAnalyzeR ( ISAR ) using a DEXSeq - based test and corrected for multiple testing using the Benjamini - Hochberg method . For AS , p - values were calculated by LeafCutter using an asymptotic Chi - squared distribution and corrected for multiple testing using the Benjamini - Hochberg method .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Fraction of expressed genes (genes with non-zero counts in DESeq2) were calculated which exhibit individual or combinations of differential gene expression (DGE), differential transcript usage (DTU) and/or alternative splicing (AS) events in HEK293 and HeLa WT cells overexpressing UPF3A using the respective computational analysis (cutoffs are indicated). AS and DTU events were collapsed on the gene level. For DGE, p-values were calculated by DESeq2 using a two-sided Wald test and corrected for multiple testing using the Benjamini-Hochberg method. For DTU, p-values were calculated by IsoformSwitchAnalyzeR (ISAR) using a DEXSeq-based test and corrected for multiple testing using the Benjamini-Hochberg method. For AS, p-values were calculated by LeafCutter using an asymptotic Chi-squared distribution and corrected for multiple testing using the Benjamini-Hochberg method.


D Volcano plot showing the differential gene expression analyses from the RNA - Seq dataset of HEK293 and HeLa WT cells overexpressing UPF3A . The log2 fold change is plotted against the - log10 adjusted p - value ( adj . p - value ) . P - values were calculated by DESeq2 using a two - sided Wald test and corrected for multiple testing using the Benjamini - Hochberg method . OE = overexpression .

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

D Volcano plot showing the differential gene expression analyses from the RNA-Seq dataset of HEK293 and HeLa WT cells overexpressing UPF3A. The log2 fold change is plotted against the -log10 adjusted p-value (adj. p-value). P-values were calculated by DESeq2 using a two-sided Wald test and corrected for multiple testing using the Benjamini-Hochberg method. OE = overexpression.


E Read coverage of SMG5 from WT HEK293 and HeLa RNA - seq data with or without induced UPF3A overexpression shown as Integrative Genomics Viewer ( IGV ) snapshot . Differential gene expression ( from DESeq2 ) is indicated as log2 fold change ( log2FC ) on the right . Schematic representation of the protein coding transcript below .

O O O O B-MEASURED_VAR 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

E Read coverage of SMG5 from WT HEK293 and HeLa RNA-seq data with or without induced UPF3A overexpression shown as Integrative Genomics Viewer (IGV) snapshot. Differential gene expression (from DESeq2) is indicated as log2 fold change (log2FC) on the right. Schematic representation of the protein coding transcript below.


A Western blot analysis of WT and UPF3A KO cells ( clones 4 , 14 and 20 ) with the indicated siRNA treatments ( n = 1 ) . UPF3A and UPF3B protein levels were detected , Tubulin serves as control . The asterisk indicates unspecific bands .

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

A Western blot analysis of WT and UPF3A KO cells (clones 4, 14 and 20) with the indicated siRNA treatments (n=1). UPF3A and UPF3B protein levels were detected, Tubulin serves as control. The asterisk indicates unspecific bands.


B Quantitative RT - PCR of the indicated cell lines treated with the indicated siRNAs for 2 or 6 days . For RSRC2 and SRSF2 the ratio of NMD isoform to canonical isoform was calculated . ZFAS1 expression was normalized to C1orf43 reference . Data points and means are plotted as log2 fold change ( log2FC ) ( n = 3 for RSRC2 and SRSF2 , n = 4 for ZFAS1 ) .

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

B Quantitative RT-PCR of the indicated cell lines treated with the indicated siRNAs for 2 or 6 days. For RSRC2 and SRSF2 the ratio of NMD isoform to canonical isoform was calculated. ZFAS1 expression was normalized to C1orf43 reference. Data points and means are plotted as log2 fold change (log2FC) (n=3 for RSRC2 and SRSF2, n=4 for ZFAS1).


C Fraction of expressed genes ( genes with non - zero counts in DESeq2 ) were calculated which exhibit individual or combinations of differential gene expression ( DGE ) , differential transcript usage ( DTU ) and / or alternative splicing ( AS ) events in the indicated conditions using the respective computational analysis ( cutoffs are indicated ) . AS and DTU events were collapsed on the gene level . For DGE , p - values were calculated by DESeq2 using a two - sided Wald test and corrected for multiple testing using the Benjamini - Hochberg method . For DTU , p - values were calculated by IsoformSwitchAnalyzeR using a DEXSeq - based test and corrected for multiple testing using the Benjamini - Hochberg method . For AS , p - values were calculated by LeafCutter using an asymptotic Chi - squared distribution and corrected for multiple testing using the Benjamini - Hochberg method .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Fraction of expressed genes (genes with non-zero counts in DESeq2) were calculated which exhibit individual or combinations of differential gene expression (DGE), differential transcript usage (DTU) and/or alternative splicing (AS) events in the indicated conditions using the respective computational analysis (cutoffs are indicated). AS and DTU events were collapsed on the gene level. For DGE, p-values were calculated by DESeq2 using a two-sided Wald test and corrected for multiple testing using the Benjamini-Hochberg method. For DTU, p-values were calculated by IsoformSwitchAnalyzeR using a DEXSeq-based test and corrected for multiple testing using the Benjamini-Hochberg method. For AS, p-values were calculated by LeafCutter using an asymptotic Chi-squared distribution and corrected for multiple testing using the Benjamini-Hochberg method.


D , E Volcano plots showing the differential gene expression analyses from the indicated RNA - Seq datasets ( D UPF3A KO clone 14 , E UPF3A KO clone 20 ) . The log2 fold change is plotted against the - log10 adjusted p - value ( adj . p - value ) . P - values were calculated by DESeq2 using a two - sided Wald test and corrected for multiple testing using the Benjamini - Hochberg

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Volcano plots showing the differential gene expression analyses from the indicated RNA-Seq datasets (D UPF3A KO clone 14, E UPF3A KO clone 20). The log2 fold change is plotted against the -log10 adjusted p-value (adj. p-value). P-values were calculated by DESeq2 using a two-sided Wald test and corrected for multiple testing using the Benjamini-Hochberg method


A Western blot analysis of WT and UPF3B KO cells ( clones 90 and 91 ) combined with the indicated knockdowns ( n = 1 ) . UPF3A and UPF3B ( AK - 141 ) protein levels were detected , Tubulin serves as control . Protein levels of UPF3A were quantified , normalized to tubulin expression , and shown as datapoints and mean . Fold - changes of relevant conditions are shown . The asterisk indicates unspecific bands .

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

A Western blot analysis of WT and UPF3B KO cells (clones 90 and 91) combined with the indicated knockdowns (n=1). UPF3A and UPF3B (AK-141) protein levels were detected, Tubulin serves as control. Protein levels of UPF3A were quantified, normalized to tubulin expression, and shown as datapoints and mean. Fold-changes of relevant conditions are shown. The asterisk indicates unspecific bands.


B Quantitative RT - PCR of the indicated cell lines with the indicated knockdowns . For RSRC2 and SRSF2 the ratio of NMD isoform to canonical isoform was calculated . Data points and means are plotted as log2 fold change ( log2FC , n = 3 ) .

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

B Quantitative RT-PCR of the indicated cell lines with the indicated knockdowns. For RSRC2 and SRSF2 the ratio of NMD isoform to canonical isoform was calculated. Data points and means are plotted as log2 fold change (log2FC, n=3).


D , E Volcano plots showing the differential transcript usage ( via IsoformSwitchAnalyzeR ) in various RNA - Seq data . Isoforms containing GENCODE ( release 33 ) annotated PTC ( red , TRUE ) , regular stop codons ( blue , FALSE ) or having no annotated open reading frame ( grey , NA ) are indicated . The change in isoform fraction ( dIF ) is plotted against the - log10 adjusted p - value ( adj . p - value ) . Density plots show the distribution of filtered isoforms in respect to the dIF , cutoffs were | dIF | > 0 . 1 and adj . p - value < 0 . 05 . P - values were calculated by IsoformSwitchAnalyzeR using a DEXSeq - based test and corrected for multiple testing using the Benjamini - Hochberg method .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Volcano plots showing the differential transcript usage (via IsoformSwitchAnalyzeR) in various RNA-Seq data. Isoforms containing GENCODE (release 33) annotated PTC (red, TRUE), regular stop codons (blue, FALSE) or having no annotated open reading frame (grey, NA) are indicated. The change in isoform fraction (dIF) is plotted against the -log10 adjusted p-value (adj. p-value). Density plots show the distribution of filtered isoforms in respect to the dIF, cutoffs were |dIF| > 0.1 and adj. p-value < 0.05. P-values were calculated by IsoformSwitchAnalyzeR using a DEXSeq-based test and corrected for multiple testing using the Benjamini-Hochberg method.


A Western blot analysis of WT , UPF3B KO and UPF3A - UPF3B double KO cells ( clones 1 and 2 ) ( n = 1 ) . UPF3A and UPF3B protein levels were detected , Tubulin serves as control . The asterisk indicates unspecific bands .

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

A Western blot analysis of WT, UPF3B KO and UPF3A-UPF3B double KO cells (clones 1 and 2) (n=1). UPF3A and UPF3B protein levels were detected, Tubulin serves as control. The asterisk indicates unspecific bands.


C Quantitative RT - PCR of the indicated samples with the indicated KDs . For RSRC2 and SRSF2 the ratio of NMD isoform to canonical isoform was calculated . ZFAS1 expression was normalized to C1orf43 reference . Data points and means are plotted as log2 fold change ( log2FC , n = 3 ) .

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

C Quantitative RT-PCR of the indicated samples with the indicated KDs. For RSRC2 and SRSF2 the ratio of NMD isoform to canonical isoform was calculated. ZFAS1 expression was normalized to C1orf43 reference. Data points and means are plotted as log2 fold change (log2FC, n=3).


E Northern blot analysis of globin reporter and xrFrag . Ethidium bromide stained 28S and 18S rRNAs are shown as controls . Quantification results are shown as data points and mean ( n = 3 ) .

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

E Northern blot analysis of globin reporter and xrFrag. Ethidium bromide stained 28S and 18S rRNAs are shown as controls. Quantification results are shown as data points and mean (n=3).


A - C Volcano plots of label free mass spectrometry - based analysis of the interaction partners of UPF2 in WT cells treated with control siRNAs and the UPF3B KO clone 90 and dKO clone 1 both treated with siRNAs targeting UPF3B ( n = 4 biologically independent samples ) . ( A ) FLAG - UPF2 in WT against FLAG - GST control in WT cells , ( B ) UPF2 in 3B KO cells against FLAG control in WT cells , ( C ) UPF2 in dKO cells against FLAG control in WT cells . Points labeled in purple indicate NMD factors ; points labeled in turquoise indicate EJC components . Cut offs : Log2 fold change ≥

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

A-C Volcano plots of label free mass spectrometry-based analysis of the interaction partners of UPF2 in WT cells treated with control siRNAs and the UPF3B KO clone 90 and dKO clone 1 both treated with siRNAs targeting UPF3B (n = 4 biologically independent samples). (A) FLAG-UPF2 in WT against FLAG-GST control in WT cells, (B) UPF2 in 3B KO cells against FLAG control in WT cells, (C) UPF2 in dKO cells against FLAG control in WT cells. Points labeled in purple indicate NMD factors; points labeled in turquoise indicate EJC components. Cut offs: Log2 fold change ≥ 0


E Representative Western blot after FLAG co - immunoprecipitation ( IP ) of FLAG - tagged GST ( control ) or UPF3B WT and single mutant constructs in UPF3B KO cells ( n = 3 ) .

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

E Representative Western blot after FLAG co-immunoprecipitation (IP) of FLAG-tagged GST (control) or UPF3B WT and single mutant constructs in UPF3B KO cells (n = 3).


F Western blot analysis of WT and UPF3B KO clone 90 with Luciferase and UPF3A KDs respectively . Monitored expression of the FLAG - tagged UPF3B rescue construct shown in ( D ) . Rescue construct protein levels were detected with anti - FLAG and anti - UPF3B ( AK - 141 ) antibodies . Tubulin serves as control ( n = 1 ) .

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

F Western blot analysis of WT and UPF3B KO clone 90 with Luciferase and UPF3A KDs respectively. Monitored expression of the FLAG-tagged UPF3B rescue construct shown in (D). Rescue construct protein levels were detected with anti-FLAG and anti-UPF3B (AK-141) antibodies. Tubulin serves as control (n=1).


G Quantitative RT - PCR of the samples from ( F ) . For RSRC2 and SRSF2 the ratio of NMD isoform to canonical isoform was calculated . ZFAS1 expression was normalized to C1orf43 reference . Data points and means are plotted as log2 fold change ( log2FC , n = 3 ) .

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

G Quantitative RT-PCR of the samples from (F). For RSRC2 and SRSF2 the ratio of NMD isoform to canonical isoform was calculated. ZFAS1 expression was normalized to C1orf43 reference. Data points and means are plotted as log2 fold change (log2FC, n=3).


B Western blot analysis of WT and UPF3B KO clone 90 with Luciferase and UPF3A KDs respectively . Monitored expression of the FLAG - tagged UPF3A and UPF3B rescue construct shown in ( A ) . Rescue construct protein levels were detected with anti - FLAG , anti - UPF3A and anti - UPF3B ( AK - 141 ) antibodies . Tubulin serves as control ( n = 1 ) . The asterisk indicates unspecific bands .

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

B Western blot analysis of WT and UPF3B KO clone 90 with Luciferase and UPF3A KDs respectively. Monitored expression of the FLAG-tagged UPF3A and UPF3B rescue construct shown in (A). Rescue construct protein levels were detected with anti-FLAG, anti-UPF3A and anti-UPF3B (AK-141) antibodies. Tubulin serves as control (n=1). The asterisk indicates unspecific bands.


C Quantitative RT - PCR of the samples from ( B ) . For RSRC2 and SRSF2 the ratio of NMD isoform to canonical isoform was calculated . Data points and means are plotted as log2 fold change ( log2FC ) ( n = 3 ) .

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

C Quantitative RT-PCR of the samples from (B). For RSRC2 and SRSF2 the ratio of NMD isoform to canonical isoform was calculated. Data points and means are plotted as log2 fold change (log2FC) (n=3).


B Representative adhesion frequency ( Pa ) vs . contact duration ( tc ) curves forNKG2D expressing NK cells ( n ≥ 3 ) in contact with RBCs ( n ≥ 3 ) coated with a ligand ( MICA in red , MICB in orange , ULBP1 in green , or ULBP3 in blue ) at different contact durations , fitted by non - linear in - situ binding kinetics model Site densities of NKG2D ( mr ) and its ligands ( ml ) are indicated . Data information : Error bars are mean ± SEM for at least three independent biological experiments where * P < 0 . 05 , * * P < 0 . 01 ( two - tailed unpaired 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 B-MEASURED_VAR O O O B-MEASURED_VAR O O O B-MEASURED_VAR 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 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

B Representative adhesion frequency (Pa) vs. contact duration (tc) curves forNKG2D expressing NK cells (n ≥ 3) in contact with RBCs (n ≥ 3) coated with a ligand (MICA in red, MICB in orange, ULBP1 in green, or ULBP3 in blue) at different contact durations, fitted by non-linear in-situ binding kinetics model Site densities of NKG2D (mr) and its ligands (ml) are indicated. Data information: Error bars are mean ± SEM for at least three independent biological experiments where *P < 0.05, **P < 0.01 (two-tailed unpaired t-test).


In - situ force - free affinities ( C ) , on - rates ( D ) of NKG2D binding with indicated ligands from mammalian cells . The in - situ force - free kinetics were obtained from fittings with in - situ binding kinetics modeling in ( B ) . Data information : Every dot represents one independent binding experiment . Error bars are mean ± SEM for at least three independent biological experiments where * P < 0 . 05 , * * P < 0 . 01 ( two - tailed unpaired t - test ) .

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

In-situ force-free affinities (C), on-rates (D) of NKG2D binding with indicated ligands from mammalian cells. The in-situ force-free kinetics were obtained from fittings with in-situ binding kinetics modeling in (B). Data information: Every dot represents one independent binding experiment. Error bars are mean ± SEM for at least three independent biological experiments where *P < 0.05, **P < 0.01 (two-tailed unpaired t-test).


In - situ force - free off - rates ( E ) of NKG2D binding with indicated ligands from mammalian cells . The in - situ force - free kinetics were obtained from fittings with in - situ binding kinetics modeling in ( B ) . Data information : Every dot represents one independent binding experiment . Error bars are mean ± SEM for at least three independent biological experiments where * P < 0 . 05 , * * P < 0 . 01 ( two - tailed unpaired t - test ) .

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

In-situ force-free off-rates (E) of NKG2D binding with indicated ligands from mammalian cells. The in-situ force-free kinetics were obtained from fittings with in-situ binding kinetics modeling in (B). Data information: Every dot represents one independent binding experiment. Error bars are mean ± SEM for at least three independent biological experiments where *P < 0.05, **P < 0.01 (two-tailed unpaired t-test).


F Detection range comparison in affinity measurement of NKG2D and indicated ligands between in - situ and in - solution assay . Bars in different colors are the ratios of the affinities of indicated ligands divided by that of ULBP1 . Data information : In - solution affinities of proteins purified from E . coli In - solution affinities of proteins purified from mammalian cells were measured by

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 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

F Detection range comparison in affinity measurement of NKG2D and indicated ligands between in-situ and in-solution assay. Bars in different colors are the ratios of the affinities of indicated ligands divided by that of ULBP1. Data information: In-solution affinities of proteins purified from E. coli In-solution affinities of proteins purified from mammalian cells were measured by BLI


IFN - γ release assay and IFN - γ production ( B ) by human peripheral NK cells stimulated with plate - coated NKG2D ligands ( MICA in red , MICB in green and ULBP3 in blue ) assessed by Cytometric Bead Array , of which are their 50 % effective concentration ( EC50 ) ( C ) . Data information : The IFN - γ release assay in ( B ) was one representative experiment of three total independent experiments . Data points in ( B ) and bars in ( C ) represent mean values . Error bars in ( B ) and ( C ) represent mean ± SEM . * * * P < 0 . 001 , * * * * P < 0 . 0001 ( two - tailed unpaired t - test ) .

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

IFN-γ release assay and IFN-γ production (B) by human peripheral NK cells stimulated with plate-coated NKG2D ligands (MICA in red, MICB in green and ULBP3 in blue) assessed by Cytometric Bead Array, of which are their 50% effective concentration (EC50) (C). Data information: The IFN-γ release assay in (B) was one representative experiment of three total independent experiments. Data points in (B) and bars in (C) represent mean values. Error bars in (B) and (C) represent mean ± SEM. ***P < 0.001, **** P <0.0001 (two-tailed unpaired t-test).


D - F Plots of reciprocals of EC50 vs . the effective in - situ affinities ( AcKa ) ( D ) , the effective in - situ on - rate ( AcKon ) ( E ) and in - situ off - rate ( koff ) ( F ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-F Plots of reciprocals of EC50 vs. the effective in-situ affinities (AcKa) (D), the effective in-situ on-rate (AcKon) (E) and in-situ off-rate (koff) (F).


C Verification of the binding specificity of NKG2D with different ligands . Data information : Every dot represents the adhesion frequency of one cell - bead pair and the experimental data came from at least two to three repeated trials . Horizontal lines represent mean values . Error bars represent mean ± SEM .

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

C Verification of the binding specificity of NKG2D with different ligands. Data information: Every dot represents the adhesion frequency of one cell-bead pair and the experimental data came from at least two to three repeated trials. Horizontal lines represent mean values. Error bars represent mean ± SEM.


E , F Force - dependent bond lifetimes of NKG2D and various ligands at 5 pN , 10 pN and 15 pN ( E ) and full force spectrum ( F ) . Data information : Bond lifetimes in ( E ) and ( F ) ( in which n = 1505 for MICA , n = 1852 for MICB , n = 531 for ULBP1 , n = 1241 for ULBP3 ) of all ligands with NKG2D came from at least 19 NK cell - bead pairs of at least four independent biological experiments . bars in ( E ) , and data points in ( F ) represent mean values . Error bars represent mean ± SEM .

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

E, F Force-dependent bond lifetimes of NKG2D and various ligands at 5 pN, 10 pN and 15 pN (E) and full force spectrum (F). Data information: Bond lifetimes in (E) and (F) (in which n = 1505 for MICA, n = 1852 for MICB, n = 531 for ULBP1, n = 1241 for ULBP3) of all ligands with NKG2D came from at least 19 NK cell-bead pairs of at least four independent biological experiments. bars in (E), and data points in (F) represent mean values. Error bars represent mean ± SEM.


C The force vs . extension trajectories for the simulations are shown in ( A ) and ( B ) . Occurrence of the sudden extension changes are indicated in the shaded area and time points correspond with the snapshots in ( A ) and ( B ) are marked .

C The force vs. extension trajectories for the simulations are shown in (A) and (B). Occurrence of the sudden extension changes are indicated in the shaded area and time points correspond with the snapshots in (A) and (B) are marked.


F Distance vs . time curves for force - induced H - bond formation between indicated residues within NKG2D - MICA binding interfaces . The dashed red lines represent H - bonds whose distances are less than 3 . 5 Å .

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F Distance vs. time curves for force-induced H-bond formation between indicated residues within NKG2D-MICA binding interfaces. The dashed red lines represent H-bonds whose distances are less than 3.5 Å.


A , B Abolishments of force - induced binding residues by 3A mutations ( MICA 3A , n = 526 ) and TAT mutations ( MICA TAT , n = 580 ) impairs NKG2D ' s catch bond with MICA WT ( n = 1505 ) ( A ) and IFN - γ release ( B ) . Data information : Bond lifetimes in ( A ) of MICA WT and mutants with NKG2D came from at least 21 pairs of cells and beads of at least three repeated experiments . IFN - γ release of NK cells in ( B ) was one representative experiment of three total independent experiments . Data points represent mean values . Error bars represent mean ± SEM for biological triplicate experiments . * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 ( two - tailed unpaired t - test ) .

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

A, B Abolishments of force-induced binding residues by 3A mutations (MICA 3A, n = 526) and TAT mutations (MICA TAT, n= 580) impairs NKG2D's catch bond with MICA WT (n = 1505) (A) and IFN-γ release (B). Data information: Bond lifetimes in (A) of MICA WT and mutants with NKG2D came from at least 21 pairs of cells and beads of at least three repeated experiments. IFN-γ release of NK cells in (B) was one representative experiment of three total independent experiments. Data points represent mean values. Error bars represent mean ± SEM for biological triplicate experiments. **P < 0.01, ***P < 0.001, **** P <0.0001 (two-tailed unpaired t-test).


50 % effective concentration ( EC50 ) of NK cells stimulated by indicated MICA mutants Data information : horizontal lines represent mean values . Error bars represent mean ± SEM for biological triplicate experiments . * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 ( two - tailed unpaired t - test ) .

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50% effective concentration (EC50) of NK cells stimulated by indicated MICA mutants Data information: horizontal lines represent mean values. Error bars represent mean ± SEM for biological triplicate experiments. **P < 0.01, ***P < 0.001, **** P <0.0001 (two-tailed unpaired t-test).


IFN - γ release of NK cells stimulated by indicated MICA mutants at a concentration of 11nM ( D ) and 22nM ( E ) . Data information : bars in ( E ) , and data point in ( F ) represent mean values . Error bars represent mean ± SEM for biological triplicate experiments . * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 ( two - tailed unpaired t - test ) .

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

IFN-γ release of NK cells stimulated by indicated MICA mutants at a concentration of 11nM (D) and 22nM (E). Data information: bars in (E), and data point in (F) represent mean values. Error bars represent mean ± SEM for biological triplicate experiments. **P < 0.01, ***P < 0.001, **** P <0.0001 (two-tailed unpaired t-test).


A - C Plots and Pearson correlation analysis of reciprocals of EC50 vs . the force - dependent in - situ koff at 5 pN ( A ) , 10 pN ( B ) 15pN ( C ) .

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A-C Plots and Pearson correlation analysis of reciprocals of EC50 vs. the force-dependent in-situ koff at 5 pN (A), 10 pN (B) 15pN (C).


I Detection range comparison in force - dependent affinity of NKG2D and indicated ligands under difference force . Bars in different colors are the ratios of the affinities of indicated ligands divided by that of ULBP1 .

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I Detection range comparison in force-dependent affinity of NKG2D and indicated ligands under difference force. Bars in different colors are the ratios of the affinities of indicated ligands divided by that of ULBP1.


D Contour plots showed probabilities of output signals at 300s produced by continuously variable initial on - rates and force - dependent off - rates .

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D Contour plots showed probabilities of output signals at 300s produced by continuously variable initial on-rates and force-dependent off-rates.


( A ) Assessment of vacuole ( arrows ) formation in neutrophils after stimulation with PMA ( II ) and rapamycin ( III ) , compared with control cells ( I : treated with PBS and serum ) ( magnification 1000 × ) .

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(A) Assessment of vacuole (arrows) formation in neutrophils after stimulation with PMA (II) and rapamycin (III), compared with control cells (I: treated with PBS and serum) (magnification 1000×).


( B ) ( I ) Presence of late autophagosomes as observed with MDC staining in cells treated with PMA and rapamycin ( RAP ) compared with control cells ( PBS ) and cells treated with PMA and 3 ‐ MA ( magnification 400 and 1000 × ) . ( II ) Percentage of PMN with acidified vacuoles as detected by MDC staining . Data are representative of six independent experiments and are presented as mean ± SD ; Wilcoxon matched ‐ pairs test , * p < 0 . 05 : PMA ( bar 2 : 86 . 16 ± 6 . 49 % ) , rapamycin ( bar 3 : 65 . 83 ± 8 . 47 % ) treated cells , cells treated with PMA and 3 ‐ MA ( bar 4 : 9 . 17 ± 0 . 75 % ) and cells treated with rapamycin and 3 ‐ MA ( bar 5 : 8 . 5 ± 1 . 87 % ) compared with control conditions ( bar 1 : 15 . 67 ± 3 . 50 % ) . The percentage of cells with acidified autophagosomes in PMN treated with PMA and 3 ‐ MA ( bar 4 ) or rapamycin and 3 ‐ MA ( bar 5 ) was significantly lower ( p < 0 . 05 ) compared with cells treated with PMA ( bar 2 ) and rapamycin ( bar 3 ) , 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 O O O O O O O O O O O O O O O O O O O O O O O O O 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) (I) Presence of late autophagosomes as observed with MDC staining in cells treated with PMA and rapamycin (RAP) compared with control cells (PBS) and cells treated with PMA and 3‐MA (magnification 400 and 1000×). (II) Percentage of PMN with acidified vacuoles as detected by MDC staining. Data are representative of six independent experiments and are presented as mean±SD; Wilcoxon matched‐pairs test, *p<0.05: PMA (bar 2: 86.16±6.49%), rapamycin (bar 3: 65.83±8.47%) treated cells, cells treated with PMA and 3‐MA (bar 4: 9.17±0.75%) and cells treated with rapamycin and 3‐MA (bar 5: 8.5±1.87%) compared with control conditions (bar 1: 15.67±3.50%). The percentage of cells with acidified autophagosomes in PMN treated with PMA and 3‐MA (bar 4) or rapamycin and 3‐MA (bar 5) was significantly lower (p<0.05) compared with cells treated with PMA (bar 2) and rapamycin (bar 3), respectively.


( C ) Analysis of LC3B ‐ I to LC3B ‐ II conversion in cells treated with PMA ( lane IV ) and rapamycin ( lane II ) , as observed by LC3B immunoblotting . Effect of 3 ‐ MA in both PMA ( lane V ) and rapamycin ( lane I ) ‐ treated cells . Lane III : control cells , and lane VI : cells treated only with 3 ‐ MA . One representative out of six independent experiments is shown .

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(C) Analysis of LC3B‐I to LC3B‐II conversion in cells treated with PMA (lane IV) and rapamycin (lane II), as observed by LC3B immunoblotting. Effect of 3‐MA in both PMA (lane V) and rapamycin (lane I)‐treated cells. Lane III: control cells, and lane VI: cells treated only with 3‐MA. One representative out of six independent experiments is shown.


( D ) Impact of PI ‐ 3K inhibition using LY294002 on LC3B conversion after PMA challenge ( lane III ) , ( lane I : control cells , lane II : PMA , lane IV : LY294002 only ) . One representative out of four independent experiments is shown .

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(D) Impact of PI‐3K inhibition using LY294002 on LC3B conversion after PMA challenge (lane III), (lane I: control cells, lane II: PMA, lane IV: LY294002 only). One representative out of four independent experiments is shown.


( E ) Immunofluorescence confocal microscopy staining of LC3B . ( I ) Control cells treated with PBS and serum , ( II ) PMA ‐ treated cells . 1 . DAPI : blue , 2 . LC3B : red and 3 . merge . ( III ) Percentage of PMN with LC3B aggregates . Control conditions ( bar 1 : 17 . 7 ± 2 . 4 % ) , 3 ‐ MA ( bar 2 : 3 ± 1 . 01 % ) , PMA ( bar 3 : 69 ± 2 . 1 % ) , PMA and 3 ‐ MA ( bar 4 : 4 ± 1 . 9 % ) , rapamycin ( bar 5 : 56 ± 1 . 6 % ) . Data are representative of five independent experiments and presented as mean ± SD ; Student ' s t ‐ test , * p < 0 . 01 compared to the control ( bar 1 ) or the indicated comparision ( bar 3 vs 4 ) .

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(E) Immunofluorescence confocal microscopy staining of LC3B. (I) Control cells treated with PBS and serum, (II) PMA‐treated cells. 1. DAPI: blue, 2. LC3B: red and 3. merge. (III) Percentage of PMN with LC3B aggregates. Control conditions (bar 1: 17.7±2.4%), 3‐MA (bar 2: 3±1.01%), PMA (bar 3: 69±2.1%), PMA and 3‐MA (bar 4: 4±1.9%), rapamycin (bar 5: 56±1.6%). Data are representative of five independent experiments and presented as mean±SD; Student's t‐test, *p<0.01 compared to the control (bar 1) or the indicated comparision (bar 3 vs 4).


ATG3 and LC3B gene expression pattern in neutrophils after stimulation with PMA or rapamycin . ATG3 mRNA expression analysis ( fold ) after PMA ( 2 . 44 ± 0 . 78 ) and rapamycin ( RAP : 1 . 81 ± 0 . 41 ) stimulation . LC3B gene alterations ( PMA : 4 . 98 ± 0 . 59 ) and rapamycin ( RAP : 1 . 7 ± 0 . 55 ) . Effect of 3 ‐ MA pre ‐ treatment , for both stimuli , was also assessed ( PMA + 3 ‐ MA : ATG3 : 1 . 36 ± 0 . 64 , LC3B : 1 . 5 ± 0 . 49 and rapamycin + 3 ‐ MA : ATG3 : 1 . 28 ± 0 . 46 , LC3B : 1 . 24 ± 0 . 57 ) . No other significant alteration in ATG genes mRNA levels was detected . Representative data of ATG7 mRNA expression analysis after treatment with PMA ( 1 . 21 ± 0 . 42 ) and rapamycin ( 0 . 94 ± 0 . 64 ) . Data are representative of six independent experiments and presented as mean relative expression ± SD . Wilcoxon matched ‐ pairs test , * p < 0 . 05 and n . s . = not significant compared with control conditions ( dot line ) as indicated in the figure . Relative expression ( folds ) of each gene is derived from substitution of DCT values ( normalized by GAPDH DCT value ) in 2DDCT equation 36 .

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

ATG3 and LC3B gene expression pattern in neutrophils after stimulation with PMA or rapamycin. ATG3 mRNA expression analysis (fold) after PMA (2.44±0.78) and rapamycin (RAP: 1.81±0.41) stimulation. LC3B gene alterations (PMA: 4.98±0.59) and rapamycin (RAP: 1.7±0.55). Effect of 3‐MA pre‐treatment, for both stimuli, was also assessed (PMA+3‐MA: ATG3: 1.36±0.64, LC3B: 1.5±0.49 and rapamycin+3‐MA: ATG3: 1.28±0.46, LC3B: 1.24±0.57). No other significant alteration in ATG genes mRNA levels was detected. Representative data of ATG7 mRNA expression analysis after treatment with PMA (1.21±0.42) and rapamycin (0.94±0.64). Data are representative of six independent experiments and presented as mean relative expression±SD. Wilcoxon matched‐pairs test, *p<0.05 and n.s.=not significant compared with control conditions (dot line) as indicated in the figure. Relative expression (folds) of each gene is derived from substitution of DCT values (normalized by GAPDH DCT value) in 2DDCT equation 36.


( A ) ( I ) MDC staining in neutrophils treated with LPS , LTA , peptidoglycan ( PGN ) , loxorubin ( LOX ) and R848 ( 400 × ) . ( II ) Percentage of PMN with acidified vacuoles as detected by MDC staining . LPS ( 50 ng / mL ) ( bar 2 : 69 . 67 ± 8 . 21 % ) , LTA ( bar 3 : 75 . 17 ± 9 . 80 % ) , peptidoglycan ( bar 4 : 77 ± 6 . 96 % ) , loxorubin ( bar 5 : 66 . 67 ± 5 . 61 % ) , R848 ( bar 6 : 69 . 5 ± 7 . 45 % ) , poly I : C ( bar 7 : 16 . 33 ± 3 . 14 % ) and flagellin ( bar 8 : 19 . 83 ± 5 . 16 % ) − treated cells compared with control conditions ( bar 1 : 14 . 5 ± 4 . 97 % ) . Data are representative of six independent experiments and presented as mean ± SD ; Wilcoxon matched − pairs test , * p < 0 . 05 , n . s . = not significant .

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 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 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

(A) (I) MDC staining in neutrophils treated with LPS, LTA, peptidoglycan (PGN), loxorubin (LOX) and R848 (400×). (II) Percentage of PMN with acidified vacuoles as detected by MDC staining. LPS (50 ng/mL) (bar 2: 69.67±8.21%), LTA (bar 3: 75.17±9.80%), peptidoglycan (bar 4: 77±6.96%), loxorubin (bar 5: 66.67±5.61%), R848 (bar 6: 69.5±7.45%), poly I:C (bar 7: 16.33±3.14%) and flagellin (bar 8: 19.83±5.16%)−treated cells compared with control conditions (bar 1: 14.5±4.97%). Data are representative of six independent experiments and presented as mean±SD; Wilcoxon matched−pairs test, *p<0.05, n.s.=not significant.


( B ) LC3B lipidation status of cells treated with LPS ( lane I ) , LTA ( lane II ) , peptidoglycan ( lane III ) , loxoribin ( lane IV ) and R848 ( lane V ) , detected by immunoblotting . One representative out of six independent experiments is shown .

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(B) LC3B lipidation status of cells treated with LPS (lane I), LTA (lane II), peptidoglycan (lane III), loxoribin (lane IV) and R848 (lane V), detected by immunoblotting. One representative out of six independent experiments is shown.


( C ) LC3B − II / LC3B - I ratio as obtained from integrated optical density measurements . LTA ( bar 2 : 1 . 75 ± 0 . 84 ) , peptidoglycan ( bar 3 : 1 . 82 ± 1 . 17 ) , LPS ( bar 4 : 1 . 9 ± 1 . 7 ) , loxorubin ( bar 5 : 1 . 32 ± 0 . 49 ) and R848 ( bar 6 : 1 . 4 ± 0 . 77 ) compared with control neutrophils ( bar 1 : 0 . 76 ± 0 . 30 ) . Data are representative of six independent experiments and are presented as mean ± SD . Wilcoxon matched − pairs test , * p < 0 . 05 .

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

(C) LC3B−II/LC3B-I ratio as obtained from integrated optical density measurements. LTA (bar 2: 1.75±0.84), peptidoglycan (bar 3: 1.82±1.17), LPS (bar 4: 1.9±1.7), loxorubin (bar 5: 1.32±0.49) and R848 (bar 6: 1.4±0.77) compared with control neutrophils (bar 1: 0.76±0.30). Data are representative of six independent experiments and are presented as mean±SD. Wilcoxon matched−pairs test, *p<0.05.


( D ) Analysis of LC3B gene expression levels in neutrophils treated with TLR agonists . LPS ( bar 1 : 2 . 27 ± 0 . 43 ) , peptidoglycan ( bar 2 : 2 . 07 ± 0 . 42 ) , LTA ( bar 3 : 2 . 1 ± 0 . 49 ) and R848 ( bar 4 : 1 . 89 ± 0 . 35 ) compared with medium − treated control cells . Dotted line represents LC3B mRNA levels of medium - treated control cells . Data are representative of six independent experiments and are presented as mean relative expression ± SD . Wilcoxon matched − pairs test , * p < 0 . 05 .

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 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 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

(D) Analysis of LC3B gene expression levels in neutrophils treated with TLR agonists. LPS (bar 1: 2.27±0.43), peptidoglycan (bar 2: 2.07±0.42), LTA (bar 3: 2.1±0.49) and R848 (bar 4: 1.89±0.35) compared with medium−treated control cells. Dotted line represents LC3B mRNA levels of medium-treated control cells. Data are representative of six independent experiments and are presented as mean relative expression±SD. Wilcoxon matched−pairs test, *p<0.05.


( E ) Assessment of ATG gene expression levels in PMN after LPS stimulation ( 50 ng / mL ) . ATG3 ( bar 1 : 2 . 94 ± 0 . 52 ) , ATG4 ( bar 2 : 1 . 72 ± 0 . 42 ) , ATG5 ( bar 3 : 1 . 84 ± 0 . 34 ) , ATG6 ( bar 4 : 1 . 8 ± 0 . 3 ) , ATG7 ( bar 5 : 3 . 13 ± 0 . 64 ) and LC3B ( bar 6 : 2 . 27 ± 0 . 43 ) compared with control cells . Dotted line represents mRNA levels of medium - treated cells . Data are representative of six independent experiments and are presented as mean relative expression ± SD . Wilcoxon matched − pairs test , * p < 0 . 05 .

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

(E) Assessment of ATG gene expression levels in PMN after LPS stimulation (50 ng/mL). ATG3 (bar 1: 2.94±0.52), ATG4 (bar 2: 1.72±0.42), ATG5 (bar 3: 1.84±0.34), ATG6 (bar 4: 1.8±0.3), ATG7 (bar 5: 3.13±0.64) and LC3B (bar 6: 2.27±0.43) compared with control cells. Dotted line represents mRNA levels of medium-treated cells. Data are representative of six independent experiments and are presented as mean relative expression±SD. Wilcoxon matched−pairs test, *p<0.05.


( A ) ( I ) Formation of acidified autophagosomes after stimulation with 10 ng / mL of IL ‐ 1β , compared with medium ‐ treated control cells . ( II ) Percentage of PMN with acidified vacuoles as detected by MDC staining . IL ‐ 1β ‐ treated cells ( bar 2 : 65 . 17 ± 10 . 23 % ) compared with control conditions ( bar 1 : 15 . 83 ± 6 . 08 % ) . Data are representative of six independent experiments and presented as mean ± SD ; Wilcoxon matched ‐ pairs test , * p < 0 . 05 .

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(A) (I) Formation of acidified autophagosomes after stimulation with 10 ng/mL of IL‐1β, compared with medium‐treated control cells. (II) Percentage of PMN with acidified vacuoles as detected by MDC staining. IL‐1β‐treated cells (bar 2: 65.17±10.23%) compared with control conditions (bar 1: 15.83±6.08%). Data are representative of six independent experiments and presented as mean±SD; Wilcoxon matched‐pairs test, *p<0.05.


( B ) LC3B conversion in neutrophils treated with 10 ng / mL ( lane II ) and 100 ng / mL ( lane III ) of IL ‐ 1β . ( lane I : medium ‐ treated control cells ) . One representative out of five independent experiments is shown .

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(B) LC3B conversion in neutrophils treated with 10 ng/mL (lane II) and 100 ng/mL (lane III) of IL‐1β. (lane I: medium‐treated control cells). One representative out of five independent experiments is shown.


( A ) Effect of 3 ‐ MA on neutrophil phagocytic capacity of opsonized E . coli as observed by flow cytometry . Percentage of phagocytosing neutrophils after 3 ‐ MA pretreatment ( III ) compared with cells incubated with opsonized bacteria ( II ) . Data are representative of six independent experiments and are presented as mean ± SD , ( I : control unstimulated group ) , ( IV ) MFI representation ( bar 1 : control : 1 ± 1 . 9 , bar 2 : E . coli : 387 . 7 ± 33 . 3 , bar 3 : E . coli + 3 ‐ MA : 25 . 13 ± 18 , Wilcoxon matched ‐ pairs test , * p < 0 . 05 ; bar 2 compared with bar 1 and bar 3 compared to bar 2 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Effect of 3‐MA on neutrophil phagocytic capacity of opsonized E. coli as observed by flow cytometry. Percentage of phagocytosing neutrophils after 3‐MA pretreatment (III) compared with cells incubated with opsonized bacteria (II). Data are representative of six independent experiments and are presented as mean±SD, (I: control unstimulated group), (IV) MFI representation (bar 1: control: 1±1.9, bar 2: E. coli: 387.7±33.3, bar 3: E. coli+3‐MA: 25.13±18, Wilcoxon matched‐pairs test, *p<0.05; bar 2 compared with bar 1 and bar 3 compared to bar 2).


( B ) ( I ) Presence of acidified structures in E . coli ‐ phagocytosing neutrophils and medium ‐ treated control cells . ( II ) Percentage of PMN with acidified vacuoles as detected by MDC staining . E . coli ‐ phagocytosing cells ( bar 2 : 79 . 33 ± 7 . 15 % ) compared with control conditions ( bar 1 : 14 . 17 ± 4 . 79 % ) . Data are representative of six independent experiments and presented as mean ± SD ; Wilcoxon matched ‐ pairs test , * p < 0 . 05 . (

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) (I) Presence of acidified structures in E. coli‐phagocytosing neutrophils and medium‐treated control cells. (II) Percentage of PMN with acidified vacuoles as detected by MDC staining. E. coli‐phagocytosing cells (bar 2: 79.33±7.15%) compared with control conditions (bar 1: 14.17±4.79%). Data are representative of six independent experiments and presented as mean±SD; Wilcoxon matched‐pairs test, *p<0.05. (


( C ) LC3B conversion analysis in E . coli ‐ phagocytosing neutrophils ( lane II ) , control neutrophils ( lane I ) and E . coli ‐ phagocytosing neutrophils after 3 ‐ MA pretreatment ( lane III ) . One representative out of six independent experiments is shown .

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(C) LC3B conversion analysis in E. coli‐phagocytosing neutrophils (lane II), control neutrophils (lane I) and E. coli‐phagocytosing neutrophils after 3‐MA pretreatment (lane III). One representative out of six independent experiments is shown.


( D ) Effect of 3 ‐ MA administration to E . coli ‐ phagocytosing neutrophils 15 min after the initiation of phagocytosis ( lane III ) as detected by LC3B conversion analysis . Lane II represents E . coli ‐ phagocytosing neutrophils and lane I control neutrophils . One representative out of four independent experiments is shown .

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(D) Effect of 3‐MA administration to E. coli‐phagocytosing neutrophils 15 min after the initiation of phagocytosis (lane III) as detected by LC3B conversion analysis. Lane II represents E. coli‐phagocytosing neutrophils and lane I control neutrophils. One representative out of four independent experiments is shown.


( E ) ( I ) Percentage of neutrophils with LC3B punctuated structures . E . coli ( bar 1 : 84 ± 2 . 2 % ) compared with E . coli + 3 ‐ MA ( bar 2 : 4 ± 1 . 95 % ) . Data are representative of six independent experiments and presented as mean ± SD . Wilcoxon matched ‐ pairs test , * p < 0 . 05 . ( II ) Formation of LC3B puncta in phagocytosing neutrophils and ( III ) in cells treated with 3 ‐ MA before incubation with opsonized E . coli , as observed by immunofluorescence confocal microscopy ( 1 . LC3B : red ; 2 . FITC conjugated E . coli : green ; 3 . DAPI : blue ; and 4 . merge ) . Solid line arrows point the colocalization of LC3B puncta with E . coli bacteria . Dashed line arrows point LC3B aggregates that are not colocalized with E . coli .

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(E) (I) Percentage of neutrophils with LC3B punctuated structures. E. coli (bar 1: 84±2.2%) compared with E. coli+3‐MA (bar 2: 4±1.95%). Data are representative of six independent experiments and presented as mean±SD. Wilcoxon matched‐pairs test, *p<0.05. (II) Formation of LC3B puncta in phagocytosing neutrophils and (III) in cells treated with 3‐MA before incubation with opsonized E. coli, as observed by immunofluorescence confocal microscopy (1. LC3B: red; 2. FITC conjugated E. coli: green; 3. DAPI: blue; and 4. merge). Solid line arrows point the colocalization of LC3B puncta with E. coli bacteria. Dashed line arrows point LC3B aggregates that are not colocalized with E. coli.


Alterations in ATG gene expression after phagocytosis of opsonized E . coli . ATG3 ( 2 . 05 ± 0 . 27 ) , ATG4 ( 2 . 20 ± 0 . 27 ) , ATG5 ( 2 . 89 ± 0 . 42 ) , ATG6 ( 2 . 04 ± 0 . 40 ) , ATG7 ( 1 . 98 ± 0 . 30 ) and LC3B ( 2 . 46 ± 0 . 39 ) gene expression ( fold ) in E . coli ‐ phagocytosing neutrophils compared with control cells . Dotted line represents mRNA expression levels of control cells . Data are representative of six independent experiments and are presented as mean relative expression ± SD Wilcoxon matched ‐ pairs test , * p < 0 . 05 .

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

Alterations in ATG gene expression after phagocytosis of opsonized E. coli. ATG3 (2.05±0.27), ATG4 (2.20±0.27), ATG5 (2.89±0.42), ATG6 (2.04±0.40), ATG7 (1.98±0.30) and LC3B (2.46±0.39) gene expression (fold) in E. coli‐phagocytosing neutrophils compared with control cells. Dotted line represents mRNA expression levels of control cells. Data are representative of six independent experiments and are presented as mean relative expression±SD Wilcoxon matched‐pairs test, *p<0.05.


( A ) LC3B conversion status after pretreatment with the ROS scavenger BHA prior to PMA or E . coli stimulation ( lane I : PMA , lane II : PMA + BHA , lane III : E . coli , lane IV : E . coli + BHA , lane V : control cells ) . One representative out of four independent experiments is shown .

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(A) LC3B conversion status after pretreatment with the ROS scavenger BHA prior to PMA or E. coli stimulation (lane I: PMA, lane II: PMA+BHA, lane III: E. coli, lane IV: E. coli+BHA, lane V: control cells). One representative out of four independent experiments is shown.


( B ) LC3B conversion status after pretreatment with the NADPH oxidase inhibitor DPI in E . coli − phagocytosing neutrophils ( lane I : PBS , lane II : E . coli , lane III : E . coli + DPI ) . One representative out of four independent experiments is shown .

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(B) LC3B conversion status after pretreatment with the NADPH oxidase inhibitor DPI in E. coli−phagocytosing neutrophils (lane I: PBS, lane II: E. coli, lane III: E. coli+DPI). One representative out of four independent experiments is shown.


( C ) ( I ) Effect of NADPH ‐ oxidase inhibition with DPI in the formation of acidified autophagosomes in E . coli ‐ phagocytosing neutrophils . ( II ) Percentage of PMN with acidified vacuoles as detected by MDC staining . E . coli − phagocytosing PMN ( 77 . 17 ± 8 . 33 % ) and PMN treated with DPI before incubation with opsonized E . coli ( 21 . 67 ± 4 . 37 % ) . Data are representative of six independent experiments and presented as mean ± SD ; Wilcoxon matched ‐ pairs test , * p < 0 .

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(C) (I) Effect of NADPH‐oxidase inhibition with DPI in the formation of acidified autophagosomes in E. coli‐phagocytosing neutrophils. (II) Percentage of PMN with acidified vacuoles as detected by MDC staining. E. coli−phagocytosing PMN (77.17±8.33%) and PMN treated with DPI before incubation with opsonized E. coli (21.67±4.37%). Data are representative of six independent experiments and presented as mean±SD; Wilcoxon matched‐pairs test, *p<0.05


( D ) ( I ) Assessment of formation of acidified autophagosomes in neutrophils pretreated with BNP before stimulation with peptidoglycan ( PGN ) , compared with cells treated with PGN . ( II ) Percentage of PMN with acidified vacuoles as detected by MDC staining . PGN ‐ treated neutrophils ( 74 . 83 ± 9 . 09 % ) and neutrophils pretreated with BNP before the addition of PGN ( 17 . 33 ± 2 . 07 % ) compared with control conditions ( 14 . 67 ± 3 . 20 % ) . Data are representative of six independent experiments and presented as mean ± SD ; Wilcoxon matched ‐ pairs test , * p < 0 . 05 , n . s . = not significant :

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(D) (I) Assessment of formation of acidified autophagosomes in neutrophils pretreated with BNP before stimulation with peptidoglycan (PGN), compared with cells treated with PGN. (II) Percentage of PMN with acidified vacuoles as detected by MDC staining. PGN‐treated neutrophils (74.83±9.09%) and neutrophils pretreated with BNP before the addition of PGN (17.33±2.07%) compared with control conditions (14.67±3.20%). Data are representative of six independent experiments and presented as mean±SD; Wilcoxon matched‐pairs test, *p<0.05, n.s.=not significant:


( E ) Alterations in LC3B conversion in BNP pretreated , peptidoglycan ‐ stimulated neutrophils ( lane III ) , ( lane I : medium ‐ treated cells , lane II : peptidoglycan ‐ treated cells ) . One representative out of four independent experiments is shown .

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(E) Alterations in LC3B conversion in BNP pretreated, peptidoglycan‐stimulated neutrophils (lane III), (lane I: medium‐treated cells, lane II: peptidoglycan‐treated cells). One representative out of four independent experiments is shown.


( F ) Effect of PI3K inhibition with 3 ‐ MA in the percentage of PMA ‐ induced oxidizing cells . PMA ( 99 . 41 ± 0 . 33 % ) and PMA + 3 ‐ MA ( 15 . 82 ± 3 . 97 % ) . Data are representative of six independent experiments and presented as mean ± SD ; Wilcoxon matched ‐ pairs test , * p < 0 . 05 .

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(F) Effect of PI3K inhibition with 3‐MA in the percentage of PMA‐induced oxidizing cells. PMA (99.41±0.33%) and PMA+3‐MA (15.82±3.97%). Data are representative of six independent experiments and presented as mean±SD; Wilcoxon matched‐pairs test, *p<0.05.


A . Expression of Dot1L in different B - cell populations ( Shi et al . , 2015 ) . B1 : B1 cells , MZB : marginal zone B , FOB : follicular B , GCB : germinal center B , SPLPB : spleen plasma blast , SPLPC : spleen plasma cells , and BMPC : Bone marrow plasma cells . Expression is shown as transcript per million ( TPM ) . Results represent the data from two biological replicates except SPLPB and BMPC which lack replicates ( WT ; n = 2 ) . . Bars indicate mean values .

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A. Expression of Dot1L in different B-cell populations (Shi et al., 2015). B1: B1 cells, MZB: marginal zone B, FOB: follicular B, GCB: germinal center B, SPLPB: spleen plasma blast, SPLPC: spleen plasma cells, and BMPC: Bone marrow plasma cells. Expression is shown as transcript per million (TPM). Results represent the data from two biological replicates except SPLPB and BMPC which lack replicates (WT; n=2)..Bars indicate mean values.


A - G . Statistical analysis of absolute number of total nucleated splenocytes , splenic B - and T - cells , and indicated mature B cell subsets in WT and KO mice . Results represent the data pooled from at least two independent experiments and numbers represent biological replicates for each group ( A : ( WT ) n = 16 , ( KO ) n = 17 ; B ( B - cells ) : ( WT ) n = 16 , ( KO ) n = 17 ; B ( T - cells ) : ( WT ) n = 8 , ( KO ) n = 9 ; C - E : ( WT ) n = 9 , ( KO ) n = 9 ; F : ( WT ) n = 6 , ( KO ) n = 6 ; G : ( WT ) n = 9 , ( KO ) n = 9 ) .

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A-G. Statistical analysis of absolute number of total nucleated splenocytes, splenic B- and T-cells, and indicated mature B cell subsets in WT and KO mice. Results represent the data pooled from at least two independent experiments and numbers represent biological replicates for each group (A: (WT) n = 16, (KO) n = 17; B (B-cells): (WT) n = 16, (KO) n = 17; B (T-cells): (WT) n = 8, (KO) n = 9; C-E: (WT) n = 9, (KO) n = 9; F: (WT) n = 6, (KO) n = 6; G: (WT) n = 9, (KO) n = 9).


D - E . Statistical analysis of switching of WT and KO naïve B cells activated for four days with LPS alone ( IgG3 switching ) ( D ) or with LPS + IL - 4 ( IgG1 switching ) ( E ) . Results represent the data pooled from at least four independent experiments and numbers represent biological replicates for each group ( D : ( WT ) n = 8 , ( KO ) n = 9 ; E : ( WT ) n = 11 , ( KO ) n = 13 ) . F . Statistical analysis of IgG1 switching of naïve B cells after four days of activation with anti - CD40 + IL - 4 in the presence of the DOT1L inhibitor Pinometostat or DMSO as a control . Results represent the data from one experiment and numbers represent biological replicates for each treatment ( n = 4 ) .

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

D-E. Statistical analysis of switching of WT and KO naïve B cells activated for four days with LPS alone (IgG3 switching) (D) or with LPS + IL-4 (IgG1 switching) (E). Results represent the data pooled from at least four independent experiments and numbers represent biological replicates for each group (D: (WT) n = 8, (KO) n = 9; E: (WT) n = 11, (KO) n = 13). F. Statistical analysis of IgG1 switching of naïve B cells after four days of activation with anti-CD40 + IL-4 in the presence of the DOT1L inhibitor Pinometostat or DMSO as a control. Results represent the data from one experiment and numbers represent biological replicates for each treatment (n = 4).


C . Statistical analysis of serum titers of LCMV - specific IgM and IgG of WT and KO mice before ( D0 ) and 14 days after ( D14 ) LCMV Armstrong infection . Results represent the data from two experiments and numbers represent biological replicates for each group ( WT ; n = 7 , KO ; n = 8 ) .

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C. Statistical analysis of serum titers of LCMV-specific IgM and IgG of WT and KO mice before (D0) and 14 days after (D14) LCMV Armstrong infection. Results represent the data from two experiments and numbers represent biological replicates for each group (WT; n = 7, KO; n = 8).


D . Statistical analysis of serum titers of NP - specific IgG1 and IgM quantified by ELISA from WT and KO mice at the indicated days following immune challenge . Adjusted p - value were calculated using two - way ANOVA . Error bars indicate mean ± standard error of mean ( SEM ) . Results represent the data from one experiment and numbers represent biological replicates for each group ( WT ; n = 6 , KO ; n = 6 ) . * * * denotes p - value < 0 . 0001 and * * denotes p - value < 0 . 001 .

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D. Statistical analysis of serum titers of NP-specific IgG1 and IgM quantified by ELISA from WT and KO mice at the indicated days following immune challenge. Adjusted p-value were calculated using two-way ANOVA. Error bars indicate mean ± standard error of mean (SEM). Results represent the data from one experiment and numbers represent biological replicates for each group (WT; n = 6, KO; n = 6).*** denotes p- value < 0.0001 and ** denotes p- value < 0.001.


A . Representative flow cytometry plots showing gating strategy to identify and compare plasma cells ( Sca - I + CD138 + ) after four days of stimulation with LPS either in the presence of DOT1L inhibitor , Pinometostat or DMSO as a control ( left panel ) and statistical analyses of their relative numbers ( right panel ) . Results represent the data from one experiment and numbers represent biological replicates for each treatment ( n = 4 ) .

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

A. Representative flow cytometry plots showing gating strategy to identify and compare plasma cells (Sca-I+CD138+) after four days of stimulation with LPS either in the presence of DOT1L inhibitor, Pinometostat or DMSO as a control (left panel) and statistical analyses of their relative numbers (right panel). Results represent the data from one experiment and numbers represent biological replicates for each treatment (n = 4).


B . Representative flow cytometry plots showing gating strategy to identify and compare activated B cells ( CD138 + Blimp1 + ) , Pre - PB cells ( CD138 - Blimp1 + ) and plasma blast ( PB ) cells after four days of stimulation with LPS either in the presence of DOT1L inhibitor , Pinometostat or DMSO as a control ( left panel ) and statistical analyses of their relative numbers ( right panel ) . Results represent the data from one experiment and numbers represent biological replicates for each treatment ( n = 4 ) .

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

B. Representative flow cytometry plots showing gating strategy to identify and compare activated B cells (CD138+ Blimp1+), Pre-PB cells (CD138- Blimp1+) and plasma blast (PB) cells after four days of stimulation with LPS either in the presence of DOT1L inhibitor, Pinometostat or DMSO as a control (left panel) and statistical analyses of their relative numbers (right panel). Results represent the data from one experiment and numbers represent biological replicates for each treatment (n = 4).


C . Representative flow cytometry plots showing the relative surface density for B220 ( CD45R ) after four days of stimulation with LPS either in the presence of DOT1L inhibitor , Pinometostat or DMSO as a control ( left panel ) and statistical analysis of MFI of B220 for each treatment ( right panel ) . Results represent the data from one experiment and numbers represent biological replicates for each treatment ( n = 4 ) .

O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O 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 flow cytometry plots showing the relative surface density for B220 (CD45R) after four days of stimulation with LPS either in the presence of DOT1L inhibitor, Pinometostat or DMSO as a control (left panel) and statistical analysis of MFI of B220 for each treatment (right panel). Results represent the data from one experiment and numbers represent biological replicates for each treatment (n = 4).


B . Differential ( FDR < 0 . 05 ) expression of Bach2 , Prdm1 ( encoding BLIMP - 1 ) , Cd138 ( a plasma - cell marker ) and Myc transcripts as indicated by counts per million after TMM normalization from WT and KO . Data was generated from three independent biological replicates for each genotype . Bars and error bars indicate mean ± SD . Statistical significance is indicated by FDR after the Benjamini - Hochberg multiple testing correction performed by edgeR package using R language .

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B. Differential (FDR < 0.05) expression of Bach2, Prdm1 (encoding BLIMP-1), Cd138 (a plasma-cell marker) and Myc transcripts as indicated by counts per million after TMM normalization from WT and KO. Data was generated from three independent biological replicates for each genotype. Bars and error bars indicate mean ± SD. Statistical significance is indicated by FDR after the Benjamini-Hochberg multiple testing correction performed by edgeR package using R language.


D . H3K79me2 methylation at the Ezh2 locus from WT activated and naïve B cells , as determined by reads per genomic content ( RPGC ) . Data represent three independent biological replicates .

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D. H3K79me2 methylation at the Ezh2 locus from WT activated and naïve B cells, as determined by reads per genomic content (RPGC). Data represent three independent biological replicates.


a - c , RFA1 : : FLAG DDC2 : : MYC cells were arrested in G2 and released in YP galactose to induce HO endonuclease . After 30 min , the culture was split in two : + VPA and - VPA . a , Samples were processed for western blot using anti - Rad53 , Srs2 and Ddc2 antibodies .

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a-c, RFA1::FLAG DDC2::MYC cells were arrested in G2 and released in YP galactose to induce HO endonuclease. After 30 min, the culture was split in two: +VPA and -VPA. a, Samples were processed for western blot using anti-Rad53, Srs2 and Ddc2 antibodies.


a - c , RFA1 : : FLAG DDC2 : : MYC cells were arrested in G2 and released in YP galactose to induce HO endonuclease . After 30 min , the culture was split in two : + VPA and − VPA . b , A schematic diagram showing probe locations with respect to the HO cut site . The resection rate was calculated as the rate of HO cut band disappearance .

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a-c, RFA1::FLAG DDC2::MYC cells were arrested in G2 and released in YP galactose to induce HO endonuclease. After 30 min, the culture was split in two: +VPA and −VPA. b, A schematic diagram showing probe locations with respect to the HO cut site. The resection rate was calculated as the rate of HO cut band disappearance.


a - c , RFA1 : : FLAG DDC2 : : MYC cells were arrested in G2 and released in YP galactose to induce HO endonuclease . After 30 min , the culture was split in two : + VPA and - VPA . c , Fold enrichment of the 0 . 2 - kb fragment was calculated after ChIP of Rfa1 - Flag , Ddc2 - Myc .

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a-c, RFA1::FLAG DDC2::MYC cells were arrested in G2 and released in YP galactose to induce HO endonuclease. After 30 min, the culture was split in two: +VPA and -VPA. c, Fold enrichment of the 0.2-kb fragment was calculated after ChIP of Rfa1-Flag, Ddc2-Myc.


d , VPA effect on ectopic recombination ( rec . ) in wild - type ( WT ) cells . Error bars represent standard deviation ( s . d . ) calculated from four independent experiments .

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d, VPA effect on ectopic recombination (rec.) in wild-type (WT) cells. Error bars represent standard deviation (s.d.) calculated from four independent experiments.


a , MRE11 : : MYC cells were treated as in Fig . 1a . Cell samples were processed for ChIP analysis and the fold enrichment of the 0 . 2 - kb fragment after ChIP of Mre11 - Myc without ( - VPA ) or with ( + VPA ) VPA was calculated .

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a, MRE11::MYC cells were treated as in Fig. 1a. Cell samples were processed for ChIP analysis and the fold enrichment of the 0.2-kb fragment after ChIP of Mre11-Myc without (-VPA) or with (+VPA) VPA was calculated.


b , EXO1 : : FLAG SAE2 : : PK MRE11 : : MYC cells were grown as in a . Cell samples were taken and processed for western blot analysis using anti - Flag , PK and Myc antibodies .

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b, EXO1::FLAG SAE2::PK MRE11::MYC cells were grown as in a. Cell samples were taken and processed for western blot analysis using anti-Flag, PK and Myc antibodies.


a , Cherry : : APE1GFP : : ATG8 cells were grown and shifted to YPD , nitrogen starvation ( SD - N ) or YPD + VPA medium for 3 h . Samples were processed for microscopy . The table shows numbers corresponding to the experiment . Percentage of fluorescence signals is presented and error bars represent the s . d . obtained from three independent experiments . DIC , differential interference contrast . Scale bars , 3 µm .

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a, Cherry::APE1GFP::ATG8 cells were grown and shifted to YPD, nitrogen starvation (SD-N) or YPD+VPA medium for 3 h. Samples were processed for microscopy. The table shows numbers corresponding to the experiment. Percentage of fluorescence signals is presented and error bars represent the s.d. obtained from three independent experiments. DIC, differential interference contrast. Scale bars, 3 µm.


b , pho8Δ60 and pho8Δ60 atg1Δ ; cells were grown as in a . Pho8Δ60 activity was calculated by measuring alkaline phosphatase levels . Error bars represent s . d . calculated from five independent experiments .

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b, pho8Δ60 and pho8Δ60 atg1Δ; cells were grown as in a. Pho8Δ60 activity was calculated by measuring alkaline phosphatase levels. Error bars represent s.d. calculated from five independent experiments.


c , GFP : : ATG8 and GFP : : ATG8atg1Δ ; cells were grown as in a . Cell samples were processed for western blot using anti - GFP antibody . Quantification is presented in Supplementary Fig . 3 .

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c, GFP::ATG8 and GFP::ATG8atg1Δ; cells were grown as in a. Cell samples were processed for western blot using anti-GFP antibody. Quantification is presented in Supplementary Fig. 3.


a , 4HA - Sae2 was immunoprecipitated ± VPA with anti - HA and subsequently ± anti - acetyl - Lysine . Eluate was analysed using anti - HA . Lane 1 : input Sae2 ; 2 : input Sae2 - HA - VPA ; 3 : as in 2 but + VPA ; 4 : 3 µl elution Sae2 - HA - VPA after anti - HAimmunoprecipitation ( IP ; input AcK - IP ) ; 5 : double amount of 4 ; 6 : IP anti - AcK elution from anti - HA IP of Sae2 - HA - VPA ; 7 : as in 6 but + VPA .

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a, 4HA-Sae2 was immunoprecipitated ± VPA with anti-HA and subsequently ± anti-acetyl-Lysine. Eluate was analysed using anti-HA. Lane 1: input Sae2; 2: input Sae2-HA -VPA; 3: as in 2 but +VPA; 4: 3 µl elution Sae2-HA -VPA after anti-HAimmunoprecipitation (IP; input AcK-IP); 5: double amount of 4; 6: IP anti-AcK elution from anti-HA IP of Sae2-HA -VPA; 7: as in 6 but +VPA.


b , SAE2 : : PKerg6Δ cells were treated as in Fig . 2a . After 30 min induction , VPA and PMSF were added or not . Samples were processed for western blot using anti - PK .

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b, SAE2::PKerg6Δ cells were treated as in Fig. 2a. After 30 min induction, VPA and PMSF were added or not. Samples were processed for western blot using anti-PK.


c , Wild - type SAE2 : : PK , SAE2 : : PKatg1Δ and SAE2 : : PKatg19Δ cells were grown as in b . After 30 min induction , VPA was added or not and samples treated as in b .

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c, Wild-type SAE2::PK, SAE2::PKatg1Δ and SAE2::PKatg19Δ cells were grown as in b. After 30 min induction, VPA was added or not and samples treated as in b.


d , Wild - type SAE2 : : PK cells were grown as in b . After 120 min induction , rapamycin ( 200 ng ml - 1 ) was added or not and samples were treated as in b .

d, Wild-type SAE2::PK cells were grown as in b. After 120 min induction, rapamycin (200 ng ml-1) was added or not and samples were treated as in b.


a , Survival of wild - type , rpd3Δ , hda1Δ and rpd3Δ hda1Δ strains after 4NQO , MMS and HU treatment . Error bars represent s . d . calculated from seven independent experiments .

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a, Survival of wild-type, rpd3Δ, hda1Δ and rpd3Δ hda1Δ strains after 4NQO, MMS and HU treatment. Error bars represent s.d. calculated from seven independent experiments.


b , HO was induced in G2 wild - type , hda1Δ , rpd3Δ and hda1Δ rpd3Δ cells and western and Southern blot analyses were performed .

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b, HO was induced in G2 wild-type, hda1Δ, rpd3Δ and hda1Δ rpd3Δ cells and western and Southern blot analyses were performed.


c , Wild - type SAE2 : : PK and hda1Δ rpd3Δ SAE2 : : PK cells were grown as in b and western blot was performed as in Fig . 4b .

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c, Wild-type SAE2::PK and hda1Δ rpd3Δ SAE2::PK cells were grown as in b and western blot was performed as in Fig. 4b.


d , e , Wild - type SAE2 : : PK and gcn5Δ SAE2 : : PK strains were grown as in b and after 30 min of HO induction either VPA ( d ) or rapamycin ( e ) was added or not . Western blot was performed .

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d, e, Wild-type SAE2::PK and gcn5Δ SAE2::PK strains were grown as in b and after 30 min of HO induction either VPA (d) or rapamycin (e) was added or not. Western blot was performed.


f , Percentage of viability of wild - type , atg1Δ , rpd3Δ hda1Δ and atg1Δ rpd3Δ hda1Δ strains . Error bars represent s . d . calculated from four independent experiments .

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f, Percentage of viability of wild-type, atg1Δ, rpd3Δ hda1Δ and atg1Δ rpd3Δ hda1Δ strains. Error bars represent s.d. calculated from four independent experiments.


a , Internalization of beads with PAM3CSK , LPS , or Alexa - Fluor - 555 - labelled BSA ( Red - BSA ; yellow arrows ) and association with GFP - LC3 in RAW cells was followed by time - lapse video for 3 h , 1 frame / 2 min ( representative frames are shown ) .

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a, Internalization of beads with PAM3CSK, LPS, or Alexa-Fluor-555-labelled BSA (Red-BSA; yellow arrows) and association with GFP-LC3 in RAW cells was followed by time-lapse video for 3 h, 1 frame/2 min (representative frames are shown).


GFP - LC3 in RAW cells translocates to phagosomes containing zymosan ( red ) , assessed as in

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GFP-LC3 in RAW cells translocates to phagosomes containing zymosan (red), assessed as in a


c , Time course of translocation of GFP - LC3 to a phagosome containing zymosan ( red ) ( see Supplementary Movie 2 ) . Time ( minutes ) is indicated in the top left of each panel .

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c, Time course of translocation of GFP-LC3 to a phagosome containing zymosan (red) (see Supplementary Movie 2). Time (minutes) is indicated in the top left of each panel.


d , LC3 translocation to the phagosome is induced by TLR signalling . GFP - LC3 was transiently transfected into wild - type or Tlr2 - / - macrophages , which were exposed to zymosan for 45 min .

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d, LC3 translocation to the phagosome is induced by TLR signalling. GFP-LC3 was transiently transfected into wild-type or Tlr2-/- macrophages, which were exposed to zymosan for 45 min.


e , The percentage of phagosomes ( n ≥ 50 per group ) with GFP - LC3 in wild - type and Tlr2 - / - macrophages . Means and ranges are shown ( 4 mice per group were used ) .

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e, The percentage of phagosomes (n ≥ 50 per group) with GFP-LC3 in wild-type and Tlr2-/- macrophages. Means and ranges are shown (4 mice per group were used).


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a , RAW cells expressing GFP - LC3 were transfected by electroporation with control or Atg5 siRNA oligonucleotides . At 24 h after transfection , cells were fed with zymosan ( yellow arrows ) and the internalization process was followed by time - lapse video microscopy . Images obtained at 5 and 30 min after zymosan engulfment are shown . b , The percentage of GFP - LC3 - associated phagosomes ( n ≥ 100 per / group ) was obtained from three independent time - lapse videos ( 2 h each ) of macrophages transfected with control or Atg5 siRNA ( means and ranges are shown ) .

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a, RAW cells expressing GFP-LC3 were transfected by electroporation with control or Atg5 siRNA oligonucleotides. At 24 h after transfection, cells were fed with zymosan (yellow arrows) and the internalization process was followed by time-lapse video microscopy. Images obtained at 5 and 30 min after zymosan engulfment are shown. b, The percentage of GFP-LC3-associated phagosomes (n ≥ 100 per /group) was obtained from three independent time-lapse videos (2 h each) of macrophages transfected with control or Atg5 siRNA (means and ranges are shown).


e , Ly294002 ( Ly29 , 10 µM ) was added 10 min before zymosan , and wortmannin was added after engulfment but before GFP - LC3 translocation in RAW - GFP - LC3 cells . Representative images are shown . f , Percentage of GFP - LC3 - associated phagosomes ( n ≥ 50 per group ) with P ( I ) 3K inhibitors assessed by time - lapse video ( means and ranges are shown ; three independent 3 h videos ) .

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e, Ly294002 (Ly29, 10 µM) was added 10 min before zymosan, and wortmannin was added after engulfment but before GFP-LC3 translocation in RAW-GFP-LC3 cells. Representative images are shown. f, Percentage of GFP-LC3-associated phagosomes (n ≥ 50 per group) with P(I)3K inhibitors assessed by time-lapse video (means and ranges are shown; three independent 3 h videos).


g , RAW cells expressing GFP - LC3 and the PX domain of p40 ( phox ) fused to mCherry ( red ) were fed with zymosan ( yellow arrows ) or with beads ( Supplementary Fig . 6 ) . Internalization was followed at 1 - min intervals ( Supplementary Movie 5 ) , representative images are shown . Single cells were followed for at least 2 h ( n ≥ 20 per group ) ( Supplementary Movies 6 , 7 and Supplementary Fig . 6 ) . Mean ± s . d . duration of zymosan - PX translocation to the zymosan for beads ( B ) and zymosan ( Z ) are shown .

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g, RAW cells expressing GFP-LC3 and the PX domain of p40(phox) fused to mCherry (red) were fed with zymosan (yellow arrows) or with beads (Supplementary Fig. 6). Internalization was followed at 1-min intervals (Supplementary Movie 5), representative images are shown. Single cells were followed for at least 2 h (n ≥ 20 per group) (Supplementary Movies 6, 7 and Supplementary Fig. 6). Mean ± s.d. duration of zymosan-PX translocation to the zymosan for beads (B) and zymosan (Z) are shown.


h , Internalization of zymosan ( yellow arrows ) was followed at 1 . 5 - min intervals for at least 2 h in RAW cells transiently transfected with GFP - BECN1 . Representative images are shown . Translocation of GFP - BECN1 to the phagosome for beads ( B ) and zymosan ( Z ) was quantified ( mean ± s . d . ) using time - lapse movies ( Supplementary Movie 8 and 9 ; n ≥ 20 cells per group ) . For g and h , time ( in minutes ) is indicated at the top left of each panel .

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h, Internalization of zymosan (yellow arrows) was followed at 1.5-min intervals for at least 2 h in RAW cells transiently transfected with GFP-BECN1. Representative images are shown. Translocation of GFP-BECN1 to the phagosome for beads (B) and zymosan (Z) was quantified (mean ± s.d.) using time-lapse movies (Supplementary Movie 8 and 9; n ≥ 20 cells per group). For g and h, time (in minutes) is indicated at the top left of each panel.


a , Internalization of control or PAM3CSK4 - covered beads was followed in RAW - GFP - LC3 cells pre - loaded with lysotracker ( red ) by time - lapse video for 3 h . Time ( in minutes ) is indicated at the top left of each panel .

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a, Internalization of control or PAM3CSK4-covered beads was followed in RAW-GFP-LC3 cells pre-loaded with lysotracker (red) by time-lapse video for 3 h. Time (in minutes) is indicated at the top left of each panel.


b , RAW cells pre - loaded with lysotracker were exposed to control or PAM3CSK - covered beads for 6 h . Acidification of the beads ( n ≥ 20 per group ) , measured as maximum lysotracker relative light units ( RLU ) on a horizontal cross - section of each bead represented in the x axis ( mean ± s . d . ; Wilcoxon - Mann - Whitney test , P 0 . 0005 ) , is shown .

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b, RAW cells pre-loaded with lysotracker were exposed to control or PAM3CSK-covered beads for 6 h. Acidification of the beads (n ≥ 20 per group), measured as maximum lysotracker relative light units (RLU) on a horizontal cross-section of each bead represented in the x axis (mean ± s.d.; Wilcoxon-Mann-Whitney test, P 0.0005), is shown.


c , d , Knockdown of ATG5 reduced zymosan - induced GFP - LC3 translocation and lysosomal fusion .

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c, d, Knockdown of ATG5 reduced zymosan-induced GFP-LC3 translocation and lysosomal fusion.


e , Primary macrophages from wild - type ( white bars ) and Atg7 - null mice ( grey bars ) were fed live yeast for 1 h and washed . Internalized yeast were extracted from the macrophages at 4 h and 8 h after re - plating . Means and ranges from six different measurements using two mice per group are shown .

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e, Primary macrophages from wild-type (white bars) and Atg7-null mice (grey bars) were fed live yeast for 1 h and washed. Internalized yeast were extracted from the macrophages at 4 h and 8 h after re-plating. Means and ranges from six different measurements using two mice per group are shown.


f , Initiation and duration of translocation of PX - mCherry to phagosomes ( yellow bar ) , GFP - BECN1 translocation ( blue bar ) , GFP - LC3 translocation ( green bar ) and Lysotracker recruitment ( acidification ) were determined during the first 2 h after internalization ( 10 cells per group , error bars indicate s . d . )

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f, Initiation and duration of translocation of PX-mCherry to phagosomes (yellow bar), GFP-BECN1 translocation (blue bar), GFP-LC3 translocation (green bar) and Lysotracker recruitment (acidification) were determined during the first 2 h after internalization (10 cells per group, error bars indicate s.d.)


A . Single grey - scale and coloured merged images of immunocytochemical staining against PSD - 95 , synaptophysin - 1 ( SYP1 ) and NPC1 proteins in a cultured hippocampal neuron from a wt mouse . Graphs show mean ± SEM of percentage of colocalization of NPC1 with PSD95 or SYP1 ( n = 12 neurons from 3 different cultures ) .

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A. Single grey-scale and coloured merged images of immunocytochemical staining against PSD-95, synaptophysin-1 (SYP1) and NPC1 proteins in a cultured hippocampal neuron from a wt mouse. Graphs show mean ± SEM of percentage of colocalization of NPC1 with PSD95 or SYP1 (n=12 neurons from 3 different cultures).


B . Electron microscopy image of immunogold labelling against NPC1 in the CA1 hippocampal region of a wt mouse . Presynaptic compartments are indicated in pink while postsynaptic in blue . White arrows show the 15 - nm gold particles linked to NPC1 ( d - dendrite , sv - synaptic vesicles , m - mitochondria ) .

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B. Electron microscopy image of immunogold labelling against NPC1 in the CA1 hippocampal region of a wt mouse. Presynaptic compartments are indicated in pink while postsynaptic in blue. White arrows show the 15-nm gold particles linked to NPC1 (d-dendrite, sv-synaptic vesicles, m-mitochondria).


C , D . Western blots against NPC1 and actin - β ( ACTB ) in total brain ( C ) and synaptosomes ( D ) from wt and NPC1nmf164 mice . Graphs show mean ± SEM NPC1 level normalized to ACTB as a percentage of control ( wt ) values ( n = 4 mice , 3 month - old , unpaired Student t test , * * psynaptosomes = 0 . 0037 ) .

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C, D. Western blots against NPC1 and actin-β (ACTB) in total brain (C) and synaptosomes (D) from wt and NPC1nmf164 mice. Graphs show mean ± SEM NPC1 level normalized to ACTB as a percentage of control (wt) values (n=4 mice, 3 month-old, unpaired Student t test, **psynaptosomes= 0.0037).


E . Western blots against NPC1 and actin - β ( ACTB ) in total brain extracts from wt and Npc1 - null mice ( NPC1KO ) containing the same amount of protein ( n = 2 mice , 6 week - old ) .

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E. Western blots against NPC1 and actin-β (ACTB) in total brain extracts from wt and Npc1-null mice (NPC1KO) containing the same amount of protein (n=2 mice, 6 week-old).


F . Graph shows mean ± SEM mRNA levels of NPC1 in total extracts and in synaptosomes from wt and NPC1nmf164 mice ( n = 4 mice , 3 month - old ) .

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F. Graph shows mean ± SEM mRNA levels of NPC1 in total extracts and in synaptosomes from wt and NPC1nmf164 mice (n=4 mice, 3 month-old).


G . Western blots against NPC1 and actin - β ( ACTB ) in synaptosomes from wt and NPC1nmf164 mice in which cLTP was induced or not in the presence or absence of the protein synthesis inhibitor cycloheximide ( CHX ) . Graphs show mean ± SEM NPC1 level normalized to ACTB as a percentage of control ( wt non cLTP induced ) values ( n = 3 mice , 3 month - old , 2 - way ANOVA , * * * p < 0 . 001 ) .

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G. Western blots against NPC1 and actin-β (ACTB) in synaptosomes from wt and NPC1nmf164 mice in which cLTP was induced or not in the presence or absence of the protein synthesis inhibitor cycloheximide (CHX). Graphs show mean ± SEM NPC1 level normalized to ACTB as a percentage of control (wt non cLTP induced) values (n=3 mice, 3 month-old, 2-way ANOVA, ***p < 0.001).