B , Metagene profiles for Pol II ( upper panel ) and Ub - H2B ( middle panel ) ChIP - seq for USP22 - dependent , USP22 - Bound genes . mRNA analyses of select Bound gene transcripts are presented ( lower panel ) . Three independent experiments are represented as mean ± SEM , with significance measured by Student ' s t - test , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 005 C , Metagene profiles for Pol II ( upper panel ) and Ub - H2B ( middle panel ) ChIP - seq for USP22 - dependent , SAGA - Unbound genes . Nonparametric Mann - Whitney analyses of 7 bins ( grey shade ) surrounding TSS ( TSS ) and the midpoint of the profiles ( Body ) in Thaps treated conditions are reported . mRNA analysis of select Unbound gene transcripts are presented ( lower panel ) . Three independent experiments are represented as mean ± SEM , with significance measured by Student ' s t - test , * * p < 0 . 01 , * * * p < 0 . 005 .

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 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 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

B, Metagene profiles for Pol II (upper panel) and Ub-H2B (middle panel) ChIP-seq for USP22-dependent, USP22-Bound genes. mRNA analyses of select Bound gene transcripts are presented (lower panel). Three independent experiments are represented as mean ± SEM, with significance measured by Student's t-test, *p<0.05, **p<0.01, ***p<0.005 C, Metagene profiles for Pol II (upper panel) and Ub-H2B (middle panel) ChIP-seq for USP22-dependent, SAGA-Unbound genes. Nonparametric Mann-Whitney analyses of 7 bins (grey shade) surrounding TSS (TSS) and the midpoint of the profiles (Body) in Thaps treated conditions are reported. mRNA analysis of select Unbound gene transcripts are presented (lower panel). Three independent experiments are represented as mean ± SEM, with significance measured by Student's t-test, **p<0.01, ***p<0.005.


C , ChIP - qPCR for total RNA Pol II , pSer5 Pol II , and pSer2 Pol II at ERP70 , GRP78 , and a gene desert ( Prom = gene promoter , Coding = downstream coding region - see Appendix Table S1 for primer sequences ) . Three independent experiments are represented as mean ± SEM . with significance measured by Student ' s t - test , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 005 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, ChIP-qPCR for total RNA Pol II, pSer5 Pol II, and pSer2 Pol II at ERP70, GRP78, and a gene desert (Prom = gene promoter, Coding = downstream coding region - see Appendix Table S1 for primer sequences). Three independent experiments are represented as mean ± SEM. with significance measured by Student's t-test, *p<0.05, **p<0.01, ***p<0.005.


H , An aliquot of cells from D ‑ G were harvested for whole - cell lysate and subjected to immunoblotting with the indicated antibodies .

O O 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, An aliquot of cells from D‑G were harvested for whole-cell lysate and subjected to immunoblotting with the indicated antibodies.


B , HCT116 cells were treated for 2 hrs with 100 nM Thapsigargin in the presence or absence of shRNA targeting USP22 . Cells were harvested , fractionated , and fractions were subjected to immunoblotting with the indicated antibodies .

O O O O O O O O O O O O O O O O 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, HCT116 cells were treated for 2 hrs with 100 nM Thapsigargin in the presence or absence of shRNA targeting USP22. Cells were harvested, fractionated, and fractions were subjected to immunoblotting with the indicated antibodies.


C , In vitro deubiquitylation UbiTest of endogenously ubiquitylated MED24 . HCT116 cells were treated with MG132 in the presence or absence of shRNA targeting USP22 . Lysates were generated using buffer containing protease cocktail inhibitor , pan - DUB inhibitor PR619 , and the JAMM protease inhibitor o - phenanthroline . Ubiquitylated proteins were purified on ubiquitin binding resin and eluates were either undigested ( lanes 1 and 2 ) or digested with USP2 to strip polyubiquitin ( lanes 3 and 4 ) and reduce target proteins to unit length . Digestion reactions were subjected to immunoblotting with the indicated antibodies .

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 B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O 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, In vitro deubiquitylation UbiTest of endogenously ubiquitylated MED24. HCT116 cells were treated with MG132 in the presence or absence of shRNA targeting USP22. Lysates were generated using buffer containing protease cocktail inhibitor, pan-DUB inhibitor PR619, and the JAMM protease inhibitor o-phenanthroline. Ubiquitylated proteins were purified on ubiquitin binding resin and eluates were either undigested (lanes 1 and 2) or digested with USP2 to strip polyubiquitin (lanes 3 and 4) and reduce target proteins to unit length. Digestion reactions were subjected to immunoblotting with the indicated antibodies.


A , Genome browser image of ChIP - seq for middle and tail Mediator subunits and H3K4me3 HiChIP tracks before and after induction of ER stress at ER stress response gene BHLHE40 . H3K4me1 and H3K27ac tracks are from the ENCODE Consortium ( GEO accessions GSM945858 and GSM945853 ) . ATF4 / Xbp1s binding sites are from publicly available datasets ( GEO accessions GSE69304 and GSE49952 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Genome browser image of ChIP-seq for middle and tail Mediator subunits and H3K4me3 HiChIP tracks before and after induction of ER stress at ER stress response gene BHLHE40. H3K4me1 and H3K27ac tracks are from the ENCODE Consortium (GEO accessions GSM945858 and GSM945853). ATF4/Xbp1s binding sites are from publicly available datasets (GEO accessions GSE69304 and GSE49952).


A Immunoblot analysis with the indicated antibodies of immunoprecipitates ( IP ) prepared from the SSU fraction of eIF3D - V5 / eIF4E - FLAG dKI HEK293T cells ( eIF3D / 4E dKI ) or wild - type ( WT ) cells with antibodies to the epitope tags . The SSU fraction ( input ) was also analyzed directly . Cells were fixed with formalin , cell lysates were treated with RNase , and the SSU and RS fractions were separated by sucrose density gradient centrifugation before immunopurification . Molecular sizes are indicated in kilodaltons .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Immunoblot analysis with the indicated antibodies of immunoprecipitates (IP) prepared from the SSU fraction of eIF3D-V5/eIF4E-FLAG dKI HEK293T cells (eIF3D/4E dKI) or wild-type (WT) cells with antibodies to the epitope tags. The SSU fraction (input) was also analyzed directly. Cells were fixed with formalin, cell lysates were treated with RNase, and the SSU and RS fractions were separated by sucrose density gradient centrifugation before immunopurification. Molecular sizes are indicated in kilodaltons.


B , C Immunoblot analysis with the indicated antibodies of immunoprecipitates prepared as in A from the SSU fraction of eIF4A1 - FLAG KI HEK293T cells ( eIF4A1 KI ) ( B ) or ASCC3 - FLAG KI HEK293T cells ( ASCC3 KI ) ( C ) .

O O O O O O O O O O O O O O O O O O O O O O O O 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, C Immunoblot analysis with the indicated antibodies of immunoprecipitates prepared as in A from the SSU fraction of eIF4A1-FLAG KI HEK293T cells (eIF4A1 KI) (B) or ASCC3-FLAG KI HEK293T cells (ASCC3 KI) (C).


Immunoblot analysis with the indicated antibodies of immunoprecipitates prepared as in A from the RS fraction of RPL7A - HA KI HEK293T cells ( RPL7A KI ) ( 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

Immunoblot analysis with the indicated antibodies of immunoprecipitates prepared as in A from the RS fraction of RPL7A-HA KI HEK293T cells (RPL7A KI) (F).


A RNA - seq results for eIF4A1 , eIF4A2 , ASCC2 , and ASCC3 in HEK293T cells transfected with siRNAs specific for eIF4A1 and eIF4A2 ( eIF4A1 / A2 dKD # 1 or # 2 ) or for ASCC3 ( ASCC3 KD # 1 or # 2 ) relative to cells transfected with a control siRNA . The dashed line indicates a fold change of 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

A RNA-seq results for eIF4A1, eIF4A2, ASCC2, and ASCC3 in HEK293T cells transfected with siRNAs specific for eIF4A1 and eIF4A2 (eIF4A1/A2 dKD #1 or #2) or for ASCC3 (ASCC3 KD #1 or #2) relative to cells transfected with a control siRNA. The dashed line indicates a fold change of 1.


B Immunoblot analysis of the siRNA - transfected cells with the indicated antibodies .

B Immunoblot analysis of the siRNA-transfected cells with the indicated antibodies.


Volcano plots for differential TE ( D ) The changes in TE and SRO for eIF4A1 / A2 dKD # 1 and # 2 and for ASCC3 KD # 1 and # 2 HEK293T cells compared with control cells were analyzed with RiboDiff . The gray dashed lines indicate an adjusted P value of 0 . 05 . P values were calculated by chi - square test and adjusted by 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

Volcano plots for differential TE (D) The changes in TE and SRO for eIF4A1/A2 dKD #1 and #2 and for ASCC3 KD #1 and #2 HEK293T cells compared with control cells were analyzed with RiboDiff. The gray dashed lines indicate an adjusted P value of 0.05. P values were calculated by chi-square test and adjusted by Benjamini-Hochberg method.


D , E Cumulative fraction for fold change in TE in eIF4A1 / A2 dKD or ASCC3 KD cells relative to control cells . Results are shown for all coding transcripts and for transcripts with a decreased ( D ) or increased ( E ) SRO . The P values were calculated with the Mann - Whitney U 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

D, E Cumulative fraction for fold change in TE in eIF4A1/A2 dKD or ASCC3 KD cells relative to control cells. Results are shown for all coding transcripts and for transcripts with a decreased (D) or increased (E) SRO. The P values were calculated with the Mann-Whitney U test.


D Cumulative fraction for fold change in TE in eIF4A1 / A2 dKD or ASCC3 KD cells relative to control cells . Results are shown for all coding transcripts and skewed transcripts . The P values were calculated with the Mann - Whitney U 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

D Cumulative fraction for fold change in TE in eIF4A1/A2 dKD or ASCC3 KD cells relative to control cells. Results are shown for all coding transcripts and skewed transcripts. The P values were calculated with the Mann-Whitney U test.


E Number of transcripts showing significant down - regulation of TE among skewed transcripts in eIF4A1 / A2 dKD and ASCC3 KD 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

E Number of transcripts showing significant down-regulation of TE among skewed transcripts in eIF4A1/A2 dKD and ASCC3 KD cells.


Luciferase assays for RADX , DNAJB6 , and LDHA constructs ( B ) in control and ASCC3 - depleted HEK293T cells . The results of the luciferase assays ( right ) and schematic representations of the constructs ( left ) are shown . Rluc , Renilla luciferase ; Fluc , firefly luciferase . Assay data are means ± s . d . for three independent experiments . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , NS ( two - sided 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

Luciferase assays for RADX, DNAJB6, and LDHA constructs (B) in control and ASCC3-depleted HEK293T cells. The results of the luciferase assays (right) and schematic representations of the constructs (left) are shown. Rluc, Renilla luciferase; Fluc, firefly luciferase. Assay data are means ± s.d. for three independent experiments. *P < 0.05, **P < 0.01, *** P < 0.001, NS (two-sided Student's t test).


Luciferase assays for corresponding deletion mutants of the RADX and DNAJB6 constructs ( C ) in control and ASCC3 - depleted HEK293T cells . The results of the luciferase assays ( right ) and schematic representations of the constructs ( left ) are shown . The deleted regions of the RADX and DNAJB6 constructs are indicated by dashed white boxes . Rluc , Renilla luciferase ; Fluc , firefly luciferase . Assay data are means ± s . d . for three independent experiments . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 .

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

Luciferase assays for corresponding deletion mutants of the RADX and DNAJB6 constructs (C) in control and ASCC3-depleted HEK293T cells. The results of the luciferase assays (right) and schematic representations of the constructs (left) are shown. The deleted regions of the RADX and DNAJB6 constructs are indicated by dashed white boxes. Rluc, Renilla luciferase; Fluc, firefly luciferase. Assay data are means ± s.d. for three independent experiments. *P < 0.05, **P < 0.01, *** P < 0.001


BrdU incorporation reflecting DNA synthesis and SA - b - gal activity as markers of CTX - induced senescence in Em - myc transgeniccontrol ; Bcl2 versus Suv39h12 ; Bcl2lymphomas in vivo . Numbers represent mean percentages of positive cells6s . d . ( n55 each ) .

O O O B-MEASURED_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 O O

BrdU incorporation reflecting DNA synthesis and SA-b-gal activity as markers of CTX-induced senescence in Em-myc transgeniccontrol;Bcl2 versus Suv39h12;Bcl2lymphomas in vivo. Numbers represent mean percentages of positive cells6s.d. (n55 each).


b , Representative whole - body FLT - and FDG - PET scans of an individual control ; Bcl2 lymphoma - bearing mouse at tumour manifestation ( untreated ) and at day 6 after a single dose of CTX . Red circles , areas of specific tracer retention ; green circles , matching areas with lost retentions . Specific tracer uptake was assessed by calculation of tumour - to - background ratios . Data represent mean ratios ± s . d . ( n = 5 mice for FLT - PET scans and n = 6 mice for FDG - PET scans ) . Insets show representative Ki67 staining and colour - calibrated ( red , highest intensities ; blue , lowest intensities ) bioluminescence images of glucose levels in tissue sections of the respective groups .

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

b, Representative whole-body FLT- and FDG-PET scans of an individual control;Bcl2 lymphoma-bearing mouse at tumour manifestation (untreated) and at day 6 after a single dose of CTX. Red circles, areas of specific tracer retention; green circles, matching areas with lost retentions. Specific tracer uptake was assessed by calculation of tumour-to-background ratios. Data represent mean ratios ± s.d. (n = 5 mice for FLT-PET scans and n = 6 mice for FDG-PET scans). Insets show representative Ki67 staining and colour-calibrated (red, highest intensities; blue, lowest intensities) bioluminescence images of glucose levels in tissue sections of the respective groups.


c , Intracellular glucose concentrations in untreated and ADR - treated control ; Bcl2 ( that is , senescent ) versus Suv39h1 - ; Bcl2 ( that is , non - senescent ) lymphomas in vitro ( as in d - g ) . Results represent means ± s . d . ( n = 5 each ) .

O O O B-MEASURED_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

c, Intracellular glucose concentrations in untreated and ADR-treated control;Bcl2 (that is, senescent) versus Suv39h1-;Bcl2 (that is, non-senescent) lymphomas in vitro (as in d-g). Results represent means ± s.d. (n = 5 each).


d , Extracellular levels of the specified metabolite - to - glucose ratios in the indicated lymphoma cells groups relative to untreated control ; Bcl2 lymphoma cells after 18 h of cultivation . Data represent mean ratios ± s . d . ( n = 4 each , measurements were carried out in duplicate ) . Note that glucose - 6 - phosphate is not detectable ( n . d . ) in the medium . Insets show ( ADR - treated relative to untreated ) intracellular levels of the respective metabolites after labelling with 13C6 - glucose for one representative lymphoma from each group . Data represent mean ratios ( see Supplementary Fig . 5 for an assessment of the heterogeneity of the TIS - associated hypermetabolism and a comparison of the metabolic parameters of individual lymphomas ) .

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 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 B-CONTROLLED_VAR 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

d, Extracellular levels of the specified metabolite-to-glucose ratios in the indicated lymphoma cells groups relative to untreated control;Bcl2 lymphoma cells after 18 h of cultivation. Data represent mean ratios ± s.d. (n = 4 each, measurements were carried out in duplicate). Note that glucose-6-phosphate is not detectable (n.d.) in the medium. Insets show (ADR-treated relative to untreated) intracellular levels of the respective metabolites after labelling with 13C6-glucose for one representative lymphoma from each group. Data represent mean ratios (see Supplementary Fig. 5 for an assessment of the heterogeneity of the TIS-associated hypermetabolism and a comparison of the metabolic parameters of individual lymphomas).


e , Immunoblot analyses of phosphorylated AMP - activated protein kinase ( pAMPK ) , total AMPK , pyruvate kinase isoforms M1 and M2 in ADR - senescent control ; Bcl2 versus ADR - treated Suv39h1 - ; Bcl2 lymphoma cells in vitro ( n = 3 each ; α - tubulin as a loading control . Gels were cropped to ease presentation ; full - length blots are provided in Supplementary Fig . 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 B-CONTROLLED_VAR 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

e, Immunoblot analyses of phosphorylated AMP-activated protein kinase (pAMPK), total AMPK, pyruvate kinase isoforms M1 and M2 in ADR-senescent control;Bcl2 versus ADR-treated Suv39h1-;Bcl2 lymphoma cells in vitro (n = 3 each; α-tubulin as a loading control. Gels were cropped to ease presentation; full-length blots are provided in Supplementary Fig. 16).


f , Mean OCRs in ADR - senescent control ; Bcl2 versus ADR - treated Suv39h1 − ; Bcl2 lymphoma cells in vitro . Results represent means ± s . d . ( n = 5 each ) .

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

f, Mean OCRs in ADR-senescent control;Bcl2 versus ADR-treated Suv39h1−;Bcl2 lymphoma cells in vitro. Results represent means ± s.d. (n = 5 each).


g , Intracellular ATP concentrations in cells as in f . Results represent means ± s . d . ( n = 5 each ) . * P < 0 . 05 .

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

g, Intracellular ATP concentrations in cells as in f. Results represent means ± s.d. (n = 5 each). *P<0.05.


a - e , Cell viability by trypan blue dye exclusion in ADR - senescent control ; Bcl2 versus ADR - treated Suv39h1 - ; Bcl2 lymphoma cells at day 5 compared to the untreated condition in various biochemical , pharmacological or genetic interventional settings in vitro . All results in this figure represent means ± s . d . ( n = 5 each ) . a , Inhibition of glucose metabolism by phloretin , cytochalasin B , and sodium oxamate .

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

a-e, Cell viability by trypan blue dye exclusion in ADR-senescent control;Bcl2 versus ADR-treated Suv39h1-;Bcl2 lymphoma cells at day 5 compared to the untreated condition in various biochemical, pharmacological or genetic interventional settings in vitro. All results in this figure represent means ± s.d. (n = 5 each). a, Inhibition of glucose metabolism by phloretin, cytochalasin B, and sodium oxamate.


b , Exposure to 2DG or switch to glucose - free medium with or without glucose replenishment .

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

b, Exposure to 2DG or switch to glucose-free medium with or without glucose replenishment.


c , Lentiviral knockdown of hexokinase 2 ( Hk2 ) by short hairpin RNA ( shHk2 ) . d , Inhibition of AMPK by compound C . e , Block of fatty acid oxidation by the carnitine palmitoyltransferase I inhibitor etomoxir . f , Inhibition of the electron transport chain by antimycin A . * P 0 . 01 , except b ( n = 8 each ) and e , in which * 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 B-CONTROLLED_VAR I-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 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

c, Lentiviral knockdown of hexokinase 2 (Hk2) by short hairpin RNA (shHk2). d, Inhibition of AMPK by compound C. e, Block of fatty acid oxidation by the carnitine palmitoyltransferase I inhibitor etomoxir. f, Inhibition of the electron transport chain by antimycin A. *P  0.01, except b (n = 8 each) and e, in which *P  0.05.


Proteostasis analysis in TIS control versus senescence - incapable lymphoma cells in vitro , as in Fig . 2 , or versus SASP - attenuated TISlymphoma cells . a , Global protein synthesis rate , measured by incorporation of green - fluorescent methionine . Mean fluorescent intensities reflect newly synthesized protein within 2 h ( DAPI used as a counterstain ) . Data represent means ± s . d . ( n = 5 each ) . b , Rate of global protein synthesis ( measured as in a ) in ADR - senescent relative to untreated ( ut ) control ; Bcl2 lymphoma cells ; either empty vector - infected cells or cells stably expressing the NF - kB super - repressor IkBaDN ( SR , ref . 18 ) . Results represent means ± s . d . ( n = 5 each ) . c , Relative intracellular glucose concentrations in cells as in b .

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

Proteostasis analysis in TIS control versus senescence-incapable lymphoma cells in vitro, as in Fig. 2, or versus SASP-attenuated TISlymphoma cells. a, Global protein synthesis rate, measured by incorporation of green-fluorescent methionine. Mean fluorescent intensities reflect newly synthesized protein within 2 h (DAPI used as a counterstain). Data represent means ± s.d. (n = 5 each). b, Rate of global protein synthesis (measured as in a) in ADR-senescent relative to untreated (ut) control;Bcl2 lymphoma cells; either empty vector-infected cells or cells stably expressing the NF-kB super-repressor IkBaDN(SR, ref. 18). Results represent means ± s.d. (n = 5 each). c, Relative intracellular glucose concentrations in cells as in b.


d , Relative OCR in cells as in b . e , Relative intracellular ATP concentrations in cells as in

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

d, Relative OCR in cells as in b. e, Relative intracellular ATP concentrations in cells as in b


f , Expression of the transcription factors Xbp1 ( by RT - PCR ( PCR with reverse transcription ; top ) ; note that the lower band reflects the UPR - related splice form ) , ATF4 , CHOP , phospho - Jun N - terminal kinase ( JNK - P - Thr183 / Thr185 ) and total JNK ( by immunoblot analysis , with a - tubulin as a loading control ; bottom ) in lymphoma cells as indicated ( n = 3 each , gels were cropped to ease presentation ; full - length blots are provided in Supplementary Fig . 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

f, Expression of the transcription factors Xbp1 (by RT-PCR (PCR with reverse transcription; top); note that the lower band reflects the UPR-related splice form), ATF4, CHOP, phospho-Jun N-terminal kinase (JNK-P-Thr183/Thr185) and total JNK(by immunoblot analysis, with a-tubulin as a loading control; bottom) in lymphoma cells as indicated (n = 3 each, gels were cropped to ease presentation; full-length blots are provided in Supplementary Fig. 16).


g , Global protein ubiquitination , expression of p62 / SQSTM1 ( whose transcript levels were also selectively induced in TIS cells by approximately 4 . 8 - fold ( data not shown ) ) and V - ATPase subunit A1 , and conversion of MAP1 - LC3 - I to MAP1 - LC3 - II by immunoblot analysis , demonstrating autophagosome formation in lymphoma cells as in f ( α - tubulin was used as a loading control , gels were cropped to ease presentation ; full - length blots are provided in Supplementary Fig . 16 ) . * 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 O O O

g, Global protein ubiquitination, expression of p62/SQSTM1 (whose transcript levels were also selectively induced in TIS cells by approximately 4.8-fold (data not shown)) and V-ATPase subunit A1, and conversion of MAP1-LC3-I to MAP1-LC3-II by immunoblot analysis, demonstrating autophagosome formation in lymphoma cells as in f (α-tubulin was used as a loading control, gels were cropped to ease presentation; full-length blots are provided in Supplementary Fig. 16). *P<0.05.


a , Cellular viability of super - repressor - or empty - vector - infected control ; Bcl2 lymphomas treated with ADR or untreated for 5 days , then exposed for 2 days to the inhibitors 2DG or bafilomycin A1 , compared to no inhibitor ( ut ) in vitro . Results represent means ± s . d . ( n = 5 each ) .

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 B-CONTROLLED_VAR 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

a, Cellular viability of super-repressor- or empty-vector-infected control;Bcl2 lymphomas treated with ADR or untreated for 5 days, then exposed for 2 days to the inhibitors 2DG or bafilomycin A1, compared to no inhibitor (ut) in vitro. Results represent means ± s.d. (n = 5 each).


b , Immunoblot analyses of ATF4 , global protein ubiquitination , p62 / SQSTM1 , and MAP1 - LC3 - I to MAP1 - LC3 - II conversion in control ; Bcl2 lymphomas with no ADR treatment , in TIS cells , and in TIS cells after exposure to 2DG or bafilomycin A1in vitro ; representative blot with α - tubulin as a loading control ( gels were cropped to ease presentation ; full - length blots are provided in Supplementary Fig . 16 ) . c , Cleavage ( activation ) of caspase 12 and detection of cleaved caspase 3 by immunoblot analysis of control ; Bcl2 lymphomas as in b ; representative blot with α - tubulin as a loading control ( gels were cropped to ease presentation ; full - length blots are provided in Supplementary Fig . 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 B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR 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 O O 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, Immunoblot analyses of ATF4, global protein ubiquitination, p62/SQSTM1, and MAP1-LC3-I to MAP1-LC3-II conversion in control;Bcl2 lymphomas with no ADR treatment, in TIS cells, and in TIS cells after exposure to 2DG or bafilomycin A1in vitro; representative blot with α-tubulin as a loading control (gels were cropped to ease presentation; full-length blots are provided in Supplementary Fig. 16). c, Cleavage (activation) of caspase 12 and detection of cleaved caspase 3 by immunoblot analysis of control;Bcl2 lymphomas as in b; representative blot with α-tubulin as a loading control (gels were cropped to ease presentation; full-length blots are provided in Supplementary Fig. 16).


d , Cellular viability of ADR - treated control ; Bcl2 lymphoma cells infected with short - hairpin RNAs against either caspase 12 ( shCasp12 ) or caspase 3 ( shCasp3 ) compared to empty vector - infected lymphoma cells treated as in a . Results represent means ± s . d . ( n = 4 each ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Cellular viability of ADR-treated control;Bcl2 lymphoma cells infected with short-hairpin RNAs against either caspase 12 (shCasp12) or caspase 3 (shCasp3) compared to empty vector-infected lymphoma cells treated as in a. Results represent means ± s.d. (n = 4 each).


e , TIS cells in primary human acute myeloid leukaemia ( AML ) samples by SA - b - gal staining of treated ( 0 . 1 µg ml − 1 ADR for 5 days ) versus untreated cells in vitro . Data represent means ± s . d . ( n = 5 each ) .

O O O O O O O O O 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

e, TIS cells in primary human acute myeloid leukaemia (AML) samples by SA-b-gal staining of treated (0.1 µg ml−1 ADR for 5 days) versus untreated cells in vitro. Data represent means ± s.d. (n = 5 each).


f , Intracellular glucose ( left ) and ATP ( right ) concentrations in senescent AML cells as in e .

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

f, Intracellular glucose (left) and ATP (right) concentrations in senescent AML cells as in e.


g , Cellular viability of primary AML samples treated as in e and exposed to inhibitors as in a . Results represent means ± s . d . ( n = 5 each ) .

O O O O O O O O O O O O O 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, Cellular viability of primary AML samples treated as in e and exposed to inhibitors as in a. Results represent means ± s.d. (n = 5 each).


h , Colour - coded whole - body fluorescence imaging to visualize green fluorescent protein ( GFP ) - tagged tumour burden in time - courses of three representative mice harbouring CTX - senescent control ; Bcl2 lymphomas at day 5 ( see Figure 1a ) , and two days later , after the administration of 2DG ( n = 3 mice ) , bafilomycin A1 ( n = 4 mice ) or mock treatment ( n = 4 mice ) starting from day 5 post CTX ( red , highest intensities ; blue , lowest intensities ) . TdT - mediated dUTP nick end labelling ( assay ) ( TUNEL ) staining of lymph node sections in situ to visualize apoptosis at day 7 . Numbers represent means ± s . d . ( n = 3 ) .

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

h, Colour-coded whole-body fluorescence imaging to visualize green fluorescent protein (GFP)-tagged tumour burden in time-courses of three representative mice harbouring CTX-senescent control;Bcl2 lymphomas at day 5 (see Figure 1a), and two days later, after the administration of 2DG (n = 3 mice), bafilomycin A1 (n = 4 mice) or mock treatment (n = 4 mice) starting from day 5 post CTX (red, highest intensities; blue, lowest intensities). TdT-mediated dUTP nick end labelling (assay) (TUNEL) staining of lymph node sections in situ to visualize apoptosis at day 7. Numbers represent means ± s.d. (n = 3).


i , Overall survival of 5 day - CTX - treated control ; Bcl2 or Suv - 39h1 - ; Bcl2 lymphoma - bearing mice randomly assigned to sequential exposure to bafilomycin A1 or mock as in h ( control ; Bcl2 : n = 12 and Suv39h1 - ; Bcl2 : n = 10 ) , presented as matched pair analyses ( P < 0 . 001 for the control ; Bcl2 comparison ; P = 0 . 221 for the Suv39h1 - ; Bcl2 comparison ) . * P < 0 . 05 .

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 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

i, Overall survival of 5 day-CTX-treated control;Bcl2 or Suv-39h1-;Bcl2 lymphoma-bearing mice randomly assigned to sequential exposure to bafilomycin A1 or mock as in h (control;Bcl2: n = 12 and Suv39h1-;Bcl2: n = 10), presented as matched pair analyses (P<0.001 for the control; Bcl2 comparison; P = 0.221 for the Suv39h1-;Bcl2 comparison). *P<0.05.


F - G ) HeLa cells were treated as in B ) and then fixed and stained with ULK1 ( green ) ( F ) or ATG13 ( red ) antibodies ( G ) respectively . Hoechst was used to stain nuclei . Graphs ( right ) show the average of both ULK1 ( F ) ( n = 3 independent experiments , n = 11 fields analysed ) or ATG13 ( n = 3 independent experiments , n ≥ 16 fields analysed ) ( G ) puncta number per cell and both ULK1 ( n = 3 independent experiments ) ( F ) or ATG13 ( n = 3 independent experiments ) ( G ) fold of increase of the average size respectively . White arrows indicate most representative puncta of analysed conditions . All data are reported as the mean value ± SEM . Statistical analyses were performed by two - way ANOVA followed by Tukey ' s multiple comparison test . * * , P < 0 , 01 ; * * * * , P < 0 . 0001 or by unpaired Student ' s t ‐ test when two groups were compared . * , P < 0 . 05 ; * * * * , P < 0 . 0001 . Scale bar 5 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-G) HeLa cells were treated as in B) and then fixed and stained with ULK1 (green) (F) or ATG13 (red) antibodies (G) respectively. Hoechst was used to stain nuclei. Graphs (right) show the average of both ULK1 (F) (n=3 independent experiments, n=11 fields analysed) or ATG13 (n=3 independent experiments, n≥16 fields analysed) (G) puncta number per cell and both ULK1 (n=3 independent experiments) (F) or ATG13 (n=3 independent experiments) (G) fold of increase of the average size respectively. White arrows indicate most representative puncta of analysed conditions. All data are reported as the mean value ± SEM. Statistical analyses were performed by two-way ANOVA followed by Tukey's multiple comparison test. **, P<0,01; ****, P<0.0001 or by unpaired Student's t‐test when two groups were compared. *, P<0.05; ****, P<0.0001. Scale bar 5 μm.


HeLa cells were transfected with specific RNAi oligonucleotides ( siTFG ) or unrelated oligonucleotides as a negative control ( siCTR ) and grown in fed or starved conditions for 1h . Cells were fixed and co - labelled with ULK1 ( red ) antibody and A ) anti - CALNEXIN ( CANX ) ( green ) antibody to highlight ER structures ; ( n = 3 independent experiments , n ≥ 16 fields analysed ) B ) GOLGIN97 ( green ) antibody to label GOLGI complex ; ( n = 3 independent experiments , n = 12 fields analysed ) C ) SEC31A ( green ) to point out COPII vesicles . ( n = 3 independent experiments , n ≥ 16 fields analysed )

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

HeLa cells were transfected with specific RNAi oligonucleotides (siTFG) or unrelated oligonucleotides as a negative control (siCTR) and grown in fed or starved conditions for 1h. Cells were fixed and co-labelled with ULK1 (red) antibody and A) anti-CALNEXIN (CANX) (green) antibody to highlight ER structures; (n=3 independent experiments, n≥16 fields analysed) B) GOLGIN97 (green) antibody to label GOLGI complex; (n=3 independent experiments, n=12 fields analysed) C) SEC31A (green) to point out COPII vesicles. (n=3 independent experiments, n≥16 fields analysed)


D ) ERGIC53 ( green ) to mark ERGIC compartment . ( n = 3 independent experiments , n ≥ 14 fields analysed ) . Analysis of ULK1 localization with each compartment is reported in each graph . Co - localization analyses were performed by ImageJ plugin Jacop . Values of Mander ' s coefficient for ULK1 are expressed in percentage as mean ± SEM . Statistical analyses were performed by two - way ANOVA followed by Tukey ' s multiple comparison test . * * , P < 0 , 01 ; * * * , P < 0 , 001 ; * * * * , P < 0 , 0001 ; ns , not significant . Scale bars 5 μm . White arrowheads point at ULK1 puncta associated to the analysed markers ; N , nucleus .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) ERGIC53 (green) to mark ERGIC compartment. (n=3 independent experiments, n≥14 fields analysed). Analysis of ULK1 localization with each compartment is reported in each graph. Co-localization analyses were performed by ImageJ plugin Jacop. Values of Mander's coefficient for ULK1 are expressed in percentage as mean ± SEM. Statistical analyses were performed by two-way ANOVA followed by Tukey's multiple comparison test. **, P<0,01; ***, P<0,001; ****, P<0,0001; ns, not significant. Scale bars 5 μm. White arrowheads point at ULK1 puncta associated to the analysed markers; N, nucleus.


F ) HeLa cells were co - transfected with control siRNA and an empty vector ( EV ) , or 3 ' UTR TFG siRNA together with an EV or HA - TFG plasmids . Cells grown for 1h in starvation medium then fixed , permeabilized , and labelled with anti - ULK1 ( green ) and anti - ERGIC53 ( red ) antibodies respectively . Hoechst was used to stain nuclei . Co - localization analysis were performed as in D ) . Values of Mander ' s coefficient for ULK1 , expressed as percentage , are mean ± SEM . Statistical analyses were performed by one - way ANOVA followed by Dunnetts ' s multiple comparison test . * , P < 0 , 05 . ( n = 3 ) ( n = 3 independent experiments , n ≥ 9 fields analysed ) . Scale bar 5 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HeLa cells were co-transfected with control siRNA and an empty vector (EV), or 3'UTR TFG siRNA together with an EV or HA-TFG plasmids. Cells grown for 1h in starvation medium then fixed, permeabilized, and labelled with anti-ULK1 (green) and anti-ERGIC53 (red) antibodies respectively. Hoechst was used to stain nuclei. Co-localization analysis were performed as in D). Values of Mander's coefficient for ULK1, expressed as percentage, are mean ± SEM. Statistical analyses were performed by one-way ANOVA followed by Dunnetts's multiple comparison test. *, P<0,05. (n=3) (n=3 independent experiments, n≥9 fields analysed). Scale bar 5 μm.


A ) HeLa cells were transfected with specific RNAi oligonucleotides ( siTFG ) or unrelated oligonucleotides as a negative control ( siCTR ) and grown in fed ( FED ) or starvation conditions ( STV ) in the presence or absence of CLQ for 1h . Cells were then fixed and stained using LC3B - II ( red ) antibody . Analysis of LC3B puncta number is reported ( bottom ) . All data are expressed as the mean value ± SEM . Statistical analysis were performed by two - way ANOVA followed by Tukey ' s multiple comparison test . * * * * , P < 0 . 0001 . ( n = 4 independent experiments , n ≥ 25 fields analysed ) . Scale bar 5 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 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

A) HeLa cells were transfected with specific RNAi oligonucleotides (siTFG) or unrelated oligonucleotides as a negative control (siCTR) and grown in fed (FED) or starvation conditions (STV) in the presence or absence of CLQ for 1h. Cells were then fixed and stained using LC3B-II (red) antibody. Analysis of LC3B puncta number is reported (bottom). All data are expressed as the mean value ± SEM. Statistical analysis were performed by two-way ANOVA followed by Tukey's multiple comparison test. ****, P<0.0001. (n=4 independent experiments, n≥25 fields analysed). Scale bar 5 μm.


B ) HeLa cells were transfected as in A ) and cultured in starvation conditions ( STV ) in the presence or absence of Bafilomycin A1 ( Baf A1 ) for 1h . Cells were then fixed and stained using LC3B - II ( green ) and LAMP1 ( red ) antibodies . Hoechst was used to mark nuclei . Co - localization analyses were performed by ImageJ plugin Jacop . Values of Mander ' s coefficient for LC3B - II are expressed as mean ± SEM . Statistical analysis were performed by two - way ANOVA followed by Tukey ' s multiple comparison test . * , P < 0 , 05 ; ( n = 3 independent experiments , n ≥ 13 fields analysed ) . Scale bar 5 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 B-CONTROLLED_VAR I-CONTROLLED_VAR 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 O O O O O O O O O O O O O O O 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) HeLa cells were transfected as in A) and cultured in starvation conditions (STV) in the presence or absence of Bafilomycin A1 (Baf A1) for 1h. Cells were then fixed and stained using LC3B-II (green) and LAMP1 (red) antibodies. Hoechst was used to mark nuclei. Co-localization analyses were performed by ImageJ plugin Jacop. Values of Mander's coefficient for LC3B-II are expressed as mean ± SEM. Statistical analysis were performed by two-way ANOVA followed by Tukey's multiple comparison test. *, P<0,05; (n=3 independent experiments, n≥13 fields analysed). Scale bar 5 μm.


C ) HeLa cells were transfected as in A ) and grown in fed ( FED ) or starvation conditions ( STV ) for 1h . Cells were then fixed and stained using ATG16L1 ( yellow ) antibody . ATG16L1 puncta number ( upper right ) and the average size of ATG16L1 as fold increase ( bottom right ) were reported . All values are expressed as mean ± SEM . Statistical analyses were performed by two - way ANOVA followed by Tukey ' s multiple comparison test . * * , P < 0 , 01 ; * * * * , P < 0 . 0001 or by unpaired Student ' s t ‐ test when two groups were compared . ns , not significant . ( n = 3 independent experiments , n ≥ 9 fields analysed ) . Scale bar 5 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HeLa cells were transfected as in A) and grown in fed (FED) or starvation conditions (STV) for 1h. Cells were then fixed and stained using ATG16L1 (yellow) antibody. ATG16L1 puncta number (upper right) and the average size of ATG16L1 as fold increase (bottom right) were reported. All values are expressed as mean ± SEM. Statistical analyses were performed by two-way ANOVA followed by Tukey's multiple comparison test. **, P<0,01; ****, P<0.0001 or by unpaired Student's t‐test when two groups were compared. ns, not significant. (n=3 independent experiments, n≥9 fields analysed). Scale bar 5 μm.


D ) HEK293 cells stably expressing GFP - DFCP1 were transfected as in A ) and cultured in starved conditions for 40 ' . Cells were visualised with cellSens microscope for live - cell imaging . Pictures show the images taken at the same number of frames for each video . Analyses of the number of DFCP1 structures per cell ( upper right ) and the mean of omegasomes structures diameter ( μm ) ( bottom right ) were reported . N , nucleus . All values are expressed as mean ± SEM . Statistical analyses were performed by unpaired Student ' s t ‐ test . * , P < 0 , 05 ; * * * , P < 0 . 001 . ( n = 3 independent experiments ) . Scale bar 5 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HEK293 cells stably expressing GFP-DFCP1 were transfected as in A) and cultured in starved conditions for 40'. Cells were visualised with cellSens microscope for live-cell imaging. Pictures show the images taken at the same number of frames for each video. Analyses of the number of DFCP1 structures per cell (upper right) and the mean of omegasomes structures diameter (μm) (bottom right) were reported. N, nucleus. All values are expressed as mean ± SEM. Statistical analyses were performed by unpaired Student's t‐test. *, P<0,05; ***, P<0.001. (n=3 independent experiments). Scale bar 5 μm.


A ) HeLa cells were transfected with specific RNAi oligonucleotides ( siTFG ) or unrelated oligonucleotides as a negative control ( siCTR ) ; after culturing cells in fed ( FED ) or starvation conditions ( STV ) for 1h , cells were fixed and labelled with the indicated antibodies . Co - localization analysis was performed by using ImageJ Jacop plugin and mean ± SEM of Mander ' s coefficients of ULK1 , expressed as percentage , is reported ( bottom ) . White arrowheads point at co - localization events between ULK1 and LC3B ; white arrows track puncta not co - localizing ; N , nucleus . Statistical analyses were performed by two - way ANOVA followed by Tukey ' s multiple comparison test . * , P < 0 , 05 ; * * , P < 0 . 01 ; * * * * , P < 0 . 0001 . ( n = 3 independent experiments , n ≥ 16 fields analysed ) . Scale bar 5 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HeLa cells were transfected with specific RNAi oligonucleotides (siTFG) or unrelated oligonucleotides as a negative control (siCTR); after culturing cells in fed (FED) or starvation conditions (STV) for 1h, cells were fixed and labelled with the indicated antibodies. Co-localization analysis was performed by using ImageJ Jacop plugin and mean ± SEM of Mander's coefficients of ULK1, expressed as percentage, is reported (bottom). White arrowheads point at co-localization events between ULK1 and LC3B; white arrows track puncta not co-localizing; N, nucleus. Statistical analyses were performed by two-way ANOVA followed by Tukey's multiple comparison test. *, P<0,05; **, P<0.01; ****, P<0.0001. (n=3 independent experiments, n≥16 fields analysed). Scale bar 5 μm.


HEK293 cells stably expressing either GFP - ATG13 ( B ) were transiently transfected with specific RNAi oligonucleotides ( siTFG ) or unrelated oligonucleotides as a negative control ( siCTR ) , together with CFP - LC3B plasmid ( visualized in red ) and cultured in starved conditions for 40 ' . Wide - field live - cell imaging of starved cells were taken by cellSens microscope . Representative images of lifespan of ATG13 ( B ) are reported . Images of ATG13 ( B ) forming association with LC3B - II are also shown . The appearance of LC3B on ATG13 ( B ) ( bottom right ) ( C ) puncta were quantified and reported . All values are expressed in seconds ( sec ) as mean ± SEM . Yellow arrowheads point at the DFCP1 and LC3B particles in the first frame from their onset , white arrowheads and white arrows show omegasomes and autophagosomes as indicated . Statistical analyses were performed by unpaired Student ' s t ‐ test . * , P < 0 , 05 ; * * , P < 0 . 01 . ( n = 3 independent experiments , n = 50 events / condition analysed ) . Scale bar 1 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

HEK293 cells stably expressing either GFP-ATG13 (B) were transiently transfected with specific RNAi oligonucleotides (siTFG) or unrelated oligonucleotides as a negative control (siCTR), together with CFP-LC3B plasmid (visualized in red) and cultured in starved conditions for 40'. Wide-field live-cell imaging of starved cells were taken by cellSens microscope. Representative images of lifespan of ATG13 (B) are reported. Images of ATG13 (B) forming association with LC3B-II are also shown. The appearance of LC3B on ATG13 (B) (bottom right) (C) puncta were quantified and reported. All values are expressed in seconds (sec) as mean ± SEM. Yellow arrowheads point at the DFCP1 and LC3B particles in the first frame from their onset, white arrowheads and white arrows show omegasomes and autophagosomes as indicated. Statistical analyses were performed by unpaired Student's t‐test. *, P<0,05; **, P<0.01.(n=3 independent experiments, n=50 events/condition analysed). Scale bar 1 μm.


HEK293 cells stably expressing either GFP - DFCP1 ( C ) were transiently transfected with specific RNAi oligonucleotides ( siTFG ) or unrelated oligonucleotides as a negative control ( siCTR ) , together with CFP - LC3B plasmid ( visualized in red ) and cultured in starved conditions for 40 ' . Wide - field live - cell imaging of starved cells were taken by cellSens microscope . Representative images of lifespan of both ATG13 ( C ) are reported . Images of DFCP1 ( C ) forming association with LC3B - II are also shown . The appearance of LC3B on on DFCP1 ( bottom right ) ( C ) puncta were quantified and reported . All values are expressed in seconds ( sec ) as mean ± SEM . Yellow arrowheads point at the DFCP1 and LC3B particles in the first frame from their onset , white arrowheads and white arrows show omegasomes and autophagosomes as indicated . Statistical analyses were performed by unpaired Student ' s t ‐ test . * , P < 0 , 05 ; * * , P < 0 . 01 . ( n = 3 independent experiments , n = 50 events / condition analysed ) . Scale bar 1 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

HEK293 cells stably expressing either GFP-DFCP1 (C) were transiently transfected with specific RNAi oligonucleotides (siTFG) or unrelated oligonucleotides as a negative control (siCTR), together with CFP-LC3B plasmid (visualized in red) and cultured in starved conditions for 40'. Wide-field live-cell imaging of starved cells were taken by cellSens microscope. Representative images of lifespan of both ATG13 (C) are reported. Images of DFCP1 (C) forming association with LC3B-II are also shown. The appearance of LC3B on on DFCP1 (bottom right) (C) puncta were quantified and reported. All values are expressed in seconds (sec) as mean ± SEM. Yellow arrowheads point at the DFCP1 and LC3B particles in the first frame from their onset, white arrowheads and white arrows show omegasomes and autophagosomes as indicated. Statistical analyses were performed by unpaired Student's t‐test. *, P<0,05; **, P<0.01.(n=3 independent experiments, n=50 events/condition analysed). Scale bar 1 μm.


D ) HeLa cells were transfected as in A ) . Cells were imaged by TEM . Representative electron micrographs ( x26500 ) are reported . The average number of abnormal structures per cell is shown ( bottom graph ) . N , nucleus ; M , mitochondria ; ER , endoplasmic reticulum . The average number of abnormal vacuoles per cell was evaluated ( upper right ) . Statistical analysis was performed by unpaired Student ' s t ‐ test . * * * , P < 0 , 001 . A minimum of 50 cells were observed . Cell count was performed ( n = 3 independent experiments , n ≥ 51 cells analysed ) . Data are presented as mean ± SEM . Scale bar 500 nm as indicated .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HeLa cells were transfected as in A). Cells were imaged by TEM. Representative electron micrographs (x26500) are reported. The average number of abnormal structures per cell is shown (bottom graph). N, nucleus; M, mitochondria; ER, endoplasmic reticulum. The average number of abnormal vacuoles per cell was evaluated (upper right). Statistical analysis was performed by unpaired Student's t‐test. ***, P<0,001. A minimum of 50 cells were observed. Cell count was performed (n=3 independent experiments, n≥51 cells analysed). Data are presented as mean ± SEM. Scale bar 500 nm as indicated.


E ) HeLa cells stably expressing GFP - ATG13 and CFP - ATG16 were transiently co - transfected with DsRed - Rab7 and TFG siRNA ( siTFG ) . Cells were cultured in starved conditions for 1h . Representative electron micrographs ( x3900 ) and merge of the three fluorophores CLEM are shown . Micrographs at higher magnification ( x13500 ) of a region showing the three fluorophores split and merged is reported . Scale bars : 5 μm upper panels , 1 μm bottom panels .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HeLa cells stably expressing GFP-ATG13 and CFP-ATG16 were transiently co-transfected with DsRed-Rab7 and TFG siRNA (siTFG). Cells were cultured in starved conditions for 1h. Representative electron micrographs (x3900) and merge of the three fluorophores CLEM are shown. Micrographs at higher magnification (x13500) of a region showing the three fluorophores split and merged is reported. Scale bars: 5 μm upper panels, 1 μm bottom panels.


G ) Control ( TFG - WT ) and patient ' s ( TFG - R106C ) fibroblasts were grown in fed or starvation conditions for 1h . Cells were fixed , permeabilized , and labelled with anti - ULK ( red ) antibody . Hoechst was used to stain nuclei . The average of ULK1 puncta number per cell is shown ( bottom ) . All data are expressed as the mean value ± SEM . Statistical analysis was performed by two - way ANOVA followed by Tukey ' s multiple comparison test . * , P < 0 . 05 ; * * * , P < 0 , 001 ; * * * * ; P < 0 , 0001 . ( n = 3 independent experiments , n ≥ 14 fields analysed ) . Scale bar 5 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Control (TFG-WT) and patient's (TFG-R106C) fibroblasts were grown in fed or starvation conditions for 1h. Cells were fixed, permeabilized, and labelled with anti-ULK (red) antibody. Hoechst was used to stain nuclei. The average of ULK1 puncta number per cell is shown (bottom). All data are expressed as the mean value ± SEM. Statistical analysis was performed by two-way ANOVA followed by Tukey's multiple comparison test. *, P<0.05; ***, P<0,001; ****; P<0,0001. (n=3 independent experiments, n≥14 fields analysed). Scale bar 5 μm.


HeLa cells stably expressing untagged TFGwtLIR or TFGmutLIR3 were transfected with 3 ' UTR TFG siRNA , starved for 1h , fixed , permeabilized and immunolabelled with ULK1 ( red ) Hoechst was used to stain nuclei . ULK1 puncta number ( G ) was analysed and reported as mean ± SEM in the graph ( right ) ( n = 3 independent experiments , n = 24 fields analysed ) . Values of Mander ' s coefficient for ULK1 , expressed as percentage , are reported as mean ± SEM ( bottom graph ) . ( n = 3 independent experiments , n = 19 fields analysed ) White arrowheads point at co - localization events between ULK1 and ERGIC . Statistical analyses were performed by unpaired Student ' s t ‐ test . * * , P < 0 , 01 ; * * * , 0 , 001 . Scale bar 5 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

HeLa cells stably expressing untagged TFGwtLIR or TFGmutLIR3 were transfected with 3'UTR TFG siRNA, starved for 1h, fixed, permeabilized and immunolabelled with ULK1 (red) Hoechst was used to stain nuclei. ULK1 puncta number (G) was analysed and reported as mean ± SEM in the graph (right) (n=3 independent experiments, n=24 fields analysed). Values of Mander's coefficient for ULK1, expressed as percentage, are reported as mean ± SEM (bottom graph). (n=3 independent experiments, n=19 fields analysed) White arrowheads point at co-localization events between ULK1 and ERGIC. Statistical analyses were performed by unpaired Student's t‐test. **, P<0,01; ***, 0,001. Scale bar 5 μm.


HeLa cells stably expressing untagged TFGwtLIR or TFGmutLIR3 were transfected with 3 ' UTR TFG siRNA , starved for 1h , fixed , permeabilized and immunolabelled with ULK1 ( red ) alone or ULK1 ( red ) and ERGIC53 ( green ) ( H ) . Hoechst was used to stain nuclei . analysed ) . Co - localization analyses ( H ) were performed by Jacop plugin . Values of Mander ' s coefficient for ULK1 , expressed as percentage , are reported as mean ± SEM ( bottom graph ) . ( n = 3 independent experiments , n = 19 fields analysed ) White arrowheads point at co - localization events between ULK1 and ERGIC . Statistical analyses were performed by unpaired Student ' s t ‐ test . * * , P < 0 , 01 ; * * * , 0 , 001 . Scale bar 5 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

HeLa cells stably expressing untagged TFGwtLIR or TFGmutLIR3 were transfected with 3'UTR TFG siRNA, starved for 1h, fixed, permeabilized and immunolabelled with ULK1 (red) alone or ULK1 (red) and ERGIC53 (green) (H). Hoechst was used to stain nuclei. analysed). Co-localization analyses (H) were performed by Jacop plugin. Values of Mander's coefficient for ULK1, expressed as percentage, are reported as mean ± SEM (bottom graph). (n=3 independent experiments, n=19 fields analysed) White arrowheads point at co-localization events between ULK1 and ERGIC. Statistical analyses were performed by unpaired Student's t‐test. **, P<0,01; ***, 0,001. Scale bar 5 μm.


M ) HeLa cells stably expressing untagged TFGwtLIR or TFGmutLIR3 were co - transfected with HA - LC3C and Myc - ULK1 plasmids as indicated . Cells were starved for 30 ' and protein extracts were immunoprecipitated using an anti - HA antibody or IgG as negative control . Protein extracts were analysed by WB to detect ULK1 , LC3C , TFG and ACTIN as indicated .

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

M) HeLa cells stably expressing untagged TFGwtLIR or TFGmutLIR3 were co-transfected with HA-LC3C and Myc-ULK1 plasmids as indicated. Cells were starved for 30' and protein extracts were immunoprecipitated using an anti-HA antibody or IgG as negative control. Protein extracts were analysed by WB to detect ULK1, LC3C, TFG and ACTIN as indicated.


The SnRK2 OST1 activates ABFs for expression of a luciferase reporter ( LUC ) in yeast . Luciferase activity is given on a Log10 scale as relative counts of light emission per second ( rcps ) normalized to the cell density . The OST1 - mediated induction factor is indicated above the columns . In the absence of ABFs , the LUC activity was 3 x 103 rcps .

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

The SnRK2 OST1 activates ABFs for expression of a luciferase reporter (LUC) in yeast. Luciferase activity is given on a Log10 scale as relative counts of light emission per second (rcps) normalized to the cell density. The OST1-mediated induction factor is indicated above the columns. In the absence of ABFs, the LUC activity was 3 x 103 rcps.


Combining the ABA signaling components ABF2 , OST1 , the PP2C ABI1 , and RCAR11 provides ABA - controlled LUC expression . The reporter is under the control of a minimal promoter that contains ABA - responsive cis elements ( ABREs ) . Core ABA signaling involves the RCAR receptor that inhibits the ABA coreceptor ( PP2C ) in the presence of ABA and allows the activation of the protein kinase SnRK2 . SnRK2 phosphorylates and activates the key transcription factor ABF , which targets promoters containing ABRE . Reporter activity was determined 16 h after exogenous ABA ( 0 . 1 mM ) administration and referred to samples expressing ABF2 alone .

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

Combining the ABA signaling components ABF2, OST1, the PP2C ABI1, and RCAR11 provides ABA-controlled LUC expression. The reporter is under the control of a minimal promoter that contains ABA-responsive cis elements (ABREs). Core ABA signaling involves the RCAR receptor that inhibits the ABA coreceptor (PP2C) in the presence of ABA and allows the activation of the protein kinase SnRK2. SnRK2 phosphorylates and activates the key transcription factor ABF, which targets promoters containing ABRE. Reporter activity was determined 16 h after exogenous ABA (0.1 mM) administration and referred to samples expressing ABF2 alone.


In yeast , the OST1 - and ABF2 - mediated reporter activity was inhibited by ABI1 ( ‑ RCAR ) . The subsequent recovery of the response was determined by co - expressed ABA receptors RCAR1 - RCAR14 belonging to three subfamilies , in the presence or absence of 30 µM ABA . The ABA response is expressed relative to the response obtained by replacing ABI1 with the catalytically inactive ABI1D177A . Regulation of an ABA - responsive LUC reporter in Arabidopsis protoplasts by ectopic expression of 1 µg ABI1 - effector in combination with 5 µg ABA receptor DNA ( ± 10 µM ABA ) per sample containing 105 protoplasts .

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

In yeast, the OST1- and ABF2-mediated reporter activity was inhibited by ABI1 (‑RCAR). The subsequent recovery of the response was determined by co-expressed ABA receptors RCAR1-RCAR14 belonging to three subfamilies, in the presence or absence of 30 µM ABA. The ABA response is expressed relative to the response obtained by replacing ABI1 with the catalytically inactive ABI1D177A. Regulation of an ABA-responsive LUC reporter in Arabidopsis protoplasts by ectopic expression of 1 µg ABI1-effector in combination with 5 µg ABA receptor DNA (± 10 µM ABA) per sample containing 105 protoplasts.


In yeast , the OST1 - and ABF2 - mediated reporter activity was inhibited by ABI1 ( ‑ RCAR ) . The subsequent recovery of the response was determined by co - expressed ABA receptors RCAR1 - RCAR14 belonging to three subfamilies , in the presence or absence of 30 µM ABA . The ABA response is expressed relative to the response obtained by replacing ABI1 with the catalytically inactive ABI1D177A . Regulation of an ABA - responsive LUC reporter in Arabidopsis protoplasts by ectopic

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

In yeast, the OST1- and ABF2-mediated reporter activity was inhibited by ABI1 (‑RCAR). The subsequent recovery of the response was determined by co-expressed ABA receptors RCAR1-RCAR14 belonging to three subfamilies, in the presence or absence of 30 µM ABA. The ABA response is expressed relative to the response obtained by replacing ABI1 with the catalytically inactive ABI1D177A. Regulation of an ABA-responsive LUC reporter in Arabidopsis protoplasts by ectopic expression


The ABA - dependent regulation of the PP2Cs ABI1 ( A , D , G ) , ABI2 ( B , E , H ) , and HAB1 ( C , F , I ) by RCAR12 and RCAR13 were analyzed in yeast ( A - C ) , Arabidopsis protoplasts ( D - F ) , and in vitro ( G - I ) . A - C ) The analysis in yeast was conducted as mentioned in Fig . 2A except that other PP2C - RCAR combinations were used and the ABA concentrations varied as indicated . D - F ) Analysis of protoplasts was performed in the ABA - deficient background aba2 - 1 in the presence of different exogenous ABA concentrations . The ABA response was approximately tenfold inhibited by ectopic expression of the respective PP2Cs ( Tischer et al , 2017 ) . The induction of the reporter gene is related to protoplasts without ectopic RCAR expression , and in the absence of exogenous ABA . G - I ) The in vitro analysis was performed by using 50 nM PP2C and 100 nM RCAR at different ABA concentrations . Maximum inhibition of PP2C activity by ABA was set to 100 % . Data information : In ( A - C ) each data point represents the mean ± s . d . ; n = 9 biological replicates derived from three independent yeast transformants . In ( D - F ) data represent the mean ± s . d . of three independent transfections . In ( G - I ) data represent the mean ± s . d . of three replicates .

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 B-CONTROLLED_VAR 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 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 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 B-CONTROLLED_VAR 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

The ABA-dependent regulation of the PP2Cs ABI1 (A,D,G), ABI2 (B,E,H), and HAB1 (C,F,I) by RCAR12 and RCAR13 were analyzed in yeast (A-C), Arabidopsis protoplasts (D-F), and in vitro (G-I). A-C) The analysis in yeast was conducted as mentioned in Fig. 2A except that other PP2C - RCAR combinations were used and the ABA concentrations varied as indicated. D-F) Analysis of protoplasts was performed in the ABA-deficient background aba2-1 in the presence of different exogenous ABA concentrations. The ABA response was approximately tenfold inhibited by ectopic expression of the respective PP2Cs (Tischer et al, 2017). The induction of the reporter gene is related to protoplasts without ectopic RCAR expression, and in the absence of exogenous ABA. G -I) The in vitro analysis was performed by using 50 nM PP2C and 100 nM RCAR at different ABA concentrations. Maximum inhibition of PP2C activity by ABA was set to 100%. Data information: In (A-C) each data point represents the mean ± s.d.; n = 9 biological replicates derived from three independent yeast transformants. In (D-F) data represent the mean ± s.d. of three independent transfections. In (G-I) data represent the mean ± s.d. of three replicates.


The ABA - dependent regulation of the PP2Cs ABI1 ( A , D , G ) , ABI2 ( B , E , H ) , and HAB1 ( C , F , I ) by RCAR12 and RCAR13 were analyzed in yeast ( A - C ) , Arabidopsis protoplasts ( D - F ) , and in vitro ( G - I ) .

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

The ABA-dependent regulation of the PP2Cs ABI1 (A,D,G), ABI2 (B,E,H), and HAB1 (C,F,I) by RCAR12 and RCAR13 were analyzed in yeast (A-C), Arabidopsis protoplasts (D-F), and in vitro (G-I).


Expression of the SnRK2s in yeast revealed ABF2 - mediated transactivation by SnRK2 . 1 , SnRK2 . 4 , SnRK2 . 5 , and SnRK2 . 10 in addition to OST1 . Inset : Immuno - detection of FLAG - tagged SnRK2s expressed in yeast . The data in ( B ) are normalized to control samples expressing ABF2 but no SnRK2

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

Expression of the SnRK2s in yeast revealed ABF2-mediated transactivation by SnRK2.1, SnRK2.4, SnRK2.5, and SnRK2.10 in addition to OST1. Inset: Immuno-detection of FLAG-tagged SnRK2s expressed in yeast. The data in (B) are normalized to control samples expressing ABF2 but no SnRK2


Expression of the SnRK2s in yeast revealed ABF2 - mediated transactivation by SnRK2 . 1 , SnRK2 . 4 , SnRK2 . 5 , and SnRK2 . 10 in addition to OST1 . Inset : Immuno - detection of FLAG - tagged SnRK2s expressed in yeast . The data in ( B ) are normalized to control samples expressing ABF2 but no

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


The active subgroup I members together with SnRK2 . 2 and SnRK2 . 3 were analyzed for differential activation of ABFs . The OST1 data shown in Fig . 1A is included for better comparison . The induction level of ABF4 relative to the empty vector control ( ev ) is indicated .

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

The active subgroup I members together with SnRK2.2 and SnRK2.3 were analyzed for differential activation of ABFs. The OST1 data shown in Fig. 1A is included for better comparison. The induction level of ABF4 relative to the empty vector control (ev) is indicated.


Analysis of the phosphorylation status in the activation loop of SnRK2s expressed in yeast . Phosphorylation occurred at serine 175 or threonine 176 , and at threonine 179 . The relative abundance is given as the ratio of signal intensity of phosphorylated versus non - phosphorylated peptide for OST1 , SnRK2 . 3 , and SnRK2 . 4 in the mass spectrometric analysis . The numbering of phosphosites corresponds to OST1 .

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

Analysis of the phosphorylation status in the activation loop of SnRK2s expressed in yeast. Phosphorylation occurred at serine 175 or threonine 176, and at threonine 179. The relative abundance is given as the ratio of signal intensity of phosphorylated versus non-phosphorylated peptide for OST1, SnRK2.3, and SnRK2.4 in the mass spectrometric analysis. The numbering of phosphosites corresponds to OST1.


Inhibition of OST1 and SnRK2 . 4 by clade A PP2Cs . The data in ( G ) to cells expressing inactive ABI1D177A instead of ABI1 .

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

Inhibition of OST1 and SnRK2.4 by clade A PP2Cs. The data in (G) to cells expressing inactive ABI1D177A instead of ABI1.


A , B ) ABF2 - mediated reporter induction by different SnRK2s was assessed in the presence of the receptor complex RCAR11 and ABI1 , and varying exogenous ABA concentrations . Data information : The ABA concentration that provides half maximum response recovery is indicated by dashed vertical lines . represent Replicates were normalized to controls without SnRK2 in ( A , B )

O O O O 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 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

A,B) ABF2-mediated reporter induction by different SnRK2s was assessed in the presence of the receptor complex RCAR11 and ABI1, and varying exogenous ABA concentrations. Data information: The ABA concentration that provides half maximum response recovery is indicated by dashed vertical lines. represent Replicates were normalized to controls without SnRK2 in (A, B)


C ) Maximum ABA response recovery of samples shown in ( A , B ) relative to the reporter activity of 0 . 1 mM ABA - treated yeast cells expressing SnRK2s but no ABI1 . Data information : Replicates were normalized to controls without ABI1 and RCAR for each SnRK2 in ( C ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Maximum ABA response recovery of samples shown in (A,B) relative to the reporter activity of 0.1 mM ABA-treated yeast cells expressing SnRK2s but no ABI1. Data information: Replicates were normalized to controls without ABI1 and RCAR for each SnRK2 in (C).


D - F ) Analysis of the ABA response mediated by OST1 and SnRK2 . 4 using the receptors RCAR1 ( D ) , RCAR8 ( E ) , and RCAR14 ( F ) . Data information : The ABA concentration that provides half maximum response recovery is indicated by dashed vertical lines . The data in ( D - F ) are the mean ± s . e . m ; n = 12 , biological replicates from four independent yeast transformants for OST1 + RCAR1 / RCAR8 , and SnRK2 . 4 + RCAR8 .

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 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

D-F) Analysis of the ABA response mediated by OST1 and SnRK2.4 using the receptors RCAR1 (D), RCAR8 (E), and RCAR14 (F). Data information: The ABA concentration that provides half maximum response recovery is indicated by dashed vertical lines. The data in (D-F) are the mean ± s.e.m; n = 12, biological replicates from four independent yeast transformants for OST1 + RCAR1/RCAR8, and SnRK2.4 + RCAR8.


FRET - FLIM analysis of GFP : OST1 or GFP : SnRK2 . 4 and the effect of ABI1 fused to the mCherry fluorophore in the presence of RCAR1 and ABA . GFP lifetimes were determined after transient expression of the Arabidopsis proteins in leaves of Nicotiana benthamiana . Representative false colour images of GFP : SnRK2 lifetimes measured in leaf tissues expressing OST1 or SnRK2 . 4 in combination with mCherry : ABI1 and RCAR1 without exogenous ABA . The bar marks 20 µm and the nucleus ( n ) and cytosol ( c ) are indicated .

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

FRET-FLIM analysis of GFP:OST1 or GFP:SnRK2.4 and the effect of ABI1 fused to the mCherry fluorophore in the presence of RCAR1 and ABA. GFP lifetimes were determined after transient expression of the Arabidopsis proteins in leaves of Nicotiana benthamiana.   Representative false colour images of GFP:SnRK2 lifetimes measured in leaf tissues expressing OST1 or SnRK2.4 in combination with mCherry:ABI1 and RCAR1 without exogenous ABA. The bar marks 20 µm and the nucleus (n) and cytosol (c) are indicated.


Analysis of SnRK2 - ABI1 interaction as mentioned in ( A ) but in the presence of RCAR14 and different exogenous ABA levels . RCAR14 requires ABA for efficient displacement of SnRK2s from ABI1 . Representative GFP lifetime false colour image of the analysis shown in C ) 2h after 30 µM exogenous ABA administration . Details are as described in ( B ) .

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

Analysis of SnRK2-ABI1 interaction as mentioned in (A) but in the presence of RCAR14 and different exogenous ABA levels. RCAR14 requires ABA for efficient displacement of SnRK2s from ABI1.   Representative GFP lifetime false colour image of the analysis shown in C) 2h after 30 µM exogenous ABA administration. Details are as described in (B).


Analysis of SnRK2 - ABI1 interaction as mentioned in ( A ) but in the presence of RCAR14 and different exogenous ABA levels . RCAR14 requires ABA for efficient displacement of SnRK2s from ABI1 .

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 B-CONTROLLED_VAR O O O O O O O O

Analysis of SnRK2-ABI1 interaction as mentioned in (A) but in the presence of RCAR14 and different exogenous ABA levels. RCAR14 requires ABA for efficient displacement of SnRK2s from ABI1.


( A ) CD127 + and CD127 - cells from Lin - CD56 - population after gating on lymphoid , singlet , live , CD45 + cells of PBMCs . Lineage ( Lin ) markers include antibodies against : CD3 , CD4 , TCRαβ , TCRγδ , CD19 , CD20 , CD22 , CD34 , FcεRIα , CD11c , CD303 , CD123 , CD1a , and CD14 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) CD127+ and CD127- cells from Lin-CD56- population after gating on lymphoid, singlet, live, CD45+ cells of PBMCs. Lineage (Lin) markers include antibodies against: CD3, CD4, TCRαβ, TCRγδ, CD19, CD20, CD22, CD34, FcεRIα, CD11c, CD303, CD123, CD1a, and CD14.


( C ) Normalized counts of Lin - CD56 - CD127 + ( red ) and Lin - CD56 - CD127 - ( blue ) cells related genes from ( B ) by DESeq2 ( n = 5 ) .

O O O O O O O O O O O O O O O O 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) Normalized counts of Lin-CD56-CD127+ (red) and Lin-CD56-CD127- (blue) cells related genes from (B) by DESeq2 (n=5).


( A ) Heatmap of differentially expressed genes by RNA - Seq , sorted CD56hiNK , CD56dimNK , CD56 - NK cells and ILCs , from PBMCs of 4 donors ( log2 fold change > 1 , p < 0 . 01 determined by

O O O O O O O O O O O O O O O O O O O O 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) Heatmap of differentially expressed genes by RNA-Seq, sorted CD56hiNK, CD56dimNK, CD56-NK cells and ILCs, from PBMCs of 4 donors (log2 fold change >1, p<0.01 determined by DESeq2


( H ) ATAC - Seq analysis of sorted CD56hiNK , CD56dimNK , CD56 - NK cells and ILCs at indicated gene loci ( representative of two donors ) .

O O O O O O 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) ATAC-Seq analysis of sorted CD56hiNK, CD56dimNK, CD56-NK cells and ILCs at indicated gene loci (representative of two donors).


C ) Representative genes for NK cell and ILC clusters . All data were generated using blood from healthy HIV - 1 - negative donors .

O O 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 genes for NK cell and ILC clusters. All data were generated using blood from healthy HIV-1-negative donors.


( G ) the expression of genes that are enriched by NK cells and / or ILCs along the pseudotime trajectory . All data were generated using blood from healthy HIV - 1 - negative donors .

O O O O O O O O O O O O O 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) the expression of genes that are enriched by NK cells and/or ILCs along the pseudotime trajectory. All data were generated using blood from healthy HIV-1-negative donors.


( A ) PBMCs were stimulated with PMA and ionomycin for 3 hrs and then intracellular AREG was detected within the different subsets of NK cells and ILCs by flow cytometry .

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

(A) PBMCs were stimulated with PMA and ionomycin for 3 hrs and then intracellular AREG was detected within the different subsets of NK cells and ILCs by flow cytometry.


( B ) PBMCs were cultured in MACS NK medium at 37 ° C in 5 % CO2 , 50 ng / ml IL - 2 , 50 ng / ml IL - 15 , 10 ng / ml IL - 12 or 50 ng / ml IL - 18 for 5 days , then AREG , IFN - γ or TNF - α from different subsets of NK cells and ILCs were detected .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) PBMCs were cultured in MACS NK medium at 37 °C in 5% CO2, 50 ng/ml IL-2, 50 ng/ml IL-15, 10 ng/ml IL-12 or 50 ng/ml IL-18 for 5 days, then AREG, IFN-γ or TNF-α from different subsets of NK cells and ILCs were detected.


( F PBMCs were treated with CHIR99021 ( 10 uM ) in RPMI 1640 at 37 ° C in 5 % CO2 for 48 hrs , then stimulated with PMA + ionomycin . The percentage of AREG + NK cells ( F ) are shown . two - tailed paired t - test . All data were generated using blood from healthy HIV - 1 - negative donors .

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

(F PBMCs were treated with CHIR99021 (10 uM) in RPMI 1640 at 37 °C in 5% CO2 for 48 hrs, then stimulated with PMA+ionomycin. The percentage of AREG+NK cells (F) are shown. two-tailed paired t-test. All data were generated using blood from healthy HIV-1-negative donors.


G ) PBMCs were treated with CHIR99021 ( 10 uM ) in RPMI 1640 at 37 ° C in 5 % CO2 for 48 hrs , then stimulated with PMA + ionomycin . The percentage of ILCs ( G ) ( n = 15 ) are shown . two - tailed paired t - test . All data were generated using blood from healthy HIV - 1 - negative donors .

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

G) PBMCs were treated with CHIR99021 (10 uM) in RPMI 1640 at 37 °C in 5% CO2 for 48 hrs, then stimulated with PMA+ionomycin. The percentage of ILCs (G) (n=15) are shown. two-tailed paired t-test. All data were generated using blood from healthy HIV-1-negative donors.


( H ) As in ( F ) , PBMCs were additionally treated with CHIR99021 and TGF - β1 ( 50 ng / ml ) before AREG + NK cells were detected by flow cytometry ( n = 8 ) . two - tailed paired t - test . All data were generated using blood from healthy HIV - 1 - negative donors .

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

(H) As in (F), PBMCs were additionally treated with CHIR99021 and TGF-β1 (50 ng/ml) before AREG+NK cells were detected by flow cytometry (n=8). two-tailed paired t-test. All data were generated using blood from healthy HIV-1-negative donors.


( I ) AREG + NK cells from control or RUNX3 knockout groups were detected after IL - 12 + IL - 15 + IL - 18 stimulation in MACS NK medium at 37 ° C in 5 % CO2 for 16 hrs ( n = two - tailed paired t - test . All data were generated using blood from healthy HIV - 1 - negative donors .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) AREG+NK cells from control or RUNX3 knockout groups were detected after IL-12+IL-15+IL-18 stimulation in MACS NK medium at 37°C in 5% CO2 for 16 hrs (n= two-tailed paired t-test. All data were generated using blood from healthy HIV-1-negative donors.


( A PBMCs from HIV - 1 - ( n = 20 ) , HIV - 1 + viremic ( n = 20 ) , HIV - 1 + ART suppressed ( n = 19 ) , and HIV - 1 + spontaneous controllers ( n = 20 ) were stimulated with PMA and ionomycin in RPMI 1640 at 37 ° C in 5 % CO2 for 3 hrs . The percentage ( A ) of AREG + cells from are shown for the indicated populations . Mann - Whitney test . ns , not significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 PBMCs from HIV-1- (n=20), HIV-1+ viremic (n=20), HIV-1+ ART suppressed (n=19), and HIV-1+ spontaneous controllers (n=20) were stimulated with PMA and ionomycin in RPMI 1640 at 37 °C in 5% CO2 for 3 hrs. The percentage (A) of AREG+ cells from are shown for the indicated populations. Mann-Whitney test. ns, not significant.


( C - F ) Correlation of the percentage of AREG + cells in CD56 - ( C ) , CD56dim ( D ) , CD56hi ( E ) or total NK cells ( F ) , from ( A ) , with the percentage of ILCs . Spearman

O O O O O O O O O O O O O O O O O O O O O O O O O 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-F) Correlation of the percentage of AREG+ cells in CD56- (C), CD56dim (D), CD56hi (E) or total NK cells (F), from (A), with the percentage of ILCs. Spearman correlation


( A ) Percentage of ILCs among Lin - CD56 - cells from HIV - 1 - negative ( n = 40 ) , HIV - 1 + viremic ( n = 40 ) , HIV - 1 + ART suppressed ( n = 37 ) , and HIV - 1 + spontaneous controllers ( n = 40 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Percentage of ILCs among Lin-CD56- cells from HIV-1-negative (n=40), HIV-1+ viremic (n=40), HIV-1+ ART suppressed (n=37), and HIV-1+ spontaneous controllers (n=40).


( E ) Percentage of CD56 - NK cells among total NK cells from PBMCs and CD4 - PBMCs after culture in RPMI 1640 at 37 ° C in 5 % CO2 for 5 days ( n = 7 ) .

O O O O O O O O O O O O O O O O O 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) Percentage of CD56-NK cells among total NK cells from PBMCs and CD4-PBMCs after culture in RPMI 1640 at 37°C in 5% CO2 for 5 days (n=7).


( F ) Percentage of CD56 - NK cells bearing the indicated proteins by flow cytometry , after detection in fresh PBMCs or after 5 days of culturing CD4 + T cell - depleted PBMCs ( n = 4 ) .

(F) Percentage of CD56-NK cells bearing the indicated proteins by flow cytometry, after detection in fresh PBMCs or after 5 days of culturing CD4+T cell-depleted PBMCs (n=4).


( G ) CD4 + T cell number ( counts / mm3 ) from the indicated groups ( n = 40 for each group ) .

(G) CD4+T cell number (counts/mm3) from the indicated groups (n=40 for each group).


( A ) CD56dim and CD56 - NK cells were assessed by flow cytometry after the indicated cell populations were cultured for 5 days : total PBMCs ( n = 7 blood donors ) , PBMCs depleted of CD4 + T cells ( n = 7 blood donors ) , NK cells mixed with autologous CD4 + T cells ( n = 3 blood donors ) , and PBMCs depleted of CD4 + T cells but supplemented with IL - 2 ( n = 7 blood donors ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) CD56dim and CD56- NK cells were assessed by flow cytometry after the indicated cell populations were cultured for 5 days: total PBMCs (n=7 blood donors), PBMCs depleted of CD4+T cells (n=7 blood donors), NK cells mixed with autologous CD4+T cells (n=3 blood donors), and PBMCs depleted of CD4+T cells but supplemented with IL-2 (n=7 blood donors).


( B ) Percentage of CD56 - NK cells among total NK cells from CD4 - PBMCs cultured as in ( A ) , in the presence or absence of IL - 2 ( 10 ng / ml ) or IL - 2 neutralizing antibody ( 4 ug / ml ) ( n = 7 ) . ( C ) Percentage of CD56 - NK cells from PBMCs or CD4 - PBMCs cultured as in ( A ) in the presence or absence of IL - 2 ( 10 ng / ml ) , or IL - 15 ( 10 ng / ml ) ( n = 8 ) . ( D ) Percentage of CD56 - NK cells among total NK cells in PBMCs after culturing as in ( A ) with isotype control or IL - 2 neutralizing antibody ( 4 ug / ml ) ( n = 10 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Percentage of CD56-NK cells among total NK cells from CD4-PBMCs cultured as in (A), in the presence or absence of IL-2 (10 ng/ml) or IL-2 neutralizing antibody (4 ug/ml) (n=7). (C) Percentage of CD56-NK cells from PBMCs or CD4-PBMCs cultured as in (A) in the presence or absence of IL-2 (10 ng/ml), or IL-15 (10 ng/ml) (n=8). (D) Percentage of CD56-NK cells among total NK cells in PBMCs after culturing as in (A) with isotype control or IL-2 neutralizing antibody (4 ug/ml) (n=10).


( E , F ) Percentage of CD56 - NK ( E ) or IFN - γ + NK ( F ) cells among total NK cells after culturing PBMCs as in ( A ) with TGF - β1 ( 50 ng / ml ) or TGF - β1 ( 50 ng / ml ) + IL - 2 ( 20 ng / ml ) ( n = 8 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Percentage of CD56-NK (E) or IFN-γ+ NK (F) cells among total NK cells after culturing PBMCs as in (A) with TGF-β1 (50 ng/ml) or TGF-β1 (50 ng/ml) + IL-2 (20 ng/ml) (n=8).


( I , J ) NSG ( n = 15 ) or human IL - 15 transgenic NSG ( NSG - huIL - 15 ) mice ( n = 15 ) were reconstituted with human CD34 + hematopoietic stem cells for 6 weeks . Blood , spleen , and liver were harvested for detection of CD56 ( I ) or IFN - γ production after 16 hrs stimulation with IL - 12 , IL - 15 , and IL - 18 ( J ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, J) NSG (n=15) or human IL-15 transgenic NSG (NSG-huIL-15) mice (n=15) were reconstituted with human CD34+ hematopoietic stem cells for 6 weeks. Blood, spleen, and liver were harvested for detection of CD56 (I) or IFN-γ production after 16 hrs stimulation with IL-12, IL-15, and IL-18 (J).


( K - L ) Plasma concentration of IL - 2 ( HIV - 1 - ( n = 12 ) , viremic ( n = 12 ) , ART suppressed ( n = 13 ) , controllers ( n = 14 ) ) ( K ) , TGF - β1 ( HIV - 1 - ( n = 20 ) , viremic ( n = 19 ) , ART suppressed ( n = 20 ) , controllers ( n = 20 ) ) ( L ) and IL - 15 ( HIV - 1 - ( n = 19 ) , viremic ( n = 19 ) , ART suppressed ( n = 20 ) , controllers ( n = 20 ) ) ( M ) , detected by Luminex .

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

(K-L) Plasma concentration of IL-2 (HIV-1- (n=12), viremic (n=12), ART suppressed (n=13), controllers (n=14)) (K), TGF-β1 (HIV-1- (n=20), viremic (n=19), ART suppressed (n=20), controllers (n=20)) (L) and IL-15 (HIV-1- (n=19), viremic (n=19), ART suppressed (n=20), controllers (n=20)) (M), detected by Luminex.


( C ) Log2 fold change of mTOR signaling - related genes , showing cells stimulated with IL - 12 , IL - 15 , and IL - 18 versus unstimulated CD56 - or CD56dim NK cells . ns , not significant . All data were generated using blood from healthy HIV - 1 - negative donors .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Log2 fold change of mTOR signaling-related genes, showing cells stimulated with IL-12, IL-15, and IL-18 versus unstimulated CD56- or CD56dim NK cells. ns, not significant. All data were generated using blood from healthy HIV-1-negative donors.


( A ) PBMCs were cultured at 37 ° C in 5 % CO2 with or without IL - 2 ( 50 ng / ml ) for 5 days , the phosphorylation of mTOR on Ser2448 , AKT on Ser473 , 4EBP1 on Thr36 and Thr45 , and S6 on Ser235 and Ser236 , and the surface CD71 from NK cells ( Lin - TBX21 + ) were detected by flow cytometry ( n = 8 ) . ( B ) As in ( A ) , PBMCs or CD4 - PBMCs were cultured for 5 days , the indicated targets from NK cells were detected ( n = 8 ) . ( C ) As in ( A ) , PBMCs were cultured with or without IL - 15 ( 50 ng / ml ) for 5 days , the indicated targets were detected ( n = 8 ) . All data were generated using blood from healthy HIV - 1 - negative donors .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) PBMCs were cultured at 37 °C in 5% CO2 with or without IL-2 (50 ng/ml) for 5 days, the phosphorylation of mTOR on Ser2448, AKT on Ser473, 4EBP1 on Thr36 and Thr45, and S6 on Ser235 and Ser236, and the surface CD71 from NK cells (Lin-TBX21+) were detected by flow cytometry (n=8). (B) As in (A), PBMCs or CD4-PBMCs were cultured for 5 days, the indicated targets from NK cells were detected (n=8). (C) As in (A), PBMCs were cultured with or without IL-15 (50 ng/ml) for 5 days, the indicated targets were detected (n=8). All data were generated using blood from healthy HIV-1-negative donors.


( E , F ) PBMCs or CD4 - PBMCs were cultured in the presence or absence of rapamycin ( 10 nM ) ( F , n = 8 ) or Torin 1 ( 250 nM ) ( G , n = 8 ) , with or without IL - 2 ( 10 ng / ml ) or IL - 15 ( 10 ng / ml ) , for 5 days . IFN - γ was detected in Lin - TBX21 + cells . All data were generated using blood from healthy HIV - 1 - negative donors .

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 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 O

(E, F) PBMCs or CD4-PBMCs were cultured in the presence or absence of rapamycin (10 nM) (F, n=8) or Torin 1 (250 nM) (G, n=8), with or without IL-2 (10 ng/ml) or IL-15 (10 ng/ml), for 5 days. IFN-γ was detected in Lin-TBX21+ cells. All data were generated using blood from healthy HIV-1-negative donors.


( G , H ) PBMCs were cultured in the presence or absence of rapamycin ( 10 nM ) ( F ) or Torin 1 ( 250 nM ) combined with or without IL - 2 ( 10 ng / ml ) ( G , n = 8 ) or IL - 15 ( 10 ng / ml ) ( H , n = 8 ) for 5 days . The indicated targets from the Lin - TBX21 + population were detected as in ( A ) . All data were generated using blood from healthy HIV - 1 - negative donors .

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

(G, H) PBMCs were cultured in the presence or absence of rapamycin (10 nM) (F) or Torin 1 (250 nM) combined with or without IL-2 (10 ng/ml) (G, n=8) or IL-15 (10 ng/ml) (H, n=8) for 5 days. The indicated targets from the Lin-TBX21+ population were detected as in (A). All data were generated using blood from healthy HIV-1-negative donors.


A ) Overview of immunofluorescence staining for centrosomes ( TUBG1 ) and cilia ( ARL13B ) in control ( Con ) ( left ) and EPZ treated sections ( right ) . Dotted lines mark either ventricular ( bottom part ) or pial ( top part ) surface of the tissue . Data information : Scale bars in all panels : 10 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 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

A) Overview of immunofluorescence staining for centrosomes (TUBG1) and cilia (ARL13B) in control (Con) (left) and EPZ treated sections (right). Dotted lines mark either ventricular (bottom part) or pial (top part) surface of the tissue . Data information: Scale bars in all panels: 10 µm.