Figure 4 ( A ) Survival of Drosophila S2 cells treated with preheating , 100 µM NAT , and 100 µM NAT with 10 mM 2 - aminobicyclo - ( 2 , 2 , 1 ) - heptane - 2 - carboxylic acid ( BCH ) after heat stress at 42oC for 60 min ( data are means ± SEM ; n = 10 ) . After pretreatment with chemicals for 12 h , those were removed by changing the medium . * P < 0 . 05 , * * P < 0 . 01 vs . PBS . ( B ) Survival of Drosophila S2 cells pretreated with 100 µM NAT , 100 µM NAT with 30 µg / ml antimycin A , and preheating with or without 30 µg / ml antimycin A 48 h after heat stress at 42oC for 60 min ( data are means ± SEM ; n = 10 ) . After pretreatment with chemicals for 12 h , they were removed by changing the medium . * P < 0 . 05 vs . PBS . ( C ) Effects of 100 µM NAT on MitoRed localization in mitochondria of S2 cells . Upper : representative images of time - dependent changes in MitoRed localization in mitochondria after adding NAT . Scale bar : 20 µm . Lower : relative intensities of MitoRed fluorescent signals in mitochondria were quantified by microplate reader . Different letters above bars represent significant differences [ P < 0 . 05 ( data are means ± SEM , n = 12 ) ] . ( D ) ROS concentrations in S2 cells after adding 100 µM NAT ( data are means ± SEM ; n = 12 ) . * P < 0 . 05 vs . zero time . ( F ) Effects of dsRNA targeting of FoxO on NAT - dependent expression of Keap1 ( data are means ± SEM ; n = 12 ) . * P < 0 . 05 , * * P < 0 . 01 vs . without NAT . Note that NAT feeding did not increase Keap1 expression in FoxO knockdown flies . ( G ) Effect of NAT on growth of HCT116 colon cancer cells in mice ( BALB / cSlc - nu / nu , 6 wks old , female ) ( data are means ± SEM ; n = 5 ) . * P < 0 . 05 , * * P < 0 . 01 vs . without NAT . Test mice began to freely drink 5 mg / ml NAT solution for 7 d before transplantation of the tumor cells on day 0 : the average amount of NAT consumed by each mouse was about 8 . 8 mg / day ( 0 . 4 - 0 . 5 g / kg weight / day ) . HCT116 cells ( 5 . 0 x 106 cells ) were transplanted into each test mouse using a 1 ml syringe with a 27G needle .

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

Figure 4(A) Survival of Drosophila S2 cells treated with preheating, 100 µM NAT, and 100 µM NAT with 10 mM 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH) after heat stress at 42oC for 60 min (data are means ± SEM; n=10). After pretreatment with chemicals for 12 h, those were removed by changing the medium. *P<0.05, **P<0.01 vs. PBS.(B) Survival of Drosophila S2 cells pretreated with 100 µM NAT, 100 µM NAT with 30 µg/ml antimycin A, and preheating with or without 30 µg/ml antimycin A 48 h after heat stress at 42oC for 60 min (data are means ± SEM; n=10). After pretreatment with chemicals for 12 h, they were removed by changing the medium. *P<0.05 vs. PBS.(C) Effects of 100 µM NAT on MitoRed localization in mitochondria of S2 cells. Upper: representative images of time-dependent changes in MitoRed localization in mitochondria after adding NAT. Scale bar: 20 µm. Lower: relative intensities of MitoRed fluorescent signals in mitochondria were quantified by microplate reader. Different letters above bars represent significant differences [P <0.05 (data are means ± SEM, n=12)].(D) ROS concentrations in S2 cells after adding 100 µM NAT (data are means ± SEM; n=12). *P<0.05 vs. zero time.(F) Effects of dsRNA targeting of FoxO on NAT-dependent expression of Keap1 (data are means ± SEM; n=12). *P<0.05, **P<0.01 vs. without NAT. Note that NAT feeding did not increase Keap1 expression in FoxO knockdown flies.(G) Effect of NAT on growth of HCT116 colon cancer cells in mice (BALB/cSlc-nu/nu, 6 wks old, female) (data are means ± SEM; n=5). *P<0.05, **P<0.01 vs. without NAT. Test mice began to freely drink 5 mg/ml NAT solution for 7 d before transplantation of the tumor cells on day 0: the average amount of NAT consumed by each mouse was about 8.8 mg/day (0.4 - 0.5 g/kg weight/day). HCT116 cells (5.0 x 106 cells) were transplanted into each test mouse using a 1 ml syringe with a 27G needle.


Figure 1A Levels of ULK1 in MEFs exposed to hypoxia ( 1 % O2 ) for the indicated times . actin , Western blotB Quantification of the ratio of ULK1 to actin in ( A ) . C Levels of ULK1 in MEFs treated with FCCP ( 20 μM ) for the indicated times . actinD Quantification of the ratio of ULK1 to actin in ( C ) . E MEFs were exposed to hypoxia ( 1 % O2 ) for 12 h or FCCP ( 20 μM ) for 6 h and then fixed by 4 % paraformaldehyde . Cells were stained with anti ‐ TIM23 ( mouse ) and anti ‐ ULK1 ( rabbit ) primary antibodies , then Alexa Fluor ‐ 488 ‐ labeled donkey anti ‐ rabbit IgG and Alexa Fluor ‐ 555 ‐ labeled donkey anti ‐ mouse IgG secondary antibodies before analysis by immunofluorescence microscopy . Scale bar , 10 μm . F Immunoblots of subcellular fractions from control MEFs or MEFs that were exposed to hypoxia ( 1 % O2 ) for 12 h . PNS , post ‐ nuclear supernatant ; Cyto , cytosol ; Mito , mitochondria . ULK1G Immunoblots of subcellular fractions from control MEFs or MEFs that were treated with FCCP ( 20 μM ) for 6 h . PNS , post ‐ nuclear supernatant ; Cyto , cytosol ; Mito , mitochondria .

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Figure 1A Levels of ULK1 in MEFs exposed to hypoxia (1% O2) for the indicated times.actin, Western blotB Quantification of the ratio of ULK1 to actin in (A).C Levels of ULK1 in MEFs treated with FCCP (20 μM) for the indicated times.actinD Quantification of the ratio of ULK1 to actin in (C).E MEFs were exposed to hypoxia (1% O2) for 12 h or FCCP (20 μM) for 6 h and then fixed by 4% paraformaldehyde. Cells were stained with anti‐TIM23 (mouse) and anti‐ULK1 (rabbit) primary antibodies, then Alexa Fluor‐488‐labeled donkey anti‐rabbit IgG and Alexa Fluor‐555‐labeled donkey anti‐mouse IgG secondary antibodies before analysis by immunofluorescence microscopy. Scale bar, 10 μm.F Immunoblots of subcellular fractions from control MEFs or MEFs that were exposed to hypoxia (1% O2) for 12 h. PNS, post‐nuclear supernatant; Cyto, cytosol; Mito, mitochondria.ULK1G Immunoblots of subcellular fractions from control MEFs or MEFs that were treated with FCCP (20 μM) for 6 h. PNS, post‐nuclear supernatant; Cyto, cytosol; Mito, mitochondria.ULK1


Figure 2A HeLa cells were co − transfected with FUNDC1 − Myc and FLAG − ULK1 . Cells were lysed and immunoprecipitated with anti − Myc antibody 24 h after transfection . B FLAG − FUNDC1 was co − transfected with GFP − vector or GFP − ULK1 for 24 h in HeLa cells , and then , cell lysates were immunoprecipitated with anti − GFP antibody . CMEFs were exposed to hypoxia ( 1 % O2 ) for 12 h or FCCP ( 20 μM ) for 3 h , followed by immunoprecipitation using anti ‐ ULK1 antibody . D The expression of FUNDC1 is induced by 10 ng / ml tetracycline ( Tet ) in FUNDC1 ‐ inducible HeLa cells . Cells were transfected with HA ‐ ULK1 or HA ‐ ULK1 ( K46N ) for 24 h . Then , the cell lysates were prepared for immunoblotting using indicated antibodies . EULK1 + / + cells were transfected with FUNDC1 ‐ Myc , and ULK1 - / - cells were transfected with FUNDC1 ‐ Myc alone , FUNDC1 ‐ Myc and HA ‐ ULK1 , or FUNDC1 ‐ Myc and HA ‐ ULK1 ( K46N ) . Cells were harvested and lysed for immunoblots24 h after transfection . F Purified GST ‐ FUNDC1 and GST ‐ FUNDC1 ( S17A ) were subjected to an in vitro kinase assay with Myc ‐ ULK1 immunoprecipitated by anti ‐ Myc antibody from Myc ‐ ULK1 ‐ transfected cells about 24 h post ‐ transfection . Phosphorylated FUNDC1 was detected by anti ‐ p ‐ S17 ‐ FUNDC1 antibody . The red asterisks mark the target bands . G Western blotULK1 , FUNDC1 , and the phosphorylated FUNDC1 in MEFs exposed to hypoxia ( 1 % O2 ) for the indicated times . HWestern blot analysis of the kinetics of ULK1 , FUNDC1 , and the phosphorylated in MEFs treated with FCCP ( 20 μM ) for the indicated times . actinI , J The single mutant which can abolish the ULK1 and FUNDC1 interaction was identified . HeLa cells were co ‐ transfected with Flag ‐ ULK1 and the indicated constructs . 24 h after transfection , cells were lysed for immunoprecipitation by anti ‐ Flag antibody . KMEFs were transfected with FUNDC1 ‐ Myc and its mutants after the endogenous FUNDC1 was knocked down by siRNA . 36 h after transfection , cells were harvested in the absence or presence of 50 nM bafilomycin A1 ( BAF1 ) and then lysed for immunoblotting with the indicated antibodies . L MEFs were co ‐ transfected with FUNDC1 ‐ Myc or its mutants and MitoDsred after the endogenous FUNDC1 was knocked down by siRNA . 24 h post ‐ transfection , cells were treated with hypoxic ( 1 % O2 ) conditions for 12 h or FCCP ( 20 μM ) for 6 h before fixed by 4 % paraformaldehyde and stained with indicated antibodies . Scale bar , 10 μm .

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Figure 2A HeLa cells were co−transfected with FUNDC1−Myc and FLAG−ULK1. Cells were lysed and immunoprecipitated with anti−Myc antibody 24 h after transfection.B FLAG−FUNDC1 was co−transfected with GFP−vector or GFP−ULK1 for 24 h in HeLa cells, and then, cell lysates were immunoprecipitated with anti−GFP antibody.CMEFs were exposed to hypoxia (1% O2) for 12 h or FCCP (20 μM) for 3 h, followed by immunoprecipitation using anti‐ULK1 antibody.D The expression of FUNDC1 is induced by 10 ng/ml tetracycline (Tet) in FUNDC1‐inducible HeLa cells. Cells were transfected with HA‐ULK1 or HA‐ULK1 (K46N) for 24 h. Then, the cell lysates were prepared for immunoblotting using indicated antibodies.EULK1+/+ cells were transfected with FUNDC1‐Myc, and ULK1-/- cells were transfected with FUNDC1‐Myc alone, FUNDC1‐Myc and HA‐ULK1, or FUNDC1‐Myc and HA‐ULK1 (K46N). Cells were harvested and lysed for immunoblots24 h after transfection.F Purified GST‐FUNDC1 and GST‐FUNDC1 (S17A) were subjected to an in vitro kinase assay with Myc‐ULK1 immunoprecipitated by anti‐Myc antibody from Myc‐ULK1‐transfected cells about 24 h post‐transfection. Phosphorylated FUNDC1 was detected by anti‐p‐S17‐FUNDC1 antibody. The red asterisks mark the target bands.G Western blotULK1, FUNDC1, and the phosphorylated FUNDC1 in MEFs exposed to hypoxia (1% O2) for the indicated times.HWestern blot analysis of the kinetics of ULK1, FUNDC1, and the phosphorylated in MEFs treated with FCCP(20 μM) for the indicated times.actinI, J The single mutant which can abolish the ULK1 and FUNDC1 interaction was identified. HeLa cells were co‐transfected with Flag‐ULK1 and the indicated constructs. 24 h after transfection, cells were lysed for immunoprecipitation by anti‐Flag antibody.KMEFs were transfected with FUNDC1‐Myc and its mutants after the endogenous FUNDC1 was knocked down by siRNA. 36 h after transfection, cells were harvested in the absence or presence of 50 nM bafilomycin A1 (BAF1) and then lysed for immunoblotting with the indicated antibodies.L MEFs were co‐transfected with FUNDC1‐Myc or its mutants and MitoDsred after the endogenous FUNDC1 was knocked down by siRNA. 24 h post‐transfection, cells were treated with hypoxic (1% O2) conditions for 12 h or FCCP (20 μM) for 6 h before fixed by 4% paraformaldehyde and stained with indicated antibodies. Scale bar, 10 μm.


Figure 3AULK1 + / + and ULK1 − / − cells were cultured under hypoxic ( 1 % O2 ) condition for 12 h or 24 h in the absence or presence of 50 nM bafilomycin A1 ( BAF1 ) . Cell lysates were immunoblotted . B ULK1 + / + and ULK1 − / − cells were treated with FCCP ( 20 μM ) for the indicated times in the absence or presence of 50 nM bafilomycin A1 ( BAF1 ) . Cell lysates were immunoblotted . C ULK1 + / + and ULK1 − / − cells were transfected with FUNDC1 − Myc for 24 h and then harvested in the absence or presence of 50 nM bafilomycin A1 ( BAF1 ) . Cell lysates were immunoblotted . D ULK1 − / − cells were transfected with HA ‐ ULK1 or HA ‐ ULK1 ( K46N ) , then cultured under hypoxic ( 1 % O2 ) conditions for 24 h in the absence or presence of 50 nM bafilomycin A1 ( BAF1 ) . Cells lysates were immunoblotted . EULK1 − / − cells were transfected with HA − ULK1 or HA − ULK1 and then treated with FCCP ( 20 μM ) for 24 h . Cells were harvested in the absence or presence of 50 nM bafilomycin A1 ( BAF1 ) . Cell lysates were immunoblotted . F FUNDC1 ‐ knockdown MEFs were transfected with FUNDC1 ‐ Myc or FUNDC1 - Myc ( S17A ) for the indicated times . Cell lysates were used for immunoblotting . G ULK1 + / + cells were transfected with FUNDC1 ‐ Myc , and ULK1 − / − cells were transfected with FUNDC1 ‐ Myc or FUNDC1 - Myc ( S17D ) . 24 h after transfection , cells were fixed with 4 % paraformaldehyde and stained with anti ‐ Myc ( mouse ) and anti ‐ LC3 ( rabbit ) primary antibodies for immunofluorescence microscopy . Scale bar , 10 μm . H ULK1 − / − cells were transfected with FUNDC1 ‐ Myc or FUNDC1 - Myc ( S17D ) for 24 h in the absence or presence of 50 nm bafilomycin A1 ( BAF1 ) for an additional 6 h before harvesting . Cell lysates were immunoblotted . I ULK1 − / − cells were transfected with FUNDC1 ‐ Myc or FUNDC1 - Myc ( S17D ) for 24 h in the absence or presence of 50 nm bafilomycin A1 ( BAF1 ) for an additional 6 h before harvesting . Cell lysates were detected with the citrate synthase activity kit . J HeLa cells were transfected with FUNDC1 ‐ Myc , FUNDC1 - Myc ( S17A ) , or FUNDC1 - Myc ( S17D ) . 24 h after transfection , cell lysates were immunoprecipitated with anti ‐ LC3 antibody .

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Figure 3AULK1+/+ and ULK1−/− cells were cultured under hypoxic (1% O2) condition for 12 h or 24 h in the absence or presence of 50 nM bafilomycin A1 (BAF1). Cell lysates were immunoblotted.B ULK1+/+ and ULK1−/− cells were treated with FCCP (20 μM) for the indicated times in the absence or presence of 50 nM bafilomycin A1 (BAF1). Cell lysates were immunoblotted.C ULK1+/+ and ULK1−/− cells were transfected with FUNDC1−Myc for 24 h and then harvested in the absence or presence of 50 nM bafilomycin A1 (BAF1). Cell lysates were immunoblotted.D ULK1−/− cells were transfected with HA‐ULK1 or HA‐ULK1 (K46N), then cultured under hypoxic (1% O2) conditions for 24 h in the absence or presence of 50 nM bafilomycin A1 (BAF1). Cells lysates were immunoblotted.EULK1−/− cells were transfected with HA−ULK1 or HA−ULK1 and then treated with FCCP (20 μM) for 24 h. Cells were harvested in the absence or presence of 50 nM bafilomycin A1 (BAF1). Cell lysates were immunoblotted.F FUNDC1‐knockdown MEFs were transfected with FUNDC1‐Myc or FUNDC1-Myc (S17A) for the indicated times. Cell lysates were used for immunoblotting.G ULK1+/+ cells were transfected with FUNDC1‐Myc, and ULK1−/− cells were transfected with FUNDC1‐Myc or FUNDC1-Myc (S17D). 24 h after transfection, cells were fixed with 4% paraformaldehyde and stained with anti‐Myc (mouse) and anti‐LC3 (rabbit) primary antibodies for immunofluorescence microscopy. Scale bar, 10 μm.H ULK1−/− cells were transfected with FUNDC1‐Myc or FUNDC1-Myc (S17D) for 24 h in the absence or presence of 50 nm bafilomycin A1 (BAF1) for an additional 6 h before harvesting. Cell lysates were immunoblotted.I ULK1−/− cells were transfected with FUNDC1‐Myc or FUNDC1-Myc (S17D) for 24 h in the absence or presence of 50 nm bafilomycin A1 (BAF1) for an additional 6 h before harvesting. Cell lysates were detected with the citrate synthase activity kit.J HeLa cells were transfected with FUNDC1‐Myc, FUNDC1-Myc (S17A), or FUNDC1-Myc (S17D). 24 h after transfection, cell lysates were immunoprecipitated with anti‐LC3 antibody.


Figure 4A ULK1 + / + or ULK1 − / − cells were cultured under hypoxic ( 1 % O2 ) or normoxic conditions in the absence or presence of 50 nM bafilomycin A1 ( BAF1 ) . ULK1 − / − cells were transfected with HA ‐ ULK1 , and ULK1 − / − FUNDC1 ‐ KD cells were transfected with HA ‐ ULK1 and FUNDC1 ‐ Myc , then cultured under hypoxic ( 1 % O2 ) conditions for 24 h in the absence or presence of 50 nM bafilomycin A1 ( BAF1 ) . Cells were harvested and lysed for immunoblotting assays . B ULK1 + / + or ULK1 − / − cells were treated with or without FCCP ( 20 μM ) in the absence or presence of 50 nM bafilomycin A1 ( BAF1 ) . ULK1 − / − cells were transfected with HA ‐ ULK1 and ULK1 − / − FUNDC1 ‐ KD cells were transfected with HA ‐ ULK1 and FUNDC1 ‐ Myc and then treated with FCCP ( 20 μM ) for 24 h in the absence or presence of 50 nM bafilomycin A1 ( BAF1 ) . Cells were harvested and lysed for immunoblotting assays . C ULK1 + / + , ULK1 − / − FUNDC1 ‐ KD , or ULK1 − / − FUNDC1 ‐ KD cells rescued by introduction of HA ‐ ULK1 and FUNDC1 ‐ Myc were cultured under hypoxic ( 1 % O2 ) conditions for 24 h or with FCCP ( 20 μM ) for 24 h . Samples were analyzed by electron microscopy . Scale bar , 2 μm . The yellow asterisks mark mitochondria . The red arrows indicate double ‐ membraned autophagic structures . The yellow arrow denotes autolysosomes . D - E The mitochondrial coverage and mitochondrial − containing AVs shown in ( C ) were quantified . Recon , reconstitution .

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

Figure 4A ULK1+/+ or ULK1−/− cells were cultured under hypoxic (1% O2) or normoxic conditions in the absence or presence of 50 nM bafilomycin A1 (BAF1). ULK1−/− cells were transfected with HA‐ULK1, and ULK1−/− FUNDC1‐KD cells were transfected with HA‐ULK1 and FUNDC1‐Myc, then cultured under hypoxic (1% O2) conditions for 24 h in the absence or presence of 50 nM bafilomycin A1 (BAF1). Cells were harvested and lysed for immunoblotting assays.B ULK1+/+ or ULK1−/− cells were treated with or without FCCP (20 μM) in the absence or presence of 50 nM bafilomycin A1 (BAF1). ULK1−/− cells were transfected with HA‐ULK1 and ULK1−/− FUNDC1‐KD cells were transfected with HA‐ULK1 and FUNDC1‐Myc and then treated with FCCP (20 μM) for 24 h in the absence or presence of 50 nM bafilomycin A1 (BAF1). Cells were harvested and lysed for immunoblotting assays.C ULK1+/+, ULK1−/− FUNDC1‐KD, or ULK1−/− FUNDC1‐KD cells rescued by introduction of HA‐ULK1 and FUNDC1‐Myc were cultured under hypoxic (1% O2) conditions for 24 h or with FCCP (20 μM) for 24 h. Samples were analyzed by electron microscopy. Scale bar, 2 μm. The yellow asterisks mark mitochondria. The red arrows indicate double‐membraned autophagic structures. The yellow arrow denotes autolysosomes.D-E The mitochondrial coverage and mitochondrial−containing AVs shown in (C) were quantified. Recon, reconstitution.


Figure 1B . Representative immunoblot of transfected missense ROS1 TKD mutations in transfected HEK - 293A lysates . C . Densitometry analysis of immunoblots ( N = 3 , biological replicates ) as represented in ( B ) . Fold - change in relative ROS1 phosphorylation is calculated by first calculating the ratio of phospho - ROS1 to total ROS1 protein for each variant and then normalizing to ROS1WT . One - way ANOVA and Sidak ' s test were used to control for multiple comparisons in C Error bars in figure represent mean ± SEM . D . Representative immunoblot of ROS1 D2113 position substitutions ( E , G , Y , and Q as indicated ) in HEK - 293A cells . E . Densitometry of immunoblots ( N = 3 , biological replicates ) as represented in ( D ) . pROS1 - phospho - ROS1 , tROS1 - total - ROS1 . One - way ANOVA and Sidak ' s test were used to control for multiple comparisons in E . Error bars in figure represent mean ± SEM .

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Figure 1B. Representative immunoblot of transfected missense ROS1 TKD mutations in transfected HEK-293A lysates.C. Densitometry analysis of immunoblots (N = 3, biological replicates) as represented in (B). Fold-change in relative ROS1 phosphorylation is calculated by first calculating the ratio of phospho-ROS1 to total ROS1 protein for each variant and then normalizing to ROS1WT. One-way ANOVA and Sidak's test were used to control for multiple comparisons in C Error bars in figure represent mean ± SEM.D. Representative immunoblot of ROS1 D2113 position substitutions (E, G, Y, and Q as indicated) in HEK-293A cells. E. Densitometry of immunoblots (N = 3, biological replicates) as represented in (D). pROS1- phospho-ROS1, tROS1- total-ROS1. One-way ANOVA and Sidak's test were used to control for multiple comparisons in E. Error bars in figure represent mean ± SEM.


Figure 2A . Immunoblot analysis of phospho - ROS1 and total ROS1 protein expression of NIH - 3T3 cells stably transduced with empty vector ( EV ) or indicated ROS1 TKD mutantsC . Soft - agar assay of NIH - 3T3 ROS1D2113N compared with NIH - 3T3 SLC - ROS1 fusion cells ( positive control ) or ROS1K1980E ( kinase - dead , negative control ) . + / - lorlatinib treated samples were compared for significance using two - way RM ANOVA ( matching across rows , N = 3 , biological replicates ) with Šídák ' s multiple comparisons test ( P values are indicated in figure ) D . Immunoblot analysis of phospho - ROS1 and total ROS1 protein expression in MCF10A cells stably transduced with indicated ROS1 variantsCell proliferation data showing effects of treatment with DMSO ( Vehicle ) , crizotinib ( 250nM ) , lorlatinib ( 250 nM ) , and NVL - 520 ( 250 nM ) in MCF10A ROS1 wildtype ( WT ) ( G ) , D2113N ( H ) , D2113G ( I ) and SLC34A2 - ROS1 ( SLC - ROS1 ) ( J ) as indicated . Ordinary One - way ANOVA with Dunnett ' s multiple comparisons test was used to determine significant differences . N = 6 wells , biological replicates ( H - I ) . Error bars in figure represent mean ± SEM .

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Figure 2A. Immunoblot analysis of phospho-ROS1 and total ROS1 protein expression of NIH-3T3 cells stably transduced with empty vector (EV) or indicated ROS1 TKD mutantsC. Soft-agar assay of NIH-3T3 ROS1D2113N compared with NIH-3T3 SLC-ROS1 fusion cells (positive control) or ROS1K1980E (kinase-dead, negative control). +/- lorlatinib treated samples were compared for significance using two-way RM ANOVA (matching across rows, N = 3, biological replicates) with Šídák's multiple comparisons test (P values are indicated in figure)D. Immunoblot analysis of phospho-ROS1 and total ROS1 protein expression in MCF10A cells stably transduced with indicated ROS1 variantsCell proliferation data showing effects of treatment with DMSO (Vehicle), crizotinib (250nM), lorlatinib (250 nM), and NVL-520 (250 nM) in MCF10A ROS1 wildtype (WT) (G), D2113N (H), D2113G (I) and SLC34A2-ROS1 (SLC-ROS1) (J) as indicated. Ordinary One-way ANOVA with Dunnett's multiple comparisons test was used to determine significant differences. N = 6 wells, biological replicates (H-I). Error bars in figure represent mean ± SEM.


Figure 4A - B . Volcano plots of statistical discoveries from IMAC - enriched ( A ) and phospho - tyrosine - enriched ( B ) phosphoproteomic spectral counts comparing ROS1D2113N data to ROS1WT . Select phosphosites linked to indicated cellular functions are annotated within the graph . Unpaired t tests of log2 transformed counts from ROS1WT and ROS1D2113N cells were conducted using Graphpad Prism with variance assumption of individual variance for each role ( N = 3 , biological replicates ) . Multiple Comparison tests used False Discovery Rate ( FDR ) with two - stage step - up ( Benjamini , Krieger , and Yekutieli ) with desired FDR of 5 % . X axis features log2 - transformed ratio of normalized intensities of ROS1D2113N relative to ROS1WT while y axis features corresponding log10 ( q ) values .

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Figure 4A-B. Volcano plots of statistical discoveries from IMAC-enriched (A) and phospho-tyrosine-enriched (B) phosphoproteomic spectral counts comparing ROS1D2113N data to ROS1WT. Select phosphosites linked to indicated cellular functions are annotated within the graph. Unpaired t tests of log2 transformed counts from ROS1WT and ROS1D2113N cells were conducted using Graphpad Prism with variance assumption of individual variance for each role (N=3, biological replicates). Multiple Comparison tests used False Discovery Rate (FDR) with two-stage step-up (Benjamini, Krieger, and Yekutieli) with desired FDR of 5%. X axis features log2-transformed ratio of normalized intensities of ROS1D2113N relative to ROS1WT while y axis features corresponding log10(q) values.


Figure 5A . Immunoblotting was performed on cell lysates generated via transient transfection of the following plasmids : pCX4 ROS1 wildtype ( WT ) , ROS1D2113G ( D2113G ) , ROS1D2113N ( D2113N ) and the ROS1 fusions , CD74 - ROS1 , EZR - ROS1 and SLC34A2 - ROS1 ( SLC - ROS1 ) . The targets interrogated are indicated in the labels on the left side of the image panels and the catalog numbers as well as dilution for antibodies are reported in Methods . B . Immunoblot densitometry graphs to quantitatively compare activation of ROS1 , SHP2 , ERK1 / 2 and STAT3 ( N = 2 , biological replicates ) . Fusions are listed only by fusion partner : CD74 - ROS1 ( CD74 ) , EZR - ROS1 ( EZR ) , SLC34A2 - ROS1 ( SLC ) . C . Immunoblotting was performed on cell lysates generated from stable HEK293A ROS1D2113N , HEK293A CD74 - ROS1 and NIH ROS1D2113N and NIH3T3 CD74 - ROS1 after treatment with ROS1 TKI . HEK293A stable cell lines were treated with DMSO , 100 nM entrectinib and 100 nM lorlatinib , and NIH3T3 stable cell lines were treated with 25 nM and 100 nM lorlatinib . The targets interrogated are indicated in the labels on the left side of the image panels in HEK293 images and on the right side of image panels in NIH3T3 images . D . Immunoblot densitometry graphs to quantitatively compare extent of inhibition ROS1 , SHP2 , ERK1 / 2 and STAT3 and AKT1 phosphorylation from two independent replicate experiments . Pixel density of phosphorylated protein signal was divded by total protein signal , and this ratio was internally normalized to DMSO ( Vehicle ) treated cell ratio . Note that only the 100 nM lorlatinib condition that was common to the HEK293A and NIH3T3 experiments is depicted in graphs .

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Figure 5A. Immunoblotting was performed on cell lysates generated via transient transfection of the following plasmids: pCX4 ROS1 wildtype (WT), ROS1D2113G (D2113G), ROS1D2113N (D2113N) and the ROS1 fusions, CD74-ROS1, EZR-ROS1 and SLC34A2-ROS1 (SLC-ROS1). The targets interrogated are indicated in the labels on the left side of the image panels and the catalog numbers as well as dilution for antibodies are reported in Methods. B. Immunoblot densitometry graphs to quantitatively compare activation of ROS1, SHP2, ERK1/2 and STAT3 (N = 2, biological replicates). Fusions are listed only by fusion partner: CD74-ROS1 (CD74), EZR-ROS1 (EZR), SLC34A2-ROS1 (SLC).C. Immunoblotting was performed on cell lysates generated from stable HEK293A ROS1D2113N, HEK293A CD74-ROS1 and NIH ROS1D2113N and NIH3T3 CD74-ROS1 after treatment with ROS1 TKI. HEK293A stable cell lines were treated with DMSO, 100 nM entrectinib and 100 nM lorlatinib, and NIH3T3 stable cell lines were treated with 25 nM and 100 nM lorlatinib. The targets interrogated are indicated in the labels on the left side of the image panels in HEK293 images and on the right side of image panels in NIH3T3 images. D. Immunoblot densitometry graphs to quantitatively compare extent of inhibition ROS1, SHP2, ERK1/2 and STAT3 and AKT1 phosphorylation from two independent replicate experiments. Pixel density of phosphorylated protein signal was divded by total protein signal, and this ratio was internally normalized to DMSO (Vehicle) treated cell ratio. Note that only the 100 nM lorlatinib condition that was common to the HEK293A and NIH3T3 experiments is depicted in graphs.


Figure 6A - C . Cell proliferation effects after treatment with SHP2 inhibitors ( TNO155 , RMC4550 ) , MEK1 / 2 inhibitors ( selumetinib , trametinib ) , and JNK1 / 2 / 3 inhibitor , SP600125 as indicated ( N = 6 , biological replicates ) . D - F . Cell proliferation effects after treatment with AKT1 / 2 / 3 inhibitor afuresertib , and MTOR1 inhibitor , everolimus , as indicated ( N = 6 , biological replicates ) . G - I . Cell proliferation effects after treatment with JAK1 / 2 inhibitor ruxolitinib , and TGFBR1 inhibitor galunisertib ( N = 6 , biological replicates ) . All inhibitors were used at 250 nM final concentration , with the exception of galunisertib , which was used at 500 nM final concentration . Note that the DMSO data are the same in for each cell line when compared to different class of inhibitors . Error bars in figure represent mean ± SEM .

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Figure 6A-C. Cell proliferation effects after treatment with SHP2 inhibitors (TNO155, RMC4550), MEK1/2 inhibitors (selumetinib, trametinib), and JNK1/2/3 inhibitor, SP600125 as indicated (N = 6, biological replicates).D-F. Cell proliferation effects after treatment with AKT1/2/3 inhibitor afuresertib, and MTOR1 inhibitor, everolimus, as indicated (N = 6, biological replicates).G-I. Cell proliferation effects after treatment with JAK1/2 inhibitor ruxolitinib, and TGFBR1 inhibitor galunisertib (N = 6, biological replicates). All inhibitors were used at 250 nM final concentration, with the exception of galunisertib, which was used at 500 nM final concentration. Note that the DMSO data are the same in for each cell line when compared to different class of inhibitors. Error bars in figure represent mean ± SEM.


Figure 7B . Kaplan - Meier survival curve of mice described in ( A ) . Comparison of survival curves using Log - rank ( Mantel - Cox ) test was used to compare difference between EV ( N = 5 ) and D2113N ( N = 5 ) , and EV and SLC34A2 - ROS1 ( N = 4 ) ; significance is indicated as P values within graph . F . Kaplan - Meier survival curve of mice described in ( C ) . G . Immunoblot analysis of the phosphorylated ( p ) and total ( t ) proteins from NIH - 3T3 ROS1D2113N P1 cell lysates prepared from cells treated with vehicle ( DMSO ) or 10 , 100 , or 500 nM of crizotinib , entrectinib , or lorlatinib for 4 hours . H - I . Immunoblot densitometry graphs to quantitatively compare extent of inhibition ROS1 , SHP2 and ERK1 / 2 phosphorylation ( panel H data ) after treatment with indicated inhibitors from two independent biological replicate experiments .

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Figure 7B. Kaplan-Meier survival curve of mice described in (A). Comparison of survival curves using Log-rank (Mantel-Cox) test was used to compare difference between EV (N = 5) and D2113N (N = 5), and EV and SLC34A2-ROS1 (N = 4); significance is indicated as P values within graph.F. Kaplan-Meier survival curve of mice described in (C).G. Immunoblot analysis of the phosphorylated (p) and total (t) proteins from NIH-3T3 ROS1D2113N P1 cell lysates prepared from cells treated with vehicle (DMSO) or 10, 100, or 500 nM of crizotinib, entrectinib, or lorlatinib for 4 hours.H-I. Immunoblot densitometry graphs to quantitatively compare extent of inhibition ROS1, SHP2 and ERK1/2 phosphorylation (panel H data) after treatment with indicated inhibitors from two independent biological replicate experiments.


Figure 1Confocal laser scanning fluorescence microscopy ( CLSM ) images showing DNA staining ( blue in the merged images ) , rDNA FISH ( green ) and NCL immunofluorescence ( red ) in untreated HeLa cells or in cells treated with α ‐ amanitin ( 50 μg / ml ) , AMD ( 50 ng / ml ) or the Pol III inhibitor ML ‐ 60218 ( 200 μM ) for 5 h . CLSM images of propidium iodide ‐ stained RNA after microinjection of buffer or nucleolar RNA into α ‐ amanitin ‐ treated HeLa cells . Nucleoli were visualized by immunofluorescence of nucleophosmin ( NPM ) . The graph represents the average number of nucleolar domains ( ± 95 % CI ) based on the analysis of 90 , 87 , 80 and 86 cells , respectively . * * P ‐ value 0 . 01 , t ‐ test .

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Figure 1Confocal laser scanning fluorescence microscopy (CLSM) images showing DNA staining (blue in the merged images), rDNA FISH (green) and NCL immunofluorescence (red) in untreated HeLa cells or in cells treated with α‐amanitin (50 μg/ml), AMD (50 ng/ml) or the Pol III inhibitor ML‐60218 (200 μM) for 5 h.CLSM images of propidium iodide‐stained RNA after microinjection of buffer or nucleolar RNA into α‐amanitin‐treated HeLa cells. Nucleoli were visualized by immunofluorescence of nucleophosmin (NPM). The graph represents the average number of nucleolar domains (± 95% CI) based on the analysis of 90, 87, 80 and 86 cells, respectively. **P‐value 0.01, t‐test.


Figure 3CLSM images showing nucleolar co ‐ localization of NCL ( immunofluorescence ) with aluRNA ( RNA FISH ) but not with L1 ‐ repeat RNA . Cells were pre ‐ treated in situ with RNase A or an RNase inhibitor . Nuclei were counterstained with DAPI . The signal intensity of nucleolar aluRNA was two ‐ fold higher compared to nucleoplasmic signal ( n = 92 , P ‐ value 0 . 05 , t ‐ test ) . Graphs representing the average number of nucleolar bodies after microinjection of in vitro transcribed RNA into HeLa cells that were pre ‐ treated with α ‐ amanitin ( 50 μg / ml ) for 5 h or left untreated ( control ) ( ± 95 % CI . * * P ‐ value 0 . 01 , n = 90 , 87 , 86 , 83 or 86 cells , respectively ) . Representative CLSM images of propidium iodide ‐ stained RNA are shown on the right side .

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Figure 3CLSM images showing nucleolar co‐localization of NCL (immunofluorescence) with aluRNA (RNA FISH) but not with L1‐repeat RNA. Cells were pre‐treated in situ with RNase A or an RNase inhibitor. Nuclei were counterstained with DAPI. The signal intensity of nucleolar aluRNA was two‐fold higher compared to nucleoplasmic signal (n = 92, P‐value 0.05, t‐test).Graphs representing the average number of nucleolar bodies after microinjection of in vitro transcribed RNA into HeLa cells that were pre‐treated with α‐amanitin (50 μg/ml) for 5 h or left untreated (control) (± 95% CI. **P‐value 0.01, n = 90, 87, 86, 83 or 86 cells, respectively). Representative CLSM images of propidium iodide‐stained RNA are shown on the right side.


Figure 4CLSMimages showing NPM ( immunofluorescence , red ) in HeLacells transfected with control ASO , aluRNAASO , or treated with transfection reagent only ( mock ) . Nuclei were counterstained with DAPI ( blue ) . A fraction of 80 ± 8 % of the cells treated with aluRNAASO ( n = 143 ) showed irregular nucleoli or dispersed nucleoli ( 53 % of the cells ) . Only 26 ± 9 % of the cells treated with control ASO presented abnormal nucleoli , with none of them showing a dispersion of nucleolar domains ( n = 123 ) , P ‐ value 0 . 01 , t ‐ test . CLSM images showing NCL localization ( immunofluorescence ) , aluRNA ( RNA FISH ) and DNA ( DAPI ) . Transfection of HeLa cells was done as in ( A ) . HeLa cells were transfected as in ( A ) , and nascent RNAs were visualized by pulse labeling with ethynyl uridine ( EU ) for 30 min . Nuclei were counterstained with DAPI ( DNA ) . Levels of 47Spre ‐ rRNA in mock ‐ , control ASO ‐ and aluRNAASO ‐ treated HeLacells . Top : Northern blot . Center : Agarose gel electrophoretic analysis of RNA . Bottom panel : Quantification from RT - qPCR levels of 47Spre ‐ rRNA normalized to 18SrRNA . Error bars represent the standard deviation ( n = 6 ) . * * P ‐ value 0 . 01 , t ‐ test .

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Figure 4CLSMimages showing NPM (immunofluorescence, red) in HeLacells transfected with control ASO, aluRNAASO, or treated with transfection reagent only (mock). Nuclei were counterstained with DAPI (blue). A fraction of 80 ± 8% of the cells treated with aluRNAASO (n = 143) showed irregular nucleoli or dispersed nucleoli (53% of the cells). Only 26 ± 9% of the cells treated with control ASO presented abnormal nucleoli, with none of them showing a dispersion of nucleolar domains (n = 123), P‐value 0.01, t‐test.CLSM images showing NCL localization (immunofluorescence), aluRNA (RNA FISH) and DNA (DAPI). Transfection of HeLa cells was done as in (A).HeLa cells were transfected as in (A), and nascent RNAs were visualized by pulse labeling with ethynyl uridine (EU) for 30 min. Nuclei were counterstained with DAPI (DNA).Levels of 47Spre‐rRNA in mock‐, control ASO‐ and aluRNAASO‐treated HeLacells. Top: Northern blot. Center: Agarose gel electrophoretic analysis of RNA. Bottom panel: Quantification from RT-qPCR levels of 47Spre‐rRNA normalized to 18SrRNA. Error bars represent the standard deviation (n = 6). **P‐value 0.01, t‐test.


Figure 5HeLa cells were transfected with an empty vector or plasmids expressing aluRNA or L1 ‐ repeat RNA and GFP from a separate promoter . Transfected cells were identified by GFP fluorescence , and nucleoli volumes were evaluated based on the immunofluorescence signal of the nucleolar marker NPM . The graphs show that the volume of nucleoli is increased by 30 % in the presence of ectopic aluRNA ( ± 95 % CI , n = 20 ) . * * P ‐ value 0 . 01 , t ‐ test . Scale bar , 10 μm . CLSM live ‐ cell imaging of RFP ‐ NCL in cells transfected with a plasmid expressing aluRNA and imaged for 10 h . The arrows indicate nucleolar domains displaying time ‐ dependent increase in the nucleolar size or fusion of two domains into one . Scale bars , 10 μm . HeLa cells were transfected as in ( A ) and incubated for 24 h . Nascent RNA was pulse ‐ labeled for 30 min , and nucleoli were visualized by immunofluorescence of NPM . Scale bars , 10 μm . Graphs represent the intensity of nucleolar fluorescence signals as percentage of the intensity of nuclear signals and reveal a 27 % increase in the presence of ectopic aluRNA ( ± 95 % CI , n = 30 ) . * * P ‐ value 0 . 01 , * P ‐ value = 0 . 01 , t ‐ test .

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Figure 5HeLa cells were transfected with an empty vector or plasmids expressing aluRNA or L1‐repeat RNA and GFP from a separate promoter. Transfected cells were identified by GFP fluorescence, and nucleoli volumes were evaluated based on the immunofluorescence signal of the nucleolar marker NPM. The graphs show that the volume of nucleoli is increased by 30% in the presence of ectopic aluRNA (± 95% CI, n = 20). **P‐value 0.01, t‐test. Scale bar, 10 μm.CLSM live‐cell imaging of RFP‐NCL in cells transfected with a plasmid expressing aluRNA and imaged for 10 h. The arrows indicate nucleolar domains displaying time‐dependent increase in the nucleolar size or fusion of two domains into one. Scale bars, 10 μm.HeLa cells were transfected as in (A) and incubated for 24 h. Nascent RNA was pulse‐labeled for 30 min, and nucleoli were visualized by immunofluorescence of NPM. Scale bars, 10 μm.Graphs represent the intensity of nucleolar fluorescence signals as percentage of the intensity of nuclear signals and reveal a 27% increase in the presence of ectopic aluRNA (± 95% CI, n = 30). **P‐value 0.01, *P‐value = 0.01, t‐test.


Figure 6CLSMimages of UBFimmunofluorescence in mouseNIH 3T3cells treated for 5 h with α ‐ amanitin to inhibit Pol II transcription show the dispersion of nucleoli , as previously also imaged via RNAstaining ( Caudron ‐ Herger et al , ) . CLSMimages showing the localization of NPM ( immunofluorescence , red ) in mouse NIH 3T3 cells treated with ASO as indicated and counterstained with DAPI ( blue ) .

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Figure 6CLSMimages of UBFimmunofluorescence in mouseNIH 3T3cells treated for 5 h with α‐amanitin to inhibit Pol II transcription show the dispersion of nucleoli, as previously also imaged via RNAstaining (Caudron‐Herger et al, ).CLSMimages showing the localization of NPM (immunofluorescence, red) in mouse NIH 3T3 cells treated with ASO as indicated and counterstained with DAPI (blue).


Figure 7CLSM images of MS2 ‐ tagged aluRNA or MS2 ‐ tagged control RNA ( a fragment from the transcript of the CDV3 gene ) . MS2 ‐ loop ‐ containing RNAs were visualized by RNA FISH using MS2 ‐ loop ‐ specific FISH probes ( green ) ; nucleoli were visualized by immunofluorescence of NCL ( red ) . The top panel depicts the experimental approach used to tether MS2 ‐ loop ‐ containing RNAs to lacO arrays via a GFP ‐ tagged LacI ‐ MS2 coat fusion protein . The lacO arrays were stably integrated in the genome of U2OS cells . In the bottom panel , CLSM images of U2OS cells are shown that were transfected with MS2 ‐ aluRNAR . They reveal the localization of LacI ‐ GFP ‐ MS2 and NCL ( immunofluorescence , red ) . The arrow indicates the lacO array , which is associated with a nucleolar domain . MS2 ‐ loop ‐ containing forward aluRNA or aluRNAL or aluRNAR were recruited to the single stably integrated lacO array in the U2OS F4IIB8 cell line . The propensity for nucleolar localization was evaluated as the average number of lacO arrays with tethered RNA detected in nucleoli ( ± 95 % CI ) and is plotted in the bar chart . Calculations are based on analysis of more than 100 cells . * * P ‐ value 0 . 01 , t ‐ test , from the analysis of two independent biological replicates . As controls , transcripts of the MS2 loops only , the MS2 ‐ CDV3 , MS2 ‐ RepA , MS2 ‐ STARD7 and MS2 ‐ CORO1C RNAs were used . The inset shows a CLSM image with MS2 ‐ GFP ‐ LacI ( green ) and NPM ( red , immunofluorescence ) after recruitment of MS2 ‐ aluRNA .

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Figure 7CLSM images of MS2‐tagged aluRNA or MS2‐tagged control RNA (a fragment from the transcript of the CDV3 gene). MS2‐loop‐containing RNAs were visualized by RNA FISH using MS2‐loop‐specific FISH probes (green); nucleoli were visualized by immunofluorescence of NCL (red).The top panel depicts the experimental approach used to tether MS2‐loop‐containing RNAs to lacO arrays via a GFP‐tagged LacI‐MS2 coat fusion protein. The lacO arrays were stably integrated in the genome of U2OS cells. In the bottom panel, CLSM images of U2OS cells are shown that were transfected with MS2‐aluRNAR. They reveal the localization of LacI‐GFP‐MS2 and NCL (immunofluorescence, red). The arrow indicates the lacO array, which is associated with a nucleolar domain.MS2‐loop‐containing forward aluRNA or aluRNAL or aluRNAR were recruited to the single stably integrated lacO array in the U2OS F4IIB8 cell line. The propensity for nucleolar localization was evaluated as the average number of lacO arrays with tethered RNA detected in nucleoli (± 95% CI) and is plotted in the bar chart. Calculations are based on analysis of more than 100 cells. **P‐value 0.01, t‐test, from the analysis of two independent biological replicates. As controls, transcripts of the MS2 loops only, the MS2‐CDV3, MS2‐RepA, MS2‐STARD7 and MS2‐CORO1C RNAs were used. The inset shows a CLSM image with MS2‐GFP‐LacI (green) and NPM (red, immunofluorescence) after recruitment of MS2‐aluRNA.


Figure 8Top : Scheme illustrating the experimental approach used to tether GFP ‐ tagged LacI fusion proteins via a GFP ‐ binding protein ( GBP ) to lacO arrays . The lacO arrays are stably integrated in the genome of U2OS cells . Bottom : CLSM images showing the localization of GBP ‐ LacI ‐ RFP , GFP ‐ NCL , GFP ‐ NPM or GFP ‐ TIP5 ‐ RBD fusion proteins relative to aluRNA that was visualized by RNA FISH . The insets contain an enlarged image of one of the lacO loci . CLSM images showing GBP ‐ LacI ‐ RFP and GFP ‐ NCL ( green ) recruited to lacO arrays as indicated by arrows . Immunofluorescence of endogenous NPM ( red ) marks nucleoli . The inset shows decondensation of lacO arrays in the nucleolus . The graph at the bottom depicts the average number of lacO loci tethered to nucleoli ( ± 95 % CI ) by the indicated proteins ( n = 72 ) . NCL ‐ ∆ RNA is an NCL deletion mutant lacking the RBD and GAR domains required for RNA binding . * * P ‐ value 0 . 01 , t ‐ test . CLSM images of UBF ( immunofluorescence , green ) or rDNA ( DNA FISH , green ) and DNA ( DAPI , blue ) after knockdown of NCL and NPM by siRNA in HeLa cells .

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Figure 8Top: Scheme illustrating the experimental approach used to tether GFP‐tagged LacI fusion proteins via a GFP‐binding protein (GBP) to lacO arrays. The lacO arrays are stably integrated in the genome of U2OS cells. Bottom: CLSM images showing the localization of GBP‐LacI‐RFP, GFP‐NCL, GFP‐NPM or GFP‐TIP5‐RBD fusion proteins relative to aluRNA that was visualized by RNA FISH. The insets contain an enlarged image of one of the lacO loci.CLSM images showing GBP‐LacI‐RFP and GFP‐NCL (green) recruited to lacO arrays as indicated by arrows. Immunofluorescence of endogenous NPM (red) marks nucleoli. The inset shows decondensation of lacO arrays in the nucleolus. The graph at the bottom depicts the average number of lacO loci tethered to nucleoli (± 95% CI) by the indicated proteins (n = 72). NCL‐∆RNA is an NCL deletion mutant lacking the RBD and GAR domains required for RNA binding. **P‐value 0.01, t‐test.CLSM images of UBF (immunofluorescence, green) or rDNA (DNA FISH, green) and DNA (DAPI, blue) after knockdown of NCL and NPM by siRNA in HeLa cells.


Figure 1V . cholerae wild - type and ΔrpoE strains carrying the indicated plasmids were grown to early stationary phase ( OD600 of 1 . 5 ) and induced with L - arabinose ( 0 . 2 % final conc . ) . Expression of MicV and VrrA was monitored on Northern blots . 5S rRNA served as loading control . V . cholerae wild - type , ΔvchM and ΔvchM ΔrpoE strains harboring PmicV : : mKate2 plasmids were grown in M9 medium . Samples were collected during various growth phases and analyzed for fluorescence . V . cholerae wild - type and ΔrpoE strains carrying PmicV : : mKate2 plasmids were cultivated in LB to exponential phase ( OD600 of 0 . 4 ) and treated with ethanol ( 3 . 5 % final conc . ) , or water . Fluorescence was determined 180 min after ethanol treatment and mKate2 levels of the mock - treated samples were set to 1 . V . cholerae wild - type and ΔrpoE strains carrying PvrrA : : mKate2 ( E ) plasmids were cultivated in LB to exponential phase ( OD600 of 0 . 4 ) and treated with ethanol ( 3 . 5 % final conc . ) , or water . Fluorescence was determined 180 min after ethanol treatment and mKate2 levels of the mock - treated samples were set to 1 . V . cholerae wild - type , ΔmicV , ΔvrrA or ΔvrrA ΔmicV strains were grown in LB to OD600 of 0 . 2 and treated with ethanol ( 3 . 5 % final conc . ) . After 5h of treatment , serial dilutions were prepared , recovered on agar plates and CFU / ml were determined .

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Figure 1V. cholerae wild-type and ΔrpoE strains carrying the indicated plasmids were grown to early stationary phase (OD600 of 1.5) and induced with L-arabinose (0.2% final conc.). Expression of MicV and VrrA was monitored on Northern blots. 5S rRNA served as loading control.V. cholerae wild-type, ΔvchM and ΔvchM ΔrpoE strains harboring PmicV::mKate2 plasmids were grown in M9 medium. Samples were collected during various growth phases and analyzed for fluorescence.V. cholerae wild-type and ΔrpoE strains carrying PmicV::mKate2 plasmids were cultivated in LB to exponential phase (OD600 of 0.4) and treated with ethanol (3.5% final conc.), or water. Fluorescence was determined 180 min after ethanol treatment and mKate2 levels of the mock-treated samples were set to 1.V. cholerae wild-type and ΔrpoE strains carrying PvrrA::mKate2 (E) plasmids were cultivated in LB to exponential phase (OD600 of 0.4) and treated with ethanol (3.5% final conc.), or water. Fluorescence was determined 180 min after ethanol treatment and mKate2 levels of the mock-treated samples were set to 1.V. cholerae wild-type, ΔmicV, ΔvrrA or ΔvrrA ΔmicV strains were grown in LB to OD600 of 0.2 and treated with ethanol (3.5% final conc.). After 5h of treatment, serial dilutions were prepared, recovered on agar plates and CFU / ml were determined.


Figure 2V . cholerae ΔvrrA ΔmicV strains carrying pBAD - micV , pBAD - vrrA or an empty vector control ( pCtr ) were cultivated to early stationary phase ( OD600 of 1 . 5 ) in LB . Cells were treated with L - arabinose ( 0 . 2 % final conc . ) and RNA samples were collected at the indicated time points after induction . Northern blot analysis was performed to determine VrrA , MicV and ompT levels . 5S rRNA served as loading control . For comparison , RNA samples of a wild - type strain carrying pCtr were collected during various growth phases , which indicated ~ 18 - fold and ~ 7 - fold higher levels of VrrA and MicV expressed from the pBAD plasmids , respectively ( see Source data for quantifications ) . V . cholerae ΔvrrA ΔmicV strains carrying the indicated reporter plasmids ( x - axis ) and either an empty vector control ( pCtr ) , the pMicV , or the pVrrA plasmid , were cultivated in M9 and GFP fluorescence was measured . Fluorescence of the control strains was set to 1 . The target genes were classified according to ( B ) : regulated by both sRNAsV . cholerae ΔvrrA ΔmicV strains carrying the indicated reporter plasmids ( x - axis ) and either an empty vector control ( pCtr ) , the pMicV , or the pVrrA plasmid , were cultivated in M9 and GFP fluorescence was measured . Fluorescence of the control strains was set to 1 . The target genes were classified according to regulated only by MicVV . cholerae ΔvrrA ΔmicV strains carrying the indicated reporter plasmids ( x - axis ) and either an empty vector control ( pCtr ) , the pMicV , or the pVrrA plasmid , were cultivated in M9 and GFP fluorescence was measured . Fluorescence of the control strains was set to 1 . The target genes were classified according to regulated only by

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Figure 2V. cholerae ΔvrrA ΔmicV strains carrying pBAD-micV, pBAD-vrrA or an empty vector control (pCtr) were cultivated to early stationary phase (OD600 of 1.5) in LB. Cells were treated with L-arabinose (0.2% final conc.) and RNA samples were collected at the indicated time points after induction. Northern blot analysis was performed to determine VrrA, MicV and ompT levels. 5S rRNA served as loading control. For comparison, RNA samples of a wild-type strain carrying pCtr were collected during various growth phases, which indicated ~18-fold and ~7-fold higher levels of VrrA and MicV expressed from the pBAD plasmids, respectively (see Source data for quantifications).V. cholerae ΔvrrA ΔmicV strains carrying the indicated reporter plasmids (x-axis) and either an empty vector control (pCtr), the pMicV, or the pVrrA plasmid, were cultivated in M9 and GFP fluorescence was measured. Fluorescence of the control strains was set to 1. The target genes were classified according to (B): regulated by both sRNAsV. cholerae ΔvrrA ΔmicV strains carrying the indicated reporter plasmids (x-axis) and either an empty vector control (pCtr), the pMicV, or the pVrrA plasmid, were cultivated in M9 and GFP fluorescence was measured. Fluorescence of the control strains was set to 1. The target genes were classified according to regulated only by MicVV. cholerae ΔvrrA ΔmicV strains carrying the indicated reporter plasmids (x-axis) and either an empty vector control (pCtr), the pMicV, or the pVrrA plasmid, were cultivated in M9 and GFP fluorescence was measured. Fluorescence of the control strains was set to 1. The target genes were classified according to regulated only by VrrA


Figure 3V . cholerae ΔvrrA ΔmicV strains carrying the ompT : : gfp or ompT M1 * : : gfp fusions ( D ) , ushA : : gfp or ushA M1 * : : gfp fusions ( E ) , or lpp : : gfp or lpp M2 * : : gfp fusions ( F ) and an empty vector control ( pCtr ) , the micV expression plasmids ( pMicV , pMicV M1 ) or the vrrA expression plasmids ( pVrrA , pVrrA M2 ) were grown in M9 and GFP fluorescence was measured . M1 and M2 denote the mutations indicated in ( A , B , C ) . Fluorescence of the control strains was set to 1 . V . cholerae ΔrpoE , ΔrpoE ΔvrrA , ΔrpoE ΔmicV or ΔrpoE ΔvrrA ΔmicV strains carrying pBAD - rpoE or an empty vector control ( pCtr ) were grown to OD600 of 1 . 5 and L - arabinose ( 0 . 2 % final conc . ) was added . RNA samples were collected at the indicated time points and monitored for ompT ( G ) , ushA ( H ) , or lpp ( I ) levels using qRT - PCR .

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Figure 3V. cholerae ΔvrrA ΔmicV strains carrying the ompT::gfp or ompT M1*::gfp fusions (D), ushA::gfp or ushA M1*::gfp fusions (E), or lpp::gfp or lpp M2*::gfp fusions (F) and an empty vector control (pCtr), the micV expression plasmids (pMicV, pMicV M1) or the vrrA expression plasmids (pVrrA, pVrrA M2) were grown in M9 and GFP fluorescence was measured. M1 and M2 denote the mutations indicated in (A, B, C). Fluorescence of the control strains was set to 1.V. cholerae ΔrpoE, ΔrpoE ΔvrrA, ΔrpoE ΔmicV or ΔrpoE ΔvrrA ΔmicV strains carrying pBAD-rpoE or an empty vector control (pCtr) were grown to OD600 of 1.5 and L-arabinose (0.2% final conc.) was added. RNA samples were collected at the indicated time points and monitored for ompT (G), ushA (H), or lpp (I) levels using qRT-PCR.


Figure 4V . cholerae ΔvrrA ΔmicV strains carrying the ompT : : 3XFLAG gene and pMicV , pVrrA , pRybB , or an empty vector control ( pCtr ) were cultivated in LB to an OD600 of 2 . 0 . RNA and protein samples were collected and analyzed for MicV , VrrA and RybB expression on Northern blots . OmpT : : 3xFLAG production was tested on Western blots . RNAPα and 5S rRNA served as loading controls for Western and Northern blots , respectively . E . coli wild - type strains carrying pMicV , pVrrA , pRybB , or an empty vector control ( pCtr ) were grown in LB to an OD600 of 2 . 0 . RNA and protein samples were collected and investigated on Northern blots and SDS - PAGE , respectively . For comparison , we included the E . coli insertional mutant strains ompA : : kanR and ompC : : kanR for specific assignment of OmpA and OmpC bands . V . cholerae wild - type and ΔrpoE strains carrying pMicV , pVrrA , pRybB , or an empty vector control ( pCtr ) were cultivated in LB to OD600 of 0 . 2 and treated with ethanol ( 3 . 5 % final conc . ) . After 5h of treatment , serial dilutions were prepared , recovered on agar plates and CFU / ml were determined .

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

Figure 4V. cholerae ΔvrrA ΔmicV strains carrying the ompT::3XFLAG gene and pMicV, pVrrA, pRybB, or an empty vector control (pCtr) were cultivated in LB to an OD600 of 2.0. RNA and protein samples were collected and analyzed for MicV, VrrA and RybB expression on Northern blots. OmpT::3xFLAG production was tested on Western blots. RNAPα and 5S rRNA served as loading controls for Western and Northern blots, respectively.E. coli wild-type strains carrying pMicV, pVrrA, pRybB, or an empty vector control (pCtr) were grown in LB to an OD600 of 2.0. RNA and protein samples were collected and investigated on Northern blots and SDS-PAGE, respectively. For comparison, we included the E. coli insertional mutant strains ompA::kanR and ompC::kanR for specific assignment of OmpA and OmpC bands.V. cholerae wild-type and ΔrpoE strains carrying pMicV, pVrrA, pRybB, or an empty vector control (pCtr) were cultivated in LB to OD600 of 0.2 and treated with ethanol (3.5% final conc.). After 5h of treatment, serial dilutions were prepared, recovered on agar plates and CFU / ml were determined.


Figure 5V . cholerae wild - type and ΔrpoE strains carrying an empty vector control ( pCtr ) , pRybB , or the sRNA library after consecutive selection experiments ( Sel1 , Sel2 , Sel3 ) were grown in LB to OD600 of 0 . 2 . Cells were treated with ethanol ( 3 . 5 % final conc . ) for 6 hours . Serial dilutions were prepared and spotted onto agar plates . R1 and R2 indicate two independent biological replicates .

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Figure 5V. cholerae wild-type and ΔrpoE strains carrying an empty vector control (pCtr), pRybB, or the sRNA library after consecutive selection experiments (Sel1, Sel2, Sel3) were grown in LB to OD600 of 0.2. Cells were treated with ethanol (3.5% final conc.) for 6 hours. Serial dilutions were prepared and spotted onto agar plates. R1 and R2 indicate two independent biological replicates.


Figure 6V . cholerae wild - type and ΔrpoE strains carrying an empty vector control ( pCtr ) , pRybB , or the sRNA library before ( input ) or after consecutive ethanol selection experiments ( Sel1 , Sel2 , Sel3 ) were cultivated in LB to OD600 of 2 . 0 . Membrane fractions were analyzed by SDS - PAGE . The indicated bands were analyzed by mass spectrometry . V . cholerae ΔvrrA ΔmicV cells expressing the ompA : : 3xFLAG gene and carrying an empty vector control ( pCtr ) , or plasmids producing the 15 most highly enriched sRNA variants ( sRNA variants 1 - 15 ) were grown in LB to an OD600 of 2 . 0 . RNA and protein samples were collected and tested for sRNA and OmpA : : 3xFLAG expression on Northern and Western blots , respectively ( with RNAPα and 5S rRNA as loading controls ) . V . cholerae ΔvrrA ΔmicV strains carrying the ompA : : gfp fusion and an empty vector control ( pCtr ) or the enriched sRNA expression plasmids were grown in M9 and GFP fluorescence was measured . Fluorescence of the control strains was set to 1 . V . cholerae wild - type , ΔrpoE , ΔompA or ΔrpoE ΔompA strains were grown in LB to OD600 of 0 . 2 and treated with ethanol ( 3 . 5 % final conc . ) . After 5h of treatment , serial dilutions were prepared , recovered on agar plates and CFU / ml were determined .

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Figure 6V. cholerae wild-type and ΔrpoE strains carrying an empty vector control (pCtr), pRybB, or the sRNA library before (input) or after consecutive ethanol selection experiments (Sel1, Sel2, Sel3) were cultivated in LB to OD600 of 2.0. Membrane fractions were analyzed by SDS-PAGE. The indicated bands were analyzed by mass spectrometry.V. cholerae ΔvrrA ΔmicV cells expressing the ompA::3xFLAG gene and carrying an empty vector control (pCtr), or plasmids producing the 15 most highly enriched sRNA variants (sRNA variants 1-15) were grown in LB to an OD600 of 2.0. RNA and protein samples were collected and tested for sRNA and OmpA::3xFLAG expression on Northern and Western blots, respectively (with RNAPα and 5S rRNA as loading controls).V. cholerae ΔvrrA ΔmicV strains carrying the ompA::gfp fusion and an empty vector control (pCtr) or the enriched sRNA expression plasmids were grown in M9 and GFP fluorescence was measured. Fluorescence of the control strains was set to 1.V. cholerae wild-type, ΔrpoE, ΔompA or ΔrpoE ΔompA strains were grown in LB to OD600 of 0.2 and treated with ethanol (3.5% final conc.). After 5h of treatment, serial dilutions were prepared, recovered on agar plates and CFU / ml were determined.


Figure 1Macrophages produce little to no inflammatory cytokines when incubated with either ( a ) whole iRBC for 24 h . One - way ANOVA with Tukey test for multiple comparisons was performed to determine statistical significance Each symbol represents the value obtained for cells from an independent donor in an independent experiment . ( a ) n = 4 ; Asterisks indicate significance ( * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 ) when values were compared with RBCMacrophages produce little to no inflammatory cytokines when incubated with either ( b ) fresh iRBCL for 24 h . One - way ANOVA with Tukey test for multiple comparisons was performed to determine statistical significance Each symbol represents the value obtained for cells from an independent donor in an independent experiment . ( b ) n = 5 Asterisks indicate significance ( * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 ) when values were compared with RBC ( c ) Macrophages were pre - incubated with IFNγ ( 1000U / ml ) for 18 h before iRBCL stimulation . One - way ANOVA with Tukey test for multiple comparisons was performed to determine statistical significance Each symbol represents the value obtained for cells from an independent donor in an independent experiment . ( c ) n = 3 Asterisks indicate significance ( * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 ) when values were compared with RBC ( d ) Phase - contrast microscopy of macrophages 24 h after the addition of RBC , iRBC or LPS . Dark hemozoin crystals can be observed within macrophages . Bar is 30 µm . ( e ) Macrophages were incubated with iRBCL 24 h before addition of LPS . Paired t - test was performed to determine statistical significance . Each symbol represents the value obtained for cells from an independent donor in an independent experiment . ( e ) n = 3 . Asterisks indicate significance ( * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 ) when values were compared RBCL ( e ) .

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Figure 1Macrophages produce little to no inflammatory cytokines when incubated with either (a) whole iRBC for 24 h. One-way ANOVA with Tukey test for multiple comparisons was performed to determine statistical significance Each symbol represents the value obtained for cells from an independent donor in an independent experiment. (a) n=4; Asterisks indicate significance (*p<0.05, **p<0.01 and ***p<0.001) when values were compared with RBCMacrophages produce little to no inflammatory cytokines when incubated with either (b) fresh iRBCL for 24 h. One-way ANOVA with Tukey test for multiple comparisons was performed to determine statistical significance Each symbol represents the value obtained for cells from an independent donor in an independent experiment. (b) n=5 Asterisks indicate significance (*p<0.05, **p<0.01 and ***p<0.001) when values were compared with RBC(c) Macrophages were pre-incubated with IFNγ (1000U/ml) for 18 h before iRBCL stimulation. One-way ANOVA with Tukey test for multiple comparisons was performed to determine statistical significance Each symbol represents the value obtained for cells from an independent donor in an independent experiment. (c) n=3 Asterisks indicate significance (*p<0.05, **p<0.01 and ***p<0.001) when values were compared with RBC(d) Phase-contrast microscopy of macrophages 24 h after the addition of RBC, iRBC or LPS. Dark hemozoin crystals can be observed within macrophages. Bar is 30 µm.(e) Macrophages were incubated with iRBCL 24 h before addition of LPS. Paired t-test was performed to determine statistical significance. Each symbol represents the value obtained for cells from an independent donor in an independent experiment. (e) n=3. Asterisks indicate significance (*p<0.05, **p<0.01 and ***p<0.001) when values were compared RBCL (e).


Figure 2Cytokine secretion of macrophages incubated with the indicated stimuli for 24 h . RBCL , iRBCL , XO were added to macrophages for 24 h . Each symbol represents the value obtained for cells from an independent donor in an independent experiment . Insets show paired samples by donor in two different conditions . Blue asterisks indicate significance when values were compared with RBCL ( a ) n = 7 One - way ANOVA with Tukey test for multiple comparisons was performed to determine statistical significance ( * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 ) . Cytokine secretion of macrophages incubated with the indicated stimuli for 24 h . , XO , Hypoxanthine ( Hyx ) were added to macrophages for 24 h . Each symbol represents the value obtained for cells from an independent donor in an independent experiment . Insets show paired samples by donor in two different conditions . Blue asterisks indicate significance when values were compared with XO Grey asterisks mark comparison with Control n = 3 . One - way ANOVA with Tukey test for multiple comparisons was performed to determine statistical significance ( * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 ) . Cytokine secretion of macrophages incubated with the indicated stimuli for 24 h . XO added to macrophages for 24 h . Febuxostat ( Feb ) was pre - incubated for 30 minutes with XO before addition to macrophages . Each symbol represents the value obtained for cells from an independent donor in an independent experiment . Insets show paired samples by donor in two different conditions . Grey asterisks mark comparison with Control n = 3 . One - way ANOVA with Tukey test for multiple comparisons was performed to determine statistical significance ( * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 ) . Cytokine secretion of macrophages incubated with the indicated stimuli for 24 h . , XO the anti - oxidants N - acetyl - L - cysteine ( NAC ) and 1 - Thioglycerol ( 1 - TG ) , and heat inactivated ( HI ) XO were added to macrophages for 24 h . Each symbol represents the value obtained for cells from an independent donor in an independent experiment . Insets show paired samples by donor in two different conditions . Blue asterisks indicate significance when values were compared with XO Grey asterisks mark comparison with Control n = 3 . One - way ANOVA with Tukey test for multiple comparisons was performed to determine statistical significance ( * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 ) . Cytokine secretion of PBMC ( e ) incubated with the indicated stimuli for 24 h . RBC , iRBC , RBCL , iRBCL , XO Each symbol represents the value obtained for cells from an independent donor in an independent experiment . Insets show paired samples by donor in two different conditions . Blue asterisks indicate significance when values were compared with RBCL n = 3 . One - way ANOVA with Tukey test for multiple comparisons was performed to determine statistical significance ( * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 ) .

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

Figure 2Cytokine secretion of macrophages incubated with the indicated stimuli for 24 h. RBCL, iRBCL, XO were added to macrophages for 24 h. Each symbol represents the value obtained for cells from an independent donor in an independent experiment. Insets show paired samples by donor in two different conditions. Blue asterisks indicate significance when values were compared with RBCL (a) n=7 One-way ANOVA with Tukey test for multiple comparisons was performed to determine statistical significance (*p<0.05, **p<0.01 and ***p<0.001).Cytokine secretion of macrophages incubated with the indicated stimuli for 24 h. , XO, Hypoxanthine (Hyx) were added to macrophages for 24 h. Each symbol represents the value obtained for cells from an independent donor in an independent experiment. Insets show paired samples by donor in two different conditions. Blue asterisks indicate significance when values were compared with XO Grey asterisks mark comparison with Control n=3. One-way ANOVA with Tukey test for multiple comparisons was performed to determine statistical significance (*p<0.05, **p<0.01 and ***p<0.001).Cytokine secretion of macrophages incubated with the indicated stimuli for 24 h. XO added to macrophages for 24 h. Febuxostat (Feb) was pre-incubated for 30 minutes with XO before addition to macrophages. Each symbol represents the value obtained for cells from an independent donor in an independent experiment. Insets show paired samples by donor in two different conditions. Grey asterisks mark comparison with Control n=3. One-way ANOVA with Tukey test for multiple comparisons was performed to determine statistical significance (*p<0.05, **p<0.01 and ***p<0.001).Cytokine secretion of macrophages incubated with the indicated stimuli for 24 h. , XO the anti-oxidants N-acetyl-L-cysteine (NAC) and 1-Thioglycerol (1-TG), and heat inactivated (HI) XO were added to macrophages for 24 h. Each symbol represents the value obtained for cells from an independent donor in an independent experiment. Insets show paired samples by donor in two different conditions. Blue asterisks indicate significance when values were compared with XO Grey asterisks mark comparison with Control n=3. One-way ANOVA with Tukey test for multiple comparisons was performed to determine statistical significance (*p<0.05, **p<0.01 and ***p<0.001).Cytokine secretion of PBMC (e) incubated with the indicated stimuli for 24 h. RBC, iRBC, RBCL, iRBCL, XO Each symbol represents the value obtained for cells from an independent donor in an independent experiment. Insets show paired samples by donor in two different conditions. Blue asterisks indicate significance when values were compared with RBCL n=3. One-way ANOVA with Tukey test for multiple comparisons was performed to determine statistical significance (*p<0.05, **p<0.01 and ***p<0.001).


Figure 3 ( a ) XO was incubated with macrophages for the indicated times , when wells were washed twice and media was replaced . Supernatants were collected for cytokine determinations 24 h later ( n = 3 ) . Kruskal - Wallis statistical method was used for statistical analysis . Blue asterisks indicate significance when values were compared with time 0 ( * p < 0 . 05 ) . ( b ) Diagram shows experimental set up with macrophages in the bottom of the well and XO added either in the dialysis cassette ( DC , blue ) or the well ( black ) . XO activity panels show that the enzyme did not leak through the DC membrane . Secretion of IL6 and TNF was measured 24 h after addition of XO in each compartment . A representative result of two independent experiments is shown .

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

Figure 3(a) XO was incubated with macrophages for the indicated times, when wells were washed twice and media was replaced. Supernatants were collected for cytokine determinations 24 h later (n=3). Kruskal-Wallis statistical method was used for statistical analysis. Blue asterisks indicate significance when values were compared with time 0 (*p<0.05).(b) Diagram shows experimental set up with macrophages in the bottom of the well and XO added either in the dialysis cassette (DC, blue) or the well (black). XO activity panels show that the enzyme did not leak through the DC membrane. Secretion of IL6 and TNF was measured 24 h after addition of XO in each compartment. A representative result of two independent experiments is shown.


Figure 4 ( a ) Plasma levels of TNF and IL8 from patients with uncomplicated ( black circles , n = 14 ) or cerebral ( red circles , n = 9 ) malaria correlate with levels of XO - produced ROS detected in each sample . ROS is expressed as fold change over plasma from healthy controls . Uncomplicated malaria , gray circles , cerebral malaria , red circles . Linear regression ( a ) were performed to determine statistical significance ( * p < 0 . 05 , * * p < 0 . 01 ) . ( b ) Levels of XO activity in malaria patients correlate with disease severity UM ( uncomplicated malaria ) , CM ( cerebral malaria ) . Mann - Whitney test ( b ) were performed to determine statistical significance ( * p < 0 . 05 , * * p < 0 . 01 ) . ( c ) Plasma from malaria patients induce macrophages to secrete cytokines , which are inhibited by a XO specific inhibitor . Plasma from a healthy control ( HP ) and from three patients : P1 ( with cerebral malaria ) , P2 and P3 ( both with uncomplicated malaria ) was pre - incubated for 30 min with febuxostat ( Feb ) or alone at 37ºC before addition to macrophages at 1 : 2 dilution in media for 30 min . Cells were washed and cytokine secretion measured in triplicates after 24 h of incubation . unpaired t - tests ( c ) were performed to determine statistical significance ( * p < 0 . 05 , * * p < 0 . 01 ) .

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

Figure 4(a) Plasma levels of TNF and IL8 from patients with uncomplicated (black circles, n=14) or cerebral (red circles, n=9) malaria correlate with levels of XO-produced ROS detected in each sample. ROS is expressed as fold change over plasma from healthy controls. Uncomplicated malaria, gray circles, cerebral malaria, red circles. Linear regression (a) were performed to determine statistical significance (*p<0.05, **p<0.01).(b) Levels of XO activity in malaria patients correlate with disease severity UM (uncomplicated malaria), CM (cerebral malaria). Mann-Whitney test (b) were performed to determine statistical significance (*p<0.05, **p<0.01).(c) Plasma from malaria patients induce macrophages to secrete cytokines, which are inhibited by a XO specific inhibitor. Plasma from a healthy control (HP) and from three patients: P1 (with cerebral malaria), P2 and P3 (both with uncomplicated malaria) was pre-incubated for 30 min with febuxostat (Feb) or alone at 37ºC before addition to macrophages at 1:2 dilution in media for 30 min. Cells were washed and cytokine secretion measured in triplicates after 24 h of incubation. unpaired t-tests (c) were performed to determine statistical significance (*p<0.05, **p<0.01).


Figure 5Cytokine secretion by macrophages after 24 h incubation with the indicated stimuli . ( b ) IL1β levels after addition of increasing concentration of RBCL or iRBCL ( 1 : 2 , 1 : 4 and 1 : 8 macrophage : iRBCL ) . One - way ANOVA with Tukey test for multiple comparisons was performed to determine statistical significance . Asterisks indicate significance when values are compared with XO + RBCL ( * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 ) . Each symbol represents the value obtained for cells from an independent donor in an independent experiment . ( a ) n = 4 ; ( b ) n = 2 Insets show paired samples by donor in two different conditions . chemokine secretion by macrophages after 24 h incubation with the indicated stimuli . One - way ANOVA with Tukey test for multiple comparisons was performed to determine statistical significance . Asterisks indicate significance when values are compared with XO + RBCL ( * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 ) . Each symbol represents the value obtained for cells from an independent donor in an independent experiment . ( c ) n = 4 . Insets show paired samples by donor in two different conditions .

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Figure 5Cytokine secretion by macrophages after 24 h incubation with the indicated stimuli. (b) IL1β levels after addition of increasing concentration of RBCL or iRBCL (1:2, 1:4 and 1:8 macrophage:iRBCL). One-way ANOVA with Tukey test for multiple comparisons was performed to determine statistical significance. Asterisks indicate significance when values are compared with XO+RBCL (*p<0.05, **p<0.01 and ***p<0.001). Each symbol represents the value obtained for cells from an independent donor in an independent experiment. (a) n=4; (b) n=2 Insets show paired samples by donor in two different conditions.chemokine secretion by macrophages after 24 h incubation with the indicated stimuli. One-way ANOVA with Tukey test for multiple comparisons was performed to determine statistical significance. Asterisks indicate significance when values are compared with XO+RBCL (*p<0.05, **p<0.01 and ***p<0.001). Each symbol represents the value obtained for cells from an independent donor in an independent experiment. (c) n=4. Insets show paired samples by donor in two different conditions.


Figure 6 ( a ) Gene expression analysis of macrophages incubated with the indicated stimuli expressed as fold change over RBCL . Dotted red line indicates 2 - fold increase in RNA expression . ( b ) Western Blot analysis of proIL1β . Relative abundance is the average of 3 independent experiments with different donors . ( c ) FLICA analysis of activated caspase - 1 . Results are representative of 2 independent experiments with different donors . ( d ) Two distinct siRNAs against NLRP3 and an irrelevant control siRNA were utilized to knock down RNA levels by lipofectamine transfection in macrophages that were later incubated with iRBCL , or RBCL as control , with or without XO , before quantification of secreted IL1β . Results are representative of 2 independent experiments with different donors .

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Figure 6(a) Gene expression analysis of macrophages incubated with the indicated stimuli expressed as fold change over RBCL. Dotted red line indicates 2-fold increase in RNA expression.(b) Western Blot analysis of proIL1β. Relative abundance is the average of 3 independent experiments with different donors.(c) FLICA analysis of activated caspase-1. Results are representative of 2 independent experiments with different donors.(d) Two distinct siRNAs against NLRP3 and an irrelevant control siRNA were utilized to knock down RNA levels by lipofectamine transfection in macrophages that were later incubated with iRBCL, or RBCL as control, with or without XO, before quantification of secreted IL1β. Results are representative of 2 independent experiments with different donors.


Figure 1B . Images of rosettes and seedlings ( 15 DAG ) of Col - 0 , arf7 / arf19 , slr , and gLBD16 - SRDX as well as the inducible lateral - root - less lines pGATA23 : : shy2 - 2 - GR and pGATA23 : : slr1 - GR grown on DMSO control medium or 30 µM Dexamethasone ( DEX ) , Scale bar 1 cm . The rosettes were stained with a Lugol ' s Iodine solution for starch accumulation at the end of the dark period ( representative images of n = 3 biological replicates ) . C . Box plots of starch quantification in rosette tissues at 15 DAG ( n = 4 biological replicates ) . Comparison between samples was performed by one - way ANOVA and post - hoc Tukey HSD Test ( α = 0 . 05 ) ; different letters indicate significant differences .

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

Figure 1B. Images of rosettes and seedlings (15 DAG) of Col-0, arf7/arf19, slr, and gLBD16-SRDX as well as the inducible lateral-root-less lines pGATA23::shy2-2-GR and pGATA23::slr1-GR grown on DMSO control medium or 30 µM Dexamethasone (DEX), Scale bar 1 cm. The rosettes were stained with a Lugol's Iodine solution for starch accumulation at the end of the dark period (representative images of n=3 biological replicates). C. Box plots of starch quantification in rosette tissues at 15 DAG (n=4 biological replicates). Comparison between samples was performed by one-way ANOVA and post-hoc Tukey HSD Test (α = 0.05); different letters indicate significant differences.


Figure 2E . Representative confocal sections of calcofluor counterstained ( E , n = 5 biological replicates ) and differential interference contrast ( DIC ) images of GUS - stained root bends ( F , n = 16 biological replicates ) in DR5 : GUS seedlings treated as indicated 48h after gravistimulation . In E , orange arrows indicate the pericycle ( marked in yellow ) . F . Fraction of root bends forming an LRP and showing DR5 GUS staining after treatment with either control or 2D containing agar blocks ( n = 16 biological replicates , ± SE ) .

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Figure 2E. Representative confocal sections of calcofluor counterstained (E, n=5 biological replicates) and differential interference contrast (DIC) images of GUS-stained root bends (F, n=16 biological replicates) in DR5:GUS seedlings treated as indicated 48h after gravistimulation. In E, orange arrows indicate the pericycle (marked in yellow). F. Fraction of root bends forming an LRP and showing DR5 GUS staining after treatment with either control or 2D containing agar blocks (n=16 biological replicates, ± SE).


Figure 3A . Representative western blot of root tissues of pUB10 : S6K1 - 3xHA treated by the indicated combination of auxin ( IAA , 10µM ) , sucrose ( Suc , 110mM ) , 2 - deoxy - d - glucose ( 2D , 20mM ) and AZD8055 ( AZD , 10µM ) and probed with anti - S6K1 / 2 , anti - HA or anti - S6K1 - T449P . Blot is one of three biological replicates . B . Quantification of the relative S6K activation . Box plots show three biological replicates , and comparison between samples was performed by one - way ANOVA and post - hoc Tukey HSD Test ( α = 0 . 05 ) ; different letters indicate significant differences . C . Representative DIC images showing RPT1B promoter expression at different stages of LR development in 10 DAG seedlings . Orange arrowheads indicate the accumulation of reporter signals in the dividing lateral root founder cells . Scale bars : 50 µm . D . Representative confocal section showing S6K1 expression in different stages of LR development in 10 DAG pS6K1 : gS6K1 - CFP seedlings . Orange arrowheads indicate the accumulation of reporter signals in the dividing lateral root founder cells . Scale bars : 50 µm .

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Figure 3A. Representative western blot of root tissues of pUB10:S6K1-3xHA treated by the indicated combination of auxin (IAA, 10µM), sucrose (Suc, 110mM), 2-deoxy-d-glucose (2D, 20mM) and AZD8055 (AZD, 10µM) and probed with anti-S6K1/2, anti-HA or anti-S6K1-T449P. Blot is one of three biological replicates. B. Quantification of the relative S6K activation. Box plots show three biological replicates, and comparison between samples was performed by one-way ANOVA and post-hoc Tukey HSD Test (α = 0.05); different letters indicate significant differences.C. Representative DIC images showing RPT1B promoter expression at different stages of LR development in 10 DAG seedlings. Orange arrowheads indicate the accumulation of reporter signals in the dividing lateral root founder cells. Scale bars: 50 µm. D. Representative confocal section showing S6K1 expression in different stages of LR development in 10 DAG pS6K1:gS6K1-CFP seedlings. Orange arrowheads indicate the accumulation of reporter signals in the dividing lateral root founder cells. Scale bars: 50 µm.


Figure 4A - C . Box plots of emerged LR in TOR - oe ( A ) , rpt1b ( B ) , and lst8 ( C ) at 14 DAG . Comparison between samples was performed by one - way ANOVA and post - hoc Tukey HSD Test ( α = 0 . 05 ) ; different letters indicate significant differences . The number of roots scored is indicated . H . Fraction of bends developing lateral root primordia and stained for GUS activity in primary root vasculature of UB10pro > > amiR - TOR / DR5 : GUS , n = 18 bends .

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Figure 4A-C. Box plots of emerged LR in TOR-oe (A), rpt1b (B), and lst8 (C) at 14 DAG. Comparison between samples was performed by one-way ANOVA and post-hoc Tukey HSD Test (α = 0.05); different letters indicate significant differences. The number of roots scored is indicated.H. Fraction of bends developing lateral root primordia and stained for GUS activity in primary root vasculature of UB10pro>>amiR-TOR/DR5:GUS, n=18 bends.