C , PTPN3 PTPN3 ( D811A ) , but not PTPN3 ( L232R ) , enhance TGF - β - induced growth inhibitory effect . HepG2 stably expressing PTPN3 , PTPN3 ( D811A ) or PTPN3 ( L232R ) were treated with or without TGF - β ( 1 ng / mL ) for indicated days . were analyzed for proliferation by MTS . Data are shown as mean ± ; n = 3 .

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

C, PTPN3 and PTPN3 (D811A), but not PTPN3 (L232R), enhance TGF-β-induced growth inhibitory effect. HepG2 cells stably expressing PTPN3, PTPN3 (D811A) or PTPN3 (L232R) were treated with or without TGF-β (1 ng/mL) for indicated days. Cells were analyzed for cell proliferation by MTS assay. Data are shown as mean ± SEM; n = 3.


D , Depletion of PTPN3 attenuates TGF - β - induced growth inhibitory effect . HepG2 stably expressing shPTPN3 were treated with or without TGF - β ( 1 ng / mL ) for indicated days . were analyzed for proliferation by using MTS . Data are shown as mean ± ; n = 3 .

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

D, Depletion of PTPN3 attenuates TGF-β-induced growth inhibitory effect. HepG2 cells stably expressing shPTPN3 were treated with or without TGF-β (1 ng/mL) for indicated days. Cells were analyzed for cell proliferation by using MTS assay. Data are shown as mean ± SEM; n = 3.


E , PTPN3 ( L232R ) attenuates TGF - β - induced growth inhibition on colony formation in HepG2 . were treated with or without TGF - β ( 1 ng / mL ) treatment for 10 days . Clonogenic of results in clonogenic assays was analyzed by Image J . Data are shown as mean ± ; n = 3 .

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

E, PTPN3 (L232R) attenuates TGF-β-induced growth inhibition on colony formation in HepG2 cells. Cells were treated with or without TGF-β (1 ng/mL) treatment for 10 days. Clonogenic assay Quantification of the results in clonogenic assays was analyzed by Image J. Data are shown as mean ± SEM; n = 3.


F , Depletion of PTPN3 attenuates TGF - β - induced growth inhibitory effect in HepG2 . with stable knockdown of PTPN3 were treated with or without TGF - β ( 1 ng / mL ) for 10 days . Clonogenic of results in clonogenic assays were analyzed by Image J . Knockdown efficiency of PTPN3 in HepG2 were analyzed by Western blotting with anti - PTPN3 antibody . Data are shown as mean ± ; n = 3 . G , Depletion of PTPN3 attenuates TGF - β - induced growth inhibitory effect in Huh7 . Experiments were similarly done as in ( F ) . Data are shown as mean ± ; n = 3 .

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

F, Depletion of PTPN3 attenuates TGF-β-induced growth inhibitory effect in HepG2 cells. Cells with stable knockdown of PTPN3 were treated with or without TGF-β (1 ng/mL) for 10 days. Clonogenic assay Quantification of the results in clonogenic assays were analyzed by Image J. Knockdown efficiency of PTPN3 in HepG2 cells were analyzed by Western blotting with the anti-PTPN3 antibody. Data are shown as mean ± SEM; n = 3. G, Depletion of PTPN3 attenuates TGF-β-induced growth inhibitory effect in Huh7 cells. Experiments and analysis were similarly done as in (F). Data are shown as mean ± SEM; n = 3.


A , PTPN3 , PTPN3 ( D811A ) PTPN3 ( L232R ) are similarly expressed in Huh7 stable lines . Expression of indicated were detected with appropriate antibodies in Western blotting .

O O B-CONTROLLED_VAR 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

A, PTPN3, PTPN3 (D811A) and PTPN3 (L232R) are similarly expressed in Huh7 stable cell lines. Expression levels of indicated proteins were detected with appropriate antibodies in Western blotting.


B , PTPN3 PTPN3 ( D811A ) , but not PTPN3 ( L232R ) , attenuates tumorigenesis . Luciferase - harboring Huh7 ( 2 × 106 / mouse ) expressing PTPN3 , PTPN3 ( D811A ) or PTPN3 ( L232R ) or empty vector ( served as negative control ) were subcutaneously injected into 5 - week - old nude mice . Twenty days after injection , mice were analyzed by bioluminescence using Xenogen IVIS imaging system . Tumors expressing luciferase is indicated by radiance bar ( p / sec / cm2 / sr ) . C , Quantitation of bioluminescence with data from ( B ) . Data are shown as mean ± ; n = 5 .

O O B-CONTROLLED_VAR I-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 B-CONTROLLED_VAR 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, PTPN3 and PTPN3 (D811A), but not PTPN3 (L232R), attenuates tumorigenesis. Luciferase-harboring Huh7 tumor cells (2 × 106 cells/mouse) expressing PTPN3, PTPN3 (D811A) or PTPN3 (L232R) or empty vector (served as negative control) were subcutaneously injected into 5-week-old nude mice. Twenty days after injection, mice were analyzed by bioluminescence using Xenogen IVIS imaging system. Tumors expressing luciferase is indicated by radiance bar (p/sec/cm2/sr). C, Quantitation of bioluminescence with data from (B). Data are shown as mean ± SEM; n = 5.


D , Depletion of PTPN3 accelerates tumorigenesis . HepG2 stably expressing shPTPN3 or shControl were subcutaneously injected into nude mice . Fifty days after implantation , tumors were dissected photographed .

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

D, Depletion of PTPN3 accelerates tumorigenesis. HepG2 cells stably expressing shPTPN3 or shControl were subcutaneously injected into nude mice. Fifty days after cell implantation, tumors were dissected and photographed.


E , Depletion of PTPN3 increased sizes . volume was measured at indicated days post - implantation . Data are shown as mean ± ; n = 6 for each group . F , Depletion of PTPN3 increased . from tumors was recorded from both control shPTPN3 groups . Data are shown as mean ± ; n = 6 for each group .

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

E, Depletion of PTPN3 increased tumor sizes. Tumor volume was measured at indicated days post-implantation. Data are shown as mean ± SEM; n = 6 for each group. F, Depletion of PTPN3 increased tumor weight. Weight from all tumors was recorded from both control and shPTPN3 groups. Data are shown as mean ± SEM; n = 6 for each group.


, PTPN3 depletion impairs TGF - β signaling in tumors . of PTPN3 , GAPDH TGF - β signaling associated , such as TβRI , P - Smad2 , P - Smad3 , Smad2 / 3 , Smad4 , PAI - 1 , c - Myc p15 in tumors were analyzed by Western blotting .

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

, PTPN3 depletion impairs TGF-β signaling in tumors. Levels of PTPN3, GAPDH and TGF-β signaling associated proteins, such as TβRI, P-Smad2, P-Smad3, Smad2/3, Smad4, PAI-1, c-Myc and p15 in tumors were analyzed by Western blotting analysis.


H , Clinical significance of PTPN3 expression in LIHC on TCGA database . In boxplots , Y - axis represents expression level of PTPN3 in normal samples . boxes show median ( horizontal in box ) ± 1 quartile ; upper lower box limits represent upper lower quartiles , respectively . whiskers ( vertical lines ) extend from hinge to smallest or larget value within 1 . 5 interquartile range from box boundaries .

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 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

H, Clinical significance of PTPN3 expression in LIHC based on TCGA database. In the boxplots, Y-axis represents the expression level of PTPN3 in normal and tumor samples. The boxes show the median (horizontal line in the box) ± 1 quartile; the upper and lower box limits represent the upper and lower quartiles, respectively. The whiskers (vertical lines) extend from the hinge to the smallest or larget value within 1.5 interquartile range from the box boundaries.


( B ) Structured illumination microscopy image of perimCherry / cytoGFP E . coli confirming localization of mCherry ( red ) in periplasm GFP ( green ) in cytosol . Scale bar = 3 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

(B) Structured illumination microscopy image of perimCherry/cytoGFP E. coli confirming localization of mCherry (red) in the periplasm and GFP (green) in the cytosol. Scale bar = 3 µm.


( C ) Outer damage ( mCherry ) inner damage ( % Sytox positive ) of perimCherry / cytoGFP E . coli exposed to ( different concentrations of ) human serum . Inner damage correlates with killing ( samples where are killed are indicated with gray shadings a cross , see CFU data in Fig EV1b ) . Data information : ( C - E ) Data represent mean ± SD of 3 independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Outer membrane damage (mCherry intensity) and inner membrane damage (% Sytox positive) of perimCherry/cytoGFP E. coli bacteria exposed to (different concentrations of) human serum. Inner membrane damage correlates with killing (samples where bacteria are killed are indicated with gray shadings and a cross, see CFU data in Fig EV1b). Data information: (C-E) Data represent mean ± SD of 3 independent experiments.


( D ) Serum - induced inner damage ( % Sytox positive ) of different Gram - negative strains depends on MAC components C5 C8 , but not on lysozyme ( 10 % serum ) . Dotted represents detection limit of . Data information : ( D , E ) cfu / ml ( Fig . 1E , 2C ) sytox measurements ( Fig . 1D , 2E ) of " Buffer " , " Serum " , " ΔC5 " , " ΔC8 " " Δlysozym " " C5b6MAC " were generated from same experiment . ( C - E ) Data represent mean ± SD of 3 independent experiments . ( D , E ) Statistical was done using a paired two - t - displayed only when significant as * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 or * * * * p ≤ 0 . 0001 .

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

(D) Serum-induced inner membrane damage (% Sytox positive) of different Gram-negative strains depends on MAC components C5 and C8, but not on lysozyme (10% serum). Dotted line represents the detection limit of the assay. Data information: (D, E) The cfu/ml (Fig. 1E, 2C) and sytox measurements (Fig. 1D, 2E) of "Buffer", "Serum", "ΔC5", "ΔC8" "Δlysozym" and "C5b6MAC" were all generated from the same experiment. (C-E) Data represent mean ± SD of 3 independent experiments. (D, E) Statistical analysis was done using a ratio paired two-tailed t-test and displayed only when significant as * p≤0.05, ** p≤0.01, *** p≤0.001 or **** p≤0.0001.


, E Serum - induced killing ( CFU / ml ) of different Gram - negative strains depends on MAC components C5 C8 , but not on lysozyme ( 10 % serum ) . Dotted represents detection limit of . Data information : ( D , E ) cfu / ml ( Fig . 1E , 2C ) sytox measurements ( Fig . 1D , 2E ) of " Buffer " , " Serum " , " ΔC5 " , " ΔC8 " " Δlysozym " " C5b6MAC " were generated from same experiment . ( C - E ) Data represent mean ± SD of 3 independent experiments . ( D , E ) Statistical was done using a paired two - t - displayed only when significant as * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 or * * * * p ≤ 0 . 0001 .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Serum-induced killing (CFU/ml) of different Gram-negative strains depends on MAC components C5 and C8, but not on lysozyme (10% serum). Dotted line represents the detection limit of the assay. Data information: (D, E) The cfu/ml (Fig. 1E, 2C) and sytox measurements (Fig. 1D, 2E) of "Buffer", "Serum", "ΔC5", "ΔC8" "Δlysozym" and "C5b6MAC" were all generated from the same experiment. (C-E) Data represent mean ± SD of 3 independent experiments. (D, E) Statistical analysis was done using a ratio paired two-tailed t-test and displayed only when significant as * p≤0.05, ** p≤0.01, *** p≤0.001 or **** p≤0.0001.


( B ) Lysis of human erythrocytes after exposure to a range of preassembled C5b6 in presence of 100 nM C7 . After washing , erythrocytes were exposed to 20 nM C8 100 nM C9 for 30 minutes after which OD405 nm of supernatant was measured . ( B - E ) Data represent mean ± SD of 3 independent experiments .

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

(B) Lysis of human erythrocytes after exposure to a concentration range of preassembled C5b6 in the presence of 100 nM C7. After washing, erythrocytes were exposed to 20 nM C8 and 100 nM C9 for 30 minutes after which the OD405 nm of the supernatant was measured. (B-E) Data represent mean ± SD of 3 independent experiments.


C ) viability of three Gram - negative strains after exposure to buffer , 10 % human serum or C5b6MAC . Buffer serum conditions are same as Fig . 1E .

O O O O O O O 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) Bacterial viability of three Gram-negative strains after exposure to buffer, 10% human serum or C5b6MAC. Buffer and serum conditions are the same as Fig. 1E.


( D ) Permeabilization of outer , but not inner of perimCherry / cytoGFP E . coli exposed to C5b6MAC ( different concentrations of C5b6 with fixed concentrations of C7 - C9 ) . ( B - E ) Data represent mean ± SD of 3 independent experiments .

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

(D) Permeabilization of the outer, but not inner membrane of perimCherry/cytoGFP E. coli cells exposed to C5b6MAC (different concentrations of C5b6 with fixed concentrations of C7-C9). (B-E) Data represent mean ± SD of 3 independent experiments.


E ) Inner damage of three Gram - negative strains exposed to buffer , 10 % serum or C5b6MAC . Buffer serum conditions are same as Fig . 1D .

O O O O O O 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) Inner membrane damage of three Gram-negative strains exposed to buffer, 10% serum or C5b6MAC. Buffer and serum conditions are the same as Fig. 1D.


( B ) viability of convertase - labeled strains exposed to buffer ( Conv ) or C5 - C9 ( Conv - MAC ) . Data information : ( B - E ) Data represent mean ± SD of 3 independent experiments . ( B , D , E ) Statistical was done using a paired two - t - displayed only when significant as * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 or * * * * p ≤ 0 . 0001 .

O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Bacterial viability of convertase-labeled bacterial strains exposed to buffer (Conv) or C5-C9 (Conv-MAC). Data information: (B-E) Data represent mean ± SD of 3 independent experiments. (B, D, E) Statistical analysis was done using a ratio paired two-tailed t-test and displayed only when significant as * p≤0.05, ** p≤0.01, *** p≤0.001 or **** p≤0.0001.


( C ) viability of convertase - labeled E . coli MG1655 exposed to a range of C5 in presence of 100 nM C6 , 100 nM C7 , 20 nM C8 100 nM C9 . ' Ctrl ' indicates that are pretreated with heat - inactivated ΔC5 serum . Dotted represents detection limit of . Data information : ( B - E ) Data represent mean ± SD of 3 independent experiments .

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

(C) Bacterial viability of convertase-labeled E. coli MG1655 exposed to a concentration range of C5 in the presence of 100 nM C6, 100 nM C7, 20 nM C8 and 100 nM C9. 'Ctrl' indicates bacteria that are pretreated with heat-inactivated ΔC5 serum. Dotted line represents the detection limit of the assay. Data information: (B-E) Data represent mean ± SD of 3 independent experiments.


( D ) viability of convertase labeled E . coli MG1655 exposed to C5 - C9 or conditions lacking one MAC component . As an extra control , convertase formation was blocked during ΔC5 serum incubation by adding 5 µM compstatin . Data information : ( B - E ) Data represent mean ± SD of 3 independent experiments . ( B , D , E ) Statistical was done using a paired two - t - displayed only when significant as * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 or * * * * p ≤ 0 . 0001 .

O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Bacterial viability of convertase labeled E. coli MG1655 exposed to C5-C9 or conditions lacking one MAC component. As an extra control, convertase formation was blocked during ΔC5 serum incubation by adding 5 µM compstatin. Data information: (B-E) Data represent mean ± SD of 3 independent experiments. (B, D, E) Statistical analysis was done using a ratio paired two-tailed t-test and displayed only when significant as * p≤0.05, ** p≤0.01, *** p≤0.001 or **** p≤0.0001.


( E ) viability of E . coli MG1655 exposed to FB depleted serum in presence of 20 µg / ml OmCI ( to deposit C4b C3b without Bb ) . After washing , were exposed to C5 - C9 in presence or absence of C1 C2 ( to generate classical pathway C5 convertases , C4b2aC3b ) . Data information : ( B - E ) Data represent mean ± SD of 3 independent experiments . ( B , D , E ) Statistical was done using a paired two - t - displayed only when significant as * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 or * * * * p ≤ 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Bacterial viability of E. coli MG1655 exposed to FB depleted serum in the presence of 20 µg/ml OmCI (to deposit C4b and C3b without Bb). After washing, bacteria were exposed to C5-C9 in the presence or absence of C1 and C2 (to generate classical pathway C5 convertases, C4b2aC3b). Data information: (B-E) Data represent mean ± SD of 3 independent experiments. (B, D, E) Statistical analysis was done using a ratio paired two-tailed t-test and displayed only when significant as * p≤0.05, ** p≤0.01, *** p≤0.001 or **** p≤0.0001.


( A ) Outer damage ( mCherry ) inner damage ( % Sytox positive ) of convertase - labeled perimCherry / cytoGFP E . coli incubated with a range of C5 fixed concentrations of C6 - C9 . Data information : ( A - C ) Data represent mean ± SD of 3 independent experiments .

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

(A) Outer membrane damage (mCherry intensity) and inner membrane damage (% Sytox positive) of convertase-labeled perimCherry/cytoGFP E. coli cells incubated with a concentration range of C5 and fixed concentrations of C6-C9. Data information: (A-C) Data represent mean ± SD of 3 independent experiments.


( B ) Inner damage of perimCherry / cytoGFP E . coli exposed to a range of ΔC5 serum , after washing , to C5 - C9 . As controls , were incubated with heat inactivated ΔC5 serum or 5 µM compstatin was added to ΔC5 serum to block C3b deposition . Data information : ( A - C ) Data represent mean ± SD of 3 independent experiments .

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

(B) Inner membrane damage of perimCherry/cytoGFP E. coli exposed to a concentration range of ΔC5 serum and, after washing, to C5-C9. As controls, bacteria were incubated with heat inactivated ΔC5 serum or 5 µM compstatin was added to the ΔC5 serum to block C3b deposition. Data information: (A-C) Data represent mean ± SD of 3 independent experiments.


( C ) Inner damage of three different convertase - labeled exposed to buffer ( Conv ) or C5 - C9 ( Conv - MAC ) . Data information : ( A - C ) Data represent mean ± SD of 3 independent experiments . ( C ) Statistical was done using a paired two - t - displayed only when significant as * p ≤ 0 . 05 or * * p ≤ 0 . 01 .

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

(C) Inner membrane damage of three different convertase-labeled bacteria exposed to buffer (Conv) or C5-C9 (Conv-MAC). Data information: (A-C) Data represent mean ± SD of 3 independent experiments. (C) Statistical analysis was done using a ratio paired two-tailed t-test and displayed only when significant as * p≤0.05 or ** p≤0.01.


( D ) Confocal microscopy of convertase - labeled perimCherry / cytoGFP E . coli exposed to buffer ( Conv ) or C5 - C9 ( Conv - MAC ) . Unlabeled exposed to 1 % serum served as control . Green = GFP , Red = To - pro - 3 dye . Scale bars = 3 µm .

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 O O O O O O O O O O O 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) Confocal microscopy images of convertase-labeled perimCherry/cytoGFP E. coli exposed to buffer (Conv) or C5-C9 (Conv-MAC). Unlabeled bacteria exposed to 1% serum served as control. Green = GFP, Red = To-pro-3 DNA dye. Scale bars = 3 µm.


( B ) viability of convertase - labeled E . coli MG1655 exposed to Buffer ( Conv ) , preassembled C5b6 ( Conv + C5b6MAC ) or a mixture of C5 C6 ( Conv - MAC ) , in presence of C7 , C8 C9 . Dotted represents detection limit of . Data information : ( B - D ) Data represent mean ± SD of 3 independent experiments . ( B ) Statistical was done using a paired two - t - displayed only when significant as * * p ≤ 0 . 01 .

O O O O O O 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 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

(B) Bacterial viability of convertase-labeled E. coli MG1655 exposed to Buffer (Conv), preassembled C5b6 (Conv + C5b6MAC) or a mixture of C5 and C6 (Conv-MAC), in the presence of C7, C8 and C9. Dotted line represents the detection limit of the assay. Data information: (B-D) Data represent mean ± SD of 3 independent experiments. (B) Statistical analysis was done using a ratio paired two-tailed t-test and displayed only when significant as ** p≤0.01.


( C ) Inner damage ( % Sytox positive ) of convertase - labeled perimCherry / cytoGFP E . coli exposed to a range of preassembled C5b6 or a mixture of C5 C6 , in presence of 100 nM C7 . After washing , were exposed to 20 nM C8 100 nM C9 . Data information : ( B - D ) Data represent mean ± SD of 3 independent experiments . ( B ) Statistical was done using a paired two - t - displayed only when significant as * * p ≤ 0 . 01 .

O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Inner membrane damage (% Sytox positive) of convertase-labeled perimCherry/cytoGFP E. coli exposed to a concentration range of preassembled C5b6 or a mixture of C5 and C6, in the presence of 100 nM C7. After washing, bacteria were exposed to 20 nM C8 and 100 nM C9. Data information: (B-D) Data represent mean ± SD of 3 independent experiments. (B) Statistical analysis was done using a ratio paired two-tailed t-test and displayed only when significant as ** p≤0.01.


( D ) outer damage ( mCherry ) of convertase - labeled perimCherry / cytoGFP E . coli exposed to a range of preassembled C5b6 or a mixture of C5 C6 , in presence of 100 nM C7 . After washing , were exposed to 20 nM C8 100 nM C9 . Data information : ( B - D ) Data represent mean ± SD of 3 independent experiments . ( B ) Statistical was done using a paired two - t - displayed only when significant as * * p ≤ 0 . 01 .

O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) outer membrane damage (mCherry) of convertase-labeled perimCherry/cytoGFP E. coli exposed to a concentration range of preassembled C5b6 or a mixture of C5 and C6, in the presence of 100 nM C7. After washing, bacteria were exposed to 20 nM C8 and 100 nM C9. Data information: (B-D) Data represent mean ± SD of 3 independent experiments. (B) Statistical analysis was done using a ratio paired two-tailed t-test and displayed only when significant as ** p≤0.01.


A , B Step - wise assembly of MAC on convertase - labeled . Convertase - labeled were incubated with C5 / C6 or C5 / C6 / C7 for 15 minutes , subsequently washed ( @ ) or treated with 10 μg / ml Eculizumab ( Ecu ) . Then , remaining MAC components ( C7 - 9 for C5 / C6 or C8 - 9 for C5 / C6 / C7 respectively ) were added to incubation mixture . In control conditions ( Conv - MAC ) , remaining MAC components were added to incubation mixture without washing or adding an inhibitor . ( A ) Outer ( mCherry ) ( B ) inner damage ( % Sytox positive ) were determined . Data information : ( A - B ) Data represent mean ± SD of 3 independent experiments . Statistical was done using a paired two - t - in which conditions were compared to Conv - MAC displayed only when significant as * p ≤ 0 . 05 .

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 O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Step-wise assembly of MAC on convertase-labeled bacteria. Convertase-labeled bacteria were incubated with C5/C6 or C5/C6/C7 for 15 minutes, and subsequently washed (@) or treated with 10 μg/ml Eculizumab (Ecu). Then, the remaining MAC components (C7-9 for C5/C6 or C8-9 for C5/C6/C7 respectively) were added to the incubation mixture. In the control conditions (Conv-MAC), the remaining MAC components were added to the incubation mixture without washing or adding an inhibitor. (A) Outer membrane (mCherry) and (B) inner membrane damage (% Sytox positive) were determined. Data information: (A-B) Data represent mean ± SD of 3 independent experiments. Statistical analysis was done using a ratio paired two-tailed t-test in which the test conditions were compared to Conv-MAC and displayed only when significant as * p≤0.05.


( A ) Outer inner damage of convertase - labeled exposed to different combinations of MAC components . ' @ ' indicates a washing step . Data information : Data represent mean ± SD of 3 independent experiments . ( A ) Statistical was done using a one - way ANOVA displayed only when significant as * * p ≤ 0 . 01 or * * * * p ≤ 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Outer and inner membrane damage of convertase-labeled bacteria exposed to different combinations of MAC components. '@' indicates a washing step. Data information: Data represent mean ± SD of 3 independent experiments. (A) Statistical analysis was done using a one-way ANOVA and displayed only when significant as ** p≤0.01 or **** p≤0.0001.


( B ) Outer inner damage of convertase - labeled exposed to C5 - C8 after washing , to a range of C9 . Data information : Data represent mean ± SD of 3 independent experiments .

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

(B) Outer and inner membrane damage of convertase-labeled bacteria exposed to C5-C8 and after washing, to a concentration range of C9. Data information: Data represent mean ± SD of 3 independent experiments.


( A ) Convertase - labeled were exposed to a range of either preassembled C5b6 ( C5b6MAC ) or a mixture of C5 C6 ( Conv - MAC ) , in presence of 100 nM C7 . After washing , 20 nM C8 100 nM C9 - Cy3 was added . Controls at 0 nM C5b6 or C5 - C6 confirm that detected C9 - Cy3 deposition is specifically related to MAC formation ( solid lines ) . Proper insertion of pores was assessed by a previously described shaving method with trypsin ( Moskovich & Fishelson , 2007 ) . were first incubated with MAC components for 30 minutes subsequently treated with 20 µg / ml trypsin for 15 min at 37 ° C ( dotted lines ) . Data information : ( A ) Data represent mean ± SD of 3 independent experiments .

O O O O O O O O O O O O O O O O O O O O O O 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 B-MEASURED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Convertase-labeled bacteria were exposed to a concentration range of either preassembled C5b6 (C5b6MAC) or a mixture of C5 and C6 (Conv-MAC), in the presence of 100 nM C7. After washing, 20 nM C8 and 100 nM C9-Cy3 was added. Controls at 0 nM C5b6 or C5-C6 confirm that the detected C9-Cy3 deposition is specifically related to MAC formation (solid lines). Proper insertion of pores was assessed by a previously described shaving method with trypsin (Moskovich & Fishelson, 2007). Bacteria were first incubated with MAC components for 30 minutes and subsequently treated with 20 µg/ml trypsin for 15 min at 37°C (dotted lines). Data information: (A) Data represent mean ± SD of 3 independent experiments.


( B ) Convertase labeled perimCherry / cytoGFP ( Green ) exposed to C5b6MAC or Conv - MAC . Conditions were similar to those in ( A ) , however C9 - Cy5 was used to detect MAC pores ( Red ) . 100 nM of C5 C6 or C5b6 were used in combination with 100 nM C7 , 20 nM C8 100 nM C9 - Cy5 . Conv + C5b6MAC conv - MAC were taken in separate experiments in which settings were adjusted to of C9 - Cy5 to properly visualize pore distribution . Scale bars = 3 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 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 B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O

(B) Convertase labeled perimCherry/cytoGFP bacteria (Green) exposed to C5b6MAC or Conv-MAC. Conditions were similar to those in (A), however C9-Cy5 was used to detect MAC pores (Red). 100 nM of C5 and C6 or C5b6 were used in combination with 100 nM C7, 20 nM C8 and 100 nM C9-Cy5. Conv + C5b6MAC and conv-MAC images were taken in separate experiments in which laser settings were adjusted to the staining intensity of C9-Cy5 to properly visualize pore distribution. Scale bars = 3 µm.


( C Atomic force microscopy of E . coli BL21 MG1655 immobilized using Poly - L - Lysine protocol . ( C ) Entire high - resolution comparisons of untreated convertase - labeled E . coli BL21 exposed to C5 - C9 ( Conv - MAC ) for 10 minutes . Scale bars : 800 nm ( left ) 30 nm ( right ) . Height Scales : 1 . 01 µm ( left ) , 8 nm ( top right ) , 22 nm ( bottom right ) . Width of magnification boxes ( left ) : 42 nm , height scales : 8 nm ( top left ) 13 nm ( bottom left ) . Arrows highlight E . coli porin structures , an asterisk highlights C5b - 7 stalk . Height profiles ( bottom ) are shown for dashed lines in .

O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Atomic force microscopy analysis of E. coli BL21 and MG1655 immobilized using the Poly-L-Lysine protocol. (C) Entire bacteria and high-resolution comparisons of untreated and convertase-labeled E. coli BL21 exposed to C5-C9 (Conv-MAC) for 10 minutes. Scale bars: 800 nm (left) and 30 nm (right). Height Scales: 1.01 µm (left), 8 nm (top right), 22 nm (bottom right). Width of magnification boxes (left): 42 nm, height scales: 8 nm (top left) and 13 nm (bottom left). Arrows highlight E. coli porin structures, an asterisk highlights the C5b-7 stalk. Height profiles (bottom) are shown for the white dashed lines in the images.


( D ) Atomic force microscopy ( height phase ) of convertase - labeled E . coli MG1655 exposed to a mixture of C5 C6 ( Conv - MAC ) or preassembled C5b6 ( Conv + C5b6MAC ) , in presence of C7 , C8 , C9 , FB FD . were generated in same experiment . Scale bars : 50 nm . Height scales : 15 nm .

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 O O O O O O 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) Atomic force microscopy (height and phase images) of convertase-labeled E. coli MG1655 exposed to a mixture of C5 and C6 (Conv-MAC) or preassembled C5b6 (Conv + C5b6MAC), in the presence of C7, C8, C9, FB and FD. Images were generated in the same experiment. Scale bars: 50 nm. Height scales: 15 nm.


BrdU incorporation reflecting synthesis 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 ) .

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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 - FLT - FDG - PET scans of an control ; Bcl2 lymphoma - bearing mouse at tumour manifestation ( untreated ) 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 n = 6 mice for FDG - PET scans ) . Insets show representative Ki67 colour - calibrated ( red , highest intensities ; blue , lowest intensities ) bioluminescence of glucose in tissue sections of respective groups .

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

b, 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 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 ) .

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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 of specified metabolite - to - glucose ratios in indicated lymphoma groups relative to untreated control ; Bcl2 lymphoma 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 medium . Insets show ( ADR - treated relative to untreated ) intracellular of 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 heterogeneity of TIS - associated hypermetabolism a comparison of metabolic parameters of lymphomas ) .

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

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 kinase ( pAMPK ) , total AMPK , pyruvate kinase isoforms M1 M2 in ADR - senescent control ; Bcl2 versus ADR - treated Suv39h1 - ; Bcl2 lymphoma in vitro ( n = 3 each ; α - tubulin as a loading control . Gels were cropped to ease presentation ; full - 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 B-CONTROLLED_VAR 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 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 in vitro . Results represent means ± s . d . ( n = 5 each ) .

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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 as in f . Results represent means ± s . d . ( n = 5 each ) . * 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

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


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

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 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

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 .

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 ( shHk2 ) . d , Inhibition of AMPK by compound C . e , Block of fatty acid oxidation by carnitine palmitoyltransferase I inhibitor etomoxir . f , Inhibition of electron transport chain by antimycin A . * P 0 . 01 , except b ( n = 8 each ) e , in which * P 0 . 05 .

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

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

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


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

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

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 ubiquitination , expression of p62 / SQSTM1 ( whose transcript were also selectively induced in TIS by approximately 4 . 8 - fold ( data not shown ) ) V - ATPase subunit A1 , conversion of MAP1 - LC3 - I to MAP1 - LC3 - II by immunoblot , demonstrating autophagosome formation in lymphoma as in f ( α - tubulin was used as a loading control , gels were cropped to ease presentation ; full - blots are provided in Supplementary Fig . 16 ) . * P < 0 . 05 .

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

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

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

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 , viability of ADR - treated control ; Bcl2 lymphoma infected with short - hairpin RNAs against either caspase 12 ( shCasp12 ) or caspase 3 ( shCasp3 ) compared to empty vector - infected lymphoma 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 B-CONTROLLED_VAR I-CONTROLLED_VAR 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

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

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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 ) ATP ( right ) concentrations in senescent AML as in e .

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


g , viability of AML samples treated as in e 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

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 - imaging to visualize green ( GFP ) - tagged tumour burden in time - courses of three representative mice harbouring CTX - senescent control ; Bcl2 lymphomas at day 5 ( see Figure 1a ) , two days later , after 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 ( ) ( TUNEL ) 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Suv39h1 - ; Bcl2 : n = 10 ) , presented as matched pair analyses ( P < 0 . 001 for control ; Bcl2 comparison ; P = 0 . 221 for Suv39h1 - ; Bcl2 comparison ) . * P < 0 . 05 .

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


A Control ( + / w ; UAS - ralIR / + ) , n = 20 , those with salivary - specific knockdown of ral ( fkh - GAL4 / w ; UAS - ralIR / + ) , n = 20 , were analyzed by histology for presence of salivary material ( red dotted circle ) 24 hours after puparium formation . B of data from ( A ) . Data are represented as means . Statistical significance was determined using a Chi - square . C Control ( + / w ; UAS - ralS25N / + ) , n = 20 , those with salivary - specific expression of dominant - negative Ral ( fkh - GAL4 / w ; UAS - ralS25N / + ) , n = 20 , were analyzed by histology for presence of salivary material ( red dotted circle ) 24 hours after puparium formation . D of data from ( C ) . Data are represented as means . Statistical significance was determined using a Chi - square . E Control ( ral35d / + ) , n = 25 , ral hypomorph mutants ( ral35d / Y ) , n = 23 , were analyzed by histology for presence of salivary material ( red dotted circle ) 24 hours after puparium formation . F of data from ( E ) . Data are represented as means . Statistical significance was determined using a Chi - square .

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

A Control animals (+/w; UAS-ralIR/+), n= 20, and those with salivary gland-specific knockdown of ral (fkh-GAL4/w; UAS-ralIR/+), n=20, were analyzed by histology for the presence of salivary gland material (red dotted circle) 24 hours after puparium formation.B Quantification of data from (A). Data are represented as means. Statistical significance was determined using a Chi-square test.C Control animals (+/w; UAS-ralS25N/+), n= 20, and those with salivary gland-specific expression of dominant-negative Ral (fkh-GAL4/w; UAS-ralS25N/+), n= 20, were analyzed by histology for the presence of salivary gland material (red dotted circle) 24 hours after puparium formation.D Quantification of data from (C). Data are represented as means. Statistical significance was determined using a Chi-square test.E Control animals (ral35d/+), n= 25, and ral hypomorph mutants (ral35d/Y), n= 23, were analyzed by histology for the presence of salivary gland material (red dotted circle) 24 hours after puparium formation.F Quantification of data from (E). Data are represented as means. Statistical significance was determined using a Chi-square test.


G Control ( + / w ; UAS - rglIR / + ) , n = 19 , those with salivary - specific knockdown of rgl ( fkh - GAL4 / w ; UAS - rglIR / + ) , n = 20 , were analyzed by histology for presence of salivary material ( red dotted circle ) 24 hours after puparium formation . H of data from ( G ) . Data are represented as means . Statistical significance was determined using a Chi - square .

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 B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 animals (+/w; UAS-rglIR/+), n= 19, and those with salivary gland-specific knockdown of rgl (fkh-GAL4/w; UAS-rglIR/+), n=20, were analyzed by histology for the presence of salivary gland material (red dotted circle) 24 hours after puparium formation.H Quantification of data from (G). Data are represented as means. Statistical significance was determined using a Chi-square test.


A ral hypomorph mutants ( ral35d / Y ; ; UAS - p35 / + ) , n = 20 , with salivary - specific expression of p35 ( ral35d / + ; ; UAS - p35 / fkh - GAL4 ) , n = 21 , ral hypomorph mutants with salivary - specific expression of p35 ( ral35d / Y ; ; UAS - p35 / fkh - GAL4 ) , n = 20 , were analyzed by histology for presence of salivary material ( red dotted circles ) 24 hours after puparium formation . B of data from ( A ) . Data are represented as means . Statistical significance was determined using a Chi - square comparing percentages of fragments .

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

A ral hypomorph mutants (ral35d/Y;; UAS-p35/+), n= 20, animals with salivary gland-specific expression of p35 (ral35d/+;; UAS-p35/fkh-GAL4), n= 21, and ral hypomorph mutants with salivary gland-specific expression of p35 (ral35d/Y;; UAS-p35/fkh-GAL4), n= 20, were analyzed by histology for the presence of salivary gland material (red dotted circles) 24 hours after puparium formation.B Quantification of data from (A). Data are represented as means. Statistical significance was determined using a Chi-square test comparing the percentages of gland fragments.


C Salivary dissected from 0 hour after puparium formation control ( + / w ; UAS - ralIR / + ) those with salivary - specific knockdown of ral ( fkh - GAL4 / w ; UAS - ralIR / + ) stained with Caspase - 3 antibody ( green ) DAPI ( blue ) . Scale bars represent 20µm . D Salivary dissected from 13 hour after puparium formation control ( + / w ; UAS - ralIR / + ) those with salivary - specific knockdown of ral ( fkh - GAL4 / w ; UAS - ralIR / + ) stained with Caspase - 3 antibody ( green ) DAPI ( blue ) . Scale bars represent 20µm . E of data from ( C D ) . Data are represented as means ± ; n ≥ 10 . Statistical significance was determined using a Student ' s t .

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

C Salivary glands dissected from 0 hour after puparium formation control animals (+/w; UAS-ralIR/+) and those with salivary gland-specific knockdown of ral (fkh-GAL4/w; UAS-ralIR/+) and stained with cleaved Caspase-3 antibody (green) and DAPI (blue). Scale bars represent 20µm.D Salivary glands dissected from 13 hour after puparium formation control animals (+/w; UAS-ralIR/+) and those with salivary gland-specific knockdown of ral (fkh-GAL4/w; UAS-ralIR/+) and stained with cleaved Caspase-3 antibody (green) and DAPI (blue). Scale bars represent 20µm.E Quantification of data from (C and D). Data are represented as means ± SEM; n≥ 10. Statistical significance was determined using a Student's t test.


A ral hypomorph mutants ( ral35d / Y ; ; UAS - Atg6IR / + ) , n = 27 , with salivary - specific expression of Atg6IR ( ral35d / + ; ; UAS - Atg6IR / fkh - GAL4 ) , n = 20 , ral hypomorph mutants with salivary - specific expression of Atg6IR ( ral35d / Y ; ; UAS - Atg6IR / fkh - GAL4 ) , n = 24 , were analyzed by histology for presence of salivary material ( red dotted circles ) 24 hours after puparium formation . B of data from ( A ) . Data are represented as means . Statistical significance was determined using a Chi - square .

O B-CONTROLLED_VAR 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 B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR 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 B-CONTROLLED_VAR 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 O O O O O O 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 ral hypomorph mutants (ral35d/Y;; UAS-Atg6IR/+), n= 27, animals with salivary gland-specific expression of Atg6IR (ral35d/+;; UAS-Atg6IR/fkh-GAL4), n= 20, and ral hypomorph mutants with salivary gland-specific expression of Atg6IR (ral35d/Y;; UAS-Atg6IR/fkh-GAL4), n= 24, were analyzed by histology for the presence of salivary gland material (red dotted circles) 24 hours after puparium formation.B Quantification of data from (A). Data are represented as means. Statistical significance was determined using a Chi-square test.


C Salivary dissected from 14 hours after puparium formation expressing mCherry - Atg8a in , ralIR specifically in GFP - marked clone ( yw , hsflp / w ; pmCherry - Atg8a / UAS - ralIR ; act < FRT , cd2 , FRT > GAL4 , UAS - GFP / + ) analyzed for mCherry - Atg8a . GFP - marked clone ralIR - expressing contain a yellow asterisk . Scale bars represent 20µm . D of data from ( C ) . Data are represented as means ± ; n ≥ 10 . Statistical significance was determined using a Student ' s t . E Salivary dissected from 14 hours after puparium formation that have ral loss of function ( ralPG89 ) specifically in non - RFP , that express GFP - Atg8a in ( yw , hs - Flp , FRT19A , Ubi - RFP / ralPG89 , FRT19A ; pGFP - Atg8a ) analyzed for GFP - Atg8a . ralPG89 mutant are marked with a yellow asterisk . Scale bars represent 20µm . F of data from ( E ) . Data are represented as means ± ; n ≥ 10 . Statistical significance was determined using a Student ' s t . G Salivary dissected from 14 hours after puparium formation that have ral loss of function ( ralPG89 ) specifically in non - RFP ( yw , hs - Flp , FRT19A , Ubi - RFP / ralPG89 , FRT19A ) are stained for Ref ( 2 ) p ( green ) DAPI ( blue ) . ralPG89 mutant are marked with a yellow asterisk . Scale bars represent 20µm . H of data from ( G ) . Data are represented as means ± ; n ≥ 10 . Statistical significance was determined using a Student ' s t .

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

C Salivary glands dissected from 14 hours after puparium formation animals expressing mCherry-Atg8a in all cells, and ralIR specifically in GFP-marked clone cells (yw, hsflp/w; pmCherry-Atg8a/UAS-ralIR; act< FRT, cd2, FRT> GAL4, UAS-GFP/+) analyzed for mCherry-Atg8a puncta. GFP-marked clone ralIR-expressing cells contain a yellow asterisk. Scale bars represent 20µm.D Quantification of data from (C). Data are represented as means ± SEM; n≥ 10. Statistical significance was determined using a Student's t test.E Salivary glands dissected from 14 hours after puparium formation animals that have ral loss of function (ralPG89) specifically in non-RFP cells, and that express GFP-Atg8a in all cells (yw, hs-Flp, FRT19A, Ubi-RFP/ ralPG89,FRT19A; pGFP-Atg8a) analyzed for GFP-Atg8a puncta. ralPG89 mutant cells are marked with a yellow asterisk. Scale bars represent 20µm.F Quantification of data from (E). Data are represented as means ± SEM; n≥ 10. Statistical significance was determined using a Student's t test.G Salivary glands dissected from 14 hours after puparium formation animals that have ral loss of function (ralPG89) specifically in non-RFP cells (yw, hs-Flp, FRT19A, Ubi-RFP/ ralPG89,FRT19A) and are stained for Ref(2)p (green) and DAPI (blue). ralPG89 mutant cells are marked with a yellow asterisk. Scale bars represent 20µm.H Quantification of data from (G). Data are represented as means ± SEM; n≥ 10. Statistical significance was determined using a Student's t test.


I Fat bodies dissected from 4 hour starved that have ral loss of function ( ralPG89 ) specifically in non - RFP , that express GFP - Atg8a in ( yw , hs - Flp , FRT19A , Ubi - RFP / ralPG89 , FRT19A ; pGFP - Atg8a ) analyzed for GFP - Atg8a . ralPG89 mutant are marked with a yellow asterisk . Scale bars represent 20µm . J of data from ( I ) . Data are represented as means ± ; n ≥ 10 . Statistical significance was determined using a Student ' s t .

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

I Fat bodies dissected from 4 hour starved animals that have ral loss of function (ralPG89) specifically in non-RFP cells, and that express GFP-Atg8a in all cells (yw, hs-Flp, FRT19A, Ubi-RFP/ ralPG89,FRT19A; pGFP-Atg8a) analyzed for GFP-Atg8a puncta. ralPG89 mutant cells are marked with a yellow asterisk. Scale bars represent 20µm.J Quantification of data from (I). Data are represented as means ± SEM; n≥ 10. Statistical significance was determined using a Student's t test.


A , C , E , G , I Fat bodies dissected from 4 hour starved expressing mCherry - Atg8a in , ( A ) sec5IR , ( C ) sec15IR , ( E ) sec3IR , ( G ) sec8IR , ( I ) exo84IR specifically in GFP - marked clone ( yw , hsflp / w ; pmCherry - Atg8a / + ; act < FRT , cd2 , FRT > GAL4 , UAS - GFP / + ) analyzed for mCherry - Atg8a . Scale bars represent 20µm . B , D , F , H , J of data from ( A , C , E , G , I ) . Data are represented as means ± ; n ≥ 10 . Statistical significance was determined using a Student ' s t .

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

A, C, E, G, I Fat bodies dissected from 4 hour starved animals expressing mCherry-Atg8a in all cells, and (A) sec5IR, (C) sec15IR, (E) sec3IR, (G) sec8IR, (I) exo84IR specifically in GFP-marked clone cells (yw, hsflp/w; pmCherry-Atg8a/+; act< FRT, cd2, FRT> GAL4, UAS-GFP/+) analyzed for mCherry-Atg8a puncta. Scale bars represent 20µm.B, D, F, H, J Quantification of data from (A, C, E, G, I). Data are represented as means ± SEM; n≥ 10. Statistical significance was determined using a Student's t test.


A Salivary dissected from 14 hours after puparium formation ( top left ) control ( fkh - GAL4 / + ; pmCherry - Atg8a / + ) , those with salivary - specific knockdown of ( middle left ) sec5 ( fkh - GAL4 / w ; pmCherry - Atg8a / + ; UAS - sec5IR / + ) , ( bottom left ) sec15 ( fkh - GAL4 / w ; pmCherry - Atg8a / + ; UAS - sec15IR / + ) , ( top right ) sec3 ( fkh - GAL4 / w ; pmCherry - Atg8a / UAS - sec3IR ) , ( middle right ) sec8 ( fkh - GAL4 / w ; pmCherry - Atg8a / UAS - sec8IR ) , ( bottom right ) exo84 ( fkh - GAL4 / w ; pmCherry - Atg8a / UAS - exo84IR ) analyzed for mCherry - Atg8a . Scale bars represent 20µm . B of data from ( A ) . Data are represented as means ± ; n ≥ 10 . Statistical significance was determined using a Student ' s t ( * * p < 0 . 001 , * * * p < 0 . 0001 ) .

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

A Salivary glands dissected from 14 hours after puparium formation (top left) control (fkh-GAL4/+; pmCherry-Atg8a/+), and those with salivary gland-specific knockdown of (middle left) sec5 (fkh-GAL4/w; pmCherry-Atg8a/+; UAS-sec5IR/+), (bottom left) sec15 (fkh-GAL4/w; pmCherry-Atg8a/+; UAS-sec15IR/+), (top right) sec3 (fkh-GAL4/w; pmCherry-Atg8a/UAS-sec3IR), (middle right) sec8 (fkh-GAL4/w; pmCherry-Atg8a/UAS-sec8IR), (bottom right) exo84 (fkh-GAL4/w; pmCherry-Atg8a/UAS-exo84IR) analyzed for mCherry-Atg8a puncta. Scale bars represent 20µm.B Quantification of data from (A). Data are represented as means ± SEM; n≥ 10. Statistical significance was determined using a Student's t test (** p< 0.001, *** p< 0.0001).


A Control ( + / w ; UAS - sec5IR / + ) , n = 19 , those with salivary - specific knockdown of sec5 ( fkh - GAL4 / w ; UAS - sec5IR / + ) , n = 20 , were analyzed by histology for presence of salivary material ( red dotted circle ) 24 hours after puparium formation . B of data from ( A ) . Data are represented as means . Statistical significance was determined using a Chi - square . C Control ( + / w ; UAS - sec15IR / + ) , n = 20 , those with salivary - specific expression sec15 ( fkh - GAL4 / w ; UAS - sec15IR / + ) , n = 20 , were analyzed by histology for presence of salivary material ( red dotted circle ) 24 hours after puparium formation . D of data from ( C ) . Data are represented as means . Statistical significance was determined using a Chi - square . E Control ( + / w ; UAS - sec3IR / + ) , n = 20 , those with salivary - specific expression sec3 ( fkh - GAL4 / w ; UAS - sec3IR / + ) , n = 20 , were analyzed by histology for presence of salivary material ( red dotted circle ) 24 hours after puparium formation . F of data from ( E ) . Data are represented as means . Statistical significance was determined using a Chi - square . G Control ( + / w ; UAS - sec8IR / + ) , n = 20 , those with salivary - specific knockdown of sec8 ( fkh - GAL4 / w ; UAS - sec8IR / + ) , n = 19 , were analyzed by histology for presence of salivary material ( red dotted circle ) 24 hours after puparium formation . H of data from ( G ) . Data are represented as means . Statistical significance was determined using a Chi - square . I Control ( + / w ; UAS - exo84IR / + ) , n = 19 , those with salivary - specific knockdown of exo84 ( fkh - GAL4 / w ; UAS - exo84IR / + ) , n = 20 , were analyzed by histology for presence of salivary material ( red dotted circle ) 24 hours after puparium formation . J of data from ( I ) . Data are represented as means . Statistical significance was determined using a Chi - square .

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A Control animals (+/w; UAS-sec5IR/+), n= 19, and those with salivary gland-specific knockdown of sec5 (fkh-GAL4/w; UAS-sec5IR/+), n=20, were analyzed by histology for the presence of salivary gland material (red dotted circle) 24 hours after puparium formation.B Quantification of data from (A). Data are represented as means. Statistical significance was determined using a Chi-square test.C Control animals (+/w; UAS-sec15IR/+), n= 20, and those with salivary gland-specific expression sec15 (fkh-GAL4/w; UAS-sec15IR/+), n= 20, were analyzed by histology for the presence of salivary gland material (red dotted circle) 24 hours after puparium formation.D Quantification of data from (C). Data are represented as means. Statistical significance was determined using a Chi-square test.E Control animals (+/w; UAS-sec3IR/+), n= 20, and those with salivary gland-specific expression sec3 (fkh-GAL4/w; UAS-sec3IR/+), n= 20, were analyzed by histology for the presence of salivary gland material (red dotted circle) 24 hours after puparium formation.F Quantification of data from (E). Data are represented as means. Statistical significance was determined using a Chi-square test.G Control animals (+/w; UAS-sec8IR/+), n= 20, and those with salivary gland-specific knockdown of sec8 (fkh-GAL4/w; UAS-sec8IR/+), n=19, were analyzed by histology for the presence of salivary gland material (red dotted circle) 24 hours after puparium formation.H Quantification of data from (G). Data are represented as means. Statistical significance was determined using a Chi-square test.I Control animals (+/w; UAS-exo84IR/+), n= 19, and those with salivary gland-specific knockdown of exo84 (fkh-GAL4/w; UAS-exo84IR/+), n=20, were analyzed by histology for the presence of salivary gland material (red dotted circle) 24 hours after puparium formation.J Quantification of data from (I). Data are represented as means. Statistical significance was determined using a Chi-square test.


A Salivary dissected from late 3rd instar larvae ( Gbe Su ( H ) - lac - Z / w - ; + ; Fkh - GAL4 / + ) show less notch activity ( left ) compared to from 14 hours after puparium formation ( APF ) ( middle ) . Down - regulation of ral in salivary from 14 hours APF ( Gbe Su ( H ) - lac - Z / w - ; UAS - ralIR / + ; Fkh - GAL4 / + ) reduces of notch activity ( right ) . Scale bar represents 20µm . B of data from ( A ) . Data are represented as means ± ; n ≥ 4 . Statistical significance was determined using a Student ' s t .

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A Salivary glands dissected from late 3rd instar larvae (Gbe Su(H)-lac-Z/w-;+;Fkh-GAL4/+) show less notch activity (left) compared to glands from 14 hours after puparium formation (APF) animals (middle). Down-regulation of ral in salivary glands from 14 hours APF animals (Gbe Su(H)-lac-Z/w-;UAS-ralIR/+;Fkh-GAL4/+) reduces the levels of notch activity (right). Scale bar represents 20µm.B Quantification of data from (A). Data are represented as means ± SEM; n≥ 4. Statistical significance was determined using a Student's t test.


C Salivary dissected from 14 hours after puparium formation expressing mCherry - Atg8a in , notchIR specifically in GFP - marked clone ( yw , hsflp / w , UAS - notchIR ; pmCherry - Atg8a / + ; act < FRT , cd2 , FRT > GAL4 , UAS - GFP / + ) analyzed for mCherry - Atg8a . Scale bar represents 20µm . D of data from ( C ) . Data are represented as means ± ; n > 10 . Statistical significance was determined using a Student ' s t .

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C Salivary glands dissected from 14 hours after puparium formation animals expressing mCherry-Atg8a in all cells, and notchIR specifically in GFP-marked clone cells (yw, hsflp/w,UAS-notchIR; pmCherry-Atg8a/+; act< FRT, cd2, FRT> GAL4, UAS-GFP/+) analyzed for mCherry-Atg8a puncta. Scale bar represents 20µm.D Quantification of data from (C). Data are represented as means ± SEM; n> 10. Statistical significance was determined using a Student's t test.


( A ) Inducible GFP ‐ S ‐ Ubqln1 293 was treated with 1 μg / ml of doxycycline for 24 h . GFP ‐ S ‐ Ubqln1 was immunoprecipitated sent for mass spectrometry . Red sequences indicate Ubqln4 peptides identified by mass spectrometry .

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(A) Inducible GFP‐S‐Ubqln1 293 cell line was treated with 1 μg/ml of doxycycline for 24 h. GFP‐S‐Ubqln1 was immunoprecipitated and sent for mass spectrometry analysis. Red sequences indicate Ubqln4 peptides identified by mass spectrometry.


( B ) 293 were co ‐ transfected with GFP ‐ S or GFP ‐ S ‐ Ubqln1 HA ‐ Ubqln4 . GFP was immunoprecipitated analysed by western blot using anti ‐ GFP anti ‐ HA antibodies . Lower molecular GFP ‐ tag ‐ containing bands that appear in GFP ‐ S ‐ Ubqln1 lane might represent degradation products .

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(B) 293 cells were co‐transfected with GFP‐S or GFP‐S‐Ubqln1 and HA‐Ubqln4. GFP was immunoprecipitated and analysed by western blot using anti‐GFP and anti‐HA antibodies. Lower molecular weight GFP‐tag‐containing bands that appear in the GFP‐S‐Ubqln1 lane might represent degradation products.


( C ) Endogenous Ubqln1 in 293 was immunoprecipitated using a rabbit Ubqln1 antibody or a control rabbit IgG . immunoprecipitates were analysed by western blotting using anti ‐ Ubqln1 anti ‐ Ubqln4 antibodies .

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(C) Endogenous Ubqln1 in 293 cells was immunoprecipitated using a rabbit Ubqln1 antibody or a control rabbit IgG. The immunoprecipitates were analysed by western blotting using anti‐Ubqln1 and anti‐Ubqln4 antibodies.


( D ) Confocal of Hela transfected with GFP ‐ S ‐ Ubqln1 RFP ‐ Ubqln4 . Linescan of in merged image indicates intensities of molecules in cross ‐ section . GFP , green ; HA , haemagglutinin ; IP , immunoprecipitation ; Ubqln , Ubiquilin ; WCL , whole ‐ lysate .

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(D) Confocal images of Hela cells transfected with GFP‐S‐Ubqln1 and RFP‐Ubqln4. Linescan of the white line in the merged image indicates the intensities of the fluorescent molecules in the cross‐section. GFP, green fluorescent protein; HA, haemagglutinin; IP, immunoprecipitation; Ubqln, Ubiquilin; WCL, whole‐cell lysate.


Ubqln4 interacts with LC3 . Confocal of ( A ) Hela transfected with GFP ‐ LC3 RFP ‐ Ubqln4 starved for 1 h

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Ubqln4 interacts with LC3. Confocal images of (A) Hela cells transfected with GFP‐LC3 and RFP‐Ubqln4 and starved for 1 h or


( B ) Hela starved for 1 h stained for endogenous LC3 Ubqln4 . squares show colocalization . insets show enlargement of indicated areas . Linescan of in merged image indicates intensities of molecules in cross ‐ section .

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(B) Hela cells starved for 1 h and stained for endogenous LC3 and Ubqln4. The squares show colocalization. The insets show the enlargement of the indicated areas. Linescan of the white line in the merged image indicates the intensities of the fluorescent molecules in the cross‐section.


( C ) 293 were transfected with GFP ‐ S , GFP ‐ S ‐ Ubqln1 or GFP ‐ S ‐ Ubqln4 treated with 50 μM chloroquine for 14 h . GFP was immunoprecipitated analysed by western blot using anti ‐ GFP anti ‐ LC3 antibodies .

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(C) 293 cells were transfected with GFP‐S, GFP‐S‐Ubqln1 or GFP‐S‐Ubqln4 and treated with 50 μM chloroquine for 14 h. GFP was immunoprecipitated and analysed by western blot using anti‐GFP and anti‐LC3 antibodies.


( D ) Recombinant His ‐ Ubqln1 , His ‐ Ubqln4 , His ‐ S5a or His ‐ p62 was mixed with LC3 covalently coupled to agarose beads . beads were washed , eluted detected by western blot using anti ‐ His antibody . extent of Ubqln1 , Ubqln4 , s5a or p62 co ‐ immunoprecipitation with LC3 was measured . Bars for Ubqln1 , Ubqln4 s5a represents average s . e . m . of four independent experiments . * P ‐ value = 0 . 0094 .

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(D) Recombinant His‐Ubqln1, His‐Ubqln4, His‐S5a or His‐p62 was mixed with LC3 covalently coupled to agarose beads. The beads were washed, eluted and detected by western blot using anti‐His antibody. The extent of Ubqln1, Ubqln4, s5a or p62 co‐immunoprecipitation with LC3 was measured. Bars for Ubqln1, Ubqln4 and s5a represents the average and s.e.m. of four independent experiments. *P‐value=0.0094.


( E ) 293 were transfected with GFP ‐ S ‐ Ubqln4 WT , GFP ‐ S ‐ Ubqln4 ΔSTI1 12 , GFP ‐ S ‐ Ubqln4 Δ between STI1 or GFP ‐ S ‐ Ubqln4 ΔSTI1 34 treated with 50 μM chloroquine for 14 h . GFP was immunoprecipitated analysed by western blot using anti ‐ GFP anti ‐ LC3 antibodies . GFP , green ; IP , immunoprecipitation ; LC3 , microtubule ‐ associated chain 3 ; Ubqln , Ubiquilin ; WCL , whole ‐ lysate ; WT , wild ‐ type .

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(E) 293 cells were transfected with GFP‐S‐Ubqln4 WT, GFP‐S‐Ubqln4 ΔSTI1 12, GFP‐S‐Ubqln4 Δ between STI1 or GFP‐S‐Ubqln4 ΔSTI1 34 and treated with 50 μM chloroquine for 14 h. GFP was immunoprecipitated and analysed by western blot using anti‐GFP and anti‐LC3 antibodies. GFP, green fluorescent protein; IP, immunoprecipitation; LC3, microtubule‐associated protein light chain 3; Ubqln, Ubiquilin; WCL, whole‐cell lysate; WT, wild‐type.


( A ) Confocal of Hela transfected with GFP ‐ S ‐ Ubqln1 also transfected with either control or Ubqln4 siRNA . were either untreated or were transferred to starvation medium for 1 h .

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(A) Confocal images of Hela cells transfected with GFP‐S‐Ubqln1 and also transfected with either control or Ubqln4 siRNA. The cells were either untreated or were transferred to starvation medium for 1 h.


( B ) of number of GFP ‐ S ‐ Ubqln1 WT per in ( A ) . Each bar represents average s . e . m . of 30 per condition in three independent experiments . * P ‐ value = 0 . 011 , * * P ‐ value = 0 . 0005 * * * P ‐ value = 0 . 0001 .

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(B) Quantification of the number of GFP‐S‐Ubqln1 WT puncta per cell in (A). Each bar represents the average and s.e.m. of 30 cells per condition in three independent experiments. *P‐value=0.011, **P‐value=0.0005 and ***P‐value=0.0001.


( C ) Depletion of Ubqln4 with siRNA in ( A ) was verified by western blot using anti ‐ Ubqln1 , anti ‐ Ubqln4 anti ‐ tubulin antibodies .

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(C) Depletion of Ubqln4 with siRNA in (A) was verified by western blot using anti‐Ubqln1, anti‐Ubqln4 and anti‐tubulin antibodies.


( D ) Hela transfected with GFP ‐ S ‐ Ubqln1 also transfected with either control or Ubqln4 siRNA were starved for 1 h . GFP was immunoprecipitated analysed by western blot using anti ‐ GFP anti ‐ LC3 antibodies .

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(D) Hela cells transfected with GFP‐S‐Ubqln1 and also transfected with either control or Ubqln4 siRNA were starved for 1 h. GFP was immunoprecipitated and analysed by western blot using anti‐GFP and anti‐LC3 antibodies.


( E ) Confocal of GFP ‐ S ‐ Ubqln1 WT in Hela transfected with RFP , RFP ‐ Ubqln4 or RFP ‐ Ubqln4 ΔUBL .

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(E) Confocal images of GFP‐S‐Ubqln1 WT in Hela cells transfected with RFP, RFP‐Ubqln4 or RFP‐Ubqln4 ΔUBL.


( F ) of number of GFP ‐ S ‐ Ubqln1 WT per in ( E ) . Each bar represents average s . e . m . of 30 per condition in three independent experiments . * P ‐ value = 0 . 0001 * * P ‐ value = 0 . 0003 . GFP , green ; IP , immunoprecipitation ; LC3 , microtubule ‐ associated chain 3 ; UBL , ubiquitin ‐ like ; Ubqln , Ubiquilin ; WCL , whole ‐ lysate ; WT , wild ‐ type .

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(F) Quantification of the number of GFP‐S‐Ubqln1 WT puncta per cell in (E). Each bar represents the average and s.e.m. of 30 cells per condition in three independent experiments. *P‐value=0.0001 and **P‐value=0.0003. GFP, green fluorescent protein; IP, immunoprecipitation; LC3, microtubule‐associated protein light chain 3; UBL, ubiquitin‐like; Ubqln, Ubiquilin; WCL, whole‐cell lysate; WT, wild‐type.


( A ) 293 were co ‐ transfected with GFP ‐ S ‐ Ubqln1 , GFP ‐ S ‐ Ubqln1 ΔUBL ( deletion of UBL domain ) or GFP ‐ S ‐ Ubqln1 ΔUBA ( deletion of UBA domain ) HA ‐ Ubqln4 . GFP was immunoprecipitated analysed by western blot using anti ‐ GFP anti ‐ HA antibodies .

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(A) 293 cells were co‐transfected with GFP‐S‐Ubqln1, GFP‐S‐Ubqln1 ΔUBL (deletion of UBL domain) or GFP‐S‐Ubqln1 ΔUBA (deletion of UBA domain) and HA‐Ubqln4. GFP was immunoprecipitated and analysed by western blot using anti‐GFP and anti‐HA antibodies.


( B ) extent of Ubqln4 co ‐ immunoprecipitation with Ubqln1 was measured of Ubqln4 to Ubqln1 was calculated for WT Ubqln1 , Ubqln1ΔUBL Ubqln1ΔUBA . numbers represent of Ubqln4 to Ubqln1 relative to WT . Each bar represents average s . e . m . of four independent experiments . * P ‐ value = 0 . 0041 .

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(B) The extent of Ubqln4 co‐immunoprecipitation with Ubqln1 was measured and the ratio of Ubqln4 to Ubqln1 was calculated for WT Ubqln1, Ubqln1ΔUBL and Ubqln1ΔUBA. The numbers represent the ratio of Ubqln4 to Ubqln1 relative to WT. Each bar represents the average and s.e.m. of four independent experiments. *P‐value=0.0041.


( C ) 293 were co ‐ transfected with GFP ‐ S or GFP ‐ S ‐ Ubqln1 HA ‐ Ubqln4 , HA ‐ Ubqln4 ΔUBL or HA ‐ Ubqln4 ΔUBA . GFP was immunoprecipitated analysed by western blot using anti ‐ GFP anti ‐ HA antibodies .

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(C) 293 cells were co‐transfected with GFP‐S or GFP‐S‐Ubqln1 and HA‐Ubqln4, HA‐Ubqln4 ΔUBL or HA‐Ubqln4 ΔUBA. GFP was immunoprecipitated and analysed by western blot using anti‐GFP and anti‐HA antibodies.


( D ) extent of Ubqln4 co ‐ immunoprecipitation with Ubqln1 was measured of Ubqln4 to Ubqln1 was calculated for WT Ubqln4 , Ubqln4ΔUBL Ubqln4ΔUBA . numbers represent of Ubqln4 to Ubqln1 relative to WT . Each bar represents average s . e . m . of four independent experiments . * P ‐ value = 0 . 0003 * * P ‐ value = 0 . 0001 .

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(D) The extent of Ubqln4 co‐immunoprecipitation with Ubqln1 was measured and the ratio of Ubqln4 to Ubqln1 was calculated for WT Ubqln4, Ubqln4ΔUBL and Ubqln4ΔUBA. The numbers represent the ratio of Ubqln4 to Ubqln1 relative to WT. Each bar represents the average and s.e.m. of four independent experiments. *P‐value=0.0003 and **P‐value=0.0001.


( E ) 293 were co ‐ transfected with GFP ‐ S ‐ Ubqln1 HA ‐ Ubqln4 , HA ‐ Ubqln4 ΔUBA or HA ‐ Ubqln4 ΔUBA I55A ( I55 in UBL domain of Ubqln4 was mutated to A ) . GFP was immunoprecipitated analysed by western blot using anti ‐ GFP anti ‐ HA antibodies .

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(E) 293 cells were co‐transfected with GFP‐S‐Ubqln1 and HA‐Ubqln4, HA‐Ubqln4 ΔUBA or HA‐Ubqln4 ΔUBA I55A (I55 in the UBL domain of Ubqln4 was mutated to A). GFP was immunoprecipitated and analysed by western blot using anti‐GFP and anti‐HA antibodies.


( F ) extent of Ubqln4 co ‐ immunoprecipitation with Ubqln1 was measured of Ubqln4 to Ubqln4 was calculated for WT Ubqln4 , Ubqln4ΔUBA Ubqln4ΔUBA I55A . numbers represent of Ubqln4 to Ubqln1 relative to WT . Each bar represents average of two independent experiments . GFP , green ; HA , haemagglutinin ; IP , immunoprecipitation ; UBA , ubiquitin ‐ associated ; UBL , ubiquitin ‐ like ; Ubqln , Ubiquilin ; WCL , whole ‐ lysate ; WT , wild ‐ type .

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(F) The extent of Ubqln4 co‐immunoprecipitation with Ubqln1 was measured and the ratio of Ubqln4 to Ubqln4 was calculated for WT Ubqln4, Ubqln4ΔUBA and Ubqln4ΔUBA I55A. The numbers represent the ratio of Ubqln4 to Ubqln1 relative to WT. Each bar represents the average of two independent experiments. GFP, green fluorescent protein; HA, haemagglutinin; IP, immunoprecipitation; UBA, ubiquitin‐associated; UBL, ubiquitin‐like; Ubqln, Ubiquilin; WCL, whole‐cell lysate; WT, wild‐type.


( A ) Confocal of Hela transfected with mCherry ‐ GFP ‐ LC3 also transfected with control , Ubqln1 or Ubqln4 siRNA . were either untreated or were transferred to starvation medium for 8 h .

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(A) Confocal images of Hela cells transfected with mCherry‐GFP‐LC3 and also transfected with control, Ubqln1 or Ubqln4 siRNA. The cells were either untreated or were transferred to starvation medium for 8 h.


( B ) of number of mCherry / GFP double ‐ positive mCherry single ‐ positive per in ( A ) . Each bar represents average s . e . m . of 30 per condition in three independent experiments . number of mCherry single ‐ positive of Ubqln1 Ubqln4 siRNA was compared with that of control siRNA . * P ‐ value = 0 . 0475 * * P ‐ value = 0 . 0373 . GFP , green ; LC3 , microtubule ‐ associated chain 3 ; Ubqln , Ubiquilin .

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(B) Quantification of the number of mCherry/GFP double‐positive and mCherry single‐positive puncta per cell in (A). Each bar represents the average and s.e.m. of 30 cells per condition in three independent experiments. The number of mCherry single‐positive puncta of Ubqln1 and Ubqln4 siRNA was compared with that of control siRNA. *P‐value=0.0475 and **P‐value=0.0373. GFP, green fluorescent protein; LC3, microtubule‐associated protein light chain 3; Ubqln, Ubiquilin.


( B ) Structures of S . cerevisiae Hrr251 - 394 K38R bound to CK1 - 7 ( Chijiwa et al , 1989 ; Xu et al , 1996 ) C . glabrata Hrr251 - 403 K38R bound to ADP , with domains colored as in ( A ) . Bound PO4 - / SO4 - ions are shown as spheres . See Figure EV2A for additional C . glabrataHrr25 structures , Figure EV3A for SO4 - ion electron density .

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(B) Structures of S. cerevisiae Hrr251-394 K38R bound to CK1-7 (Chijiwa et al, 1989; Xu et al, 1996) and C. glabrata Hrr251-403 K38R bound to ADP, with domains colored as in (A). Bound PO4-/SO4- ions are shown as spheres. See Figure EV2A for additional C. glabrataHrr25 structures, and Figure EV3A for SO4- ion electron density.


( C ) Overlay of kinase domain of C . glabrataHrr25 ( Apo form , crystallized with 1 . 2 M PO4 - ) with ratCK1δ crystallized in presence of tungstate ions ( WO4 ; PDB ID 1CKJ , ( Longenecker et al , 1996 ) ) See Figure EV2B for detailed views of ion binding .

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(C) Overlay of the kinase domain of C. glabrataHrr25 (Apo form, crystallized with 1.2 M PO4-) with ratCK1δ crystallized in the presence of tungstate ions (WO4; PDB ID 1CKJ, (Longenecker et al, 1996)) See Figure EV2B for detailed views of ion binding.


( D ) Stereo view of CK1 - 7 binding to S . cerevisiae Hrr25 . Bound drug is positioned identically to a previous structure of S . pombe Cki1 bound to CK1 - 7 ( Xu et al , 1996 ) .

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(D) Stereo view of CK1-7 binding to S. cerevisiae Hrr25. Bound drug is positioned identically to a previous structure of S. pombe Cki1 bound to CK1-7 (Xu et al, 1996).


( E ) Stereo view of ADP binding to C . glabrata Hrr25 ( formate structure ; SO4 - structure is equivalent , but GxGxxG motif is disordered in that structure ) . active - site residues shown in panels ( D ) ( E ) are conserved between S . cerevisiae C . glabrata Hrr25 .

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(E) Stereo view of ADP binding to C. glabrata Hrr25 (formate structure; SO4- structure is equivalent, but the GxGxxG motif is disordered in that structure). All active-site residues shown in panels (D) and (E) are conserved between S. cerevisiae and C. glabrata Hrr25.


( A ) Two views of C . glabrata Hrr25 ( Apo form ) with domains colored as in Figure 1 , showing bound PO4 - ions previously - identified Mam1 - binding residues His25 Glu34 ( Petronczki et al , 2006 ) .

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(A) Two views of C. glabrata Hrr25 (Apo form) with domains colored as in Figure 1, showing bound PO4- ions and the previously-identified Mam1-binding residues His25 and Glu34 (Petronczki et al, 2006).