Left : Representative immunofluorescence ( IF ) images showing primary cilium formation in RPE cells upon starvation after infection with shLuc ( above ) or shGPR56strong ( below ) . The non - motile primary cilium is visualized using antibodies against γ - tubulin , which stains the basal body , and the ciliary membrane marker ARL13B . DAPI indicates the nucleus . Images on the right show selected cells at 3 x magnification . Brightness was increased in all images to enhance visibility of cilia . Right : Percentage of RPE - 1 cells with primary cilium in shLuc vs . shGPR56strong RPE - 1 cells at different time points after the start of serum starvation . Unpaired t - test , bars and error bars represent mean and stdev of three biological replicate wells . * * * * p < 0 . 0001 , * * * p < 0 . 0005 , * * p < 0 . 005 , * p < 0 . 05 .

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

Left: Representative immunofluorescence (IF) images showing primary cilium formation in RPE cells upon starvation after infection with shLuc (above) or shGPR56strong (below). The non-motile primary cilium is visualized using antibodies against γ-tubulin, which stains the basal body, and the ciliary membrane marker ARL13B. DAPI indicates the nucleus. Images on the right show selected cells at 3 x magnification. Brightness was increased in all images to enhance visibility of cilia. Right: Percentage of RPE-1 cells with primary cilium in shLuc vs. shGPR56strong RPE-1 cells at different time points after the start of serum starvation. Unpaired t-test, bars and error bars represent mean and stdev of three biological replicate wells. **** p<0.0001, *** p<0.0005, ** p<0.005, * p<0.05.


B Relative ( left ) and absolute ( right ) amount of 13C - Lys - labeled and unlabeled Aβ in APPtg mouse brains after labeling from 75 - 125 d ( n = 1 ) , 75 - 175 d ( n = 4 ) , 50 - 100 d ( n = 5 ) , 50 - 125 d ( n = 2 ) , 50 - 150 ( n = 4 ) or 50 - 175 d ( n = 9 ) of age . Error bars show mean ± SD .

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B Relative (left) and absolute (right) amount of 13C-Lys-labeled and unlabeled Aβ in APPtg mouse brains after labeling from 75-125 d (n = 1), 75-175 d (n = 4), 50-100 d (n = 5), 50-125 d (n = 2), 50-150 (n = 4) or 50-175 d (n = 9) of age. Error bars show mean ± SD.


C Representative chromatogram and mass spectra of doubly charged ions of unlabeled Aβ17 - 28 ( 663 . 3 m / z ) and 13C - Lys Aβ17 - 28 ( 666 . 3 m / z ) in an APPtg brain after labeling from 75 - 175 d . % - ion intensity , m / z - mass - to - charge . Data were acquired in data - independent analysis mode ( MSE ) . D Representative chromatograms of unlabeled Aβ17 - 28 ( left ) and 13C - Lys Aβ17 - 28 ( right ) in an APPtg brain after labeling from 50 - 175 d . The intensities of 4 transitions ( y10 , y9 , y8 , and y7 ) were assessed . Data were acquired by MRM in nanoflow mode .

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C Representative chromatogram and mass spectra of doubly charged ions of unlabeled Aβ17-28 (663.3 m/z) and 13C-Lys Aβ17-28 (666.3 m/z) in an APPtg brain after labeling from 75-175 d. %- ion intensity, m/z- mass-to-charge. Data were acquired in data-independent analysis mode (MSE). D Representative chromatograms of unlabeled Aβ17-28 (left) and 13C-Lys Aβ17-28 (right) in an APPtg brain after labeling from 50-175 d. The intensities of 4 transitions (y10, y9, y8, and y7) were assessed. Data were acquired by MRM in nanoflow mode.


F Representative chromatograms of unlabeled Aβ17 - 28 ( left ) and 13C - Lys Aβ17 - 28 ( right ) in a non - APPtg brain after labeling from 90 - 100 d . Data were acquired by MRM in microflow mode .

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F Representative chromatograms of unlabeled Aβ17-28 (left) and 13C-Lys Aβ17-28 (right) in a non-APPtg brain after labeling from 90-100 d. Data were acquired by MRM in microflow mode.


A MALDI - TOF mass spectrum of trypsin - digested , synthetic Aβ1 - 42 ( 10 pmol ) . The Aβ17 - 28 [ MH ] + fragment was detected with the highest intensity . Measurements in reflectron positive mode . The sequence of Aβ1 - 42 with the tryptic cleavage sites is indicated at the

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A MALDI-TOF mass spectrum of trypsin-digested, synthetic Aβ1-42 (10 pmol). The Aβ17-28 [MH]+ fragment was detected with the highest intensity. Measurements in reflectron positive mode. The sequence of Aβ1-42 with the tryptic cleavage sites is indicated at the top


B MS / MS spectrum of unlabeled Aβ17 - 28 and 13C15N - Lys Aβ17 - 28 synthetic peptides in the 50 fmol range . m / z of precursors ions ( m / z 663 . 34 for Aβ17 - 28 [ MH ] 2 + , m / z 667 . 36 for 13C15N - Lys Aβ17 - 28 [ MH ] 2 + ) and their derived fragments are indicated as a series of y ions ( y4 - y10 ) . The peptides were analyzed in data independent acquisition MSE

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B MS/MS spectrum of unlabeled Aβ17-28 and 13C15N-Lys Aβ17-28 synthetic peptides in the 50 fmol range. m/z of precursors ions (m/z 663.34 for Aβ17-28 [MH]2+, m/z 667.36 for 13C15N-Lys Aβ17-28 [MH]2+) and their derived fragments are indicated as a series of y ions (y4-y10). The peptides were analyzed in data independent acquisition MSE mode


C Decreasing quantities of synthetic 13C15N - Lys Aβ1 - 42 were trypsin - digested and analyzed by MRM . The intensities of 4 transitions ( y10 , y9 , y8 , and y7 ) were assessed for 13C15N - Lys Aβ17 - 28 , showing a detection limit level of 25 amol . Measurements in technical triplicates , normalization to total ion current . Error bars show mean ±

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C Decreasing quantities of synthetic 13C15N-Lys Aβ1-42 were trypsin-digested and analyzed by MRM. The intensities of 4 transitions (y10, y9, y8, and y7) were assessed for 13C15N-Lys Aβ17-28, showing a detection limit level of 25 amol. Measurements in technical triplicates, normalization to total ion current. Error bars show mean ± SD


D Decreasing quantities of synthetic 13C15N - Lys Aβ1 - 42 were spiked into the insoluble fraction of APPtg brains , immunoprecipitated , trypsin - digested , and analyzed by MRM . Assessment of the intensities of y10 , y9 , y8 and y7 showed a detection limit level of 50 amol for 13C15N - Lys Aβ17 - 28 and detected an excess of brain - derived unlabeled Aβ17 - 28 . Measurements in technical triplicates , normalization to total ion current . Error bars show mean ±

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D Decreasing quantities of synthetic 13C15N-Lys Aβ1-42 were spiked into the insoluble fraction of APPtg brains, immunoprecipitated, trypsin-digested, and analyzed by MRM. Assessment of the intensities of y10, y9, y8 and y7 showed a detection limit level of 50 amol for 13C15N-Lys Aβ17-28 and detected an excess of brain-derived unlabeled Aβ17-28. Measurements in technical triplicates, normalization to total ion current. Error bars show mean ± SD


B Temporal MRM analysis of 13C - Lys Aβ17 - 28 and unlabeled Aβ17 - 28 in the liver , spleen , mesenteric lymph nodes , and peritoneal cells after 13C - Lys - APPtg brain extract i . p . injection ( n = 3 mice per time point and tissue at 1 - 10 d , except for n = 2 mice for peritoneal cells at 3 d ; n = 4 mice per time point and tissue at 25 - 100 d , except for n = 3 mice for spleen at 100 d ) . Measurements in technical triplicates , normalization to 13C15N - Lys Aβ17 - 28 intensity as a spiked internal standard and additionally to organ weight ( mg ) for lymph nodes . Error bars show median ± interquartile range .

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B Temporal MRM analysis of 13C-Lys Aβ17-28 and unlabeled Aβ17-28 in the liver, spleen, mesenteric lymph nodes, and peritoneal cells after 13C-Lys-APPtg brain extract i.p. injection (n = 3 mice per time point and tissue at 1-10 d, except for n = 2 mice for peritoneal cells at 3 d; n = 4 mice per time point and tissue at 25-100 d, except for n = 3 mice for spleen at 100 d). Measurements in technical triplicates, normalization to 13C15N-Lys Aβ17-28 intensity as a spiked internal standard and additionally to organ weight (mg) for lymph nodes. Error bars show median ± interquartile range.


C Temporal 4G8 immunoassay quantification of Aβ42 in the liver , spleen , mesenteric lymph nodes , and peritoneal cells after unlabeled APPtg ( n = 8 mice at 5 h - 7 d , except for n = 5 mice for peritoneal lavage , n = 5 mice at 10 - 14 d , n = 4 mice at 21 d ) or non - tg brain extract injection ( n = 2 mice at 5 h and 3 d , n = 3 mice at 1 d , n = 1 mouse at 5 and 7 d , except for peritoneal cells where n = 1 mouse at 5 h and 3 d , and n = 2 mice at 1 d ) . Error bars show median ± interquartile range . D Normalization of APPtg extract group data presented in ( C ) to organ weight ( mg ) and injected Aβ amount ( range 1 , 027 - 1 , 426 ng ) . Error bars show median ± interquartile range .

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C Temporal 4G8 immunoassay quantification of Aβ42 in the liver, spleen, mesenteric lymph nodes, and peritoneal cells after unlabeled APPtg (n = 8 mice at 5 h - 7 d, except for n = 5 mice for peritoneal lavage, n = 5 mice at 10-14 d, n = 4 mice at 21 d) or non-tg brain extract injection (n = 2 mice at 5 h and 3 d, n = 3 mice at 1 d, n = 1 mouse at 5 and 7 d, except for peritoneal cells where n = 1 mouse at 5 h and 3 d, and n = 2 mice at 1 d). Error bars show median ± interquartile range. D Normalization of APPtg extract group data presented in (C) to organ weight (mg) and injected Aβ amount (range 1,027-1,426 ng). Error bars show median ± interquartile range.


A Temporal MRM analysis of unlabeled Aβ17 - 28 and 13C - Lys Aβ17 - 28 in the soluble brain fraction after 13C - Lys APPtg brain extract i . p . injection ( n = 3 mice per time point at 1 - 10 d , n = 4 mice per time point at 25 - 100 d ) . Data information : measurements in technical triplicates , normalization to 13C15N - Lys Aβ17 - 28 intensity as a spiked internal standard and brain weight ( mg ) . All error bars show mean ± SD .

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A Temporal MRM analysis of unlabeled Aβ17-28 and 13C-Lys Aβ17-28 in the soluble brain fraction after 13C-Lys APPtg brain extract i.p. injection (n = 3 mice per time point at 1-10 d, n = 4 mice per time point at 25-100 d). Data information: measurements in technical triplicates, normalization to 13C15N-Lys Aβ17-28 intensity as a spiked internal standard and brain weight (mg). All error bars show mean ± SD.


Comparative MRM analysis of 13C - Lys Aβ17 - 28 ( B ) in the insoluble brain fraction after 13C - Lys - APPtg brain extract ( donors labeled for 50 - 125 d ) and 13C - Lys - non - tg brain extract ( donors labeled for 10 d ) i . p . injection ( n = 3 mice per group and time point at 1 - 10 d , n = 4 mice per group and time point at 25 , 50 , 100 d , n = 3 mice in the APPtg extract group and n = 4 in the non - tg extract group at 75 d ) , right side zoom for time points 1 - 10 d . Unpaired two - tailed t - test * p < 0 . 05 ( P = 0 . 0129 at 3 d , P = 0 . 0156 at 10 d , P = 0 . 0109 at 25 d ) , * * p < 0 . 01 ( P = 0 . 0091 at 1 d , P = 0 . 0011 at 5 d ) , * * * p < 0 . 001 ( P = 0 . 0006 at 7 d ) , 13C - Lys APPtg extract - injected vs 13C - Lys non - tg extract - injected for each time point . Data information : measurements in technical triplicates , normalization to 13C15N - Lys Aβ17 - 28 intensity as a spiked internal standard and brain weight ( mg ) . All error bars show mean ± SD .

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

Comparative MRM analysis of 13C-Lys Aβ17-28 (B) in the insoluble brain fraction after 13C-Lys-APPtg brain extract (donors labeled for 50-125 d) and 13C-Lys-non-tg brain extract (donors labeled for 10 d) i.p. injection (n = 3 mice per group and time point at 1-10 d, n = 4 mice per group and time point at 25, 50, 100 d, n = 3 mice in the APPtg extract group and n = 4 in the non-tg extract group at 75 d), right side zoom for time points 1-10 d. Unpaired two-tailed t-test * p<0.05 (P=0.0129 at 3 d, P=0.0156 at 10 d, P=0.0109 at 25 d), ** p<0.01 (P=0.0091 at 1 d, P=0.0011 at 5 d), *** p<0.001 (P=0.0006 at 7 d), 13C-Lys APPtg extract-injected vs 13C-Lys non-tg extract-injected for each time point. Data information: measurements in technical triplicates, normalization to 13C15N-Lys Aβ17-28 intensity as a spiked internal standard and brain weight (mg). All error bars show mean ± SD.


C Comparative MRM analysis of unlabeled Aβ17 - 28 ( C ) in the insoluble brain fraction after 13C - Lys - APPtg brain extract ( donors labeled for 50 - 125 d ) and 13C - Lys - non - tg brain extract ( donors labeled for 10 d ) i . p . injection ( n = 3 mice per group and time point at 1 - 10 d , n = 4 mice per group and time point at 25 , 50 , 100 d , n = 3 mice in the APPtg extract group and n = 4 in the non - tg extract group at 75 d ) , right side zoom for time points 1 - 10 d . Unpaired two - tailed t - test * p < 0 . 05 ( P = 0 . 0129 at 3 d , P = 0 . 0156 at 10 d , P = 0 . 0109 at 25 d ) , * * p < 0 . 01 ( P = 0 . 0091 at 1 d , P = 0 . 0011 at 5 d ) , * * * p < 0 . 001 ( P = 0 . 0006 at 7 d ) , 13C - Lys APPtg extract - injected vs 13C - Lys non - tg extract - injected for each time point . Data information : measurements in technical triplicates , normalization to 13C15N - Lys Aβ17 - 28 intensity as a spiked internal standard and brain weight ( mg ) . All error bars show mean ± SD .

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

C Comparative MRM analysis of unlabeled Aβ17-28 (C) in the insoluble brain fraction after 13C-Lys-APPtg brain extract (donors labeled for 50-125 d) and 13C-Lys-non-tg brain extract (donors labeled for 10 d) i.p. injection (n = 3 mice per group and time point at 1-10 d, n = 4 mice per group and time point at 25, 50, 100 d, n = 3 mice in the APPtg extract group and n = 4 in the non-tg extract group at 75 d), right side zoom for time points 1-10 d. Unpaired two-tailed t-test * p<0.05 (P=0.0129 at 3 d, P=0.0156 at 10 d, P=0.0109 at 25 d), ** p<0.01 (P=0.0091 at 1 d, P=0.0011 at 5 d), *** p<0.001 (P=0.0006 at 7 d), 13C-Lys APPtg extract-injected vs 13C-Lys non-tg extract-injected for each time point. Data information: measurements in technical triplicates, normalization to 13C15N-Lys Aβ17-28 intensity as a spiked internal standard and brain weight (mg). All error bars show mean ± SD.


D Comparative 4G8 immunoassay quantification of Aβ42 in the insoluble brain fraction after 13C - Lys APPtg or 13C - Lys non - tg brain extract i . p . injection ( n = 3 mice per group and time point at 1 - 10 d , n = 4 mice per group and time point at 25 - 100 d ) , right side zoom for time points 1 - 10 d . Normalization to protein concentration .

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D Comparative 4G8 immunoassay quantification of Aβ42 in the insoluble brain fraction after 13C-Lys APPtg or 13C-Lys non-tg brain extract i.p. injection (n = 3 mice per group and time point at 1-10 d, n = 4 mice per group and time point at 25-100 d), right side zoom for time points 1-10 d. Normalization to protein concentration.


E Representative chromatograms of unlabeled Aβ17 - 28 ( left ) and 13C - Lys Aβ17 - 28 ( right ) in the brain of an unlabeled 50 - day - old APPtg mouse not injected with labeled brain extract . Note the absence of 13C - Lys Aβ17 - 28 .

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E Representative chromatograms of unlabeled Aβ17-28 (left) and 13C-Lys Aβ17-28 (right) in the brain of an unlabeled 50-day-old APPtg mouse not injected with labeled brain extract. Note the absence of 13C-Lys Aβ17-28.


F MRM analysis of unlabeled and 13C - Lys - labeled Aβ17 - 28 in APPtg mouse brains after 13C - Lys - labeled diet for 1 or 3 d ( n = 3 mice per time point ) . Data information : measurements in technical triplicates , normalization to 13C15N - Lys Aβ17 - 28 intensity as a spiked internal standard and brain weight ( mg ) . All error bars show mean ± SD .

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F MRM analysis of unlabeled and 13C-Lys-labeled Aβ17-28 in APPtg mouse brains after 13C-Lys-labeled diet for 1 or 3 d (n = 3 mice per time point). Data information: measurements in technical triplicates, normalization to 13C15N-Lys Aβ17-28 intensity as a spiked internal standard and brain weight (mg). All error bars show mean ± SD.


G Comparative MRM analysis of 13C - Lys Aβ17 - 28 in the insoluble brain fraction after 13C - Lys APPtg and 13C - Lys non - tg brain extract ( all donors labeled for 100 d ) i . p . injection ( n = 6 mice per group ) . Data information : measurements in technical triplicates , normalization to 13C15N - Lys Aβ17 - 28 intensity as a spiked internal standard and brain weight ( mg ) . All error bars show mean ± SD .

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G Comparative MRM analysis of 13C-Lys Aβ17-28 in the insoluble brain fraction after 13C-Lys APPtg and 13C-Lys non-tg brain extract (all donors labeled for 100 d) i.p. injection (n = 6 mice per group). Data information: measurements in technical triplicates, normalization to 13C15N-Lys Aβ17-28 intensity as a spiked internal standard and brain weight (mg). All error bars show mean ± SD.


H MRM analysis of unlabeled and 13C - Lys - labeled Aβ17 - 28 in non - APPtg mouse brains after 13C - Lys - labeled diet for 1 d ( n = 3 mice ) or at 1 d after 13C - Lys - APPtg brain extract i . p . injection ( n = 3 mice ) . Data information : measurements in technical triplicates , normalization to 13C15N - Lys Aβ17 - 28 intensity as a spiked internal standard and brain weight ( mg ) . All error bars show mean ± SD .

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

H MRM analysis of unlabeled and 13C-Lys-labeled Aβ17-28 in non-APPtg mouse brains after 13C-Lys-labeled diet for 1 d (n = 3 mice) or at 1 d after 13C-Lys-APPtg brain extract i.p. injection (n = 3 mice). Data information: measurements in technical triplicates, normalization to 13C15N-Lys Aβ17-28 intensity as a spiked internal standard and brain weight (mg). All error bars show mean ± SD.


( A ) Representative 5 - day - old seedlings of wild - type Arabidopsis thaliana Col - 0 and TF mutants gata12 and wri3 grown in the dark . Scale bar , 1 mm .

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(A) Representative 5-day-old seedlings of wild-type Arabidopsis thaliana Col-0 and TF mutants gata12 and wri3 grown in the dark. Scale bar, 1 mm.


( B ) Heat map indicates which TFs significantly affects hypocotyl length , root length and the composite trait of root to total length in dark - grown seedlings and whether the effects of TFs are dependent on condition . TFs are hierarchically clustered using Euclidean distance .

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(B) Heat map indicates which TFs significantly affects hypocotyl length, root length and the composite trait of root to total length in dark-grown seedlings and whether the effects of TFs are dependent on condition. TFs are hierarchically clustered using Euclidean distance.


( C ) Heat map of the average relative effects of TF mutant alleles on hypocotyl length , root length and the ratio of root to total length reveal that TF lesions significantly perturbed TCA cycle - dependent growth . Mutant alleles are listed in rows and traits in columns . Cells of TF mutant alleles in heat map are colored if the AGI or AGI : TCA Metabolite linear model terms for each trait are statistically significant ( P < 0 . 05 , two - way ANOVA , 16 - 20 seedlings per genotype per condition across two experiments per genotype ) . Mutant alleles are hierarchically clustered using Euclidean distance .

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(C) Heat map of the average relative effects of TF mutant alleles on hypocotyl length, root length and the ratio of root to total length reveal that TF lesions significantly perturbed TCA cycle-dependent growth. Mutant alleles are listed in rows and traits in columns. Cells of TF mutant alleles in heat map are colored if the AGI or AGI:TCA Metabolite linear model terms for each trait are statistically significant (P < 0.05, two-way ANOVA, 16-20 seedlings per genotype per condition across two experiments per genotype). Mutant alleles are hierarchically clustered using Euclidean distance.


( A ) Heat map indicates which TFs significantly affects rosette area , growth rate , shoot biomass , flowering time and seed yield and whether the effects of TFs are dependent on salt treatment . TFs are hierarchically clustered using Euclidean distance .

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(A) Heat map indicates which TFs significantly affects rosette area, growth rate, shoot biomass, flowering time and seed yield and whether the effects of TFs are dependent on salt treatment. TFs are hierarchically clustered using Euclidean distance.


( C ) Rosette area of mutant allele of BPC4 from day 7 to day 21 post - germination . Linear model for two - way ANOVA considers AGI , salt treatment , day post - germination and their interactions . The AGI and AGI : Salt Condition terms are statistically signification ( P < 0 . 001 , P < 0 . 01 , respectively ) . Heat map under line plot indicates which term in linear model is statistically significant ( P < 0 . 05 ) using two - way ANOVA for each day from 9 biological replicates per condition . Solid lines , Col - 0 ; dashed line , bpc4 ; circle , control condition ; triangle , 50 mM NaCl .

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(C) Rosette area of mutant allele of BPC4 from day 7 to day 21 post-germination. Linear model for two-way ANOVA considers AGI, salt treatment, day post-germination and their interactions. The AGI and AGI:Salt Condition terms are statistically signification (P < 0.001, P < 0.01, respectively). Heat map under line plot indicates which term in linear model is statistically significant (P < 0.05) using two-way ANOVA for each day from 9 biological replicates per condition. Solid lines, Col-0; dashed line, bpc4; circle, control condition; triangle, 50 mM NaCl.


C ) Western Blot showing the levels of the PgP and control ( α - TUBB ) in RPE - 1 parental and Taxol resistant clones .

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C) Western Blot showing the levels of the PgP and control (α-TUBB) in RPE-1 parental and Taxol resistant clones.


E ) Representative smRNA - FISH images of RPE - 1 Parental and clones for the ABCB1 gene and DAPI . The images are projections of 0 , 5μm sections and a total 5 μm in thickness . Scale bar , 15μm .

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E) Representative smRNA-FISH images of RPE-1 Parental and clones for the ABCB1 gene and DAPI. The images are projections of 0,5μm sections and a total 5 μm in thickness. Scale bar, 15μm.


A ) TLA analysis for ABCB1 contacts in RPE - 1 parental and Taxol resistant clone sg6C9 covering the whole genome . Green arrow in sg6C9 shows a de novo interaction found between ABCB1 and a region in chromosome 6 . B ) TLA analysis for RPE - 1 Parental and sg6C9 . Zoom in in the region of chr6 with de novo interaction for sg6C9 . Image modified from IGV viewer .

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A) TLA analysis for ABCB1 contacts in RPE-1 parental and Taxol resistant clone sg6C9 covering the whole genome. Green arrow in sg6C9 shows a de novo interaction found between ABCB1 and a region in chromosome 6. B) TLA analysis for RPE-1 Parental and sg6C9. Zoom in in the region of chr6 with de novo interaction for sg6C9. Image modified from IGV viewer.


D ) PCR products using the primers in C ( 1 ) over the ABCB1 and EEF1A1 regions in RPE - 1 Parental and the different Taxol resistant clones . E ) PCR products using the primers in C ( 2 ) over the specific break site in the ABCB1 promoter in RPE - 1 Parental and the different Taxol resistant clones .

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D) PCR products using the primers in C(1) over the ABCB1 and EEF1A1 regions in RPE-1 Parental and the different Taxol resistant clones. E) PCR products using the primers in C(2) over the specific break site in the ABCB1 promoter in RPE-1 Parental and the different Taxol resistant clones.


G ) Percentage of RPE - 1 cells targeted with the ABCB1 promoter sgRNAs found with vector integration . Percentages were calculated from data in Fig 1A . Each dot represents a CFA replicate experiment . Horizontal bars represent the mean of lentiviral integratation per sgRNA .

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G) Percentage of RPE-1 cells targeted with the ABCB1 promoter sgRNAs found with vector integration. Percentages were calculated from data in Fig 1A. Each dot represents a CFA replicate experiment. Horizontal bars represent the mean of lentiviral integratation per sgRNA.


A ) H3K9me3 ChIP - sequencing tracks from the q21 . 12 arm of chromosome 7 in RPE - 1 cells ( Parental and Taxol - resistant clones ) . The ABCB1 gene region is shown in pink . Each colored track shows the H3k9me3 profile of a different Taxol - resistant clone .

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A) H3K9me3 ChIP-sequencing tracks from the q21.12 arm of chromosome 7 in RPE-1 cells (Parental and Taxol-resistant clones). The ABCB1 gene region is shown in pink. Each colored track shows the H3k9me3 profile of a different Taxol-resistant clone.


B ) H3K9me3 ChIP - qPCR in the ABCB1 promoter region . Bar graph show primer pairs amplifying the sgRNA # 6 break and spanning this region ( + / - base pairs ) for each Taxol - resistant clone compared to the parental . Bar plots show the mean of H3K9me3 relative enrichment . Each dot represents a technical replicate ( n = 3 ) .

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B) H3K9me3 ChIP-qPCR in the ABCB1 promoter region. Bar graph show primer pairs amplifying the sgRNA#6 break and spanning this region (+/- base pairs) for each Taxol-resistant clone compared to the parental. Bar plots show the mean of H3K9me3 relative enrichment. Each dot represents a technical replicate (n=3).


( a ) vBcl - 2 binding proteins . After transfection , 293T cells were used for GST - pulldown and the purified proteins were detected by silver staining .

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(a) vBcl-2 binding proteins. After transfection, 293T cells were used for GST-pulldown and the purified proteins were detected by silver staining.


( c ) UVRAG binds Beclin1 through its CCD . After transfection with Beclin1 - V5 , together with full - length or fragments of Flag - UVRAG , 293T whole cell lysates ( WCLs ) were used for immunoprecipitation ( IP ) with anti - Flag followed by immunoblotting ( IB ) with anti - V5 .

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(c) UVRAG binds Beclin1 through its CCD. After transfection with Beclin1-V5, together with full-length or fragments of Flag-UVRAG, 293T whole cell lysates (WCLs) were used for immunoprecipitation (IP) with anti-Flag followed by immunoblotting (IB) with anti-V5.


( d ) Interactions between endogenous UVRAG and Beclin1 and between endogenous UVRAG and PI ( 3 ) KC3 . 293T cell lysates were used for immunoprecipitation with preimmune sera or anti - Beclin1 , followed by immunoblotting with anti - UVRAG or anti - Beclin1 , or for immunoprecipitation with preimmune sera or anti - UVRAG , followed by immunoblotting with anti - PI ( 3 ) KC3 .

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(d) Interactions between endogenous UVRAG and Beclin1 and between endogenous UVRAG and PI(3)KC3. 293T cell lysates were used for immunoprecipitation with preimmune sera or anti-Beclin1, followed by immunoblotting with anti-UVRAG or anti-Beclin1, or for immunoprecipitation with preimmune sera or anti-UVRAG, followed by immunoblotting with anti-PI(3)KC3.


( e ) UVRAGCCD is required for Beclin1 interaction . After transfection with Beclin1 - V5 ( lanes 2 and 4 ) together with Flag - UVRAGΔCCD ( lanes 1 and 2 ) or Flag - UVRAGΔC2 ( lanes 3 and 4 ) , WCLs were used for immunoprecipitation with anti - Flag followed by immunoblotting with anti - V5 .

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(e) UVRAGCCD is required for Beclin1 interaction. After transfection with Beclin1-V5 (lanes 2 and 4) together with Flag-UVRAGΔCCD (lanes 1 and 2) or Flag-UVRAGΔC2 (lanes 3 and 4), WCLs were used for immunoprecipitation with anti-Flag followed by immunoblotting with anti-V5.


( f ) Beclin1 binds UVRAG through its CCD . After transfection with Flag - UVRAG together with full - length or fragments of Beclin1 - V5 , WCLs were used for immunoprecipitation with anti - V5 followed by immunoblotting with anti - Flag .

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(f) Beclin1 binds UVRAG through its CCD. After transfection with Flag-UVRAG together with full-length or fragments of Beclin1-V5, WCLs were used for immunoprecipitation with anti-V5 followed by immunoblotting with anti-Flag.


( g ) An increase in UVRAG stability induced by Beclin1 . After transfection with Flag - UVRAG and empty vector or Flag - UVRAG and Beclin1 - V5 , WCLs were treated with cycloheximide ( CHX , 20 μg ml − 1 ) for the indicated time , followed by immunoblotting with anti - Flag .

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(g) An increase in UVRAG stability induced by Beclin1. After transfection with Flag-UVRAG and empty vector or Flag-UVRAG and Beclin1-V5, WCLs were treated with cycloheximide (CHX, 20 μg ml−1) for the indicated time, followed by immunoblotting with anti-Flag.


( h ) UVRAG directly interacts with Beclin1 . MBP - UVRAGCCD ( lane 1 ) , His6 - Beclin1CCD ( lane 2 ) or mixtures of both proteins in a concentration dependent manner ( lanes 3 - 8 ) , were subjected to native gel electrophoresis . The numbers indicate the concentration ( nmol ) of protein . Arrows indicate the purified MBP - UVRAGCCD , His6 - Beclin1CCD and complex between these two .

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(h) UVRAG directly interacts with Beclin1. MBP-UVRAGCCD (lane 1), His6-Beclin1CCD (lane 2) or mixtures of both proteins in a concentration dependent manner (lanes 3-8), were subjected to native gel electrophoresis. The numbers indicate the concentration (nmol) of protein. Arrows indicate the purified MBP-UVRAGCCD, His6-Beclin1CCD and complex between these two.


( i ) Beclin1CCD and UVRAGCCD associate into a α - helical heterodimer . After removing the N - terminal His6 and MBP tags , the Beclin1CCD and the preformed Beclin1CCD - UVRAGCCD complex were subjected to circular dichroism spectroscopic analysis . The raw data for this experiment are presented in the Supplementary Information , Fig . S5 .

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(i) Beclin1CCD and UVRAGCCD associate into a α-helical heterodimer. After removing the N-terminal His6 and MBP tags, the Beclin1CCD and the preformed Beclin1CCD-UVRAGCCD complex were subjected to circular dichroism spectroscopic analysis. The raw data for this experiment are presented in the Supplementary Information, Fig. S5.


( a ) The Bcl - 2 binding domain of Beclin1 is sufficient to bind vBcl - 2 . At 48 h posttransfection with wild - type and mutant Beclin1 - V5 , together with HA - vBcl - 2 , WCLs of 293T cells were used for immunoprecipitation with anti - V5 followed by immunoblotting with anti - HA .

(a) The Bcl-2 binding domain of Beclin1 is sufficient to bind vBcl-2. At 48 h posttransfection with wild-type and mutant Beclin1-V5, together with HA-vBcl-2, WCLs of 293T cells were used for immunoprecipitation with anti-V5 followed by immunoblotting with anti-HA.


( b ) The loss of Beclin1 interaction of vBcl - 2AAA mutant . At 48 h posttransfection with wild - type HA - vBcl - 2 or vBcl - 2AAA mutant , together with Beclin1 - V5 , WCLs of 293T cells were used for immunoprecipitation with anti - V5 , followed by immunoblotting with anti - HA .

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(b) The loss of Beclin1 interaction of vBcl-2AAA mutant. At 48 h posttransfection with wild-type HA-vBcl-2 or vBcl-2AAA mutant, together with Beclin1-V5, WCLs of 293T cells were used for immunoprecipitation with anti-V5, followed by immunoblotting with anti-HA.


( c ) vBcl - 2 interaction with wild - type UVRAG in the presence of Beclin1 . At 48 h posttransfection with Flag - UVRAG together with Beclin1 - V5 and HA - vBcl - 2 or vBcl - 2AAA mutant , WCLs of 293T cells were used for immunoprecipitation with anti - HA followed by immunoblotting with anti - Flag .

(c) vBcl-2 interaction with wild-type UVRAG in the presence of Beclin1. At 48 h posttransfection with Flag-UVRAG together with Beclin1-V5 and HA-vBcl-2 or vBcl-2AAA mutant, WCLs of 293T cells were used for immunoprecipitation with anti-HA followed by immunoblotting with anti-Flag.


( d ) vBcl - 2 interaction with PI ( 3 ) KC3 in the presence of Beclin1 . At 48 h posttransfection with HA - vBcl - 2 together with Flag - PI ( 3 ) KC3 and Beclin1 - V5 , WCLs of 293T cells were used for immunprecipitation with anti - HA followed by immunoblotting with anti - Flag .

(d) vBcl-2 interaction with PI(3)KC3 in the presence of Beclin1. At 48 h posttransfection with HA-vBcl-2 together with Flag-PI(3)KC3 and Beclin1-V5, WCLs of 293T cells were used for immunprecipitation with anti-HA followed by immunoblotting with anti-Flag.


( e ) PI ( 3 ) KC3 N - terminal C2 domain binds Beclin1 . At 48 h posttransfection with Beclin1 - V5 together with full - length or fragments of Flag - PI ( 3 ) KC3 , WCLs of 293T cells were used for immunoprecipitation with anti - Flag followed by immunoblotting with anti - V5 . CAT , catalytic domain .

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(e) PI(3)KC3 N-terminal C2 domain binds Beclin1. At 48 h posttransfection with Beclin1-V5 together with full-length or fragments of Flag-PI(3)KC3, WCLs of 293T cells were used for immunoprecipitation with anti-Flag followed by immunoblotting with anti-V5. CAT, catalytic domain.


( f ) Beclin1CCD and Beclin1ECD are sufficient for binding PI ( 3 ) KC3 . At 48 h posttransfection with Flag - PI ( 3 ) KC3 together with full - length or fragments of Beclin1 - V5 , WCLs of 293T cells were used for immunoprecipitation with anti - V5 followed by immunoblotting with anti - Flag . CT , C terminus ( aa 337 - 448 ) .

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(f) Beclin1CCD and Beclin1ECD are sufficient for binding PI(3)KC3. At 48 h posttransfection with Flag-PI(3)KC3 together with full-length or fragments of Beclin1-V5, WCLs of 293T cells were used for immunoprecipitation with anti-V5 followed by immunoblotting with anti-Flag. CT, C terminus (aa 337-448).


( g ) PI ( 3 ) KC3 interaction with UVRAG in the presence of Beclin1 . At 48 h posttransfection with Flag - UVRAGΔCCD or UVRAGCCD together with HA - PI ( 3 ) KC3 and Beclin1 - V5 , WCLs of 293T cells were used for immunoprecipitation with anti - Flag followed by immunoblotting with anti - HA .

(g) PI(3)KC3 interaction with UVRAG in the presence of Beclin1. At 48 h posttransfection with Flag-UVRAGΔCCD or UVRAGCCD together with HA- PI(3)KC3 and Beclin1-V5, WCLs of 293T cells were used for immunoprecipitation with anti-Flag followed by immunoblotting with anti-HA.


( a ) UVRAG expression and LC3 process in HCT116 cells . The same protein amounts of 293T , NIH3T3 , HCT116 and MCF7 cells were used for immunoblotting with anti - UVRAG and anti - tubulin . WCLs of 293T cells transfected with Flag - tagged UVRAG were included as controls . WCLs of HCT116 . vector and HCT116 . UVRAG were incubated with or without starvation conditions and used for immunoblotting with anti - LC3 and anti - tubulin . The raw data for this experiment is presented in the Supplementary information , Fig . S5 .

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(a) UVRAG expression and LC3 process in HCT116 cells. The same protein amounts of 293T, NIH3T3, HCT116 and MCF7 cells were used for immunoblotting with anti-UVRAG and anti-tubulin. WCLs of 293T cells transfected with Flag-tagged UVRAG were included as controls. WCLs of HCT116.vector and HCT116.UVRAG were incubated with or without starvation conditions and used for immunoblotting with anti-LC3 and anti-tubulin. The raw data for this experiment is presented in the Supplementary information, Fig. S5.


( b ) Electron microscopy of autophagy in HCT116 cells . HCT116 . vector and HCT116 . UVRAG cells were incubated under normal or starvation conditions for 4 h and subjected to scanning electron microscopy . Scale bars ( from left to right panels ) represent 2 nm , 0 . 5 nm , 0 . 2 nm and 0 . 2 nm . The arrows indicate autophagosome or autolysosome .

(b) Electron microscopy of autophagy in HCT116 cells. HCT116.vector and HCT116.UVRAG cells were incubated under normal or starvation conditions for 4 h and subjected to scanning electron microscopy. Scale bars (from left to right panels) represent 2 nm, 0.5 nm, 0.2 nm and 0.2 nm. The arrows indicate autophagosome or autolysosome.


( c ) UVRAG induces autophagosome formation in HCT116 cells . After transfection with GFP - LC3 vector , HCT116 . vector and HCT116 . UVRAG cells were maintained under normal conditions or starved for 3 h . GFP - LC3 was detected using an inverted fluorescence microscope . Arrows indicate autophagosomes . The scale bars represent 10 μm .

(c) UVRAG induces autophagosome formation in HCT116 cells. After transfection with GFP-LC3 vector, HCT116.vector and HCT116.UVRAG cells were maintained under normal conditions or starved for 3 h. GFP-LC3 was detected using an inverted fluorescence microscope. Arrows indicate autophagosomes. The scale bars represent 10 μm.


( d ) After transfection with GFP - LC3 vector , HCT116 . vector and HCT116 . UVRAG cells were maintained under normal conditions , starved for 3 h in the absence or presence of 10 mM 3 - methyladenine or treated with or without 2 μM rapamycin or rapamycin + 10 mM 3 - methyladenine . Autophagome was quantified as the mean ± s . d . of combined results from three independent experiments .

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(d) After transfection with GFP-LC3 vector, HCT116.vector and HCT116.UVRAG cells were maintained under normal conditions, starved for 3 h in the absence or presence of 10 mM 3-methyladenine or treated with or without 2 μM rapamycin or rapamycin + 10 mM 3-methyladenine. Autophagome was quantified as the mean ± s.d. of combined results from three independent experiments.


( a ) HCT116 . vector and HCT116 . UVRAG cells were labelled with LysoTracker and subjected to flow cytometry analysis . HCT116 . vector , HCT116 . UVRAG , and HCT116 . UVRAG ΔCCD cells were treated with or without wortmannin ( 1 μM ) for 2 h and their lysates were used to measure the lysosomal acid phosphatase enzymatic activity . Data represents mean ± s . d . of three experiments .

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(a) HCT116.vector and HCT116.UVRAG cells were labelled with LysoTracker and subjected to flow cytometry analysis. HCT116.vector, HCT116.UVRAG, and HCT116.UVRAG ΔCCD cells were treated with or without wortmannin (1 μM) for 2 h and their lysates were used to measure the lysosomal acid phosphatase enzymatic activity. Data represents mean ± s.d. of three experiments.


( b ) UVRAG induces autophagosome formation in NIH3T3 cells . After transfection with GFP - LC3 vector , NIH3T3 . vector and NIH3T3 . UVRAG cells were maintained under normal conditions , starved for 3 h in the absence or presence of 10 mM 3 - methyladenine or treated with or without 2 μM rapamycin or rapamycin + 10 mM 3 - methyladenine . Data represents mean ± s . d . of three experiments .

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(b) UVRAG induces autophagosome formation in NIH3T3 cells. After transfection with GFP-LC3 vector, NIH3T3.vector and NIH3T3.UVRAG cells were maintained under normal conditions, starved for 3 h in the absence or presence of 10 mM 3-methyladenine or treated with or without 2 μM rapamycin or rapamycin + 10 mM 3-methyladenine. Data represents mean ± s.d. of three experiments.


( a ) Autophagic activity of UVRAG mutants . HCT116 cells expressing wild - type or mutant UVRAG were transfected with GFP - LC3 expression vector and incubated under normal or starvation conditions for 3 h . Autophagy was quantified as described in Fig . 3 .

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(a) Autophagic activity of UVRAG mutants. HCT116 cells expressing wild-type or mutant UVRAG were transfected with GFP-LC3 expression vector and incubated under normal or starvation conditions for 3 h. Autophagy was quantified as described in Fig. 3.


( b ) Effect of wild - type and mutant UVRAG on Beclin1 - mediated autophagy . MCF7 . vector , MCF7 . Beclin1 and MCF7 . Beclin1ΔECD cells were cotransfected with wild - type UVRAG or its mutant expression vector together with GFP - LC3 expression vector . At 16 - 18 h posttransfection , cells were incubated under normal or starvation conditions for 4 h . Autophagy was quantified as described in Fig . 3 . The scale bars represent 20 μm .

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(b) Effect of wild-type and mutant UVRAG on Beclin1-mediated autophagy. MCF7.vector, MCF7.Beclin1 and MCF7.Beclin1ΔECD cells were cotransfected with wild-type UVRAG or its mutant expression vector together with GFP-LC3 expression vector. At 16-18 h posttransfection, cells were incubated under normal or starvation conditions for 4 h. Autophagy was quantified as described in Fig. 3. The scale bars represent 20 μm.


( c , d ) Interdependence between UVRAG and Beclin1 for autophagosome induction . HCT116 . vector and HCT116 . UVRAG cells were transfected with control siRNA , Beclin1 siRNA or UVRAG siRNA together with GFP - LC3 expression vector as indicated . Autophagy was quantified as described in Fig . 3 . WCLs of these cells were used for immunoblotting with anti - UVRAG , anti - Beclin1 or anti - tubulin . The scale bars represent 20 μm . In all figures , data represents mean ± s . d . of three experiments . The raw data for c and d are presented in the Supplementary Information , Fig S5 .

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(c, d) Interdependence between UVRAG and Beclin1 for autophagosome induction. HCT116.vector and HCT116.UVRAG cells were transfected with control siRNA, Beclin1 siRNA or UVRAG siRNA together with GFP-LC3 expression vector as indicated. Autophagy was quantified as described in Fig. 3. WCLs of these cells were used for immunoblotting with anti-UVRAG, anti-Beclin1 or anti-tubulin. The scale bars represent 20 μm. In all figures, data represents mean ± s.d. of three experiments. The raw data for c and d are presented in the Supplementary Information, Fig S5.


( c , d ) Interdependence between UVRAG and Beclin1 for autophagosome induction . MCF7 . vector , MCF7 . Beclin1 and MCF7 . Beclin1ΔECD cells ( d ) were transfected with control siRNA , Beclin1 siRNA or UVRAG siRNA together with GFP - LC3 expression vector as indicated . Autophagy was quantified as described in Fig . 3 . WCLs of these cells were used for immunoblotting with anti - UVRAG , anti - Beclin1 or anti - tubulin . The scale bars represent 20 μm . In all figures , data represents mean ± s . d . of three experiments . The raw data for c and d are presented in the Supplementary Information , Fig S5 .

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

(c, d) Interdependence between UVRAG and Beclin1 for autophagosome induction.MCF7.vector, MCF7.Beclin1 and MCF7.Beclin1ΔECD cells (d) were transfected with control siRNA, Beclin1 siRNA or UVRAG siRNA together with GFP-LC3 expression vector as indicated. Autophagy was quantified as described in Fig. 3. WCLs of these cells were used for immunoblotting with anti-UVRAG, anti-Beclin1 or anti-tubulin. The scale bars represent 20 μm. In all figures, data represents mean ± s.d. of three experiments. The raw data for c and d are presented in the Supplementary Information, Fig S5.


( a ) Increased Beclin1 - PI ( 3 ) KC3 interaction by UVRAG and continued interaction of UVRAG with Beclin1 on stress conditions . HCT116 . vector , HCT116 . UVRAG and HCT116 . UVRAGΔCCD cells were used for immunoprecipitation with anti - PI ( 3 ) KC3 followed by immunoblotting with anti - Beclin1 . WCLs were used for immunoblotting with anti - Beclin1 and anti - PI ( 3 ) KC3 . HCT116 . UVRAG cells expressing Flag - tagged UVRAG were incubated under nutrient - rich conditions ( lanes 1 and 2 ) , starvation for 4 h ( lane 3 ) , or 2 μM rapamycin treatment for 2 h ( lane 4 ) . WCLs were then used for immunoprecipitation with anti - Beclin1 ( lanes 2 , 3 and 4 ) or preimmune antibody ( lane 1 ) followed by immunoblotting with anti - Flag . The raw data for this experiment are presented in the Supplementary Information , Fig . S5 .

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

(a) Increased Beclin1-PI(3)KC3 interaction by UVRAG and continued interaction of UVRAG with Beclin1 on stress conditions. HCT116.vector, HCT116.UVRAG and HCT116.UVRAGΔCCD cells were used for immunoprecipitation with anti-PI(3)KC3 followed by immunoblotting with anti-Beclin1. WCLs were used for immunoblotting with anti-Beclin1 and anti-PI(3)KC3. HCT116.UVRAG cells expressing Flag-tagged UVRAG were incubated under nutrient-rich conditions (lanes 1 and 2), starvation for 4 h (lane 3), or 2 μM rapamycin treatment for 2 h (lane 4). WCLs were then used for immunoprecipitation with anti-Beclin1 (lanes 2, 3 and 4) or preimmune antibody (lane 1) followed by immunoblotting with anti-Flag. The raw data for this experiment are presented in the Supplementary Information, Fig. S5.


( b ) Increased PI ( 3 ) KC3 activity by UVRAG . At 48 h posttransfection with Flag - tagged PI ( 3 ) KC3 with Beclin1 ( lane 1 ) , with Beclin1 + UVRAG ( lane 2 ) or with Beclin1 + UVRAGΔCCD vector ( lane 3 ) , WCLs of 293T cells were used for immunoprecipitation with anti - Flag followed by an assay for PI ( 3 ) KC3 lipid kinase activity , as described in the Methods . At 16 h posttransfection with p40 ( phox ) PX - EGFP fusion , HCT116 . vector and HCT116 . UVRAG cells were detected using an inverted fluorescence microscope . p40 ( phox ) PX - EGFP - positive vesicles were quantified as the mean ± s . d . of combined results from three independent experiments . The scale bars represent 10 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Increased PI(3)KC3 activity by UVRAG. At 48 h posttransfection with Flag-tagged PI(3)KC3 with Beclin1 (lane 1), with Beclin1 + UVRAG (lane 2) or with Beclin1 + UVRAGΔCCD vector (lane 3), WCLs of 293T cells were used for immunoprecipitation with anti-Flag followed by an assay for PI(3)KC3 lipid kinase activity, as described in the Methods. At 16 h posttransfection with p40(phox) PX-EGFP fusion, HCT116.vector and HCT116.UVRAG cells were detected using an inverted fluorescence microscope. p40(phox) PX-EGFP-positive vesicles were quantified as the mean ± s.d. of combined results from three independent experiments. The scale bars represent 10 μm.


( c ) Suppression of autophagy by γHV68 vBcl - 2 . At 18 h posttransfection with GFP - LC3 vector , NIH3T3 . vector , NIH3T3 . vBcl - 2 and NIH3T3 . vBcl - 2AAA , cells were incubated under normal or starvation conditions for 4 h . Subsequently , GFP - LC3 was detected using an inverted fluorescence microscope and autophagy was quantified as mean ± s . d . of combined results from three independent experiments .

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

(c) Suppression of autophagy by γHV68 vBcl-2. At 18 h posttransfection with GFP-LC3 vector, NIH3T3.vector, NIH3T3.vBcl-2 and NIH3T3.vBcl-2AAA, cells were incubated under normal or starvation conditions for 4 h. Subsequently, GFP-LC3 was detected using an inverted fluorescence microscope and autophagy was quantified as mean ± s.d. of combined results from three independent experiments.


( a ) Proliferation rates . HCT116 . vector , HCT116 . UVRAG , HCT116 . UVRAGΔC2 , HCT116 . UVRAGΔCCD and HCT116 . UVRAGCCD cells ( 1 × 105 per well ) were seeded into 6 - well microtitre plates . Cell numbers were counted on the days indicated . The results represent means of three independent assays .

(a) Proliferation rates. HCT116.vector, HCT116.UVRAG, HCT116.UVRAGΔC2, HCT116.UVRAGΔCCD and HCT116.UVRAGCCD cells (1 × 105 per well) were seeded into 6-well microtitre plates. Cell numbers were counted on the days indicated. The results represent means of three independent assays.


( b ) Anchorage independence . HCT116 . vector , HCT116 . UVRAG , HCT116 . UVRAGΔC2 , HCT116 . UVRAGΔCCD and HCT116 . UVRAGCCD cells ( 1 × 104 ) were incubated in soft agar as described in the Methods . After ten days , colonies were photographed and counted . The scale bars represent 50 μm .

(b) Anchorage independence. HCT116.vector, HCT116.UVRAG, HCT116.UVRAGΔC2, HCT116.UVRAGΔCCD and HCT116.UVRAGCCD cells (1 × 104) were incubated in soft agar as described in the Methods. After ten days, colonies were photographed and counted. The scale bars represent 50 μm.


( c ) Tumorigenicity . HCT116 . vector , HCT116 . UVRAG and HCT116 . UVRAGΔCCD cells ( 1 × 107 ) were subcutaneously injected into 5 nude mice , separately . At 16 days postinoculation , mice and tumours were photographed and tumour weights were measured . Arrows indicate location of tumours .

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(c) Tumorigenicity. HCT116.vector, HCT116.UVRAG and HCT116.UVRAGΔCCD cells (1 × 107) were subcutaneously injected into 5 nude mice, separately. At 16 days postinoculation, mice and tumours were photographed and tumour weights were measured. Arrows indicate location of tumours.


C . qRT - PCR data showing induction of each gene in the iniBAC operon in WT Mtb cultured in low iron and in the virR mutant strain cultured in high iron , expressed as fold change relative to WT Mtb cultured in iron sufficient conditions . RNA extracted from biological triplicates was analyzed . The data are presented as means + / - SEM . * * P ≤ 0 . 01 * * * P ≤ 0 . 001 ( Student ' s t - test ) .

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

C. qRT-PCR data showing induction of each gene in the iniBAC operon in WT Mtb cultured in low iron and in the virR mutant strain cultured in high iron, expressed as fold change relative to WT Mtb cultured in iron sufficient conditions. RNA extracted from biological triplicates was analyzed. The data are presented as means +/- SEM. **P≤ 0.01 ***P ≤ 0.001 (Student's t-test).


A . MEV quantitation based on protein and lipid content in vesicle preparations isolated from the same number of Mtb cells treated with INH 50 ng . mlL - 1 for 24 and 48 h relative to untreated cells . Three independent cultures were analyzed . Data are presented as mean ± SEM * * * P ≤ 0 . 001 ( Student ' s t - test ) .

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 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

A. MEV quantitation based on protein and lipid content in vesicle preparations isolated from the same number of Mtb cells treated with INH 50 ng.mlL-1 for 24 and 48 h relative to untreated cells. Three independent cultures were analyzed. Data are presented as mean ± SEM ***P ≤ 0.001 (Student's t-test).


B . Immunodot blot assay detecting MEVs obtained from three independent cultures ( 1 , 2 , 3 ) and derived from the same number of Mtb exposed to 50 ng . mlL - 1 INH for 24 and 48 h . A polyclonal antiserum raised against purified MEV ( Prados - Rosales et al . , 2014a ) was used as a primary antibody .

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B. Immunodot blot assay detecting MEVs obtained from three independent cultures (1, 2, 3) and derived from the same number of Mtb exposed to 50 ng.mlL-1 INH for 24 and 48 h. A polyclonal antiserum raised against purified MEV (Prados-Rosales et al., 2014a) was used as a primary antibody.


E . Protein and lipid content in vesicle preparations isolated from the same number of WT and Msm overexpressing iniACMtb ( Msm iniACMtb ) . Three independent cultures were analyzed . Data are presented as mean ± SEM * P ≤ 0 . 05 ; * * * P ≤ 0 . 001 ( Student ' s t - test ) .

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E. Protein and lipid content in vesicle preparations isolated from the same number of WT and Msm overexpressing iniACMtb (Msm iniACMtb). Three independent cultures were analyzed. Data are presented as mean ± SEM *P≤ 0.05; *** P≤ 0.001 (Student's t-test).


H . The graph shows the avarage of nanoparticle size distribution in three independent vesicle preparations derived from WT ( black ) and Msm iniACMtb ( grey ) .

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H. The graph shows the avarage of nanoparticle size distribution in three independent vesicle preparations derived from WT (black) and Msm iniACMtb (grey).


A . Quantitation of MEVs released by Mtb strains in low iron cultures based on vesicle protein and lipid content normalized to CFUs . Three biological replicates were processed . Data are presented as mean ± SD * P ≤ 0 . 05 , * * P ≤ 0 . 005 ( Student ' s t - test ) .

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A. Quantitation of MEVs released by Mtb strains in low iron cultures based on vesicle protein and lipid content normalized to CFUs. Three biological replicates were processed. Data are presented as mean ± SD *P ≤0.05, **P≤ 0.005 (Student's t-test).


C . Protein in vesicle isolates derived from three independent cultures of Mtb WT and iniA : : hyg mutant grown in low ( LI ) or high iron ( HI ) MM with or without 50 ng · mL - 1 INH . Data are presented as mean ± SEM * P ≤ 0 . 05 . * * * P ≤ 0 . 001 ( Student ' s t - test )

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 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

C. Protein in vesicle isolates derived from three independent cultures of Mtb WT and iniA::hyg mutant grown in low (LI) or high iron (HI) MM with or without 50 ng·mL-1 INH. Data are presented as mean ± SEM *P ≤ 0.05. ***P≤ 0.001 (Student's t-test)


D . Lipid in vesicle isolates derived from three independent cultures of Mtb WT and iniA : : hyg mutant cultured in low ( LI ) or high iron ( HI ) MM with or without 50 ng · mL - 1 INH . Data are presented as mean ± SD * P ≤ 0 . 05 ( Student ' s t - test )

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 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

D. Lipid in vesicle isolates derived from three independent cultures of Mtb WT and iniA::hyg mutant cultured in low (LI) or high iron (HI) MM with or without 50 ng·mL-1 INH. Data are presented as mean ± SD *P ≤ 0.05 (Student's t-test)


B . Mycobactin synthesized by Mtb WT and iniA : : hyg grown in low iron conditions . Data are presented as the mean of mycobactin extracted from two independent cultures .

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

B. Mycobactin synthesized by Mtb WT and iniA::hyg grown in low iron conditions. Data are presented as the mean of mycobactin extracted from two independent cultures.


C . The Mtb siderophore synthesis mutant ( ST142 ) was cultured in low iron MM ( medium ) plain or supplemented with MEV preparations derived from equal number of Mtb WT , iniA : : hyg and complemented strains grown in low iron MM . ST142 growth was determined based on the number of viable cells detected by the resazurine cell viability assay . MEV obtained from three independent cultures were tested .

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

C. The Mtb siderophore synthesis mutant (ST142) was cultured in low iron MM (medium) plain or supplemented with MEV preparations derived from equal number of Mtb WT, iniA::hyg and complemented strains grown in low iron MM. ST142 growth was determined based on the number of viable cells detected by the resazurine cell viability assay. MEV obtained from three independent cultures were tested.


A . Representative Immunodot - blot of bacterial antigens associated with EVs isolated from the culture supernatants of THP - 1 cells infected with Mtb WT , iniA : hyg and complemented strains versus uninfected cells ( UI ) . Anti - MEV specific antiserum was used for detection . The decreasing black bar indicates that increasing dilutions were spotted onto the membrane . The experiment was repeated three times with independent cultures .

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

A. Representative Immunodot-blot of bacterial antigens associated with EVs isolated from the culture supernatants of THP-1 cells infected with Mtb WT, iniA:hyg and complemented strains versus uninfected cells (UI). Anti-MEV specific antiserum was used for detection. The decreasing black bar indicates that increasing dilutions were spotted onto the membrane. The experiment was repeated three times with independent cultures.


B . Number of nanoparticles per mL determined by Zeta View NTA in three EV preparations derived from the culture supernatans of THP - 1 cells infected with Mtb WT , iniA : hyg and iniA : : hyg complemented strains .

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

B. Number of nanoparticles per mL determined by Zeta View NTA in three EV preparations derived from the culture supernatans of THP-1 cells infected with Mtb WT, iniA:hyg and iniA::hyg complemented strains.


C . Representative Western blot of total EVs isolated from the CSF of THP - 1 cells infected with Mtb WT , iniA : : hyg and complemented strains versus uninfected cells ( UI ) detecting the exosome marker CD9 . The experiment was repeated twice with similar results .

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

C. Representative Western blot of total EVs isolated from the CSF of THP-1 cells infected with Mtb WT, iniA::hyg and complemented strains versus uninfected cells (UI) detecting the exosome marker CD9. The experiment was repeated twice with similar results.


B . Changes in the expression of cytokines in THP1 macrophages ( left ) and PBMCs ( right ) upon treatment with full - length S1 subunit at 1 µg / ml for 24 hr . Data information ,

O O O O O O O O O O O O O O O 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. Changes in the expression of cytokines in THP1 macrophages (left) and PBMCs (right) upon treatment with full-length S1 subunit at 1 µg/ml for 24 hr. Data information,


E . Effect of an anti - RBD antibody on S1 subunit stimulated changes in the expression of cytokines in PBMCs . Left , a schematic showing the domain structure of S1 spike protein and anti - RBD antibody . Right , changes in cytokine gene expression in response to S1 spike protein in the presence or abscene of anti - RBD antibody . Gene expression was measured by qPCR . Cytokine release in the conditioned media was measured by Luminex . Full - length S1 and S1 subunits are purified from HEK293 cells . Data information , data are shown as the mean of three technical replicates ; in ( D ) , data are shown as the mean of 20 individual donors . Error bars denote SEM . ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 , Multiple t - test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Effect of an anti-RBD antibody on S1 subunit stimulated changes in the expression of cytokines in PBMCs. Left, a schematic showing the domain structure of S1 spike protein and anti-RBD antibody. Right, changes in cytokine gene expression in response to S1 spike protein in the presence or abscene of anti-RBD antibody. Gene expression was measured by qPCR. Cytokine release in the conditioned media was measured by Luminex. Full-length S1 and S1 subunits are purified from HEK293 cells. Data information, data are shown as the mean of three technical replicates; in (D), data are shown as the mean of 20 individual donors. Error bars denote SEM. ∗p < 0.05, ∗∗p < 0.01, Multiple t-test.


C . Validation of predicted kinases as drivers of cytokine release by siRNAs in THP1 cells . Cytokine release was measured by Luminex and normalized to scrambled siRNA .

O O O O O 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. Validation of predicted kinases as drivers of cytokine release by siRNAs in THP1 cells. Cytokine release was measured by Luminex and normalized to scrambled siRNA.


C . Comparison of kinase inhibition profile of Ponatinib and Baricitinib .

C. Comparison of kinase inhibition profile of Ponatinib and Baricitinib.


B . Dose - response curves of ponatinib treatment on SARS - Cov2 NTD ( Wuhan ) - mediated cytokine release in PBMCs . Right , a table showing EC50 ( nM ) values of ponatinib - mediated inhibition of cytokine release in response to indicated SARS - Cov2 variant NTD treatments in PBMCs .

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B. Dose-response curves of ponatinib treatment on SARS-Cov2 NTD (Wuhan)-mediated cytokine release in PBMCs. Right, a table showing EC50 (nM) values of ponatinib-mediated inhibition of cytokine release in response to indicated SARS-Cov2 variant NTD treatments in PBMCs.


A . Comparison of LPS and full length S1 spike protein - mediated changes in cytokines in THP1 macrophages . Data information , , data are shown as the mean of three technical replicates . Error bars denote SEM .

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

A. Comparison of LPS and full length S1 spike protein-mediated changes in cytokines in THP1 macrophages. Data information, , data are shown as the mean of three technical replicates. Error bars denote SEM.


D . Representative H & E images showing Ponatinib alleviates LPS - induced inflammatory cell infiltration , septal thickening , alveolar edema in mouse lungs . Scale bars 100 µm ( upper ) and 50 µm ( lower ) .

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D. Representative H&E images showing Ponatinib alleviates LPS-induced inflammatory cell infiltration, septal thickening, alveolar edema in mouse lungs. Scale bars 100 µm (upper) and 50 µm (lower).


E . Plots showing pre - treatment ( 60 min ) with Ponatinib ( 35 mg / Kg ) inhibits LPS - induced cytokine release in BALF . Cytokine levels were measured using Luminex . Data information , data are mean of five biological replicates from two independent studies , error bars denote SEM . ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 , Welch ' s t - test .

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E. Plots showing pre-treatment (60 min) with Ponatinib (35 mg/Kg) inhibits LPS-induced cytokine release in BALF. Cytokine levels were measured using Luminex. Data information, data are mean of five biological replicates from two independent studies, error bars denote SEM. ∗p < 0.05, ∗∗p < 0.01, Welch's t-test.


( A ) 293T cells were transfected with Flag - ULK1 or control empty vector and subjected to immunoprecipitation with anti - Flag antibody , followed by immunoblot analysis with the indicated antibodies .

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

(A) 293T cells were transfected with Flag-ULK1 or control empty vector and subjected to immunoprecipitation with anti-Flag antibody, followed by immunoblot analysis with the indicated antibodies.


( B ) 293T cells were co - transfected with Flag - AMPKα2 and HA - ULK1 . Cell lysates were subjected to immunoprecipitation using anti - Flag antibody followed by SDS - PAGE / immunoblot analysis with anti - HA and anti - Flag antibodies .

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(B) 293T cells were co-transfected with Flag-AMPKα2 and HA-ULK1. Cell lysates were subjected to immunoprecipitation using anti-Flag antibody followed by SDS-PAGE/immunoblot analysis with anti-HA and anti-Flag antibodies.


( C ) 293T cells were transiently transfected with Flag - AMPKα2 or Flag - AMPKβ1 . After 24 h , immunoprecipitation was performed using anti - Flag or control anti - HA antibodies and analyzed by immunoblotting with anti - ULK1 and anti - Flag polyclonal antibodies .

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

(C) 293T cells were transiently transfected with Flag-AMPKα2 or Flag-AMPKβ1. After 24 h, immunoprecipitation was performed using anti-Flag or control anti-HA antibodies and analyzed by immunoblotting with anti-ULK1 and anti-Flag polyclonal antibodies.


( D ) 293T cell lysates were subjected to immunoprecipitation with anti - ULK1 polyclonal antibody or control preimmune rabbit serum ( NRS ) , followed by immunoblot analysis with anti - ULK1 and anti - AMPKα antibodies .

(D) 293T cell lysates were subjected to immunoprecipitation with anti-ULK1 polyclonal antibody or control preimmune rabbit serum (NRS), followed by immunoblot analysis with anti-ULK1 and anti-AMPKα antibodies.


( E ) 293T cells were infected with ULK1 shRNA or control shRNA lentiviruses and subjected to immunoprecipitation with control rabbit IgG or anti - AMPKα antibody followed by immunoblotting with anti - ULK1 and anti - AMPKα antibodies .

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(E) 293T cells were infected with ULK1 shRNA or control shRNA lentiviruses and subjected to immunoprecipitation with control rabbit IgG or anti-AMPKα antibody followed by immunoblotting with anti-ULK1 and anti-AMPKα antibodies.


( F ) Purified His6 - AMPKα1 / β1 / γ1 fusion proteins ( 250 ng ) were mixed with purified His6 - tagged ULK1 ( 1 µg ) or Bcl - XL ( 200 ng ) proteins in 1 % Triton X - 100 lysis buffer containing protease inhibitors and subjected to immunoprecipitation with anti - ULK1 antibody . The resulting protein complexes and 10 % of the input proteins were analyzed by immunoblotting with anti - His - Tag polyclonal antibody .

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(F) Purified His6-AMPKα1/β1/γ1 fusion proteins (250 ng) were mixed with purified His6-tagged ULK1 (1 µg) or Bcl-XL (200 ng) proteins in 1% Triton X-100 lysis buffer containing protease inhibitors and subjected to immunoprecipitation with anti-ULK1 antibody. The resulting protein complexes and 10% of the input proteins were analyzed by immunoblotting with anti-His-Tag polyclonal antibody.


( B , C ) 293T cells were transfected with empty vector or HA - tagged wild type ( WT ) or deletion mutants of ULK1 and subjected to immunoprecipitation with anti - HA antibody . The resulting immune complexes were analyzed by immunoblotting with antibodies specific for mTOR , raptor , AMPKα or HA - tag .

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(B, C) 293T cells were transfected with empty vector or HA-tagged wild type (WT) or deletion mutants of ULK1 and subjected to immunoprecipitation with anti-HA antibody. The resulting immune complexes were analyzed by immunoblotting with antibodies specific for mTOR, raptor, AMPKα or HA-tag.


( A ) 293T cells were transfected with empty vector or HA - tagged wild type ( WT ) or deletion mutant ( Δ654 - 828 ) ULK1 and subjected to immunoprecipitation with anti - HA antibody followed by immunoblotting with the indicated antibodies .

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(A) 293T cells were transfected with empty vector or HA-tagged wild type (WT) or deletion mutant (Δ654-828) ULK1 and subjected to immunoprecipitation with anti-HA antibody followed by immunoblotting with the indicated antibodies.


( B ) U - 2OS cells stably expressing GFP - LC3 were infected with lentivirus expressing human Ulk1 shRNA ( shhUlk1 ) or scrambled shRNA ( shScr ) and subjected to selection with 1 µg / ml puromycin for 5 days . The cells were then transfected with wild type mouse Ulk1 ( mUlk1 ) , mUlk1 Δ654 - 828 or control empty vector for 24 h , incubated in serum - free DMEM overnight , and cultured in complete medium with or without 10 mM metformin for 20 h . The images were obtained using a fluorescence microscope . The scale bars represent 10 µm .

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(B) U-2OS cells stably expressing GFP-LC3 were infected with lentivirus expressing human Ulk1 shRNA (shhUlk1) or scrambled shRNA (shScr) and subjected to selection with 1 µg/ml puromycin for 5 days. The cells were then transfected with wild type mouse Ulk1 (mUlk1), mUlk1 Δ654-828 or control empty vector for 24 h, incubated in serum-free DMEM overnight, and cultured in complete medium with or without 10 mM metformin for 20 h. The images were obtained using a fluorescence microscope. The scale bars represent 10 µm.


( C ) The number of GFP - LC3 dots per GFP - positive cell in ( B ) was quantified ( mean ± s . d . ; n = 81 ) .

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(C) The number of GFP-LC3 dots per GFP-positive cell in (B) was quantified (mean ± s.d.; n = 81).


( D ) U - 2OS cells stably expressing GFP - LC3 were transfected with 0 , 2 or 4 µg of pcDNA3 encoding HA - tagged ULK1 deletion mutant ( 554 - 828 ) . The total amount of plasmid DNA used for each transfection was normalized at 4 µg with empty vector . After 24 h transfection , the cells were starved in nutrient - free medium for 1 . 5 h , fixed in 4 % paraformaldehyde , and analyzed by fluorescence microscopy . The number of GFP - LC3 dots per GFP - positive cell was quantified ( mean ± s . d . ; n = 100 ) . Statistical significance was determined by Student ' s t - test and the asterisks indicate P < 0 . 0001 .

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(D) U-2OS cells stably expressing GFP-LC3 were transfected with 0, 2 or 4 µg of pcDNA3 encoding HA-tagged ULK1 deletion mutant (554-828). The total amount of plasmid DNA used for each transfection was normalized at 4 µg with empty vector. After 24 h transfection, the cells were starved in nutrient-free medium for 1.5 h, fixed in 4% paraformaldehyde, and analyzed by fluorescence microscopy. The number of GFP-LC3 dots per GFP-positive cell was quantified (mean ± s.d.; n = 100). Statistical significance was determined by Student's t-test and the asterisks indicate P<0.0001.


293 cells stably expressing HA - ULK1 were treated with 1 mM AICAR for the indicated times and subjected to immunoprecipitation with anti - HA or control anti - FLAG antibodies . The resulting immune complexes were analyzed by immunoblotting with the indicated antibodies .

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293 cells stably expressing HA-ULK1 were treated with 1 mM AICAR for the indicated times and subjected to immunoprecipitation with anti-HA or control anti-FLAG antibodies. The resulting immune complexes were analyzed by immunoblotting with the indicated antibodies.


( A ) TSC2 − / − , p53 − / − MEFs stably reconstituted with wild - type raptor or S722A / S792A mutant raptor were transfected with GFP - LC3 , treated with 2 mM AICAR or control vehicle for 4 h , and then analyzed by fluorescent microscopy . The number of GFP - LC3 dots per GFP - positive cell was counted ( mean ± s . d . ; n = 60 ) . The scale bars represent 10 µm .

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(A) TSC2−/−, p53−/− MEFs stably reconstituted with wild-type raptor or S722A/S792A mutant raptor were transfected with GFP-LC3, treated with 2 mM AICAR or control vehicle for 4 h, and then analyzed by fluorescent microscopy. The number of GFP-LC3 dots per GFP-positive cell was counted (mean ± s.d.; n = 60). The scale bars represent 10 µm.


( B ) TSC2 − / − , p53 − / − MEFs stably reconstituted with wild - type raptor or S722A / S792A mutant raptor were treated with 2 mM AICAR for the indicated times . Cell lysates were prepared in RIPA buffer and analyzed by immunoblotting with the indicated antibodies . The levels of p62 are listed relative to those of untreated WT raptor cells , which were set as 1 .

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(B) TSC2−/−, p53−/− MEFs stably reconstituted with wild-type raptor or S722A/S792A mutant raptor were treated with 2 mM AICAR for the indicated times. Cell lysates were prepared in RIPA buffer and analyzed by immunoblotting with the indicated antibodies. The levels of p62 are listed relative to those of untreated WT raptor cells, which were set as 1.


( C ) Clonal cell lines expressing p53 - Venus were engineered to monitor p53 expression dynamics ( " input " ) and two canonically regulated p53 target promoters expressing mCherry ( " output " ) in response to Nutlin - 3 treatment .

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(C) Clonal cell lines expressing p53-Venus were engineered to monitor p53 expression dynamics ("input") and two canonically regulated p53 target promoters expressing mCherry ("output") in response to Nutlin-3 treatment.


( D ) Representative phase contrast and yellow fluorescent ( indicating p53 - Venus levels ) images at the indicated time points for a cell exposed to the " natural dynamics " Nutlin - 3 dosing regimen .

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(D) Representative phase contrast and yellow fluorescent (indicating p53-Venus levels) images at the indicated time points for a cell exposed to the "natural dynamics" Nutlin-3 dosing regimen.


( E - F ) p53 - Venus expression in response to the " natural dynamics " Nutlin - 3 dosing regimen . Single cell traces ( gray ; E ) and the mean ( red ; E ) are shown for p53 - Venus expression in response to each Nutlin - 3 regimen . Heat map ( F ) shows an alternative representation of all traces as shown in ( E ) . N = 51 cells .

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(E-F) p53-Venus expression in response to the "natural dynamics" Nutlin-3 dosing regimen. Single cell traces (gray; E) and the mean (red; E) are shown for p53-Venus expression in response to each Nutlin-3 regimen. Heat map (F) shows an alternative representation of all traces as shown in (E). N = 51 cells.


( A ) Representative phase contrast , yellow fluorescent ( indicating p53 - Venus levels ) , and red fluorescent ( indicating reporter mCherry levels ) images at the indicated time points for MDM2 promoter reporter cells exposed to the " natural dynamics " Nutlin - 3 dosing regimen .

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(A) Representative phase contrast, yellow fluorescent (indicating p53-Venus levels), and red fluorescent (indicating reporter mCherry levels) images at the indicated time points for MDM2 promoter reporter cells exposed to the "natural dynamics" Nutlin-3 dosing regimen.


( B - G ) Single cell traces of mCherry expression for the " responding " ( light gray ) or " not responding " ( dark gray ) MDM2 promoter or CDKN1A promoter cells exposed to the natural dynamics ( B ) , low amplitude ( D ) , high amplitude ( D ) , high frequency ( E ) , low frequency / short duration ( F ) , or long duration ( G ) Nutlin - 3 dosing regimens . The average trace for responding and not responding cells is shown in red and blue , respectively . Heat maps show alternative representation of all single cell traces below each associated time course plot .

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(B-G) Single cell traces of mCherry expression for the "responding" (light gray) or "not responding" (dark gray) MDM2 promoter or CDKN1A promoter cells exposed to the natural dynamics (B), low amplitude (D), high amplitude (D), high frequency (E), low frequency/short duration (F), or long duration (G) Nutlin-3 dosing regimens. The average trace for responding and not responding cells is shown in red and blue, respectively. Heat maps show alternative representation of all single cell traces below each associated time course plot.


Dose - response curves representing the rate of mCherry expression from the MDM2 ( pink dots ) and CDKN1A ( purple dots ) promoters as a function of total p53 levels . Values of expression levels were averaged across all cells in the first 15 - h response to the long duration Nutlin - 3 dosing regimen . Solid black lines represent the best fit to a Hill function model of promoter activation . Dashed lines indicate the half - maximal threshold of p53 levels for promoter activation and the half maximal promoter activity for each promoter .

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Dose-response curves representing the rate of mCherry expression from the MDM2 (pink dots) and CDKN1A (purple dots) promoters as a function of total p53 levels. Values of expression levels were averaged across all cells in the first 15-h response to the long duration Nutlin-3 dosing regimen. Solid black lines represent the best fit to a Hill function model of promoter activation. Dashed lines indicate the half-maximal threshold of p53 levels for promoter activation and the half maximal promoter activity for each promoter.


( B - G ) Effects of p53 duration modulation on target promoter activation in terms of the mean timing ( B , E ) , magnitude ( C , F ) , and rate ( D , G ) for the MDM2 ( B - D ) and CDKN1A ( E - G ) promoters in response to short ( light green ) and long ( dark green ) p53 durations . N = at least 45 cells per condition , error bars = SEM .

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(B-G) Effects of p53 duration modulation on target promoter activation in terms of the mean timing (B, E), magnitude (C, F), and rate (D, G) for the MDM2 (B-D) and CDKN1A (E-G) promoters in response to short (light green) and long (dark green) p53 durations. N = at least 45 cells per condition, error bars = SEM.


( H ) Mean nuclear MDM2 - YFP levels in single cells at times corresponding to the first pulse ( 5 . 5 h ) or second pulse ( 11 h ) of p53 expression in response to natural and long duration Nutlin - 3 dosing regimens . N = at least 40 cells per condition , error bars = SEM .

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(H) Mean nuclear MDM2-YFP levels in single cells at times corresponding to the first pulse (5.5 h) or second pulse (11 h) of p53 expression in response to natural and long duration Nutlin-3 dosing regimens. N = at least 40 cells per condition, error bars = SEM.


( A ) Effects of p53 frequency modulation on target promoter activation in terms of the mean timing ( A , D ) , magnitude ( B , E ) , and rate ( C , F ) for the MDM2 ( A - C ) and CDKN1A ( D - F ) promoters in response to low ( light blue ) , natural ( medium blue ) , high ( dark blue ) p53 frequencies . N = at least 45 cells per condition , error bars = SEM .

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(A) Effects of p53 frequency modulation on target promoter activation in terms of the mean timing (A, D), magnitude (B, E), and rate (C, F) for the MDM2 (A-C) and CDKN1A (D-F) promoters in response to low (light blue), natural (medium blue), high (dark blue) p53 frequencies. N = at least 45 cells per condition, error bars = SEM.


( H ) Percentage of responding cells for the MDM2 and CDKN1A promoters in response to low , natural , and high frequency p53 pulses .

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(H) Percentage of responding cells for the MDM2 and CDKN1A promoters in response to low, natural, and high frequency p53 pulses.


( A - B ) Representative phase contrast , red fluorescence ( indicating geminin - mCherry levels ) , and yellow fluorescence ( indicating p53 - Venus levels ) images of cells in response to a low ( A ) or high ( B ) frequency Nutlin - 3 dosing regimen over 40 h .

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(A-B) Representative phase contrast, red fluorescence (indicating geminin-mCherry levels), and yellow fluorescence (indicating p53-Venus levels) images of cells in response to a low (A) or high (B) frequency Nutlin-3 dosing regimen over 40 h.


( C ) Geminin - mCherry traces in single cells treated with the low , natural , or high frequency Nutlin - 3 dosing regimens . N = at least 50 cells per condition .

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(C) Geminin-mCherry traces in single cells treated with the low, natural, or high frequency Nutlin-3 dosing regimens. N = at least 50 cells per condition.


Percentage of cells undergoing cell division ( D ) within the 40 h imaging in response to the low , natural , and high frequency Nutlin - 3 dosing

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Percentage of cells undergoing cell division (D) within the 40 h imaging in response to the low, natural, and high frequency Nutlin-3 dosing regimens


Percentage of cells positive for geminin - mCherry expression ( E ) within the 40 h imaging in response to the low , natural , and high frequency Nutlin - 3 dosing

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Percentage of cells positive for geminin-mCherry expression (E) within the 40 h imaging in response to the low, natural, and high frequency Nutlin-3 dosing regimens


B HeLa cells expressing the indicated GFP - tagged Nups were semi - permeabilized and incubated with mitotic HeLa cell extract at 30ºC . NPC disassembly was followed by monitoring the fluorescence intensity of the respective Nups at the nuclear rim and influx of TRITC - dextran . Scale bar , 30 μm .

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B HeLa cells expressing the indicated GFP-tagged Nups were semi-permeabilized and incubated with mitotic HeLa cell extract at 30ºC. NPC disassembly was followed by monitoring the fluorescence intensity of the respective Nups at the nuclear rim and influx of TRITC-dextran. Scale bar, 30 μm.


C Quantification of dextran - positive nuclei over time of the experiments in ( B ) . N = 3 biological replicates , n > 100 cells . Error bars , SEM .

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C Quantification of dextran-positive nuclei over time of the experiments in (B). N = 3 biological replicates, n > 100 cells. Error bars, SEM.


E Quantification of 3GFP - Nup133 , GFP - Nup107 and 2GFP - Nup58 intensity at the NE over time of the experiments in ( B ) . Error bars , SEM .

E Quantification of 3GFP-Nup133, GFP-Nup107 and 2GFP-Nup58 intensity at the NE over time of the experiments in (B). Error bars, SEM.


B Input , light and dense fractions were analyzed by immunoblotting using the indicated antibodies . The samples were derived from the same experiment but processed using several gels and blots .

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B Input, light and dense fractions were analyzed by immunoblotting using the indicated antibodies. The samples were derived from the same experiment but processed using several gels and blots.


C Quantification of the percentage of Nups in the light fractions relative to the total amount of Nups over all fractions . N = 4 biological replicates , except for Nup214 ( N = 2 biological replicates ) . Error bars , SEM .

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C Quantification of the percentage of Nups in the light fractions relative to the total amount of Nups over all fractions. N = 4 biological replicates, except for Nup214 (N = 2 biological replicates). Error bars, SEM.


B Confocal images of metaphase - arrested HeLa cells expressing 3GFP - Nup133 , GFP - Nup107 , GFP - Nup53 or 2GFP - Nup58 that were either directly fixed ( untreated ) or first semi - permeabilized and then fixed . The ER was visualized by immunostaining with an antibody targeting calnexin ; DNA was visualized with Hoechst ( visible only in the merged channels ) . Representative images of 3 biological replicates . Scale bar , 10 μm and inset scale bar , 1 μm .

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B Confocal images of metaphase-arrested HeLa cells expressing 3GFP-Nup133, GFP-Nup107, GFP-Nup53 or 2GFP-Nup58 that were either directly fixed (untreated) or first semi-permeabilized and then fixed. The ER was visualized by immunostaining with an antibody targeting calnexin; DNA was visualized with Hoechst (visible only in the merged channels). Representative images of 3 biological replicates. Scale bar, 10 μm and inset scale bar, 1 μm.


A , B 2D dSTORM images of interphase ( A ) or metaphase ( B ) U2OS cells expressing SNAP - tagged Nup96 . Cells were semi - permeabilized with digitonin and Nup96 was labelled with the BG - AF647 SNAP dye . The SMLM data was reconstructed using Thunderstorm . Left panels were rendered to black and white and enhanced for viewing purposes . Insets ( yellow dashed boxes ) are displayed to the right . Insets in ( A ) show ring - shaped structures built by Y - complexes in interphase cells . Insets in ( B ) represents mitotic clusters outside the spindle area ( class 1 , inset 1 ) and at kinetochores ( class 2 ) . Scale bar ( left panels ) , 10 μm . Representative images of 4 biological replicates . Inset scale bars , 200 nm .

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A, B 2D dSTORM images of interphase (A) or metaphase (B) U2OS cells expressing SNAP-tagged Nup96. Cells were semi-permeabilized with digitonin and Nup96 was labelled with the BG-AF647 SNAP dye. The SMLM data was reconstructed using Thunderstorm. Left panels were rendered to black and white and enhanced for viewing purposes. Insets (yellow dashed boxes) are displayed to the right. Insets in (A) show ring-shaped structures built by Y-complexes in interphase cells. Insets in (B) represents mitotic clusters outside the spindle area (class 1, inset 1) and at kinetochores (class 2). Scale bar (left panels), 10 μm. Representative images of 4 biological replicates. Inset scale bars, 200 nm.


A 2D dSTORM images of a metaphase - arrested U2OS cell expressing Nup96 with a SNAP - tag , labelled with BG - Alexa Fluor 647 ( orange ) , in combination with confocal imaging of the ER immunostained for Climp63 ( red ) and phospho - histone H3 ( cyan ) to label DNA . Inset 1 ( mitotic clusters ) and inset 2 ( kinetochores ) are indicated with white dashed boxes . SMLM data was reconstructed using Thunderstorm . Representative images of N = 4 biological replicates . Scale bars , 10 μm . Inset scale bars , 200 nm .

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A 2D dSTORM images of a metaphase-arrested U2OS cell expressing Nup96 with a SNAP-tag, labelled with BG-Alexa Fluor 647 (orange), in combination with confocal imaging of the ER immunostained for Climp63 (red) and phospho-histone H3 (cyan) to label DNA. Inset 1 (mitotic clusters) and inset 2 (kinetochores) are indicated with white dashed boxes. SMLM data was reconstructed using Thunderstorm. Representative images of N = 4 biological replicates. Scale bars, 10 μm. Inset scale bars, 200 nm.