A Maximum intensity projections of smFISH fluorescence microscopy z - stacks of Jurkat T cells stained for the indicated genes after 1 - hour ( Nfkbia , Tnfaip3 , Tnf , Il8 ) or 4 - hour ( Il6 , Csf2 ) treatment with 20 ng / mL TNF . Nfkbia , Tnfaip3 , Tnf , and Il6 were labeled with Quasar 670 , and Il8 and Csf2 were labeled with fluorescein . All images were filtered as described in Materials and Methods . Brightness and contrast were enhanced for visualization . Scale bars : 10 μm . B Bar graphs of mean of smFISH distributions before and after TNF treatment for the indicated genes . Cells were combined from three replicates ( Nfkbia 1h ; Tnfaip3 1h ; Tnf 1h , 2h ; and Il6 2h , 4h ) two replicates ( Nfkbia 2h ) or one replicate ( Tnfaip3 2h ; Il8 1h , 2h ; Csf2 2h , 4h ) . Basal data are same as in Fig . 1E . Error bars indicate bootstrapped 95 % CIs for the samples in ( C ) . Significant differences indicated by non - overlapping CIs . C

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A Maximum intensity projections of smFISH fluorescence microscopy z-stacks of Jurkat T cells stained for the indicated genes after 1-hour (Nfkbia, Tnfaip3, Tnf, Il8) or 4-hour (Il6, Csf2) treatment with 20 ng/mL TNF. Nfkbia, Tnfaip3, Tnf, and Il6 were labeled with Quasar 670, and Il8 and Csf2 were labeled with fluorescein. All images were filtered as described in Materials and Methods. Brightness and contrast were enhanced for visualization. Scale bars: 10 μm. B Bar graphs of mean of smFISH distributions before and after TNF treatment for the indicated genes. Cells were combined from three replicates (Nfkbia 1h; Tnfaip3 1h; Tnf 1h, 2h; and Il6 2h, 4h) two replicates (Nfkbia 2h) or one replicate (Tnfaip3 2h; Il8 1h, 2h; Csf2 2h, 4h). Basal data are same as in Fig. 1E. Error bars indicate bootstrapped 95% CIs for the samples in (C). Significant differences indicated by non-overlapping CIs. C


B - C Burst size ( B ) and burst frequency ( C ) parameter fits from the two - state model in the basal state and after treatment with 20 ng / mL TNF for 1 , 2 or 4 hours . Error bars indicate bootstrapped 95 % CIs . Significant differences indicated by non - overlapping CIs . The fit for Nfkbia ( 1h TNF ) was unstable and thus is not

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B-C Burst size (B) and burst frequency (C) parameter fits from the two-state model in the basal state and after treatment with 20 ng/mL TNF for 1, 2 or 4 hours. Error bars indicate bootstrapped 95% CIs. Significant differences indicated by non-overlapping CIs. The fit for Nfkbia (1h TNF) was unstable and thus is not reported


A Enrichment of histone H3 , AcH3 , total RNPII , ser5 - p RNPII , ser2 - p RNPII , and NELF - E in the basal state ( 0 hours ) and after treatment ( 2 and 4 hours ) with 20 ng / ml TNF quantified using ChIP and shown as % input ( non - IP control ) . Data are presented as mean ± standard error of the mean ( s . e . m . ) of three biological replicates . Significance calculated by Dunnett ' s multiple comparison test ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 ) .

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A Enrichment of histone H3, AcH3, total RNPII, ser5-p RNPII, ser2-p RNPII, and NELF-E in the basal state (0 hours) and after treatment (2 and 4 hours) with 20 ng/ml TNF quantified using ChIP and shown as % input (non-IP control). Data are presented as mean ± standard error of the mean (s.e.m.) of three biological replicates. Significance calculated by Dunnett's multiple comparison test (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).


C Bar graphs of mean mRNA level for basal condition , 1h TNF , 1h TNF + 4h pretreatment with 300nM A - 485 , and 1h TNF + 62 . 5nM JQ1 cotreatment measured by smFISH for Tnfaip3 ( left ) and Tnf ( right ) . Cells were combined from two replicates ( Tnfaip3 A - 485 , JQ1 ; Tnf A - 485 ) or one replicate ( Tnf JQ1 ) . Error bars indicated bootstrapped 95 % CIs . Samples with non - overlapping CIs are significant . D Change in enrichment of total and Serine - 5 - phosphorylated RNAPII after 20ng / mL TNF treatment for 1 hour with 62 . 5nM JQ1 measured by ChIP - qPCR and shown as % input ( non - IP control ) normalized to the uninhibited control for each gene . Data are presented as mean for two biological replicates . E

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C Bar graphs of mean mRNA level for basal condition, 1h TNF, 1h TNF + 4h pretreatment with 300nM A-485, and 1h TNF+62.5nM JQ1 cotreatment measured by smFISH for Tnfaip3 (left) and Tnf (right). Cells were combined from two replicates (Tnfaip3 A-485, JQ1; Tnf A-485) or one replicate (Tnf JQ1). Error bars indicated bootstrapped 95% CIs. Samples with non-overlapping CIs are significant. D Change in enrichment of total and Serine-5-phosphorylated RNAPII after 20ng/mL TNF treatment for 1 hour with 62.5nM JQ1 measured by ChIP-qPCR and shown as % input (non-IP control) normalized to the uninhibited control for each gene. Data are presented as mean for two biological replicates. E


Ratiometric and mTFP1 donor images of EKAR2G in an EGF ‐ stimulated PC ‐ 12 cell at the indicated time points , with t = 0 ' corresponding to EGF application . Upper panel : FRET ratio image is color ‐ coded for ERK activity . Lower panel : raw mTFP1 donor image in black / white contrast . Scale bar = 20 μm .

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Ratiometric and mTFP1 donor images of EKAR2G in an EGF‐stimulated PC‐12 cell at the indicated time points, with t = 0' corresponding to EGF application. Upper panel: FRET ratio image is color‐coded for ERK activity. Lower panel: raw mTFP1 donor image in black/white contrast. Scale bar = 20 μm.


Population average of ERK activation dynamics measured by Western blot using a phosphoERK antibody .

Population average of ERK activation dynamics measured by Western blot using a phosphoERK antibody.


Population average of ERK activation dynamics cell ‐ averaged EKAR2G emission ratios ( ERs ) from n = at least 111 cells . StDev are shown .

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Population average of ERK activation dynamics cell‐averaged EKAR2G emission ratios (ERs) from n= at least 111 cells. StDev are shown.


Selected EKAR2G ratio time series illustrating , from top to bottom , sustained , oscillatory , or transient ERK activity dynamics . ER is color ‐ coded as in bar . Scale bar = 20 μm .

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Selected EKAR2G ratio time series illustrating, from top to bottom, sustained, oscillatory, or transient ERK activity dynamics. ER is color‐coded as in bar. Scale bar = 20 μm.


Single ‐ cell ERK activity trajectories . Cell ‐ averaged ERs for n = 10 cells , standard deviation range ( StDev ) , population average for the indicated GFs and dosages . Experimental time courses were normalized to the mean of 5 time points immediately preceding GF application .

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Single‐cell ERK activity trajectories. Cell‐averaged ERs for n = 10 cells, standard deviation range (StDev), population average for the indicated GFs and dosages. Experimental time courses were normalized to the mean of 5 time points immediately preceding GF application.


Waterfall plots of single ‐ cell ERK activity trajectories . Cell ‐ averaged ER trajectories are color ‐ coded ( n = 78 cells ) , population average ( bottom ) . Vertical dotted line indicates GF application .

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Waterfall plots of single‐cell ERK activity trajectories. Cell‐averaged ER trajectories are color‐coded (n = 78 cells), population average (bottom). Vertical dotted line indicates GF application.


Trajectories from all sustained GF stimulation experiments ( n = 307 cells , same ERK activation trajectories as in Fig 1 ) were pooled and five representative trajectories were identified using k ‐ means clustering with squared Euclidean distance . Raw ( color ‐ coded by cluster ) and cluster representative trajectories ( black ) .

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Trajectories from all sustained GF stimulation experiments (n = 307 cells, same ERK activation trajectories as in Fig 1) were pooled and five representative trajectories were identified using k‐means clustering with squared Euclidean distance. Raw (color‐coded by cluster) and cluster representative trajectories (black).


Overlaid cluster representative trajectories .

Overlaid cluster representative trajectories.


Population distribution of representative ERK activity trajectories in response to different GF dosages . Data representative of n = 3 experiments .

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Population distribution of representative ERK activity trajectories in response to different GF dosages. Data representative of n = 3 experiments.


Peak emission ratio intensity for each cluster . Boxplots with median , interquartile ( box ) and 1 . 5 IQR ( whiskers ) range , and raw datasets are shown . Boxplot notches extend 1 . 58 IQR / √ Nobs , which gives approximately 95 % confidence interval for comparing medians .

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Peak emission ratio intensity for each cluster. Boxplots with median, interquartile (box) and 1.5 IQR (whiskers) range, and raw datasets are shown. Boxplot notches extend 1.58 IQR / √Nobs, which gives approximately 95% confidence interval for comparing medians.


A - DSingle GF pulse experiments . Cell ‐ averaged ERs , population average , and StDev range for n = 10 cells . Pulse application indicated by black bars . 3 ′ ( A , C ) and 10 ′ ( B , D ) pulse . High ( A , B ) and low ( C , D ) GF concentrations .

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A-DSingle GF pulse experiments. Cell‐averaged ERs, population average, and StDev range for n = 10 cells. Pulse application indicated by black bars. 3′ (A, C) and 10′ (B, D) pulse. High (A, B) and low (C, D) GF concentrations.


ERationale for measuring amplitude and duration of 1st ERK activity peak in response to GF stimulation . Peak amplitude was measured as the ER change from when the GF stimulation starts until the highest ERK activity before adaptation occurs . Peak duration was estimated as the time between the first point before reaching the half ‐ maximum of the peak in the ascending phase , and the first point after the half ‐ maximum in the descending phase .

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ERationale for measuring amplitude and duration of 1st ERK activity peak in response to GF stimulation. Peak amplitude was measured as the ER change from when the GF stimulation starts until the highest ERK activity before adaptation occurs. Peak duration was estimated as the time between the first point before reaching the half‐maximum of the peak in the ascending phase, and the first point after the half‐maximum in the descending phase.


FAmplitude of 1st ERK activity peak in response to different sustained or pulsed GF stimulation experiments . Notched boxplots of 1st ERK activity peak amplitude with median , interquartile range ( box ) , and data within 1 . 5 IQR range of the lower and upper quartiles ( whiskers ) are shown ( n = 20 cells per experiment ) .

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FAmplitude of 1st ERK activity peak in response to different sustained or pulsed GF stimulation experiments. Notched boxplots of 1st ERK activity peak amplitude with median, interquartile range (box), and data within 1.5 IQR range of the lower and upper quartiles (whiskers) are shown (n = 20 cells per experiment).


GDuration of 1st ERK activity peak in response to sustained and pulsed GF stimulation . Boxplots of 1st ERK activity peak duration with median , interquartile range ( box ) , and data within 1 . 5 IQR range of the lower and upper quartiles ( whiskers ) are shown ( n = 20 cells per experiment ) .

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GDuration of 1st ERK activity peak in response to sustained and pulsed GF stimulation. Boxplots of 1st ERK activity peak duration with median, interquartile range (box), and data within 1.5 IQR range of the lower and upper quartiles (whiskers) are shown (n = 20 cells per experiment).


A - CDouble ‐ pulse GF experiments . ( A ) 3 ′ GF / 3 ′ pause / 3 ′ GF . ( B ) 3 ′ GF / 10 ′ pause / 3 ′ GF . ( C ) 3 ′ GF / 20 ′ pause / 3 ′ GF . Cell ‐ averaged ERs , population average , and StDev range for n = 10 cells .

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A-CDouble‐pulse GF experiments. (A) 3′ GF/3′ pause/3′ GF. (B) 3′ GF/10′ pause/3′ GF. (C) 3′ GF/20′ pause/3′ GF. Cell‐averaged ERs, population average, and StDev range for n = 10 cells.


D - GMulti ‐ pulse GF experiments . Population average ERs ( n = at least 30 cells ) for different EGF / NGF dosages . ( D ) 3 ′ GF / 3 ′ pause . ( E ) 3 ′ GF / 10 ′ pause . ( F ) 3 ′ GF / 20 ′ pause . ( G ) 3 ′ GF / 60 ′ pause .

D-GMulti‐pulse GF experiments. Population average ERs (n= at least 30 cells) for different EGF/NGF dosages. (D) 3′ GF/3′pause. (E) 3′ GF/10′ pause. (F) 3′ GF/20′ pause. (G) 3′ GF/60′ pause.


HDecay kinetics of ERK activity maxima from ( D - G ) . Boxplots with median , interquartile ( box ) , and 1 . 5 IQR ( whiskers ) range are shown for at least n = 30 cells .

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HDecay kinetics of ERK activity maxima from (D-G). Boxplots with median, interquartile (box), and 1.5 IQR (whiskers) range are shown for at least n = 30 cells.


ARepresentative micrographs of tubulin ‐ stained PC ‐ 12 NS ‐ 1 subclone cells stimulated with sustained and multi ‐ pulse GF regimes . Scale bar = 20 μm . B , CQuantification of neuronal differentiation fate . ( B ) Total neurite outgrowth per cell normalized to the major axis length of cell soma for sustained and multi ‐ pulse GF regimes . ( C ) Differentiation ratio calculated as the fraction of cells with normalized total neurite outgrowth per cell larger than 1 . At least 5 , 000 cells quantified in at least 2 independent experiments for each condition . Boxplots with median , interquartile ( Box ) , and 1 . 5 IQR ( Whiskers ) are shown .

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ARepresentative micrographs of tubulin‐stained PC‐12 NS‐1 subclone cells stimulated with sustained and multi‐pulse GF regimes. Scale bar = 20 μm.B, CQuantification of neuronal differentiation fate. (B) Total neurite outgrowth per cell normalized to the major axis length of cell soma for sustained and multi‐pulse GF regimes. (C) Differentiation ratio calculated as the fraction of cells with normalized total neurite outgrowth per cell larger than 1. At least 5,000 cells quantified in at least 2 independent experiments for each condition. Boxplots with median, interquartile (Box), and 1.5 IQR (Whiskers) are shown.


( B ) The concentration - effect relationships of PARP1 inhibition by thioparib , talazoparib , and olaparib , as assayed by histone - based ELISA . Data are obtained from three biological replicates and depicted as mean ± SEM . ( C ) Concentration - dependent increase in PARP1 - DNA binding by three PARP inhibitors using DSB FA assays . FA was measured 60 min after adding NAD + , and the ΔFA values represent the changes in PARP1 - DNA binding . Data from four biological replicates are presented as mean ± SEM . (

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(B) The concentration-effect relationships of PARP1 inhibition by thioparib, talazoparib, and olaparib, as assayed by histone-based ELISA. Data are obtained from three biological replicates and depicted as mean ± SEM. (C) Concentration-dependent increase in PARP1-DNA binding by three PARP inhibitors using DSB FA assays. FA was measured 60 min after adding NAD+, and the ΔFA values represent the changes in PARP1-DNA binding. Data from four biological replicates are presented as mean ± SEM. (


( F ) and ( G ) Effects of thioparib on BRCA1 - deficient MDA - MB - 436 ( F ) and BRCA2 - deficient Capan - 1 ( G ) xenografts . Mice bearing MDA - MB - 436 tumors were dosed orally with 10 mg / kg thioparib , 100 mg / kg olaparib , or vehicle ( Veh ) once daily for 3 weeks ; and mice bearing Capan - 1 tumors were dosed orally with 5 or 25 mg / kg thioparib , 0 . 3 mg / kg talazoparib , or vehicle ( Veh ) once daily for 3 weeks ( n = 6 ) . Data are shown as mean ± SEM . Statistical analysis was performed by two - way ANOVA . * * * P < 0 . 0001 . Representative images of xenograft tumors are shown , and mice tails represent complete regression of tumors .

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(F) and (G) Effects of thioparib on BRCA1-deficient MDA-MB-436 (F) and BRCA2-deficient Capan-1 (G) xenografts. Mice bearing MDA-MB-436 tumors were dosed orally with 10 mg/kg thioparib, 100 mg/kg olaparib, or vehicle (Veh) once daily for 3 weeks; and mice bearing Capan-1 tumors were dosed orally with 5 or 25 mg/kg thioparib, 0.3 mg/kg talazoparib, or vehicle (Veh) once daily for 3 weeks (n = 6). Data are shown as mean ± SEM. Statistical analysis was performed by two-way ANOVA. ***P < 0.0001. Representative images of xenograft tumors are shown, and mice tails represent complete regression of tumors.


( C ) The IC50 values of thioparib in six acquired PARPi - resistant cell lines . Numbers 1 - 6 represent the PARPi - resistant Capan - 1 / OP , Capan - 1 / TP , MDA - MB - 436 / OP , MDA - MB - 436 / TP , U251 / OP , and U251 / TP cells , respectively . Cells were treated with the indicated PARP inhibitors for 7 days and then subjected to SRB assay . The IC50 values are expressed as the mean ± SD from three separate experiments .

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(C) The IC50 values of thioparib in six acquired PARPi-resistant cell lines. Numbers 1-6 represent the PARPi-resistant Capan-1/OP, Capan-1/TP, MDA-MB-436/OP, MDA-MB-436/TP, U251/OP, and U251/TP cells, respectively. Cells were treated with the indicated PARP inhibitors for 7 days and then subjected to SRB assay. The IC50 values are expressed as the mean ± SD from three separate experiments.


( E ) Efficacy of thioparib in the PDX model BR - 05 - 0028 ( BRCA1 - deficient ) . Mice bearing BR - 05 - 0028 tumors derived from a patient with breast cancer ( n = 6 ) were treated with 10 or 30 mg / kg thioparib or 100 mg / kg olaparib for 6 weeks . Data are depicted as mean ± SEM . * * * P < 0 . 0001 .

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(E) Efficacy of thioparib in the PDX model BR-05-0028 (BRCA1-deficient). Mice bearing BR-05-0028 tumors derived from a patient with breast cancer (n = 6) were treated with 10 or 30 mg/kg thioparib or 100 mg/kg olaparib for 6 weeks. Data are depicted as mean ± SEM. ***P < 0.0001.


( B ) Concentration - dependent effects of thioparib on the viability of malignant hematologic cells . Cells were treated with thioparib or other PARP inhibitors for 72 h and then subjected to CCK8 assay . Data from three independent experiments were presented as mean ± SEM .

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(B) Concentration-dependent effects of thioparib on the viability of malignant hematologic cells. Cells were treated with thioparib or other PARP inhibitors for 72 h and then subjected to CCK8 assay. Data from three independent experiments were presented as mean ± SEM.


( D ) Kaplan - Meier survival curve of M - NSG mice transplanted with JeKo - 1 cells treated with 10 mg / kg thioparib , 0 . 3 mg / kg talazoparib , or vehicle ( n = 10 ) . Statistical analysis was performed by log - rank ( Mantel - Cox ) test . * * * P < 0 . 0001 , ns : P = 0 . 3526 .

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(D) Kaplan-Meier survival curve of M-NSG mice transplanted with JeKo-1 cells treated with 10 mg/kg thioparib, 0.3 mg/kg talazoparib, or vehicle (n = 10). Statistical analysis was performed by log-rank (Mantel-Cox) test. ***P < 0.0001, ns: P = 0.3526.


( B ) Western blotting of γH2AX and phosphor - RPA32 in cells exposed to thioparib , talazoparib , or olaparib for 12 h .

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(B) Western blotting of γH2AX and phosphor-RPA32 in cells exposed to thioparib, talazoparib, or olaparib for 12 h.


( E ) and ( F ) . Thioparib caused changes in apoptosis - related proteins in JeKo - 1 or THP - 1 cells , as determined by western blotting . Cells were exposed to thioparib , talazoparib , olaparib , or Cpd 391 for 24 h and then subjected to western blotting .

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(E) and (F). Thioparib caused changes in apoptosis-related proteins in JeKo-1 or THP-1 cells, as determined by western blotting. Cells were exposed to thioparib, talazoparib, olaparib, or Cpd 391 for 24 h and then subjected to western blotting.


( G ) and ( H ) . Depletion of PARP1 in HT - 29 and Capan - 1 / TP cells resulted in a decrease in DNA damage and thioparib resistance . The upper panel shows the changes in γH2AX and phospho - RPA32 in HT - 29 PARP1 knockout ( KO ) clones ( G ) or Capan - 1 / TP PARP1 KO clones ( H ) and their parental cells after exposure to thioparib for 24 h . The lower panel shows the IC50 values of thioparib in these PARP1 KO clones and parental cells . Cells were treated with thioparib for 5 or 7 d and the IC50 values were determined by SRB assay from three separate experiments . Data are presented as the mean ± SD . ThP : Thioparib , OP : Olaparib , TP : Talazoparib , 391 : Cpd 391 .

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(G) and (H). Depletion of PARP1 in HT-29 and Capan-1/TP cells resulted in a decrease in DNA damage and thioparib resistance. The upper panel shows the changes in γH2AX and phospho-RPA32 in HT-29 PARP1 knockout (KO) clones (G) or Capan-1/TP PARP1 KO clones (H) and their parental cells after exposure to thioparib for 24 h. The lower panel shows the IC50 values of thioparib in these PARP1 KO clones and parental cells. Cells were treated with thioparib for 5 or 7 d and the IC50 values were determined by SRB assay from three separate experiments. Data are presented as the mean ± SD. ThP: Thioparib, OP: Olaparib, TP: Talazoparib, 391: Cpd 391.


( A ) HR repair assays in U2OS - DR - GFP cells treated with increasing concentrations of thioparib . Cells were incubated with the indicated drugs at the time of I - SceI introduction for 48 h . GFP - positive cells were analyzed after I - SceI transfection for 72 h by flow cytometry . The ATR inhibitor VE - 821 was used as a positive control . Three independent experiments were performed and data are expressed as mean ± SD . Statistical analysis was performed by one - way ANOVA , and all groups were compared with control . * * * P < 0 . 0001 , * P = 0 . 0205 , 0 . 0101 ( from left to right ) .

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(A) HR repair assays in U2OS-DR-GFP cells treated with increasing concentrations of thioparib. Cells were incubated with the indicated drugs at the time of I-SceI introduction for 48 h. GFP-positive cells were analyzed after I-SceI transfection for 72 h by flow cytometry. The ATR inhibitor VE-821 was used as a positive control. Three independent experiments were performed and data are expressed as mean ± SD. Statistical analysis was performed by one-way ANOVA, and all groups were compared with control. ***P < 0.0001, *P = 0.0205, 0.0101 (from left to right).


( C ) Representative images of RAD51 foci in U2OS - DR - GFP cells with or without radiation . Graphs ( right panel ) show quantification of cells with ≥ 5 RAD51 foci in vehicle - and drug - treated cells . Scale bar : 5 μm . Data are obtained from three biological replicates and presented as mean ± SD . Statistical analysis was performed by one - way ANOVA . Left to right : P < 0 . 0001 , = 0 . 0033 , < 0 . 0001 , = 0 . 0006 , = 0 . 0031 , = 0 . 0015 .

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(C) Representative images of RAD51 foci in U2OS-DR-GFP cells with or without radiation. Graphs (right panel) show quantification of cells with ≥ 5 RAD51 foci in vehicle- and drug-treated cells. Scale bar: 5 μm. Data are obtained from three biological replicates and presented as mean ± SD. Statistical analysis was performed by one-way ANOVA. Left to right: P < 0.0001, = 0.0033, < 0.0001, = 0.0006, = 0.0031, = 0.0015.


( H ) HR repair assay . U2OS - DR - GFP ( WT ) and PARP1 KO cells ( KO1 and KO2 ) were treated with the indicated drugs for 48 h and collected after 72 h of I - SceI transfection as in ( A ) . VE - 821 served as a positive control . PARP1 knockout efficiency was evaluated by western blotting . Data from three biological replicates are presented as mean ± SD . Statistical analysis was performed by one - way ANOVA , and each group was compared with the control . * * * P < 0 . 0001 , * P = 0 . 0143 , 0 . 0483 , ns : P = 0 . 3061 , 0 . 8269 , 0 . 9997 , 0 . 3906 ( from left to right ) .

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(H) HR repair assay. U2OS-DR-GFP (WT) and PARP1 KO cells (KO1 and KO2) were treated with the indicated drugs for 48 h and collected after 72 h of I-SceI transfection as in (A). VE-821 served as a positive control. PARP1 knockout efficiency was evaluated by western blotting. Data from three biological replicates are presented as mean ± SD. Statistical analysis was performed by one-way ANOVA, and each group was compared with the control. ***P < 0.0001, *P = 0.0143, 0.0483, ns: P = 0.3061, 0.8269, 0.9997, 0.3906 (from left to right).


( C ) Effects of thioparib on CXCL9 , CXCL10 , and IL15 mRNA in JeKo - 1 cells . Data from three independent experiments are shown as the mean ± SD . Statistical analysis was performed by two - way ANOVA . Left panel : * P = 0 . 0367 , * * P = 0 . 0011 ; median panel : * * * P = 0 . 0002 , < 0 . 0001 , < 0 . 0001 ; right panel : * * * P = 0 . 0005 , 0 . 0009 , 0 . 0001 ( from left to right ) .

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(C) Effects of thioparib on CXCL9, CXCL10, and IL15 mRNA in JeKo-1 cells. Data from three independent experiments are shown as the mean ± SD. Statistical analysis was performed by two-way ANOVA. Left panel: *P = 0.0367, **P = 0.0011; median panel: ***P = 0.0002, < 0.0001, < 0.0001; right panel: ***P = 0.0005, 0.0009, 0.0001 (from left to right).


( D ) The protein levels of p - STAT1 , p - TBK1 , γH2AX , and phospho - RPA32 in HT - 29 parent , PARP1 − / − ( # KO1 ) , and PARP7 − / − cell lines after 24 or 48 h thioparib treatment .

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(D) The protein levels of p-STAT1, p-TBK1, γH2AX, and phospho-RPA32 in HT-29 parent, PARP1−/− (#KO1), and PARP7−/− cell lines after 24 or 48 h thioparib treatment.


( F ) Western blot analysis of p - STAT1 , p - TBK1 , and γH2AX in HT - 29 parent , STING − / − , and TBK1 − / − cells after 24 or 48 h thioparib treatment .

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(F) Western blot analysis of p-STAT1, p-TBK1, and γH2AX in HT-29 parent, STING−/−, and TBK1−/−cells after 24 or 48 h thioparib treatment.


( H ) Immunohistochemistry staining of CD4 , CD8 , and CD45 expression in MC38 tumor tissue sections . Quantification of cells positive of CD4 , CD8 , CD45 were shown . Scale bar : 25 μm . Data from ten technical replicates from each of the two mice are presented as mean ± SD . Statistical analysis was performed by unpaired t - test . * * * P < 0 . 0001 , ns : P = 0 . 1958 .

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(H) Immunohistochemistry staining of CD4, CD8, and CD45 expression in MC38 tumor tissue sections. Quantification of cells positive of CD4, CD8, CD45 were shown. Scale bar: 25 μm. Data from ten technical replicates from each of the two mice are presented as mean ± SD. Statistical analysis was performed by unpaired t-test. ***P < 0.0001, ns: P = 0.1958.


B Representative pictures of different CTCs from the initial CellSearch ® analysis of the metastatic breast cancer patient who gave rise to the breast CTC line . The detected tumor cells display clear keratin and DAPI staining , CD45 - negativity as well as lack of , or very weak ( 4 , 8 ) , ERBB2 expression . Cells of small ( about 5 µm in diameter , 1 , 2 ) and large size ( larger than 10µm in diameter , 2 , 3 ) were detected . While some CTCs displayed dot - like perinuclear keratin signals ( 1 , 2 ) , the majority showed diffuse keratin staining . Additionally , CTC clusters of 4 - 8 cells were present ( 5 , 6 ) . Some CTCs showed multiple / lobed nuclei ( 4 , 7 , 8 ) .

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B Representative pictures of different CTCs from the initial CellSearch® analysis of the metastatic breast cancer patient who gave rise to the breast CTC line. The detected tumor cells display clear keratin and DAPI staining, CD45-negativity as well as lack of, or very weak (4, 8), ERBB2 expression. Cells of small (about 5 µm in diameter, 1, 2) and large size (larger than 10µm in diameter, 2,3) were detected. While some CTCs displayed dot-like perinuclear keratin signals (1, 2), the majority showed diffuse keratin staining. Additionally, CTC clusters of 4-8 cells were present (5, 6). Some CTCs showed multiple/lobed nuclei (4, 7, 8).


C Bright field images of CTC - ITB - 01 cells growing adherently . D Bright field images of CTC - ITB - 01 cells growing non - adherently ( relation between adherent and non - adherent cells : 80 / 20 % ) .

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C Bright field images of CTC-ITB-01 cells growing adherently. D Bright field images of CTC-ITB-01 cells growing non-adherently (relation between adherent and non-adherent cells: 80/20 %).


Unsupervised hierarchical clustering ( Ward linkage with Euclidean distance ) of CNA profiles generated from patient primary tumors , the vaginal metastasis , isolated single and pooled CTCs from the primary blood collection and CTC - ITB - 01 ( early and later passage ) and leukocytes . " CL " represents the cell line , followed by the passage analyzed ( p04 / p30 ) . " CTC " marks circulating tumor cells isolated from the initial blood draw , followed by 01 / 05 / 10 indicating single ( 01 ) or pooled cells ( 5 or 10 , respectively ) . Light and dark grey bars to the left signify chromosomes , starting from the top with chromosome 1 . Red color represents chromosome loss , blue represents gain . All copy number aberrations were calculated using the patient ' s own germline DNA extracted from leukocytes as reference to eliminate copy number variations ( CNVs ) .

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Unsupervised hierarchical clustering (Ward linkage with Euclidean distance) of CNA profiles generated from patient primary tumors, the vaginal metastasis, isolated single and pooled CTCs from the primary blood collection and CTC-ITB-01 (early and later passage) and leukocytes. "CL" represents the cell line, followed by the passage analyzed (p04/p30). "CTC" marks circulating tumor cells isolated from the initial blood draw, followed by 01/05/10 indicating single (01) or pooled cells (5 or 10, respectively). Light and dark grey bars to the left signify chromosomes, starting from the top with chromosome 1. Red color represents chromosome loss, blue represents gain. All copy number aberrations were calculated using the patient's own germline DNA extracted from leukocytes as reference to eliminate copy number variations (CNVs).


Direct comparisons of CNA profiles . Percentages of gene copy number gains and losses across the chromosomes for different sample types . Copy number gains are displayed in green , losses in red . A CNAs generated from CTCs from the primary blood collection ( upper panel ) and CTC - ITB - 01 , indicated as " CL " ( medium panel ) . Significant distinctions between both samples were calculated using Fisher ' s exact test ( Joosse et al , 2018 ) and are indicated in lowest panel ( blue ) . Increased blue color intensity represents lower p - value and therefore higher statistical significance .

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Direct comparisons of CNA profiles. Percentages of gene copy number gains and losses across the chromosomes for different sample types. Copy number gains are displayed in green, losses in red. A CNAs generated from CTCs from the primary blood collection (upper panel) and CTC-ITB-01, indicated as "CL" (medium panel). Significant distinctions between both samples were calculated using Fisher's exact test (Joosse et al, 2018) and are indicated in lowest panel (blue). Increased blue color intensity represents lower p-value and therefore higher statistical significance.


Direct comparisons of CNA profiles . Percentages of gene copy number gains and losses across the chromosomes for different sample types . Copy number gains are displayed in green , losses in red . B CNA comparison between early ( p04 ) and late ( p30 ) CTC - ITB - 01 passages ( upper and medium panel ) . Significant distinctions between both samples were calculated using Fisher ' s exact test ( Joosse et al , 2018 ) and are indicated in lowest panel ( in blue ) . Increased blue color intensity represents lower p - value and therefore higher statistical significance .

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Direct comparisons of CNA profiles. Percentages of gene copy number gains and losses across the chromosomes for different sample types. Copy number gains are displayed in green, losses in red. B CNA comparison between early (p04) and late (p30) CTC-ITB-01 passages (upper and medium panel). Significant distinctions between both samples were calculated using Fisher's exact test (Joosse et al, 2018) and are indicated in lowest panel (in blue). Increased blue color intensity represents lower p-value and therefore higher statistical significance.


A Western blot analysis of selected protein markers , including ERα , EGFR , ERBB2 , EpCAM , K18 , K19 , K8 , E - cadherin , N - cadherin , vimentin , CD44 , CD24 , SNAIL , SLUG , TWIST and α - tubulin ( as a loading control ) ( n = 3 replicates ) . CTC - ITB - 01 was compared to more mesenchymal ER - Hs578t and epithelial ER + MCF - 7 breast cancer cell lines .

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A Western blot analysis of selected protein markers, including ERα, EGFR, ERBB2, EpCAM, K18, K19, K8, E-cadherin, N-cadherin, vimentin, CD44, CD24, SNAIL, SLUG, TWIST and α-tubulin (as a loading control) (n=3 replicates). CTC-ITB-01 was compared to more mesenchymal ER- Hs578t and epithelial ER+ MCF-7 breast cancer cell lines.


B ICC staining of CTC - ITB - 01 for pan - keratin ( orange ) , ER ( green ) , and DAPI ( blue ) . The scale bar corresponds to 20 µm . Two representative panels are shown for CTC - ITB - 01 ( 1 ) and ( 2 ) . MCF - 7 cells are depicted as reference cell line .

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B ICC staining of CTC-ITB-01 for pan-keratin (orange), ER (green), and DAPI (blue). The scale bar corresponds to 20 µm. Two representative panels are shown for CTC-ITB-01 (1) and (2). MCF-7 cells are depicted as reference cell line.


C PAM50 classifier results showing probabilities of pooled CTC - ITB - 01 cells matching specific molecular breast cancer subtypes . Starting from lowest probability , CTC - ITB - 01 was classified as 1 . 02 % ( s = 2 . 5 % ) normal - like , 3 . 11 % ( s = 6 . 6 % ) Basal - like , 13 . 11 % ( s = 9 . 3 % ) ERBB2 - positive , 16 . 77 % ( s = 23 . 7 % ) luminal A , and 65 . 22 % ( s = 16 . 6 % ) luminal B breast cancer subtype . Data was generated from n = 3 replicates .

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C PAM50 classifier results showing probabilities of pooled CTC-ITB-01 cells matching specific molecular breast cancer subtypes. Starting from lowest probability, CTC-ITB-01 was classified as 1.02% (s=2.5%) normal-like, 3.11% (s=6.6%) Basal-like, 13.11% (s=9.3%) ERBB2-positive, 16.77% (s=23.7%) luminal A, and 65.22% (s=16.6%) luminal B breast cancer subtype. Data was generated from n=3 replicates.


D PAM50 classifier results showing probabilities of the non - adherent and adherent CTC - ITB - 01 fractions matching molecular subtypes . Selected bars are not visible due to extremely low probability ( close to zero ) . Both fractions show greatest alignment with a luminal B subtype . Starting from lowest probability , the adherent fraction of CTC - ITB - 01 was classified as 0 % ( ± 0 % ) normal - like , 6 . 22 % ( ± 9 . 0 % ) Basal - like , 17 . 85 % ( ± 8 . 6 % ) ERBB2 - positive , 0 % ( s = 0 % ) luminal A , and 75 . 92 % ( s = 0 . 7 % ) luminal B breast cancer subtype . The suspension cell fraction of CTC - ITB - 01 was classified as 2 . 04 % ( s = 3 . 5 % ) normal - like , 0 % ( s = 0 % ) Basal - like , 17 . 32 % ( s = 9 . 7 % ) ERBB2 - positive , 33 . 5 % ( s = 23 . 8 % ) luminal A , and 68 . 67 % ( s = 18 . 6 % ) luminal B breast cancer subtype , respectively . Data was generated from n = 3 replicates .

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D PAM50 classifier results showing probabilities of the non-adherent and adherent CTC-ITB-01 fractions matching molecular subtypes. Selected bars are not visible due to extremely low probability (close to zero). Both fractions show greatest alignment with a luminal B subtype. Starting from lowest probability, the adherent fraction of CTC-ITB-01 was classified as 0% (±0%) normal-like, 6.22% (±9.0%) Basal-like, 17.85% (±8.6%) ERBB2-positive, 0% (s=0%) luminal A, and 75.92% (s=0.7%) luminal B breast cancer subtype. The suspension cell fraction of CTC-ITB-01 was classified as 2.04% (s=3.5%) normal-like, 0% (s=0%) Basal-like, 17.32% (s=9.7%) ERBB2-positive, 33.5% (s=23.8%) luminal A, and 68.67% (s=18.6%) luminal B breast cancer subtype, respectively. Data was generated from n=3 replicates.


( E ) ALDH activity measurement on viable cells via flow cytometry using the ALDEFLUOR ™ assay . CTC - ITB - 01 is compared to A549 as recommended control cell line . An internal ( DEAB ) control is also depicted . 77 . 92 % of the gated CTC - ITB - 01 population are ALDH + .

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(E) ALDH activity measurement on viable cells via flow cytometry using the ALDEFLUOR™ assay. CTC-ITB-01 is compared to A549 as recommended control cell line. An internal (DEAB) control is also depicted. 77.92% of the gated CTC-ITB-01 population are ALDH+.


A ICC staining of CTC - ITB - 01 for ß - catenin ( green ) , E - cadherin ( orange ) , and DAPI ( blue ) . The scale bar corresponds to 20 µm . Two representative panels are shown for CTC - ITB - 01 .

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A ICC staining of CTC-ITB-01 for ß-catenin (green), E-cadherin (orange), and DAPI (blue). The scale bar corresponds to 20 µm. Two representative panels are shown for CTC-ITB-01.


B ICC staining of CTC - ITB - 01 for α - catenin ( green ) , visualization of actin filaments by phalloidin ( orange ) , and nuclei by DAPI ( blue ) . The scale bar corresponds to 20 µm . Two representative panels are shown for CTC - ITB - 01 .

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B ICC staining of CTC-ITB-01 for α-catenin (green), visualization of actin filaments by phalloidin (orange), and nuclei by DAPI (blue). The scale bar corresponds to 20 µm. Two representative panels are shown for CTC-ITB-01.


C Generic EMT score of the adherent and non - adherently growing CTC - ITB - 01 fractions in relation to tumor samples from different cancer entities . Scaling ranges from 1 ( completely epithelial ) to - 1 ( entirely mesenchymal ) . The adherent fraction is shown with a black dot , the non - adherent fraction with a black triangle , and the mean score per cancer entity by a black line . D Generic EMT score of the adherent and non - adherently growing CTC - ITB - 01 fractions in relation to established cancer cell lines . Scaling ranges from 1 ( completely epithelial ) to - 1 ( entirely mesenchymal ) . The adherent fraction is shown with a black dot , the non - adherent fraction with a black triangle , and the mean score per cancer entity by a black line .

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C Generic EMT score of the adherent and non-adherently growing CTC-ITB-01 fractions in relation to tumor samples from different cancer entities. Scaling ranges from 1 (completely epithelial) to -1 (entirely mesenchymal). The adherent fraction is shown with a black dot, the non-adherent fraction with a black triangle, and the mean score per cancer entity by a black line. D Generic EMT score of the adherent and non-adherently growing CTC-ITB-01 fractions in relation to established cancer cell lines. Scaling ranges from 1 (completely epithelial) to -1 (entirely mesenchymal). The adherent fraction is shown with a black dot, the non-adherent fraction with a black triangle, and the mean score per cancer entity by a black line.


A In vivo bioluminescence imaging of CTC - ITB - 01 in four NSG mice . Blue indicates low radiance , red indicates high radiance . B Logarithmic plotting of in vivo radiance increase over time . Curves represent means ± SEM of measurements performed on multiple glands ( n = 8 ) .

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A In vivo bioluminescence imaging of CTC-ITB-01 in four NSG mice. Blue indicates low radiance, red indicates high radiance. B Logarithmic plotting of in vivo radiance increase over time. Curves represent means ± SEM of measurements performed on multiple glands (n=8).


C H & E staining of xenografted mouse mammary glands show formation of primary tumors .

C H&E staining of xenografted mouse mammary glands show formation of primary tumors.


D Stereoscope image of a mammary gland of immunodeficient NSG mouse . Fluorescently tagged CTC - ITB - 01 cells in green .

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D Stereoscope image of a mammary gland of immunodeficient NSG mouse. Fluorescently tagged CTC-ITB-01 cells in green.


E IHC staining for ER , confirms retained in vivo ER - positivity of formed tumors . Representative images of ER staining in higher ( left panel ) and lower ( right panel ) magnification of 2 different areas of a xenografted mammary gland .

E IHC staining for ER, confirms retained in vivo ER-positivity of formed tumors. Representative images of ER staining in higher (left panel) and lower (right panel) magnification of 2 different areas of a xenografted mammary gland.


F Representative ex vivo luminescence images of indicated organs from engrafted mice . Organs include brain ( 1 ) , lung ( 2 ) , liver ( 3 ) and bones ( 4 ) . G Scatter blot showing ex vivo radiance intensity of metastatic organs in three different mice ( M1 , M2 , M3 ) , indicating highest intensity in liver and bone . Data represent means ± SD of measurements performed on metastatic organs derived from three different mice .

F Representative ex vivo luminescence images of indicated organs from engrafted mice. Organs include brain (1), lung (2), liver (3) and bones (4). G Scatter blot showing ex vivo radiance intensity of metastatic organs in three different mice (M1, M2, M3), indicating highest intensity in liver and bone. Data represent means ± SD of measurements performed on metastatic organs derived from three different mice.


A Equal numbers of CTC - ITB - 01 cells were seeded on 6 - well culture plates and grown in the presence or absence of different 17 - β - estradiol concentrations as indicated . After 10 days cells were fixed and stained with Coomassie blue staining ( right panel ) . Macroscopic photos are shown of the fixed cell colonies from cells grown in the absence or presence of the indicated E2 concentrations ( right panel ) . Scale bar 200 µm .

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A Equal numbers of CTC-ITB-01 cells were seeded on 6-well culture plates and grown in the presence or absence of different 17-β-estradiol concentrations as indicated. After 10 days cells were fixed and stained with Coomassie blue staining (right panel). Macroscopic photos are shown of the fixed cell colonies from cells grown in the absence or presence of the indicated E2 concentrations (right panel). Scale bar 200 µm.


B Stable knockdown of ER - alpha within CTC - ITB - 01 cells was performed by using lentiviral transfer of non - targeted ( scramble ) or two different targeted shRNAs against ER - alpha . CTC - ITB - 01 cells were seeded on 6 - well plates . After 10 days cells were fixed and stained ( left upper panel ) . Macroscopic photos are shown of the fixed cells ( left lower panel ) , scale bar 200 µm . Western blots of lysates from knockdown of ER - alpha expression in CTC - ITB - 01 cells transduced with the same amounts of lentiviral vectors were probed with the indicated antibodies . The extracts were obtained 72h after lentiviral transduction ( right panel ) . Where indicated , western blots were visualized at long exposure ( l . e . ) and / or short exposure ( s . e . ) .

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B Stable knockdown of ER-alpha within CTC-ITB-01 cells was performed by using lentiviral transfer of non-targeted (scramble) or two different targeted shRNAs against ER-alpha. CTC-ITB-01 cells were seeded on 6-well plates. After 10 days cells were fixed and stained (left upper panel). Macroscopic photos are shown of the fixed cells (left lower panel), scale bar 200 µm. Western blots of lysates from knockdown of ER-alpha expression in CTC-ITB-01 cells transduced with the same amounts of lentiviral vectors were probed with the indicated antibodies. The extracts were obtained 72h after lentiviral transduction (right panel). Where indicated, western blots were visualized at long exposure (l.e.) and/or short exposure (s.e.).


C CTC - ITB - 01 and MCF - 7 cells were grown under the same culture conditions in the presence or absence of E2 as indicated . 24h after E2 deprivation or E2 - treatment cells were harvested . Western blots of the protein lysates were probed with antibodies targeting ER - alpha , FOXM1 , Bcl - 2 , PR - A , PR - B and ID1 , actin was used as loading control , visualized at long exposure ( l . e . ) and / or short exposure ( s . e . ) .

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C CTC-ITB-01 and MCF-7 cells were grown under the same culture conditions in the presence or absence of E2 as indicated. 24h after E2 deprivation or E2-treatment cells were harvested. Western blots of the protein lysates were probed with antibodies targeting ER-alpha, FOXM1, Bcl-2, PR-A, PR-B and ID1, actin was used as loading control, visualized at long exposure (l.e.) and/or short exposure (s.e.).


( D ) Growth curves of CTC - ITB - 01 cells under varying concentrations of Palbociclib treatment were measured using an IncuCyte Zoom live cell imaging system . The mean values from three technical triplicates ( one experiment ) with standard deviation are shown .

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(D) Growth curves of CTC-ITB-01 cells under varying concentrations of Palbociclib treatment were measured using an IncuCyte Zoom live cell imaging system. The mean values from three technical triplicates (one experiment) with standard deviation are shown.


E ) Growth curves of MCF - 7 cells under varying concentrations of Palbociclib treatment were measured using an IncuCyte Zoom live cell imaging system . The mean values from three technical triplicates ( one experiment ) with standard deviation are shown .

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E) Growth curves of MCF-7 cells under varying concentrations of Palbociclib treatment were measured using an IncuCyte Zoom live cell imaging system. The mean values from three technical triplicates (one experiment) with standard deviation are shown.


( F ) Influence of Palbociclib on CTC - ITB - 01 growth after 300 hours Data was chosen from one representative time point during the exponential growth phase . Statistical significance was analyzed with a one - way ANOVA with Dunnett ' s multiple comparisons test and compared to the vehicle substance dimethyl sulfoxide ( DMSO ) - . The mean values from three technical triplicates ( one experiment ) with standard deviation are shown .

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(F) Influence of Palbociclib on CTC-ITB-01 growth after 300 hours Data was chosen from one representative time point during the exponential growth phase. Statistical significance was analyzed with a one-way ANOVA with Dunnett's multiple comparisons test and compared to the vehicle substance dimethyl sulfoxide (DMSO)-. The mean values from three technical triplicates (one experiment) with standard deviation are shown.


G ) Influence of Palbociclib on MCF - 7 after 80 hours . Data was chosen from one representative time point during the exponential growth phase . Statistical significance was analyzed with a one - way ANOVA with Dunnett ' s multiple comparisons test and compared to the vehicle substance dimethyl sulfoxide ( DMSO ) - . The mean values from three technical triplicates ( one experiment ) with standard deviation are shown .

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G) Influence of Palbociclib on MCF-7 after 80 hours. Data was chosen from one representative time point during the exponential growth phase. Statistical significance was analyzed with a one-way ANOVA with Dunnett's multiple comparisons test and compared to the vehicle substance dimethyl sulfoxide (DMSO)-. The mean values from three technical triplicates (one experiment) with standard deviation are shown.


( H ) Effect of Palbociclib on the growth of CTC - ITB - 01 cells Concentrations were transformed to common logarithm . Three - parameter non - linear logistic regression was used to determine the IC50 . The mean values from three technical triplicates ( one experiment ) with standard deviation are shown . Error bars for standard deviation smaller than the symbols are not displayed .

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(H) Effect of Palbociclib on the growth of CTC-ITB-01 cells Concentrations were transformed to common logarithm. Three-parameter non-linear logistic regression was used to determine the IC50. The mean values from three technical triplicates (one experiment) with standard deviation are shown. Error bars for standard deviation smaller than the symbols are not displayed.


I ) Effect of Palbociclib on the growth of MCF - 7 cells . Concentrations were transformed to common logarithm . Three - parameter non - linear logistic regression was used to determine the IC50 . The mean values from three technical triplicates ( one experiment ) with standard deviation are shown . Error bars for standard deviation smaller than the symbols are not displayed .

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I) Effect of Palbociclib on the growth of MCF-7 cells. Concentrations were transformed to common logarithm. Three-parameter non-linear logistic regression was used to determine the IC50. The mean values from three technical triplicates (one experiment) with standard deviation are shown. Error bars for standard deviation smaller than the symbols are not displayed.


Representative immunofluorescence images of SCOTIN - KO cells expressing Str - Ii - SCOTIN - SBP - EGFP with 40 μM D - biotin treatment for the indicated times . B Upper , localization of RUSH reporters retained on the ER membrane with Sec61β ( red ) . Bottom , localization of released RUSH reporters after 240 min of biotin treatment . The arrow indicates the EGFP signal on the ER . The arrowhead indicates the EGFP signal on vesicles . The empty arrowhead indicates the EGFP signal on the plasma membrane . Scale bar : 20 μm . Scale bars in the magnified images : 5 μm .

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Representative immunofluorescence images of SCOTIN-KO cells expressing Str-Ii-SCOTIN-SBP-EGFP with 40 μM D-biotin treatment for the indicated times. B Upper, localization of RUSH reporters retained on the ER membrane with Sec61β (red). Bottom, localization of released RUSH reporters after 240 min of biotin treatment. The arrow indicates the EGFP signal on the ER. The arrowhead indicates the EGFP signal on vesicles. The empty arrowhead indicates the EGFP signal on the plasma membrane. Scale bar: 20 μm. Scale bars in the magnified images: 5 μm.


Representative immunofluorescence images of SCOTIN - KO cells expressing Str - Ii - VSVG - SBP - EGFP ( green ) with 40 μM D - biotin treatment for the indicated times . , Upper , localization of RUSH reporters retained on the ER membrane with Sec61β ( red ) . Bottom , localization of released RUSH reporters after 240 min of biotin treatment . The arrow indicates the EGFP signal on the ER . The arrowhead indicates the EGFP signal on vesicles . The empty arrowhead indicates the EGFP signal on the plasma membrane . Scale bar : 20 μm . Scale bars in the magnified images : 5 μm .

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Representative immunofluorescence images of SCOTIN-KO cells expressing Str-Ii-VSVG-SBP-EGFP (green) with 40 μM D-biotin treatment for the indicated times. , Upper, localization of RUSH reporters retained on the ER membrane with Sec61β (red). Bottom, localization of released RUSH reporters after 240 min of biotin treatment. The arrow indicates the EGFP signal on the ER. The arrowhead indicates the EGFP signal on vesicles. The empty arrowhead indicates the EGFP signal on the plasma membrane. Scale bar: 20 μm. Scale bars in the magnified images: 5 μm.


( F ) The colocalization of EGFP and Sec61 or CD63 was calculated as the Pearson correlation coefficient from the experimental data in Figure 1E . The error bars indicate the SEMs . n : number of cells analyzed . Data are shown as bars with dots and represent the means ± SEMs . P values were determined using a two - tailed unpaired t test . Significant differences are labeled , * * < 0 . 01 and * * * * P < 0 . 0001 .

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(F) The colocalization of EGFP and Sec61 or CD63 was calculated as the Pearson correlation coefficient from the experimental data in Figure 1E. The error bars indicate the SEMs. n: number of cells analyzed. Data are shown as bars with dots and represent the means±SEMs. P values were determined using a two-tailed unpaired t test. Significant differences are labeled, **<0.01 and ****P<0.0001.


( A ) Immunoblot analysis of proteinase K protection assay . HeLa cells were transfected with SCOTIN - Myc ( Total ) and digitonin - permeabilized . The supernatants ( Cytosol ) and pellet ( PNSs ) were separated by centrifugation . The PNSs were incubated with PBS or with proteinase K ( + PK ) in the presence or absence of Triton X - 100 ( + PK + Triton X ) .

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(A) Immunoblot analysis of proteinase K protection assay. HeLa cells were transfected with SCOTIN-Myc (Total) and digitonin-permeabilized. The supernatants (Cytosol) and pellet (PNSs) were separated by centrifugation. The PNSs were incubated with PBS or with proteinase K (+ PK) in the presence or absence of Triton X-100 (+PK +Triton X).


( B ) Representative high - resolution microscopy images of Rab5 Q79L - EGFP - and SCOTIN - DsRed - expressing HeLa cells . Magnified images show enlarged endosomes . Ⅰ : SCOTIN on the endosomal membrane , Ⅱ : SCOTIN on the endosomal membrane and luminal side . Scale bar : 10 μm . Scale bars in the magnified images : 5 μm . ( C ) Plot analysis of SCOTIN relative to the Rab5 Q79L signal shown . The graph indicates the fluorescence intensity of each channel on lines Ⅰ and Ⅱ from Figure 2B . The arrow indicates the boundary of the endosomal membrane .

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(B) Representative high-resolution microscopy images of Rab5 Q79L-EGFP- and SCOTIN-DsRed-expressing HeLa cells. Magnified images show enlarged endosomes. Ⅰ: SCOTIN on the endosomal membrane, Ⅱ: SCOTIN on the endosomal membrane and luminal side. Scale bar: 10 μm. Scale bars in the magnified images: 5 μm. (C) Plot analysis of SCOTIN relative to the Rab5 Q79L signal shown. The graph indicates the fluorescence intensity of each channel on lines Ⅰ and Ⅱ from Figure 2B. The arrow indicates the boundary of the endosomal membrane.


( F ) Left : Representative images of Rab5 Q79L - mCherry - expressing SCOTIN - FP11 knock - in cells . Right : Plot analysis of SCOTIN - FP11 relative to the Rab5 Q79L signal shown . The graph indicates the fluorescence intensity of each channel on the lines . The arrow indicates the boundary of the endosomal membrane . Scale bars : 10 μm . Scale bars in the magnified images : 5 μm .

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(F) Left: Representative images of Rab5 Q79L-mCherry-expressing SCOTIN-FP11 knock-in cells. Right: Plot analysis of SCOTIN-FP11 relative to the Rab5 Q79L signal shown. The graph indicates the fluorescence intensity of each channel on the lines. The arrow indicates the boundary of the endosomal membrane. Scale bars : 10 μm. Scale bars in the magnified images: 5 μm.


( B ) Representative images from a PLA with VAPA and Rab7 in WT and SCOTIN - Dsred - overexpressing HeLa cells . Only one primary antibody ( anti - Rab7 antibody alone ) served as a negative control . The PLA color legends are inverted . Asterisks indicate transfected cells . All scale bars : 20 μm . ( C ) The number of PLA signals in each cell was quantified by particle analysis with ImageJ . Data information : n : number of cells analyzed . Data are shown as bars with dots and represent the means ± SEMs . P values were determined using a two - tailed unpaired t test . Significant differences are labeled , * < 0 . 05 and * * * * P < 0 . 0001 .

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(B) Representative images from a PLA with VAPA and Rab7 in WT and SCOTIN-Dsred-overexpressing HeLa cells. Only one primary antibody (anti-Rab7 antibody alone) served as a negative control. The PLA color legends are inverted. Asterisks indicate transfected cells. All scale bars: 20 μm. (C) The number of PLA signals in each cell was quantified by particle analysis with ImageJ. Data information: n: number of cells analyzed. Data are shown as bars with dots and represent the means±SEMs. P values were determined using a two-tailed unpaired t test. Significant differences are labeled, *<0.05 and ****P<0.0001.


( E ) The number of PLA signals in each cell was quantified by particle analysis with ImageJ . Data information : n : number of cells analyzed . Data are shown as bars with dots and represent the means ± SEMs . P values were determined using a two - tailed unpaired t test . Significant differences are labeled , * < 0 . 05 and * * * * P < 0 . 0001 .

(E) The number of PLA signals in each cell was quantified by particle analysis with ImageJ. Data information: n: number of cells analyzed. Data are shown as bars with dots and represent the means±SEMs. P values were determined using a two-tailed unpaired t test. Significant differences are labeled, *<0.05 and ****P<0.0001.


( F ) Immunoblot analysis of WT and SCOTIN - KO HeLa cell lysates for VAPA , Rab7 , SCOTIN , and α - tubulin .

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(F) Immunoblot analysis of WT and SCOTIN-KO HeLa cell lysates for VAPA, Rab7, SCOTIN, and α-tubulin.


( G ) Representative images from a PLA with VAPA and Rab7 in SCOTIN - , TMPRD - , or PRD - Myc - transfected SCOTIN - KO HeLa cells . The PLA color legends are inverted . All scale bars : 20 μm . ( H ) The number of PLA signals in each cell was quantified by particle analysis with ImageJ . Data information : n : number of cells analyzed . Data are shown as bars with dots and represent the means ± SEMs . P values were determined using a two - tailed unpaired t test . Significant differences are labeled , * < 0 . 05 and * * * * P < 0 . 0001 .

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(G) Representative images from a PLA with VAPA and Rab7 in SCOTIN-, TMPRD-, or PRD-Myc-transfected SCOTIN-KO HeLa cells. The PLA color legends are inverted. All scale bars: 20 μm. (H) The number of PLA signals in each cell was quantified by particle analysis with ImageJ. Data information: n: number of cells analyzed. Data are shown as bars with dots and represent the means±SEMs. P values were determined using a two-tailed unpaired t test. Significant differences are labeled, *<0.05 and ****P<0.0001.


SCOTIN PRD - mediated tethering of SUVs and GUVs . E , Representative confocal images of SCOTIN PRD - induced membrane tethering between GUVs and SUVs . PRD - SUV aligned to the PRD - GUV membrane ( arrowhead , type I in the model ) or accumulated at the surface of GUVs ( arrow , type Ⅱ in the model ) .

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SCOTIN PRD-mediated tethering of SUVs and GUVs. E, Representative confocal images of SCOTIN PRD-induced membrane tethering between GUVs and SUVs. PRD-SUV aligned to the PRD-GUV membrane (arrowhead, type I in the model) or accumulated at the surface of GUVs (arrow, type Ⅱ in the model).


( B ) Representative images of VAPA - Rab7 PLA signals taken from Str - Ii - SCOTIN - SBP - EGFP transfected SCOTIN - KO cells after untreated ( - Biotin ) or biotin treatment ( + Biotin 4 h ) . For the PLA signal , the color legends are inverted . Asterisks indicate transfected cells . All scale bars : 20 μm . ( C ) The PLA signals in the indicated samples were quantified by particle analysis with ImageJ . Data information : n : number of cells analyzed . Data are shown as bars with dots and represent the means ± SEMs . P values were determined using a two - tailed unpaired t test . Significant differences are labeled , * * < 0 . 01 , * * * < 0 . 001 , and * * * * P < 0 . 0001 .

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(B) Representative images of VAPA-Rab7 PLA signals taken from Str-Ii-SCOTIN-SBP-EGFP transfected SCOTIN-KO cells after untreated (-Biotin) or biotin treatment (+ Biotin 4 h). For the PLA signal, the color legends are inverted. Asterisks indicate transfected cells. All scale bars: 20 μm. (C) The PLA signals in the indicated samples were quantified by particle analysis with ImageJ. Data information: n: number of cells analyzed. Data are shown as bars with dots and represent the means±SEMs. P values were determined using a two-tailed unpaired t test. Significant differences are labeled, **<0.01, ***<0.001, and ****P<0.0001.


( D ) Representative images of Sec61β - Emerald - or Sec61β - PRD - Emerald - transfected SCOTIN - KO cells . Cells were stained with RTN4 ( red ) . Arrowheads indicate the accumulated ER membrane . All scale bars : 20 μm .

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(D) Representative images of Sec61β-Emerald- or Sec61β-PRD-Emerald-transfected SCOTIN-KO cells. Cells were stained with RTN4 (red). Arrowheads indicate the accumulated ER membrane. All scale bars: 20 μm.


( G ) The PLA signals in the indicated samples were quantified by particle analysis with ImageJ . Data information : n : number of cells analyzed . Data are shown as bars with dots and represent the means ± SEMs . P values were determined using a two - tailed unpaired t test . Significant differences are labeled , * * < 0 . 01 , * * * < 0 . 001 , and * * * * P < 0 . 0001 .

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(G) The PLA signals in the indicated samples were quantified by particle analysis with ImageJ. Data information: n: number of cells analyzed. Data are shown as bars with dots and represent the means±SEMs. P values were determined using a two-tailed unpaired t test. Significant differences are labeled, **<0.01, ***<0.001, and ****P<0.0001.


( C ) HEK293 cells were transfected with SCOTIN - GST along with empty ( pcDNA3 . 1 - Myc ) , SCOTIN - Myc , or SCOTIN ( Δ150 - 177 ) - Myc plasmids for 48 h . SCOTIN - GST was pulled down from total cell lysates using glutathione - Sepharose beads , and the interacting proteins were analyzed by immunoblotting .

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(C) HEK293 cells were transfected with SCOTIN-GST along with empty (pcDNA3.1-Myc), SCOTIN-Myc, or SCOTIN(Δ150-177)-Myc plasmids for 48 h. SCOTIN-GST was pulled down from total cell lysates using glutathione-Sepharose beads, and the interacting proteins were analyzed by immunoblotting.


( D ) Representative images from a PLA with VAPA and Rab7 in SCOTIN - KO HeLa cells reconstituted with FL SCOTIN , SCOTIN ( ΔPRD ) , or SCOTIN ( Δ150 - 177 ) - DsRed . The PLA color legends are inverted . Asterisks indicate transfected cells . All scale bars : 20 μm .

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(D) Representative images from a PLA with VAPA and Rab7 in SCOTIN-KO HeLa cells reconstituted with FL SCOTIN, SCOTIN(ΔPRD), or SCOTIN(Δ150-177)-DsRed. The PLA color legends are inverted. Asterisks indicate transfected cells. All scale bars: 20 μm.


( G ) Representative images of VAPA - VAPA PLA signals taken from Str - Ii - SCOTIN ( Δ150 - 177 ) - SBP - EGFP transfected SCOTIN - KO cells after untreated ( - Biotin ) or biotin - treated ( + Bition 4 h ) . For the PLA signal , the color legends are inverted . Asterisks indicate transfected cells . All scale bars : 20 μm . ( H ) The PLA signals in the indicated samples were quantified by particle analysis with ImageJ . Data information : n : number of cells analyzed . Data are shown as bars with dots and represent the means ± SEMs . P values were determined using a two - tailed unpaired t test . Significant differences are labeled , * * * * P < 0 . 0001 .

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(G) Representative images of VAPA-VAPA PLA signals taken from Str-Ii-SCOTIN(Δ150-177)-SBP-EGFP transfected SCOTIN-KO cells after untreated (-Biotin) or biotin-treated (+Bition 4 h). For the PLA signal, the color legends are inverted. Asterisks indicate transfected cells. All scale bars: 20 μm. (H) The PLA signals in the indicated samples were quantified by particle analysis with ImageJ. Data information: n: number of cells analyzed. Data are shown as bars with dots and represent the means±SEMs. P values were determined using a two-tailed unpaired t test. Significant differences are labeled, ****P<0.0001.


( A - D ) TEM images of the ER and endosomes of HeLa ( A ) , SCOTIN - KO HeLa ( B ) , SCOTIN - DsRed transfected SCOTIN - KO HeLa ( C ) , and SCOTIN Δ150 - 177 - DsRed transfected SCOTIN - KO HeLa ( D ) . DsRed - positive cells were processed for EM quantification .

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(A-D) TEM images of the ER and endosomes of HeLa (A), SCOTIN-KO HeLa (B), SCOTIN-DsRed transfected SCOTIN-KO HeLa (C), and SCOTIN Δ150-177-DsRed transfected SCOTIN-KO HeLa (D). DsRed-positive cells were processed for EM quantification.


( A ) Distribution of late endosomes ( CD63 ) in WT or SCOTIN - KO HeLa cells . Representative Z projections of confocal images are shown . The color legends are inverted . Scale bars : 20 μm . ( B ) Spatial distribution of late endosomes ( CD63 ) expressed as the fractional distance of fluorescent pixels along a straight line drawn through the endolysosomal cloud from the nuclear center ( 0 ) to the plasma membrane ( 1 . 0 ) from the experimental data in Figure 8A . ( C ) Quantification of the number of CD63 - positive vesicles per cell from the experimental data in Figure 8A . Data information : n : number of cells analyzed , with # of data points analyzed per condition given in parentheses . Data are shown as bars with dots and represent the means ± SEMs . P values were determined using a two - tailed unpaired t test . Significant differences are labeled , * * * * P < 0 . 0001 .

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(A) Distribution of late endosomes (CD63) in WT or SCOTIN-KO HeLa cells. Representative Z projections of confocal images are shown. The color legends are inverted. Scale bars: 20 μm. (B) Spatial distribution of late endosomes (CD63) expressed as the fractional distance of fluorescent pixels along a straight line drawn through the endolysosomal cloud from the nuclear center (0) to the plasma membrane (1.0) from the experimental data in Figure 8A. (C) Quantification of the number of CD63-positive vesicles per cell from the experimental data in Figure 8A. Data information: n: number of cells analyzed, with # of data points analyzed per condition given in parentheses. Data are shown as bars with dots and represent the means±SEMs. P values were determined using a two-tailed unpaired t test. Significant differences are labeled, ****P<0.0001.


( D ) Live - image analysis of WT and SCOTIN - KO HeLa cells expressing Rab7 - EGFP . Left : confocal images taken at time 0 . Right : time - lapse images of Rab7 - EGFP in WT and SCOTIN - KO HeLa cells , as shown by temporal color - coded representations . Each confocal image of late endosomes was color coded according to its acquisition time point , with images taken at 12 - sec intervals over 5 min . A total of 24 color - coded images were projected to form a single image that demonstrates the dynamics of Rab7 - labeled late endosomes . White = static late endosomes . Multicolored = motile late endosomes . Boxed , magnified images highlight selected perinuclear and peripheral regions . Scale bar : 30 μm . Scale bars in the magnified images : 3 μm . ( E ) Quantification of the fractional distance of Rab7 - EGFP - positive late endosomes in WT and SCOTIN - KO HeLa cells from the experimental data in Figure 8D . Error bars indicate the SEMs . ( F ) Quantification of Rab7 - EGFP - positive late endosome vesicles per cell from the experimental data in Figure 8D . Data information : n : number of cells analyzed , with # of data points analyzed per condition given in parentheses . Data are shown as bars with dots and represent the means ± SEMs . P values were determined using a two - tailed unpaired t test . Significant differences are labeled , * * * * P < 0 . 0001 .

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(D) Live-image analysis of WT and SCOTIN-KO HeLa cells expressing Rab7-EGFP. Left: confocal images taken at time 0. Right: time-lapse images of Rab7-EGFP in WT and SCOTIN-KO HeLa cells, as shown by temporal color-coded representations. Each confocal image of late endosomes was color coded according to its acquisition time point, with images taken at 12-sec intervals over 5 min. A total of 24 color-coded images were projected to form a single image that demonstrates the dynamics of Rab7-labeled late endosomes. White = static late endosomes. Multicolored = motile late endosomes. Boxed, magnified images highlight selected perinuclear and peripheral regions. Scale bar: 30 μm. Scale bars in the magnified images: 3 μm. (E) Quantification of the fractional distance of Rab7-EGFP-positive late endosomes in WT and SCOTIN-KO HeLa cells from the experimental data in Figure 8D. Error bars indicate the SEMs. (F) Quantification of Rab7-EGFP-positive late endosome vesicles per cell from the experimental data in Figure 8D. Data information: n: number of cells analyzed, with # of data points analyzed per condition given in parentheses. Data are shown as bars with dots and represent the means±SEMs. P values were determined using a two-tailed unpaired t test. Significant differences are labeled, ****P<0.0001.


( G , H ) Live - image analysis of Rab7 - EGFP in SCOTIN - KO HeLa cells reconstituted with SCOTIN - DsRed ( G ) or SCOTIN ( Δ150 - 177 ) - DsRed ( H ) taken at time 0 or for 5 min ( color - coded images ) . Lower , boxed , magnified images of selected perinuclear and peripheral regions . Scale bar : 20 μm . Scale bars in the magnified images : 2 μm . ( I ) Quantification of the fractional distance of Rab7 - EGFP in SCOTIN - KO cells reconstituted with SCOTIN - DsRed or SCOTIN ( Δ150 - 177 ) - DsRed from the experimental data in Figure 8G and 8H . Data information : n : number of cells analyzed , with # of data points analyzed per condition given in parentheses . Data are shown as bars with dots and represent the means ± SEMs . P values were determined using a two - tailed unpaired t test . Significant differences are labeled , * * * * P < 0 . 0001 .

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(G, H) Live-image analysis of Rab7-EGFP in SCOTIN-KO HeLa cells reconstituted with SCOTIN-DsRed (G) or SCOTIN(Δ150-177)-DsRed (H) taken at time 0 or for 5 min (color-coded images). Lower, boxed, magnified images of selected perinuclear and peripheral regions. Scale bar: 20 μm. Scale bars in the magnified images: 2 μm. (I) Quantification of the fractional distance of Rab7-EGFP in SCOTIN-KO cells reconstituted with SCOTIN-DsRed or SCOTIN(Δ150-177)-DsRed from the experimental data in Figure 8G and 8H. Data information: n: number of cells analyzed, with # of data points analyzed per condition given in parentheses. Data are shown as bars with dots and represent the means±SEMs. P values were determined using a two-tailed unpaired t test. Significant differences are labeled, ****P<0.0001.


( b ) MAPPIT screen identified RAL - interacting proteins . A MAPPIT array covering close to 8 , 500 preys was screened with RALA - G23V as bait . Data are shown in a volcano plot of the log ratio of normalized MAPPIT luciferase activity versus P value Source data can be found in Supplementary Table S1 .

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(b) MAPPIT screen identified RAL-interacting proteins. A MAPPIT array covering close to 8,500 preys was screened with RALA-G23V as bait. Data are shown in a volcano plot of the log ratio of normalized MAPPIT luciferase activity versus P value Source data can be found in Supplementary Table S1.


( c ) MAPPIT assay confirmed interaction between the RAL proteins and USP33 . RALA - G23V or RALB - G23V baits were introduced into HEK293T cells together with the indicated preys . The results are expressed as a mean of normalized luciferase activity ± s . d ( leptin - treated cells versus leptin - untreated cells ) for three independent experiments . Statistics source data can be found in Supplementary Table S1 .

(c) MAPPIT assay confirmed interaction between the RAL proteins and USP33. RALA-G23V or RALB-G23V baits were introduced into HEK293T cells together with the indicated preys. The results are expressed as a mean of normalized luciferase activity ± s.d (leptin-treated cells versus leptin-untreated cells) for three independent experiments. Statistics source data can be found in Supplementary Table S1.


( d , e ) USP33 interacts with both RALA and RALB . At 48 h post - transfection with tagged USP33 and RAL expression constructs , the indicated proteins were then immunoprecipitated with anti - Flag ( M2 ) or anti - HA - agarose , followed by immunoblotting using anti - Flag or anti - HA antibodies . WCL , whole - cell lysate ; IP , immunoprecipitates .

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(d,e) USP33 interacts with both RALA and RALB. At 48 h post-transfection with tagged USP33 and RAL expression constructs, the indicated proteins were then immunoprecipitated with anti-Flag (M2) or anti-HA-agarose, followed by immunoblotting using anti-Flag or anti-HA antibodies. WCL, whole-cell lysate; IP, immunoprecipitates.


( f ) Endogenous USP33 interacts with RALB . Flag - RALB was immunoprecipitated using anti - Flag ( M2 ) agarose and analysed for co - precipitation with endogenous USP33 using anti - USP33 antibody . Cells expressing USP33 shRNA were used as a negative control .

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(f) Endogenous USP33 interacts with RALB. Flag-RALB was immunoprecipitated using anti-Flag (M2) agarose and analysed for co-precipitation with endogenous USP33 using anti-USP33 antibody. Cells expressing USP33 shRNA were used as a negative control.


( g ) GTP binding is not required for interaction between USP33 and the RAL proteins . The GST - tagged RALA and RALB were isolated with glutathione - agarose and mixed with lysates derived from HEK293T cells expressing Flag - USP33 in the presence of GTPγS or excess of GDP followed by immunoblotting with Flag - specific antibody .

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(g) GTP binding is not required for interaction between USP33 and the RAL proteins. The GST-tagged RALA and RALB were isolated with glutathione-agarose and mixed with lysates derived from HEK293T cells expressing Flag-USP33 in the presence of GTPγS or excess of GDP followed by immunoblotting with Flag-specific antibody.


( h ) USP33 affects ubiquitylation of RALB , but not RALA . 6xHis - tagged ubiquitin and Flag - RAL - G23V mutants were introduced into HEK293T cells stably expressing GFP shRNA or USP33 shRNA . Ubiquitylated RAL proteins were purified by Co2 + metal affinity chromatography and detected by antibodies specific to RALA or RALB . Uncropped images of blots are shown in Supplementary Fig . S7 .

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(h) USP33 affects ubiquitylation of RALB, but not RALA. 6xHis-tagged ubiquitin and Flag-RAL-G23V mutants were introduced into HEK293T cells stably expressing GFP shRNA or USP33 shRNA. Ubiquitylated RAL proteins were purified by Co2+ metal affinity chromatography and detected by antibodies specific to RALA or RALB. Uncropped images of blots are shown in Supplementary Fig. S7.


( g ) GTP binding is not required for interaction between USP33 and the RAL proteins . The USP33 - tagged RALA and RALB were isolated with glutathione - agarose and mixed with lysates derived from HEK293T cells expressing Flag - USP33 in the presence of GTPγS or excess of GDP followed by immunoblotting with Flag - specific antibody .

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(g) GTP binding is not required for interaction between USP33 and the RAL proteins. The USP33-tagged RALA and RALB were isolated with glutathione-agarose and mixed with lysates derived from HEK293T cells expressing Flag-USP33 in the presence of GTPγS or excess of GDP followed by immunoblotting with Flag-specific antibody.


( a ) Tandem affinity purification of ubiquitylated RALB . 6xHis - ubiquitin and Flag - RALB - G23V were co - transfected into HEK293T cells , and ubiquitylated RALB was purified using anti - Flag resin followed by Co2 + metal affinity chromatography . Isolated RALB was visualized by immunoblotting using anti - RALB antibody .

(a) Tandem affinity purification of ubiquitylated RALB. 6xHis-ubiquitin and Flag-RALB-G23V were co-transfected into HEK293T cells, and ubiquitylated RALB was purified using anti-Flag resin followed by Co2+ metal affinity chromatography. Isolated RALB was visualized by immunoblotting using anti-RALB antibody.


( b ) RALB undergoes ubiquitylation on multiple sites . Schematic diagram of the RALB domains ( upper panel ) . RALB ubiquitylation sites were identified in two independent tandem mass spectrometry analyses with a total coverage of 63 % of the RALB amino acid sequence ( Supplementary Fig . S4a ) . The major identified ubiquitylation sites , Lys 47 and Lys 160 , are located in the effector - interacting domain ( green ) and the G5 box ( red ) , respectively .

(b) RALB undergoes ubiquitylation on multiple sites. Schematic diagram of the RALB domains (upper panel). RALB ubiquitylation sites were identified in two independent tandem mass spectrometry analyses with a total coverage of 63% of the RALB amino acid sequence (Supplementary Fig. S4a). The major identified ubiquitylation sites, Lys 47 and Lys 160, are located in the effector-interacting domain (green) and the G5 box (red), respectively.


( c ) Substitution of arginine for lysine at position 47 significantly inhibits ubiquitylation of RALB . (

(c) Substitution of arginine for lysine at position 47 significantly inhibits ubiquitylation of RALB. (


( d ) USP33 regulates RALB ubiquitylation at Lys 47 . ( e ) The GTP - GDP status of RALB affects RALB ubiquitylation at Lys 47 . For c - e , 6xHis - tagged ubiquitin and the indicated RALB mutants were introduced into HEK293T cells expressing GFP shRNA or USP33 shRNA . Ubiquitylated RALB was purified by Co2 + metal affinity chromatography and detected by antibodies specific to RALB . WT , wild type .

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(d) USP33 regulates RALB ubiquitylation at Lys 47. (e) The GTP-GDP status of RALB affects RALB ubiquitylation at Lys 47. For c-e, 6xHis-tagged ubiquitin and the indicated RALB mutants were introduced into HEK293T cells expressing GFP shRNA or USP33 shRNA. Ubiquitylated RALB was purified by Co2+ metal affinity chromatography and detected by antibodies specific to RALB. WT, wild type.


( f ) Lack of ubiquitylation at Lys 47 inhibits RALB binding to SEC5 . The indicated Flag - tagged RALB mutants were overexpressed in HEK293T cells and then immunoprecipitated with anti - Flag ( M2 ) agarose followed by immunoblotting using anti - SEC5 antibody .

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(f) Lack of ubiquitylation at Lys 47 inhibits RALB binding to SEC5. The indicated Flag-tagged RALB mutants were overexpressed in HEK293T cells and then immunoprecipitated with anti-Flag (M2) agarose followed by immunoblotting using anti-SEC5 antibody.


g ) Ubiquitylation at Lys 47 impairs RALB binding to EXO84 . Flag - tagged RALB mutants were overexpressed in HEK293T cells and then immunoprecipitated with anti - Flag ( M2 ) agarose followed by immunoblotting using anti - EXO84 antibody .

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g) Ubiquitylation at Lys 47 impairs RALB binding to EXO84. Flag-tagged RALB mutants were overexpressed in HEK293T cells and then immunoprecipitated with anti-Flag (M2) agarose followed by immunoblotting using anti-EXO84 antibody.


( h ) Ubiquitylation at Lys 47 does not affect RALB activity . The activity of RALB mutants was determined by a RALBP1 - RBD binding assay .

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(h) Ubiquitylation at Lys 47 does not affect RALB activity. The activity of RALB mutants was determined by a RALBP1-RBD binding assay.


( i ) The Rosetta v3 protocol described in ref . was used for ubiquitin - docking simulations . The plot represents the energies of the produced decoys distributed by the root - mean - square deviation ( RMSD ) of ubiquitin position relative to RALA from an arbitrarily chosen decoy .

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(i) The Rosetta v3 protocol described in ref. was used for ubiquitin-docking simulations. The plot represents the energies of the produced decoys distributed by the root-mean-square deviation (RMSD) of ubiquitin position relative to RALA from an arbitrarily chosen decoy.


( j ) Rosetta modelling of ubiquitylated - RALA - SEC5 - RBD and ubiquitylated - RALA - EXO84 - RBD complexes . Superposition of the top ten refined solutions of ubiquitylated - RALA - SEC5 - RBD complex , selected by combined scores of two independent docking simulations of ubiquitylated - RALA - SEC5 - RBD and ubiquitylated - RALA ( left ) . Simulated ubiquitin positions linked to Lys 47 of RALA were combined with the RALA - EXO84 complex ( right ) . Uncropped images of blots are shown in Supplementary Fig . S7 .

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(j) Rosetta modelling of ubiquitylated-RALA-SEC5-RBD and ubiquitylated-RALA-EXO84-RBD complexes. Superposition of the top ten refined solutions of ubiquitylated-RALA-SEC5-RBD complex, selected by combined scores of two independent docking simulations of ubiquitylated-RALA-SEC5-RBD and ubiquitylated-RALA (left). Simulated ubiquitin positions linked to Lys 47 of RALA were combined with the RALA-EXO84 complex (right). Uncropped images of blots are shown in Supplementary Fig. S7.


( a ) USP33 does not affect RALB activity . The activity of RALB was determined in HEK293T cells expressing GFP shRNA or USP33 shRNA and empty vector ( V ) or HA - tagged USP33 by RALBP1 - RBD binding assay .

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(a) USP33 does not affect RALB activity. The activity of RALB was determined in HEK293T cells expressing GFP shRNA or USP33 shRNA and empty vector (V) or HA-tagged USP33 by RALBP1-RBD binding assay.


( b ) USP33 modulates interaction between RALB and SEC5 . Flag - tagged RALB was overexpressed in HEK293T cells expressing GFP shRNA , USP33 shRNA , empty vector ( V ) or HA - tagged USP33 . The indicated proteins were then immunoprecipitated with anti - Flag ( M2 ) agarose followed by immunoblotting using anti - Flag or anti - SEC5 antibodies .

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(b) USP33 modulates interaction between RALB and SEC5. Flag-tagged RALB was overexpressed in HEK293T cells expressing GFP shRNA, USP33 shRNA, empty vector (V) or HA-tagged USP33. The indicated proteins were then immunoprecipitated with anti-Flag (M2) agarose followed by immunoblotting using anti-Flag or anti-SEC5 antibodies.


( c ) USP33 regulates RALB binding to EXO84 . Flag - tagged RALB was immunoprecipitated using anti - Flag ( M2 ) agarose from HEK293T cells expressing the indicated constructs . The presence of EXO84 in RALB complexes was analysed by immunoblotting using anti - HA or anti - EXO84 antibody .

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(c) USP33 regulates RALB binding to EXO84. Flag-tagged RALB was immunoprecipitated using anti-Flag (M2) agarose from HEK293T cells expressing the indicated constructs. The presence of EXO84 in RALB complexes was analysed by immunoblotting using anti-HA or anti-EXO84 antibody.


( d ) Activated RALB co - localizes with both EXO84 and SEC5 . At 24 h after co - transfection with RALB - G23V - EGFP and HA - EXO84 , HeLa cells were immunostained with anti - HA and anti - SEC5 antibodies . Scale bars , 10 μm . The outlined areas are shown at higher magnification in the bottom panels .

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(d) Activated RALB co-localizes with both EXO84 and SEC5. At 24 h after co-transfection with RALB-G23V-EGFP and HA-EXO84, HeLa cells were immunostained with anti-HA and anti-SEC5 antibodies. Scale bars, 10 μm. The outlined areas are shown at higher magnification in the bottom panels.