( A ) Flow cytometry analysis of control and miR - 17 transduced CD34 + thymocytes , cultured in ATOs for 3 weeks . Numbers in contour plots indicate percentage of cells for the corresponding populations . Histograms show TCRγδ expression in CD3 + TCRαβ + ( blue ) and CD3 + TCRαβ - ( red ) cells . ( B ) Graphs show the mean frequency of CD4 + CD8β + DP , CD3 + TCRαβ + and CD3 + TCRγδ + thymocytes of control ( black bar ) or miR - 17 ( grey bar ) transduced CD34 + thymocytes in these cultures ( n = 6 and n = 8 independent experiments for control and miR - 17 , respectively . Error bars indicate SEM and ns ( not significant ) indicates p > 0 . 05 in a non - parametric Mann - Whitney U test ) . ( C ) Graphs show the mean absolute cell numbers of total , CD4 + CD8β + DP , CD3 + TCRαβ + and CD3 + TCRγδ + thymocytes that developed in these cultures ( n = 6 and n = 8 independent experiments for control and miR - 17 , respectively . Error bars indicate SEM and * indicates p < 0 . 05 in a non - parametric Mann - Whitney U test ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-CELL_TYPE O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD O B-CELL_TYPE O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-CELL_TYPE O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O 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) Flow cytometry analysis of control and miR-17 transduced CD34+ thymocytes, cultured in ATOs for 3 weeks. Numbers in contour plots indicate percentage of cells for the corresponding populations. Histograms show TCRγδ expression in CD3+TCRαβ+ (blue) and CD3+TCRαβ- (red) cells. (B) Graphs show the mean frequency of CD4+CD8β+ DP, CD3+TCRαβ+ and CD3+TCRγδ+ thymocytes of control (black bar) or miR-17 (grey bar) transduced CD34+ thymocytes in these cultures (n=6 and n=8 independent experiments for control and miR-17, respectively. Error bars indicate SEM and ns (not significant) indicates p>0.05 in a non-parametric Mann-Whitney U test). (C) Graphs show the mean absolute cell numbers of total, CD4+CD8β+ DP, CD3+TCRαβ+ and CD3+TCRγδ+ thymocytes that developed in these cultures (n=6 and n=8 independent experiments for control and miR-17, respectively. Error bars indicate SEM and * indicates p<0.05 in a non-parametric Mann-Whitney U test).


( A ) Flow cytometry analysis of control and miR - 17 transduced CD34 + thymocytes , cultured in ATOs for 3 weeks . Numbers in contour plots indicate percentage of cells for the corresponding populations . Histograms show TCRγδ expression in CD3 + TCRαβ + ( blue ) and CD3 + TCRαβ - ( red ) cells . ( B ) Graphs show the mean frequency of CD4 + CD8β + DP , CD3 + TCRαβ + and CD3 + TCRγδ + thymocytes of control ( black bar ) or miR - 17 ( grey bar ) transduced CD34 + thymocytes in these cultures ( n = 6 and n = 8 independent experiments for control and miR - 17 , respectively . Error bars indicate SEM and ns ( not significant ) indicates p > 0 . 05 in a non - parametric Mann - Whitney U test ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-CELL_TYPE O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD O B-CELL_TYPE O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O 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) Flow cytometry analysis of control and miR-17 transduced CD34+ thymocytes, cultured in ATOs for 3 weeks. Numbers in contour plots indicate percentage of cells for the corresponding populations. Histograms show TCRγδ expression in CD3+TCRαβ+ (blue) and CD3+TCRαβ- (red) cells. (B) Graphs show the mean frequency of CD4+CD8β+ DP, CD3+TCRαβ+ and CD3+TCRγδ+ thymocytes of control (black bar) or miR-17 (grey bar) transduced CD34+ thymocytes in these cultures (n=6 and n=8 independent experiments for control and miR-17, respectively. Error bars indicate SEM and ns (not significant) indicates p>0.05 in a non-parametric Mann-Whitney U test).


( A ) Flow cytometry analysis showing mean of Median Fluorescence Intensity ( MFI ) of BCL11B protein levels in Jurkat cells , 72h post transduction with control shRNA ( black bar ) , BCL11B shRNA1 or BCL11B shRNA2 ( grey bars ) , each normalized to the FMO sample ( BCL11B stained - BCL11B unstained sample ) . ( n = 2 independent experiments ; error bars indicate SEM - the two data points are shown ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O B-GENEPROD 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) Flow cytometry analysis showing mean of Median Fluorescence Intensity (MFI) of BCL11B protein levels in Jurkat cells, 72h post transduction with control shRNA (black bar), BCL11B shRNA1 or BCL11B shRNA2 (grey bars), each normalized to the FMO sample (BCL11B stained - BCL11B unstained sample). (n=2 independent experiments; error bars indicate SEM - the two data points are shown).


( B ) Flow cytometry analysis showing mean of MFI of BCL11B protein levels in CD4 + CD8β + DP ( black bar ) and CD4 - CD8β - DN CD3 + TCRγδ + ( grey bar ) ex vivo thymocytes , normalized to FMO sample ( BCL11B stained - BCL11B unstained sample ) ( n = 4 independent experiments with different donors , error bars indicate SEM and * indicates p < 0 . 05 in a paired t - test ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O O B-CELL_TYPE O O O O O O B-GENEPROD B-EXP_ASSAY O B-GENEPROD O O O O O O O O O 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) Flow cytometry analysis showing mean of MFI of BCL11B protein levels in CD4+CD8β+ DP (black bar) and CD4-CD8β- DN CD3+TCRγδ+ (grey bar) ex vivo thymocytes, normalized to FMO sample (BCL11B stained - BCL11B unstained sample) (n=4 independent experiments with different donors, error bars indicate SEM and * indicates p<0.05 in a paired t-test).


( C ) Mean total cell numbers of control shRNA ( black bar ) , BCL11B shRNA1 or BCL11B shRNA2 ( grey bars ) transduced CD34 + thymocytes , cultured in ATOs for 3 weeks ( n = 5 independent experiments with different donors , error bars indicate SEM and * indicates p < 0 . 05 in a non - parametric paired Wilcoxon test ) .

O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O B-GENEPROD O B-CELL_TYPE O O O O O O O O O O O O O O O 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) Mean total cell numbers of control shRNA (black bar), BCL11B shRNA1 or BCL11B shRNA2 (grey bars) transduced CD34+ thymocytes, cultured in ATOs for 3 weeks (n=5 independent experiments with different donors, error bars indicate SEM and * indicates p<0.05 in a non-parametric paired Wilcoxon test).


( D ) Flow cytometry analysis of CD4 + CD8β + DP , CD3 + TCRαβ + ( blue ) and CD3 + TCRαβ - ( red ) thymocytes that developed in corresponding cultures from C . Numbers in contour plots indicate the percentage of cells for the corresponding populations . Histograms show TCRγδ expression in CD3 + TCRαβ + ( blue ) and CD3 + TCRαβ - ( red ) cells . ( E ) Graphs show the mean frequency of CD3 + TCRαβ + and CD3 + TCRγδ + thymocytes that developed in these cultures ( n = 5 independent experiments with different donors , error bars indicate SEM and * indicates p < 0 . 05 in a paired t - test ) . ( F - G ) Mean absolute cell numbers of ( F ) CD3 + TCRαβ + and ( G ) CD3 + TCRγδ + thymocytes that developed in these cultures ( n = 5 independent experiments with different donors , error bars indicate SEM and * indicates p < 0 . 05 in a non - parametric paired Wilcoxon test ) . ( H ) CD3 + TCRγδ + / CD3 + TCRαβ + ratio from cultures depicted in D ( n = 5 independent experiments with different donors , error bars indicate SEM and * indicates p < 0 . 05 in a paired t - test ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O B-CELL_TYPE 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-GENEPROD B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-CELL_TYPE O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O B-CELL_TYPE O O O O O O O O O O O O O O O O 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-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Flow cytometry analysis of CD4+CD8β+ DP, CD3+TCRαβ+ (blue) and CD3+TCRαβ- (red) thymocytes that developed in corresponding cultures from C. Numbers in contour plots indicate the percentage of cells for the corresponding populations. Histograms show TCRγδ expression in CD3+TCRαβ+ (blue) and CD3+TCRαβ- (red) cells. (E) Graphs show the mean frequency of CD3+TCRαβ+ and CD3+TCRγδ+ thymocytes that developed in these cultures (n=5 independent experiments with different donors, error bars indicate SEM and * indicates p<0.05 in a paired t-test). (F-G) Mean absolute cell numbers of (F) CD3+TCRαβ+ and (G) CD3+TCRγδ+ thymocytes that developed in these cultures (n=5 independent experiments with different donors, error bars indicate SEM and * indicates p<0.05 in a non-parametric paired Wilcoxon test). (H) CD3+TCRγδ+/CD3+TCRαβ+ ratio from cultures depicted in D (n=5 independent experiments with different donors, error bars indicate SEM and * indicates p<0.05 in a paired t-test).


( D ) Flow cytometry analysis of CD4 + CD8β + DP , CD3 + TCRαβ + ( blue ) and CD3 + TCRαβ - ( red ) thymocytes that developed in corresponding cultures from C . Numbers in contour plots indicate the percentage of cells for the corresponding populations . Histograms show TCRγδ expression in CD3 + TCRαβ + ( blue ) and CD3 + TCRαβ - ( red ) cells .

O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O B-CELL_TYPE 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-GENEPROD B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O O

(D) Flow cytometry analysis of CD4+CD8β+ DP, CD3+TCRαβ+ (blue) and CD3+TCRαβ- (red) thymocytes that developed in corresponding cultures from C. Numbers in contour plots indicate the percentage of cells for the corresponding populations. Histograms show TCRγδ expression in CD3+TCRαβ+ (blue) and CD3+TCRαβ- (red) cells.


( D ) Fragment length distributions of human - ( pink ) and mouse - ( blue ) specific reads from a DBS sample . Two vertical lines indicate 146 and 166 bp , the observed peaks for ctDNA and cfDNA , respectively .

O O O O O O O B-ORGANISM O O O O O B-ORGANISM 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-SUBCELLULAR O B-SUBCELLULAR O O O

(D) Fragment length distributions of human- (pink) and mouse- (blue) specific reads from a DBS sample. Two vertical lines indicate 146 and 166 bp, the observed peaks for ctDNA and cfDNA, respectively.


( E ) Example of an absolute copy number ( ACN ) profile successfully generated from human - specific reads from a DBS collected from a PDX mouse of patient line 828 . ( F ) Matching ACN profile generated from sWGS of PDX tumour tissue .

O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-ORGANISM O O O O O O O O O O B-ORGANISM O B-ORGANISM O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O O O O O

(E) Example of an absolute copy number (ACN) profile successfully generated from human-specific reads from a DBS collected from a PDX mouse of patient line 828. (F) Matching ACN profile generated from sWGS of PDX tumour tissue.


( B ) xTF change from baseline during the first 29 days since treatment start for each PDX patient line . xTFs were normalised to baseline ( day 1 ) xTF values for each mouse ( dashed lines ) . Carboplatin - treated mice are shown in purple , control mice are shown in teal . Bold lines show the linear - model fitted line across all mice within the same treatment and patient group . Horizontal dashed lines at y = 1 indicate normalised baseline .

O O O B-EXP_ASSAY O O O O O O O O O O O O O O B-ORGANISM O O B-EXP_ASSAY O O O O O O O O B-EXP_ASSAY O O O B-ORGANISM O O O O O B-SMALL_MOLECULE O O B-ORGANISM O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O B-ORGANISM O O O O O B-ORGANISM O O O O O O O O O O O O O

(B) xTF change from baseline during the first 29 days since treatment start for each PDX patient line. xTFs were normalised to baseline (day 1) xTF values for each mouse (dashed lines). Carboplatin-treated mice are shown in purple, control mice are shown in teal. Bold lines show the linear-model fitted line across all mice within the same treatment and patient group. Horizontal dashed lines at y=1 indicate normalised baseline.


( C ) Fraction of blood spot samples in which putative driver amplifications were detected over time . The fraction of samples with detected gene amplifications decreases in carboplatin treated group , while increasing in the control group over time .

O O O O O B-TISSUE O O O O O O B-EXP_ASSAY O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-SMALL_MOLECULE O O O O O O O O O O O O

(C) Fraction of blood spot samples in which putative driver amplifications were detected over time. The fraction of samples with detected gene amplifications decreases in carboplatin treated group, while increasing in the control group over time.


( D ) Correlation between xTF values and tumour volumes of the nearest matched time - point for both untreated ( Pearson ' s R = 0 . 45 , p = 0 . 00018 ) , and carboplatin - treated ( Pearson ' s R = 0 . 056 , p = 0 . 78 ) PDX mice .

O O O O O B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O O O O 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-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O B-ORGANISM O

(D) Correlation between xTF values and tumour volumes of the nearest matched time-point for both untreated (Pearson's R = 0.45, p = 0.00018), and carboplatin-treated (Pearson's R = 0.056, p = 0.78) PDX mice.


( A - D ) Weekly measured tumour volumes ( mm3 ) for PDX mice over time . Treatment start is indicated by grey solid vertical line . Solid coloured lines show modelled tumour growth curves using heteroscedastic point - wise random intercept linear mixed models ( see Methods ) . Growth curve inflection points were determined ( see Methods ) to estimate the start of treatment effect and tumour regrowth ( dashed vertical purple lines labelled t1 and t2 , respectively ) .

O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O

(A-D) Weekly measured tumour volumes (mm3) for PDX mice over time. Treatment start is indicated by grey solid vertical line. Solid coloured lines show modelled tumour growth curves using heteroscedastic point-wise random intercept linear mixed models (see Methods). Growth curve inflection points were determined (see Methods) to estimate the start of treatment effect and tumour regrowth (dashed vertical purple lines labelled t1 and t2, respectively).


( E ) The mean xTF slope was estimated for each treatment group across the four patient lines ( Fig . 2B ) and compared to the mean time to endpoint ( Pearson ' s R = - 0 . 73 , p = 0 . 039 ) and tumour regrowth ( Pearson ' s R = - 0 . 97 , p = 0 . 025 ) . The four different patient lines are indicated by different colours .

O O O O O B-EXP_ASSAY O O O O O O O O O O B-ORGANISM O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O

(E) The mean xTF slope was estimated for each treatment group across the four patient lines (Fig. 2B) and compared to the mean time to endpoint (Pearson's R = -0.73, p = 0.039) and tumour regrowth (Pearson's R = -0.97, p = 0.025). The four different patient lines are indicated by different colours.


G - I ) Experimental validation of collective survival . All populations grew to high cell densities in the absence of antibiotics ( Blue curves ) . In the presence of antibiotics , only populations with initial densities greater than NCT grew . NCT was increased with the concentration of antibiotics . Engineered bacteria with the QS - BlaM circuit exhibited a significantly higher NCT than those with the BlaM or the QS - CAT circuits . Carbenicillin was used for the BlaM and QS - BlaM circuits ; chloramphenicol for the QS - CAT circuit . Each error bar represents the standard deviation from triplicate measurements .

O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O 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-ORGANISM O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O O O B-GENEPROD O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O

G-I) Experimental validation of collective survival. All populations grew to high cell densities in the absence of antibiotics (Blue curves). In the presence of antibiotics, only populations with initial densities greater than NCT grew. NCT was increased with the concentration of antibiotics. Engineered bacteria with the QS-BlaM circuit exhibited a significantly higher NCT than those with the BlaM or the QS-CAT circuits. Carbenicillin was used for the BlaM and QS-BlaM circuits; chloramphenicol for the QS-CAT circuit. Each error bar represents the standard deviation from triplicate measurements.


O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O O O O O O O O O O O


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


( G - I ) Experimental validation of collective survival . All populations grew to high cell densities in the absence of antibiotics ( Blue curves ) . In the presence of antibiotics , only populations with initial densities greater than NCT grew . NCT was increased with the concentration of antibiotics . Engineered bacteria with the QS - BlaM circuit exhibited a significantly higher NCT than those with the BlaM or the QS - CAT circuits . Carbenicillin was used for the BlaM and QS - BlaM circuits ; chloramphenicol for the QS - CAT circuit . Each error bar represents the standard deviation from triplicate measurements .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O

(G-I) Experimental validation of collective survival. All populations grew to high cell densities in the absence of antibiotics (Blue curves). In the presence of antibiotics, only populations with initial densities greater than NCT grew. NCT was increased with the concentration of antibiotics. Engineered bacteria with the QS-BlaM circuit exhibited a significantly higher NCT than those with the BlaM or the QS-CAT circuits. Carbenicillin was used for the BlaM and QS-BlaM circuits; chloramphenicol for the QS-CAT circuit. Each error bar represents the standard deviation from triplicate measurements.


Configuration of the two - compartment model . The microenvironment of microbial swarmbot is separated from that of the chamber by its permeable hydrogel shell ( orange ) , resulting in two separate compartments , defined as swarmbot and environment ( chamber ) . Four major components involved in the population dynamics are explored in the model , including cell density ( n ) , nutrient ( s ) , carbenicillin ( a ) and BlaM concentration ( b ) . Each component has a different transport rate , depending on the molecule size . Simulated microbial swarmbot dynamics under a static condition . a = 0 . 4 , a microbial swarmbot with a low n0 cannot initiate growth ( red lines ) but one with a high n0 leads to growth both in the swarmbot and , after a delay , in the chamber ( green lines ) . While ( C ) swarmbots with both low and high initial densities lead to eventual full growth in the chamber in the absence of the antibiotic ( a = 0 ) . Solid lines : cell density inside swarmbots ; dashed lines : cell density in the chambers . ( D ) A microfluidic device for controlling and monitoring population dynamics of microbial swarmbot . The device consists of an input channel leading to a culturing chamber and a trapping slot . The chamber is used for culturing engineered bacterial populations both inside and outside the swarmbot , while the trapping slot is for trapping the swarmbot due to its smaller size but allowing the medium to flow through . Input flow patterns ( static or pulsing ) are controlled by a programmable syringe pump . ( E ) Experimental validation of model prediction . At 100µg / ml carbenicillin , a microbial swarmbot with a high initial density led to robust growth in the capsule and , after a delay , in the chamber ( green lines ) . While a swarmbot with a low initial cell density did not exhibit growth either in the swarmbot or in the chamber at the same carbenicillin concentration ( red lines ) . ( F ) In contrast , both the swarmbot and culturing chamber reached high cell densities in absence of carbenicillin , regardless of the initial cell densities .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Configuration of the two-compartment model. The microenvironment of microbial swarmbot is separated from that of the chamber by its permeable hydrogel shell (orange), resulting in two separate compartments, defined as swarmbot and environment (chamber). Four major components involved in the population dynamics are explored in the model, including cell density (n), nutrient (s), carbenicillin (a) and BlaM concentration (b). Each component has a different transport rate, depending on the molecule size.Simulated microbial swarmbot dynamics under a static condition. a = 0.4, a microbial swarmbot with a low n0 cannot initiate growth (red lines) but one with a high n0 leads to growth both in the swarmbot and, after a delay, in the chamber (green lines). While (C) swarmbots with both low and high initial densities lead to eventual full growth in the chamber in the absence of the antibiotic (a = 0). Solid lines: cell density inside swarmbots; dashed lines: cell density in the chambers.(D) A microfluidic device for controlling and monitoring population dynamics of microbial swarmbot. The device consists of an input channel leading to a culturing chamber and a trapping slot. The chamber is used for culturing engineered bacterial populations both inside and outside the swarmbot, while the trapping slot is for trapping the swarmbot due to its smaller size but allowing the medium to flow through. Input flow patterns (static or pulsing) are controlled by a programmable syringe pump.(E) Experimental validation of model prediction. At 100µg/ml carbenicillin, a microbial swarmbot with a high initial density led to robust growth in the capsule and, after a delay, in the chamber (green lines). While a swarmbot with a low initial cell density did not exhibit growth either in the swarmbot or in the chamber at the same carbenicillin concentration (red lines). (F) In contrast, both the swarmbot and culturing chamber reached high cell densities in absence of carbenicillin, regardless of the initial cell densities.


( G ) Time - lapse images of microbial swarmbot dynamics . The population initiated growth inside the swarmbot and subsequently in the chamber at a high initial density . But no growth occurred for a swarmbot with a low initial density at the same antibiotic concentration ( rightmost ) . The concentration of carbenicillin was 100 µg / ml and the scale bar represents 200 µm . ( H ) The control strain lacking BlaM circuit . The population could not survive at the same concentration of carbenicillin at the high initial density .

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(G) Time-lapse images of microbial swarmbot dynamics. The population initiated growth inside the swarmbot and subsequently in the chamber at a high initial density. But no growth occurred for a swarmbot with a low initial density at the same antibiotic concentration (rightmost). The concentration of carbenicillin was 100 µg/ml and the scale bar represents 200 µm.(H) The control strain lacking BlaM circuit. The population could not survive at the same concentration of carbenicillin at the high initial density.


Time courses of swarmbots treated with varying concentrations of carbenicillin and glucose . Solid lines indicate cell densities inside swarmbots ; dotted lines indicate those in the chamber . Safeguard was established for lower nutrient levels ( 0 . 004 % or 0 . 04 % glucose ) along with carbenicillin treatment . At a high nutrient level ( 0 . 4 % glucose ) , the safeguard was lost due to either growth in the chamber ( panels 3 and 6 ) or gradual killing of the population inside swarmbot ( solid line in panel 9 ) . These results are qualitatively consistent with the model predictions ( Fig 4 ) .

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Time courses of swarmbots treated with varying concentrations of carbenicillin and glucose. Solid lines indicate cell densities inside swarmbots; dotted lines indicate those in the chamber. Safeguard was established for lower nutrient levels (0.004% or 0.04% glucose) along with carbenicillin treatment. At a high nutrient level (0.4% glucose), the safeguard was lost due to either growth in the chamber (panels 3 and 6) or gradual killing of the population inside swarmbot (solid line in panel 9). These results are qualitatively consistent with the model predictions (Fig 4).


Snapshots of safeguard performance at the end time point of ( A ) . The red arrow in panel 6 indicates significant growth in the chamber ; the dimmer fluorescence of the swarmbot in panel 9 indicates killing of the population .

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Snapshots of safeguard performance at the end time point of (A). The red arrow in panel 6 indicates significant growth in the chamber; the dimmer fluorescence of the swarmbot in panel 9 indicates killing of the population.


Figure 6 . Experimental control of microbial swarmbot safeguard by other circuits . Demonstration of modular safeguard performance of swarmbots with different collective survival systems . For the swarmbots containing cells carrying the QS - CAT circuit , pulsing flow of medium containing 100µg / ml chloramphenicol established safeguard control but not for the static condition . For the swarmbots with the QS - BlaM circuit , the static condition of 100 µg / ml carbenicillin was sufficient for safeguard control . For all the conditions , safeguard did not occur in the absence of antibiotic ( top panels from each condition ) . All the images were from the swarmbots cultured at 37 ˚ C for 16 hours . The scale bar is 250 µm .

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Figure 6. Experimental control of microbial swarmbot safeguard by other circuits.Demonstration of modular safeguard performance of swarmbots with different collective survival systems. For the swarmbots containing cells carrying the QS-CAT circuit, pulsing flow of medium containing 100µg/ml chloramphenicol established safeguard control but not for the static condition. For the swarmbots with the QS-BlaM circuit, the static condition of 100 µg/ml carbenicillin was sufficient for safeguard control. For all the conditions, safeguard did not occur in the absence of antibiotic (top panels from each condition). All the images were from the swarmbots cultured at 37˚C for 16 hours. The scale bar is 250 µm.


( a ) Immunostaining of TCF20 , NESTIN , and merged with DAPI in E13 and E16 embryonic brain sections . TCF20 is widely expressed in the brain . Data information : Scale bar in ( A ) e13 , 50 μm , e16 100

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(a) Immunostaining of TCF20, NESTIN, and merged with DAPI in E13 and E16 embryonic brain sections. TCF20 is widely expressed in the brain. Data information: Scale bar in (A) e13, 50 μm, e16 100 μm


( b ) Costaining with the NSC markers NESTIN , SOX2 , and PAX6 reveals that TCF20 is expressed in NSCs .

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(b) Costaining with the NSC markers NESTIN, SOX2, and PAX6 reveals that TCF20 is expressed in NSCs.


( c ) Absolute quantitative PCR of TCF20 in different developmental stages from E12 to P0 . n = 4 independently repeated tests .

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(c) Absolute quantitative PCR of TCF20 in different developmental stages from E12 to P0. n=4 independently repeated tests.


( d ) Western blotting results show the changes in TCF20 protein expression level during brain development . ( e ) Statistical analysis of normalized band intensity in the western blot results . n = 4 samples for each group .

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(d) Western blotting results show the changes in TCF20 protein expression level during brain development. (e) Statistical analysis of normalized band intensity in the western blot results. n= 4 samples for each group.


( f ) In utero electroporation of TCF20 shRNAs in the E13 cerebral cortex led to abnormal cell distribution . These electroporated embryos were collected at E16 . ( g ) Statistical analysis of the distributions of GFP - positive cells in different brain regions . The percentage of GFP - positive cells in the CP ( cortical plate ) , IZ ( intermediate zone ) , and VZ / SVZ ( ventricle zone and subventricle zone ) was analyzed . n = 7 brains . Data information : Error bars represent the means ± S . E . M . Two tailed unpaired T - tests were used to analyze the data , n . s . ( no significant difference ) , p < 0 . 01 ( * * ) , p < 0 . 001 ( * * * ) . Scale bar in ( F ) 50 μm .

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(f) In utero electroporation of TCF20 shRNAs in the E13 cerebral cortex led to abnormal cell distribution. These electroporated embryos were collected at E16. (g) Statistical analysis of the distributions of GFP-positive cells in different brain regions. The percentage of GFP-positive cells in the CP (cortical plate), IZ (intermediate zone), and VZ/SVZ (ventricle zone and subventricle zone) was analyzed. n=7 brains. Data information: Error bars represent the means ± S.E.M. Two tailed unpaired T-tests were used to analyze the data, n.s. (no significant difference), p<0.01(**), p<0.001(***). Scale bar in (F) 50 μm.


( b ) Immunostaining of BrdU . White arrows represent BrdU GFP double - positive cells . Scale bar : 50 μm .

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(b) Immunostaining of BrdU. White arrows represent BrdU GFP double-positive cells. Scale bar: 50 μm.


( c ) Statistical analysis of 2 - hour BrdU labeling experiments . n = 6 brains Data information : Error bars represent the means ± S . E . M . Two tailed unpaired T - tests were used to analyze the data , n . s . ( no significant difference ) , p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) , p < 0 . 001 ( * * * ) .

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(c) Statistical analysis of 2-hour BrdU labeling experiments. n=6 brains Data information: Error bars represent the means ± S.E.M. Two tailed unpaired T-tests were used to analyze the data, n.s. (no significant difference), p<0.05(*), p<0.01(**), p<0.001(***).


( d ) Western blotting analysis indicated an increase in the proliferation marker Sox2 in TCF20 depleted NSCs .

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(d) Western blotting analysis indicated an increase in the proliferation marker Sox2 in TCF20 depleted NSCs.


( e ) Immunostaining with pax6 in E16 electroporated embryo brains . White arrows represent PAX6 , GFP double - positive cells . 1 , 2 are 2x enlarged as the dashed boxes showed . Scale bar : 50 μm . ( f ) Statistical analysis of the percentage of PAX6 + GFP + cells among the total GFP + cells . n = 3 brains for each group . Data information : Error bars represent the means ± S . E . M . Two tailed unpaired T - tests were used to analyze the data , n . s . ( no significant difference ) , p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) , p < 0 . 001 ( * * * ) .

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(e) Immunostaining with pax6 in E16 electroporated embryo brains. White arrows represent PAX6, GFP double-positive cells. 1,2 are 2x enlarged as the dashed boxes showed. Scale bar: 50 μm. (f) Statistical analysis of the percentage of PAX6+GFP+ cells among the total GFP+ cells. n=3 brains for each group. Data information: Error bars represent the means ± S.E.M. Two tailed unpaired T-tests were used to analyze the data, n.s. (no significant difference), p<0.05(*), p<0.01(**), p<0.001(***).


( g ) Immunostaining with sox2 and pax6 in E16 electroporated embryo brains . White arrows represent GFP , sox2 double positive cells ; Red arrows represent GFP , TBR2 double positive cells . area . Scale bar : 50 μm . ( h ) Statistical analysis of the percentage of SOX2 + GFP + cells among the total GFP + cells . n = 8 brains for each group . ( i ) Statistical analysis of the percentage of TBR2 + GFP + cells among the total GFP + cells . n = 5 brains for each group . Data information : Error bars represent the means ± S . E . M . Two tailed unpaired T - tests were used to analyze the data , n . s . ( no significant difference ) , p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) , p < 0 . 001 ( * * * ) .

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(g) Immunostaining with sox2 and pax6 in E16 electroporated embryo brains. White arrows represent GFP, sox2 double positive cells; Red arrows represent GFP, TBR2 double positive cells. area. Scale bar: 50 μm. (h) Statistical analysis of the percentage of SOX2+GFP+ cells among the total GFP+ cells. n=8 brains for each group. (i) Statistical analysis of the percentage of TBR2+GFP+ cells among the total GFP+ cells. n=5 brains for each group. Data information: Error bars represent the means ± S.E.M. Two tailed unpaired T-tests were used to analyze the data, n.s. (no significant difference), p<0.05(*), p<0.01(**), p<0.001(***).


( a ) SATB2 staining revealed a reduction in SATB2 and GFP colocalization when TCF20 was depleted . White arrows represent SATB2 , GFP double - positive cells . ( b ) Statistical analysis of the SATB2 GFP double - positive cell ratio in GFP positive cells . n = 3 brains . Data information : Error bars represent the means ± S . E . M . Two tailed unpaired T - tests were used to analyze the data , n . s . ( no significant difference ) , p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) , p < 0 . 001 ( * * * ) . Scale bar : 25

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(a) SATB2 staining revealed a reduction in SATB2 and GFP colocalization when TCF20 was depleted. White arrows represent SATB2, GFP double-positive cells. (b) Statistical analysis of the SATB2 GFP double-positive cell ratio in GFP positive cells. n=3 brains. Data information: Error bars represent the means ± S.E.M. Two tailed unpaired T-tests were used to analyze the data, n.s. (no significant difference), p<0.05(*), p<0.01(**), p<0.001(***). Scale bar: 25 μm


( c ) Staining with CTIP2 and TBR1 antibodies shows TCF20 knockdown decreases the colocalization of CTIP2 and TBR1 with GFP in E13 . 5 - E16 . 5 brains . White arrows represent CTIP2 , GFP double - positive cells . White arrow heads represent TBR1 , GFP double - positive cells . ( d , e ) Statistical analysis of CTIP2 , GFP and TBR1 , GFP double - positive cell ratio in GFP positive cells . n = 3 brains . Data information : Error bars represent the means ± S . E . M . Two tailed unpaired T - tests were used to analyze the data , n . s . ( no significant difference ) , p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) , p < 0 . 001 ( * * * ) . Scale bar : ( d ) 50

O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O B-TISSUE O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Staining with CTIP2 and TBR1 antibodies shows TCF20 knockdown decreases the colocalization of CTIP2 and TBR1 with GFP in E13.5-E16.5 brains. White arrows represent CTIP2, GFP double-positive cells. White arrow heads represent TBR1, GFP double-positive cells. (d, e) Statistical analysis of CTIP2, GFP and TBR1, GFP double-positive cell ratio in GFP positive cells. n=3 brains. Data information: Error bars represent the means ± S.E.M. Two tailed unpaired T-tests were used to analyze the data, n.s. (no significant difference), p<0.05(*), p<0.01(**), p<0.001(***). Scale bar: (d) 50 μm


( f ) A diagram of the BrdU labeling time point in electroporation experiments . ( g ) Immunostaining of BrdU . White arrows represent BrdU GFP double - positive cells . ( h ) Percentage of BrdU and GFP double - positive cells in GFP - positive cells n = 3 brains for each group Data information : Error bars represent the means ± S . E . M . Two tailed unpaired T - tests were used to analyze the data , n . s . ( no significant difference ) , p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) , p < 0 . 001 ( * * * ) . Scale bar : f ) 25

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(f) A diagram of the BrdU labeling time point in electroporation experiments. (g) Immunostaining of BrdU. White arrows represent BrdU GFP double-positive cells. (h) Percentage of BrdU and GFP double-positive cells in GFP-positive cells n=3 brains for each group Data information: Error bars represent the means ± S.E.M. Two tailed unpaired T-tests were used to analyze the data, n.s. (no significant difference), p<0.05(*), p<0.01(**), p<0.001(***). Scale bar: f) 25 μm


( b ) Western blotting analysis of TCF20 in TCF20 WT , HET , KO mice reveals that TCF20 was deleted . ( c ) Statistical analysis of the normalized band intensity of TCF20 . n = 3 samples for each genotype . Data information : Error bars represent the means ± S . E . M . Two tailed unpaired T - tests were used to analyze the data , n . s . ( no significant difference ) , p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) , p < 0 . 001 ( * * * ) .

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(b) Western blotting analysis of TCF20 in TCF20 WT, HET, KO mice reveals that TCF20 was deleted. (c) Statistical analysis of the normalized band intensity of TCF20. n=3 samples for each genotype. Data information: Error bars represent the means ± S.E.M. Two tailed unpaired T-tests were used to analyze the data, n.s. (no significant difference), p<0.05(*), p<0.01(**), p<0.001(***).


( d ) RT - qPCR analysis of TCF20 in TCF20 WT , HET , and KO mice reveals that TCF20 was deleted . n = 4 samples . Data information : Error bars represent the means ± S . E . M . Two tailed unpaired T - tests were used to analyze the data , n . s . ( no significant difference ) , p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) , p < 0 . 001 ( * * * ) .

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(d) RT-qPCR analysis of TCF20 in TCF20 WT, HET, and KO mice reveals that TCF20 was deleted. n=4 samples. Data information: Error bars represent the means ± S.E.M. Two tailed unpaired T-tests were used to analyze the data, n.s. (no significant difference), p<0.05(*), p<0.01(**), p<0.001(***).


( e ) Immunostaining of E16 WT and KO mouse brain slices revealed that TCF20 was successfully knocked out . Scale bar : 50 μm . ( f ) Statistical analysis of the normalized fluorescence intensity of TCF20 . n = 6 samples for each genotype . Data information : Error bars represent the means ± S . E . M . Two tailed unpaired T - tests were used to analyze the data , n . s . ( no significant difference ) , p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) , p < 0 . 001 ( * * * ) .

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(e) Immunostaining of E16 WT and KO mouse brain slices revealed that TCF20 was successfully knocked out. Scale bar: 50 μm. (f) Statistical analysis of the normalized fluorescence intensity of TCF20. n=6 samples for each genotype. Data information: Error bars represent the means ± S.E.M. Two tailed unpaired T-tests were used to analyze the data, n.s. (no significant difference), p<0.05(*), p<0.01(**), p<0.001(***).


( g ) Western blotting analysis of neural proliferation and differentiation markers in WT HET and KO mice showed that TCF20 deficits promote cell proliferation and inhibit cell differentiation .

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(g) Western blotting analysis of neural proliferation and differentiation markers in WT HET and KO mice showed that TCF20 deficits promote cell proliferation and inhibit cell differentiation.


( h ) Immunostaining of different cortical layer markers TBR1 , CTIP2 , and SATB2 , White dash line represents the positive area . Scale bar : 50 μm . ( i - k ) Statistical analysis of different cells layers in TCF20 WT , HET and KO mice . n = 7 brains of each group . Data information : Error bars represent the means ± S . E . M . Two tailed unpaired T - tests were used to analyze the data , n . s . ( no significant difference ) , p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) , p < 0 . 001 ( * * * ) .

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(h) Immunostaining of different cortical layer markers TBR1, CTIP2, and SATB2, White dash line represents the positive area. Scale bar: 50 μm. (i-k) Statistical analysis of different cells layers in TCF20 WT, HET and KO mice. n=7 brains of each group. Data information: Error bars represent the means ± S.E.M. Two tailed unpaired T-tests were used to analyze the data, n.s. (no significant difference), p<0.05(*), p<0.01(**), p<0.001(***).


( l ) TCF20 deletion impairs neurogenesis and results in abnormal cell distribution . Scale bar : 50 μm . ( m ) Statistical analysis of GFP + distribution in different zones . n = 3 brains for each genotype . Data information : Error bars represent the means ± S . E . M . Two tailed unpaired T - tests were used to analyze the data , n . s . ( no significant difference ) , p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) , p < 0 . 001 ( * * * ) .

O O O B-GENEPROD O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O B-GENEPROD O B-EXP_ASSAY O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(l) TCF20 deletion impairs neurogenesis and results in abnormal cell distribution. Scale bar: 50 μm. (m) Statistical analysis of GFP+ distribution in different zones. n=3 brains for each genotype. Data information: Error bars represent the means ± S.E.M. Two tailed unpaired T-tests were used to analyze the data, n.s. (no significant difference), p<0.05(*), p<0.01(**), p<0.001(***).


( a ) Diagram of pup ultrasonic vocalization at the frequency level after Fourier transforms ( b - d ) ( b ) Total call durations , ( c ) Call durations and ( d ) Call numbers of WT , HET , and KO mouse pups . nWT = 13 mice , nHET = 16 mice , and nKO = 18 mice . Data information : Error bars represent the means ± S . E . M . Two tailed unpaired T - tests were used to analyze the data , n . s . ( no significant difference ) , p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) , p < 0 . 001 ( * * * ) .

O O O O O B-ORGANISM B-EXP_ASSAY I-EXP_ASSAY O O O O 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-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-ORGANISM I-ORGANISM O O O O B-ORGANISM O O O O B-ORGANISM O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Diagram of pup ultrasonic vocalization at the frequency level after Fourier transforms (b-d) (b) Total call durations, (c) Call durations and (d) Call numbers of WT, HET, and KO mouse pups. nWT=13 mice, nHET=16 mice, and nKO=18 mice. Data information: Error bars represent the means ± S.E.M. Two tailed unpaired T-tests were used to analyze the data, n.s. (no significant difference), p<0.05(*), p<0.01(**), p<0.001(***).


( e ) A sketch of the mouse trace in the open field test . ( f - h ) ( f ) Statistical analysis of time spent in the center area , ( g ) Statistical analysis of total mouse movement distance in 5 min , ( h ) The ratio of mice in the center area movement distance to the total distance . n for WT = 15 adult mice , n for HET = 12 adult mice . Data information : Error bars represent the means ± S . E . M . Two tailed unpaired T - tests were used to analyze the data , n . s . ( no significant difference ) , p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) , p < 0 . 001 ( * * * ) .

O O O O O O O B-ORGANISM O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O 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-ORGANISM O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) A sketch of the mouse trace in the open field test. (f-h) (f)Statistical analysis of time spent in the center area, (g) Statistical analysis of total mouse movement distance in 5 min, (h) The ratio of mice in the center area movement distance to the total distance. n for WT=15 adult mice, n for HET=12 adult mice. Data information: Error bars represent the means ± S.E.M. Two tailed unpaired T-tests were used to analyze the data, n.s. (no significant difference), p<0.05(*), p<0.01(**), p<0.001(***).


( i ) Statistical analysis of the Y maze test . S , O , and N represent the start Arm , old Arm and new Arm . The percent of time that mice stayed in each arm was recorded and analyzed ; n for WT = 11 adult mice , n for HET = 11 adult mice . Whiskers represent the minimum to maximum value ; central lines represent median values ; boxes represent the values between upper quartile and lower quartile . ( j ) Results of the elevated - plus maze . Time that mice spent in open arms ; n for WT = 8 adult mice , and n for HET = 11 adult mice . ( k ) Results of elevated - plus maze . Time that mice spent in closed arms ; n for WT = 8 adult mice , and n for HET = 11 adult mice . Data information : Error bars represent the means ± S . E . M . Two tailed unpaired T - tests were used to analyze the data , n . s . ( no significant difference ) , p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) , p < 0 . 001 ( * * * ) .

O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O 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-ORGANISM O O O O O O O O O O O B-ORGANISM O O O O O O O O B-ORGANISM O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O B-ORGANISM O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(i) Statistical analysis of the Y maze test. S, O, and N represent the start Arm, old Arm and new Arm. The percent of time that mice stayed in each arm was recorded and analyzed; n for WT=11 adult mice, n for HET=11 adult mice. Whiskers represent the minimum to maximum value; central lines represent median values; boxes represent the values between upper quartile and lower quartile. (j) Results of the elevated-plus maze. Time that mice spent in open arms; n for WT=8 adult mice, and n for HET=11 adult mice. (k) Results of elevated-plus maze. Time that mice spent in closed arms ; n for WT=8 adult mice, and n for HET=11 adult mice.Data information: Error bars represent the means ± S.E.M. Two tailed unpaired T-tests were used to analyze the data, n.s. (no significant difference), p<0.05(*), p<0.01(**), p<0.001(***).


( l ) Numbers of marbles buried in the marble burying test ; n for WT = 10 adult mice , and n for HET = 10 adult mice . Data information : Error bars represent the means ± S . E . M . Two tailed unpaired T - tests were used to analyze the data , n . s . ( no significant difference ) , p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) , p < 0 . 001 ( * * * ) .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-ORGANISM O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(l) Numbers of marbles buried in the marble burying test; n for WT=10 adult mice, and n for HET=10 adult mice. Data information: Error bars represent the means ± S.E.M. Two tailed unpaired T-tests were used to analyze the data, n.s. (no significant difference), p<0.05(*), p<0.01(**), p<0.001(***).


( m ) Diagram of the three - chamber social interaction test and mouse movement trace . HET mice did not show preference or stay longer in the stranger1 or stranger2 chambers than the WT mice did in the two test phases . ( n ) Statistical analysis of the percentage of time spent in each chamber with one empty chamber and another chamber containing stranger1 ; n for WT = 16 adult mice , n for HET = 13 adult mice . ( o ) Comparison between the time that WT and HET mice spent in stranger1 chamber . n for WT = 16 adult mice , n for HET = 13 adult mice . ( p ) Time spent in each chamber when one contains familiar mouse stranger1 , as well as new mouse stranger2 . n for WT = 16 adult mice , n for HET = 13 adult mice . ( q ) Comparison between the time of WT and HET mice staying in the stranger2 chamber . n for WT = 16 adult mice , n for HET = 13 adult mice . Data information : Error bars represent the means ± S . E . M . Two tailed unpaired T - tests were used to analyze the data , n . s . ( no significant difference ) , p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) , p < 0 . 001 ( * * * ) .

O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-ORGANISM O O O O B-ORGANISM O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O 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-ORGANISM O O O O O O O B-ORGANISM O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O B-ORGANISM O O O O O O O B-ORGANISM O O O O O O O O O O O O O B-ORGANISM O O O O O O B-ORGANISM O O O O O O O O B-ORGANISM O O O O O O O B-ORGANISM O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O B-ORGANISM O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(m) Diagram of the three-chamber social interaction test and mouse movement trace. HET mice did not show preference or stay longer in the stranger1 or stranger2 chambers than the WT mice did in the two test phases. (n) Statistical analysis of the percentage of time spent in each chamber with one empty chamber and another chamber containing stranger1; n for WT=16 adult mice, n for HET=13 adult mice. (o) Comparison between the time that WT and HET mice spent in stranger1 chamber. n for WT=16 adult mice, n for HET=13 adult mice. (p)Time spent in each chamber when one contains familiar mouse stranger1, as well as new mouse stranger2. n for WT=16 adult mice, n for HET=13 adult mice. (q) Comparison between the time of WT and HET mice staying in the stranger2 chamber. n for WT=16 adult mice, n for HET=13 adult mice. Data information: Error bars represent the means ± S.E.M. Two tailed unpaired T-tests were used to analyze the data, n.s. (no significant difference), p<0.05(*), p<0.01(**), p<0.001(***).


( c ) Heat map of significantly different genes .

(c) Heat map of significantly different genes.


( d ) TDG is significantly decreased after TCF20 deletion .

(d) TDG is significantly decreased after TCF20 deletion.


( e ) Volcano map showing that TDG is significantly decreased .

(e) Volcano map showing that TDG is significantly decreased.


( f , g ) Electroporation of GFP at E13 . 5 to E16 . 5 in TCF20 KO mice impairs cell distribution . n = 3 brains . Scalebar 25 μm Data information : Error bars represent the means ± S . E . M . Two tailed unpaired T - tests were used to analyze the data , n . s . ( no significant difference ) , p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) .

O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O B-GENEPROD O B-ORGANISM O B-EXP_ASSAY I-EXP_ASSAY O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(f, g) Electroporation of GFP at E13.5 to E16.5 in TCF20 KO mice impairs cell distribution. n=3 brains. Scalebar 25 μm Data information: Error bars represent the means ± S.E.M. Two tailed unpaired T-tests were used to analyze the data, n.s. (no significant difference), p<0.05(*), p<0.01(**).


( h ) Chip - qPCR of TCF20 binding to the TDG promoter . n = 4 biological repeats . Data information : Error bars represent the means ± S . E . M . Two tailed unpaired T - tests were used to analyze the data , n . s . ( no significant difference ) , p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) .

O O O B-EXP_ASSAY O B-EXP_ASSAY O B-GENEPROD B-EXP_ASSAY O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(h) Chip-qPCR of TCF20 binding to the TDG promoter. n= 4 biological repeats. Data information: Error bars represent the means ± S.E.M. Two tailed unpaired T-tests were used to analyze the data, n.s. (no significant difference), p<0.05(*), p<0.01(**).


( b ) TDG ChIP - qPCR revealed that TDG mainly binds to TCF - 4 CpG islands . Seven regions ( from - 5 kbp to - 200 bp ) of the promoter were analyzed . - 500 bp and - 200 bp regions were located in the CpG island of the TCF - 4 promoter . ( n = 4 biological repeats ) . Data information : Error bars represent the means ± S . E . M . Two tailed unpaired T - tests were used to analyze the data , n . s . ( no significant difference ) , p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) . and p < 0 . 001 ( * * * ) .

O O O B-GENEPROD B-EXP_ASSAY O B-EXP_ASSAY O O B-GENEPROD O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O 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-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) TDG ChIP-qPCR revealed that TDG mainly binds to TCF-4 CpG islands. Seven regions (from -5 kbp to -200 bp) of the promoter were analyzed. -500 bp and -200 bp regions were located in the CpG island of the TCF-4 promoter. (n=4 biological repeats). Data information: Error bars represent the means ± S.E.M. Two tailed unpaired T-tests were used to analyze the data, n.s. (no significant difference), p<0.05(*), p<0.01(**). and p<0.001(***).


( c - f ) MeDIP - qPCR showed that 5fC , 5caC , and 5mC are increased in the TCF - 4 promoter , while 5hmC is decreased when TCF20 is insufficient . The MeDIP assay measure the region - 500 bp from the TCF - 4 promoter . ( n = 4 biological repeats ) Data information : Error bars represent the means ± S . E . M . Two tailed unpaired T - tests were used to analyze the data , n . s . ( no significant difference ) , p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) . and p < 0 . 001 ( * * * ) .

O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-SMALL_MOLECULE O O O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(c-f) MeDIP-qPCR showed that 5fC, 5caC, and 5mC are increased in the TCF-4 promoter, while 5hmC is decreased when TCF20 is insufficient. The MeDIP assay measure the region -500 bp from the TCF-4 promoter. (n=4 biological repeats) Data information: Error bars represent the means ± S.E.M. Two tailed unpaired T-tests were used to analyze the data, n.s. (no significant difference), p<0.05(*), p<0.01(**). and p<0.001(***).


( g ) NSCs were isolated from C57B6 / J E13 . 5 embryonic brains and then were infected with a lentivirus containing a TCF20 shRNA or with a control plasmids . Immunostaining of 5fC shows that TCF20 depletion increased 5fC levels . White dashed lines show the cells infected with lentivirus . Scalebar 25 μm ( h ) Statistical analysis of 5fC fluorescence level . n = 20 cells , and P < 0 . 0001 Data information : Error bars represent the means ± S . E . M . Two tailed unpaired T - tests were used to analyze the data , n . s . ( no significant difference ) , p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) . and p < 0 . 001 ( * * * ) .

O O O B-CELL_TYPE O O O O O O O O O B-TISSUE I-TISSUE O O O O O O B-ORGANISM O O B-GENEPROD O O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O B-GENEPROD O O B-SMALL_MOLECULE B-EXP_ASSAY O O O O O O O O O B-ORGANISM O O O O O O O O O O B-SMALL_MOLECULE B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(g) NSCs were isolated from C57B6/J E13.5 embryonic brains and then were infected with a lentivirus containing a TCF20 shRNA or with a control plasmids. Immunostaining of 5fC shows that TCF20 depletion increased 5fC levels. White dashed lines show the cells infected with lentivirus. Scalebar 25 μm (h)Statistical analysis of 5fC fluorescence level. n=20 cells, and P<0.0001 Data information: Error bars represent the means ± S.E.M. Two tailed unpaired T-tests were used to analyze the data, n.s. (no significant difference), p<0.05(*), p<0.01(**). and p<0.001(***).


( i ) Immunostaining of 5caC shows that TCF20 depletion increased 5caC level . White dashed lines show the cells infected with lentivirus . Scalebar 25 μm . ( j ) Statistical analysis of the 5caC fluorescence level . n = 20 cells , and P < 0 . 0001 . Data information : Error bars represent the means ± S . E . M . Two tailed unpaired T - tests were used to analyze the data , n . s . ( no significant difference ) , p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) . and p < 0 . 001 ( * * * ) .

O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O B-GENEPROD O O B-SMALL_MOLECULE B-EXP_ASSAY O O O O O O O O O B-ORGANISM O O O O O O O O O O O O B-SMALL_MOLECULE B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(i) Immunostaining of 5caC shows that TCF20 depletion increased 5caC level. White dashed lines show the cells infected with lentivirus. Scalebar 25 μm. (j) Statistical analysis of the 5caC fluorescence level. n=20 cells, and P<0.0001. Data information: Error bars represent the means ± S.E.M. Two tailed unpaired T-tests were used to analyze the data, n.s. (no significant difference), p<0.05(*), p<0.01(**). and p<0.001(***).


( k , l ) Overexpression of TDG and TCF - 4 rescued the TCF20 knockdown phenotype in brains ; n = 3 brains . Scalebar 50 μm Data information : Error bars represent the means ± S . E . M . Two tailed unpaired T - tests were used to analyze the data , n . s . ( no significant difference ) , p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) . and p < 0 . 001 ( * * * ) .

O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O B-EXP_ASSAY O B-TISSUE O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(k, l) Overexpression of TDG and TCF-4 rescued the TCF20 knockdown phenotype in brains; n=3 brains. Scalebar 50 μm Data information: Error bars represent the means ± S.E.M. Two tailed unpaired T-tests were used to analyze the data, n.s. (no significant difference), p<0.05(*), p<0.01(**). and p<0.001(***).


B Yeast two - hybrid analysis for interaction between the above - shown Rabaptin5 segments ( Rbpt5 # , fused to LexA on the bait plasmid ) and residues 257 - 444 of FIP200 ( FIP , fused to the Gal4 activation domain on the prey plasmid ) to drive HIS3 expression . Three different clones each were replica - plated on medium with His or without His , but containing 3 - amino - 1 , 2 , 4 - triazole ( 3AT ; an inhibitor of His synthesis to increase stringency ) and grown in the absence of Trp and leucine as a control . As negative controls , empty bait or prey plasmids were used . The asterisk indicates a clone invalidated by recombination .

O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O O O O O B-GENEPROD O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O 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 Yeast two-hybrid analysis for interaction between the above-shown Rabaptin5 segments (Rbpt5#, fused to LexA on the bait plasmid) and residues 257-444 of FIP200 (FIP, fused to the Gal4 activation domain on the prey plasmid) to drive HIS3 expression. Three different clones each were replica-plated on medium with His or without His, but containing 3-amino-1,2,4-triazole (3AT; an inhibitor of His synthesis to increase stringency) and grown in the absence of Trp and leucine as a control. As negative controls, empty bait or prey plasmids were used. The asterisk indicates a clone invalidated by recombination.


D FIP200 was immunoprecipitated ( IP ) from lysates of HeLa or HEK293A cells and probed for FIP200 , Rabaptin5 ( Rbpt5 ) , and EEA1 ( early endosome antigen 1 ) by immunoblotting . Input lysate ( 10 % ) was immunoblotted blotted parallel . As a negative control , the immunoprecipitation was performed using an anti - GAPDH antibody . E - H Lysates of HeLa cells transiently transfected with full - length FIP200 - mCherry ( FIP200 - mCh ) or a deletion mutant lacking the segment interacting with Rabaptin5 ( ∆ 280 - 440 ) were immuprecipitated with anti - mCherry ( IP FIP200 - mCh ) or , as a control , with anti - FLAG antibodies ( IP FLAG ) . Immunoprecipitates and input lysates ( 10 % ) were immunoblotted for mCherry and Rabaptin5 ( E ) , ATG13 ( F ) , or ULK1 ( G ) . Co - immunoprecipitation of Rabaptin5 , ATG13 , and ULK1 with FIP200 ∆ 280 - 440 ( FIP ∆ ) was quantified in comparison to that with wild - type FIP200 ( H ; signals normalized to that of the immunoprecipitated protein ;

O B-GENEPROD O B-EXP_ASSAY O B-EXP_ASSAY O O O O B-CELL_LINE O B-CELL_LINE O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O B-EXP_ASSAY O O O O O O O O B-EXP_ASSAY O O O O O O O O O B-EXP_ASSAY O O O O O O B-GENEPROD O O O O O O O B-CELL_LINE O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O B-EXP_ASSAY B-GENEPROD O B-GENEPROD O O O O O O O O O O B-GENEPROD O O B-EXP_ASSAY B-GENEPROD O O B-EXP_ASSAY O O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O O O O O B-EXP_ASSAY O O

D FIP200 was immunoprecipitated (IP) from lysates of HeLa or HEK293A cells and probed for FIP200, Rabaptin5 (Rbpt5), and EEA1 (early endosome antigen 1) by immunoblotting. Input lysate (10%) was immunoblotted blotted parallel. As a negative control, the immunoprecipitation was performed using an anti-GAPDH antibody. E-H Lysates of HeLa cells transiently transfected with full-length FIP200-mCherry (FIP200-mCh) or a deletion mutant lacking the segment interacting with Rabaptin5 (∆280-440) were immuprecipitated with anti-mCherry (IP FIP200-mCh) or, as a control, with anti-FLAG antibodies (IP FLAG). Immunoprecipitates and input lysates (10%) were immunoblotted for mCherry and Rabaptin5 (E), ATG13 (F), or ULK1 (G). Co-immunoprecipitation of Rabaptin5, ATG13, and ULK1 with FIP200∆280-440 (FIP∆) was quantified in comparison to that with wild-type FIP200 (H; signals normalized to that of the immunoprecipitated protein;


To more easily visualize Rabaptin5 , a stable HEK293A cell line overexpressing Rabaptin5 ( HEK + Rbpt5 ) was generated . Immunofluorescence microscopy of Rabaptin5 and transferrin receptor ( TfR ) showed swelling of early endosomes upon treatment with 60 µM chloroquine for 30 min ( + CQ , panel A ) compared to untreated cells ( - CQ , A '

O O O O B-GENEPROD O O O B-CELL_LINE O O O B-GENEPROD O B-CELL_LINE O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O

To more easily visualize Rabaptin5, a stable HEK293A cell line overexpressing Rabaptin5 (HEK+Rbpt5) was generated. Immunofluorescence microscopy of Rabaptin5 and transferrin receptor (TfR) showed swelling of early endosomes upon treatment with 60 µM chloroquine for 30 min (+CQ, panel A) compared to untreated cells (-CQ, A'


HEK + Rbpt5 cells , untransfected or 24 h after transfection with mCherry - galectin3 ( mCh - Gal3 ) , mRuby3 - galectin8 ( mRuby - Gal8 ) , mCherry - FIP200 , or - ATG16L1 were analyzed upon chloroquine treatment by immunofluorescence microscopy for Rabaptin5 and mCherry - galectin3 or mRuby3 - galectin8 ( C ) , ubiquitin ( Ub ; D ) , p62 ( E ) , mCherry - FIP200 ( F ) , WIPI2 ( G ) , mCherry - ATG16L1 ( H ) , or LC3B ( I ) . Scale bar , 10 µm . In the enlarged insets , arrowheads point out chloroquine - induced enlarged , ring - like early endosomes . Rabaptin5 - positive enlarged endosomes positive for mCherry - galectin3 ( Gal3 ) or mRuby3 - galectin8 ( Gal8 ) were quantified ( C ' ; mean ± S . D .

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HEK+Rbpt5 cells, untransfected or 24 h after transfection with mCherry-galectin3 (mCh-Gal3), mRuby3-galectin8 (mRuby-Gal8), mCherry-FIP200, or -ATG16L1 were analyzed upon chloroquine treatment by immunofluorescence microscopy for Rabaptin5 and mCherry-galectin3 or mRuby3-galectin8 (C), ubiquitin (Ub; D), p62 (E), mCherry-FIP200 (F), WIPI2 (G), mCherry-ATG16L1 (H), or LC3B (I). Scale bar, 10 µm. In the enlarged insets, arrowheads point out chloroquine-induced enlarged, ring-like early endosomes. Rabaptin5-positive enlarged endosomes positive for mCherry-galectin3 (Gal3) or mRuby3-galectin8 (Gal8) were quantified (C'; mean ± S.D.


J , K HEK + Rbpt5 cells , untransfected or 24 h after transfection with mCherry - ATG16L1 , were treated with 60 µM chloroquine for 0 , 15 and 30 min and stained for Rabaptin5 and either WIPI2 or mCherry - ATG16L1 . Manders ' colocalization coefficients were determined , M1 showing the fraction of Rabaptin5 - positive structures also positive for WIPI2 ( J ) or mCherry - ATG16L1 ( K ) , and M2 showing the respective inverse ( mean ± S . D . of three independent experiments ;

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J, K HEK+Rbpt5 cells, untransfected or 24 h after transfection with mCherry-ATG16L1, were treated with 60 µM chloroquine for 0, 15 and 30 min and stained for Rabaptin5 and either WIPI2 or mCherry-ATG16L1. Manders' colocalization coefficients were determined, M1 showing the fraction of Rabaptin5-positive structures also positive for WIPI2 (J) or mCherry-ATG16L1 (K), and M2 showing the respective inverse (mean ± S.D. of three independent experiments;


A HEK293A cells were transfected with nontargeting siRNA ( siCtr ) or siRNAs silencing Rabaptin5 ( siRbpt5 ) or FIP200 ( siFIP200 ) for 72 h and treated without ( - ) or with 60 µM chloroquine ( + CQ ) or 250 nM Torin1 for 150 min . Scale bar , 10 µm . Below , the efficiency of Rabaptin5 and FIP200 knockdown was assayed by immunoblotting using tubulin ( Tub ) as a loading control . B WIPI2 or LC3B puncta per cell were quantified for each condition ( mean ± S . D . of three independent experiments ; ANOVA : * P < 0 . 05 , * * P < 0 . 01 ) .

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A HEK293A cells were transfected with nontargeting siRNA (siCtr) or siRNAs silencing Rabaptin5 (siRbpt5) or FIP200 (siFIP200) for 72 h and treated without (-) or with 60 µM chloroquine (+CQ) or 250 nM Torin1 for 150 min. Scale bar, 10 µm. Below, the efficiency of Rabaptin5 and FIP200 knockdown was assayed by immunoblotting using tubulin (Tub) as a loading control. B WIPI2 or LC3B puncta per cell were quantified for each condition (mean ± S.D. of three independent experiments; ANOVA: *P<0.05, **P<0.01). C


C HEK293A cells were transfected with siCtr or siRbpt5 as in A and incubated without or with 280 µM LLOMe for 150 min to induce lysophagy . Cells were fixed and immunostained for endogenous WIPI2 and LC3B . Scale bar , 10 µm . D WIPI2 or LC3B puncta per cell were quantified ( mean ± S . D . of three independent experiments ) .

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C HEK293A cells were transfected with siCtr or siRbpt5 as in A and incubated without or with 280 µM LLOMe for 150 min to induce lysophagy. Cells were fixed and immunostained for endogenous WIPI2 and LC3B. Scale bar, 10 µm. D WIPI2 or LC3B puncta per cell were quantified (mean ± S.D. of three independent experiments).


A HEK293A cells were transfected with nontargeting siRNA ( siCtr ) or siRNAs silencing ATG13 ( siATG13 ) for 72 h , treated without ( - ) or with 60 µM chloroquine ( + CQ ) for 150 minutes , and fixed and immunostained for endogenous WIPI2 and LC3B . Scale bar , 10 µm . Efficiency of ATG13 knockdown was assayed by immunoblotting below on the right using GAPDH as loading controls . B WIPI2 of LC3B puncta per cell were quantified for each condition ( mean ± S . D . of three independent experiments ; ANOVA : * * P < 0 . 01 ) .

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A HEK293A cells were transfected with nontargeting siRNA (siCtr) or siRNAs silencing ATG13 (siATG13) for 72 h, treated without (-) or with 60 µM chloroquine (+CQ) for 150 minutes, and fixed and immunostained for endogenous WIPI2 and LC3B. Scale bar, 10 µm. Efficiency of ATG13 knockdown was assayed by immunoblotting below on the right using GAPDH as loading controls. B WIPI2 of LC3B puncta per cell were quantified for each condition (mean ± S.D. of three independent experiments; ANOVA: **P<0.01). C


HEK293A cells were transfected with nontargeting siRNA ( siCtr ) or siRNAs silencing ULK1 ( siULK1 ) , ULK2 ( siULK2 ) , or both ( siULK1 + 2 ) for 72 h , treated without ( - ) or with 60 µM chloroquine ( + CQ ) for 150 minutes , and fixed and immunostained for endogenous WIPI2 and LC3B . Scale bar , 10 µm . Efficiency of ULK1 knockdown was assayed by immunoblotting The efficiency of ULK2 knockdown was assayed by quantitative RT - PCR and the result is shown in panel C D WIPI2 of LC3B puncta per cell were quantified for each condition ( mean ± S . D . of three independent

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HEK293A cells were transfected with nontargeting siRNA (siCtr) or siRNAs silencing ULK1 (siULK1), ULK2 (siULK2), or both (siULK1+2) for 72 h, treated without (-) or with 60 µM chloroquine (+CQ) for 150 minutes, and fixed and immunostained for endogenous WIPI2 and LC3B. Scale bar, 10 µm. Efficiency of ULK1 knockdown was assayed by immunoblotting The efficiency of ULK2 knockdown was assayed by quantitative RT-PCR and the result is shown in panel C D WIPI2 of LC3B puncta per cell were quantified for each condition (mean ± S.D. of three independent experiments


E HEK293A cells were incubated without ( - ) , with 60 µM chloroquine ( + CQ ) , or with chloroquine and 5 µM MRT68921 ( + CQ + MRT ) for 150 min and fixed and stained for WIPI2 and LC3B . F WIPI2 of LC3B puncta per cell were quantified for each condition as in panel B ( mean ± S . D . of three independent experiments ) .

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E HEK293A cells were incubated without (-), with 60 µM chloroquine (+CQ), or with chloroquine and 5 µM MRT68921 (+CQ +MRT) for 150 min and fixed and stained for WIPI2 and LC3B. F WIPI2 of LC3B puncta per cell were quantified for each condition as in panel B (mean ± S.D. of three independent experiments).


B Co - immunoprecipitation was performed as in panel A using parental HEK293A cells and CRISPR / Cas9 knockout cells lacking FIP200 ( FIP - KO ) . Anti - HA antibodies were used as a control ( IP HA ) . On the right , HEK293A and FIP200 knockout cells were immunoblotted for FIP200 and as a loading control of tubulin ( Tub ) . Signals were quantified and the ratios of mCherry - ATG16L1 / Rabaptin5 normalized to that of HEK293A cells without chloroquine

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B Co-immunoprecipitation was performed as in panel A using parental HEK293A cells and CRISPR/Cas9 knockout cells lacking FIP200 (FIP-KO). Anti-HA antibodies were used as a control (IP HA). On the right, HEK293A and FIP200 knockout cells were immunoblotted for FIP200 and as a loading control of tubulin (Tub). Signals were quantified and the ratios of mCherry-ATG16L1/Rabaptin5 normalized to that of HEK293A cells without chloroquine treatment


Lysates of HEK293A or HeLa cells transiently transfected with full - length mCherry - ATG16L1 ( wt ) or a mutant lacking the WD domain ( ∆ WD ; residues 1 - 319 of ATG16L1 , precisely deleting only the WD40 repeats residues 320 - 607 ) were immunoprecipitated with anti - Rabaptin5 or anti - FLAG antibodies , and immunoblotted for Rabaptin5 and mCherry - ATG16L1 , or immunoprecipitated with anti - FIP200 or anti - HA antibodies Co - immunoprecipitation of ATG16L1 ∆ WD with Rabaptin5 ( C ) was reduced to 6 . 6 ± 2 . 1 % and 3 . 4 ± 2 . 1 % in HEK293A and HeLa cells , respectively , relative to that of full - length ATG16L1 ( signals normalized to the immunoprecipitated protein ; mean ± S . D . deviation of three independent experiments each ) .

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Lysates of HEK293A or HeLa cells transiently transfected with full-length mCherry-ATG16L1 (wt) or a mutant lacking the WD domain (∆WD; residues 1-319 of ATG16L1, precisely deleting only the WD40 repeats residues 320-607) were immunoprecipitated with anti-Rabaptin5 or anti-FLAG antibodies, and immunoblotted for Rabaptin5 and mCherry-ATG16L1 , or immunoprecipitated with anti-FIP200 or anti-HA antibodies Co-immunoprecipitation of ATG16L1∆WD with Rabaptin5 (C) was reduced to 6.6±2.1% and 3.4±2.1% in HEK293A and HeLa cells, respectively, relative to that of full-length ATG16L1 (signals normalized to the immunoprecipitated protein; mean ± S.D. deviation of three independent experiments each).


F Lysates of HeLa cells transiently transfected with myc - tagged wild - type Rabaptin5 ( wt ) or triple - alanine mutant ( AAA ) were immunoprecipitated with anti - myc ( IP myc ) or anti - FLAG antibodies ( IP FLAG ) , and immunoblotted for myc and ATG16L1 . Co - immunoprecipitation of ATG16L1 with Rabaptin5 - AAA triple mutant was reduced to 1 . 5 ± 1 . 2 % relative to that with wild - type Rabaptin5 ( signals normalized to that of the immunoprecipitated protein ; mean ± S . D . of three independent experiments ) .

O O O B-CELL_LINE O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O B-EXP_ASSAY B-GENEPROD O O O O B-GENEPROD O O B-EXP_ASSAY B-GENEPROD O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O

F Lysates of HeLa cells transiently transfected with myc-tagged wild-type Rabaptin5 (wt) or triple-alanine mutant (AAA) were immunoprecipitated with anti-myc (IP myc) or anti-FLAG antibodies (IP FLAG), and immunoblotted for myc and ATG16L1. Co-immunoprecipitation of ATG16L1 with Rabaptin5-AAA triple mutant was reduced to 1.5±1.2% relative to that with wild-type Rabaptin5 (signals normalized to that of the immunoprecipitated protein; mean ± S.D. of three independent experiments).


A By immunoblot analysis , the levels of Rabaptin5 and as a loading control of tubulin ( Tub ) were assessed in wild - type HEK293A cells , HEK + Rbpt5 cells stably overexpressing Rabaptin5 , and Rabaptin5 - knockout cells without ( Rbpt5 - KO ) or with stable re - expression of wild - type ( Rbpt5 - KO + wt ) or AAA - mutant Rabaptin5 ( Rbpt5 - KO + AAA ) . B The same stable HEK293A - derived cell lines were treated without ( - CQ ) or with 60 µM chloroquine for 150 min ( + CQ ) and analyzed by immunofluorescence microscopy for WIPI2 or LC3B . Scale bar , 10 µm .

O O B-EXP_ASSAY O O O O O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-CELL_LINE O O B-CELL_LINE O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-CELL_LINE O O O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O

A By immunoblot analysis, the levels of Rabaptin5 and as a loading control of tubulin (Tub) were assessed in wild-type HEK293A cells, HEK+Rbpt5 cells stably overexpressing Rabaptin5, and Rabaptin5-knockout cells without (Rbpt5-KO) or with stable re-expression of wild-type (Rbpt5-KO+wt) or AAA-mutant Rabaptin5 (Rbpt5-KO+AAA). B The same stable HEK293A-derived cell lines were treated without (-CQ) or with 60 µM chloroquine for 150 min (+CQ) and analyzed by immunofluorescence microscopy for WIPI2 or LC3B. Scale bar, 10 µm. C


D , E The HEK293A - derived cell lines were treated with 60 µM chloroquine for 30min and non - lipidated and lipidated LC3B ( I and II , resp . ) were assayed by immunoblot analysis ( D ) . The increase of the LC3B - II fraction of total LC3B upon chloroquine treatment was quantified ( mean ± S . D . of three independent experiments ;

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D, E The HEK293A-derived cell lines were treated with 60 µM chloroquine for 30min and non-lipidated and lipidated LC3B (I and II, resp.) were assayed by immunoblot analysis (D). The increase of the LC3B-II fraction of total LC3B upon chloroquine treatment was quantified (mean ± S.D. of three independent experiments;


F , G HEK + Rbpt5 cells and Rabaptin5 - knockout cells stably re - expressing wild - type ( Rbpt5 - KO + wt ) or AAA - mutant Rabaptin5 ( Rbpt5 - KO + AAA ) were transfected with mCherry - ATG16L1 , treated with 60 µM chloroquine ( CQ ) for 0 , 15 and 30 min , and analyzed by immunofluorescence microscopy for Rabaptin5 , mCherry - ATG16L1 , and WIPI2 . Manders ' colocalization coefficients were determined , M1 showing the fraction of Rabaptin5 - positive structures also positive for mCherry - ATG16L1 ( F ) or WIPI2 ( G ) and M2 showing the inverse ( mean ± S . D . of three independent

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F, G HEK+Rbpt5 cells and Rabaptin5-knockout cells stably re-expressing wild-type (Rbpt5-KO+wt) or AAA-mutant Rabaptin5 (Rbpt5-KO+AAA) were transfected with mCherry-ATG16L1, treated with 60 µM chloroquine (CQ) for 0, 15 and 30 min, and analyzed by immunofluorescence microscopy for Rabaptin5, mCherry-ATG16L1, and WIPI2. Manders' colocalization coefficients were determined, M1 showing the fraction of Rabaptin5-positive structures also positive for mCherry-ATG16L1 (F) or WIPI2 (G) and M2 showing the inverse (mean ± S.D. of three independent experiments


H Wild - type HEK293A cells , HEK + Rbpt5 cells , Rabaptin5 - knockout cells without ( Rbpt5 - KO ) or with stable re - expression of wild - type ( Rbpt5 - KO + wt ) or AAA - mutant Rabaptin5 ( Rbpt5 - KO + AAA ) were stained with lysotracker and with DAPI for nuclei . Scale bar , 10 µm . I The number of lysosomes ( lysotracker - positive structures ) per cell was quantified from cells as in panel C ( mean ± S . D . of three independent experiments ) . Images for at least 15 cells per sample were quantified . The value for HEK + Rbpt5 cells is an underestimate , because the density of puncta makes them difficult to distinguish as separate structures .

O O O O B-CELL_LINE O O B-CELL_LINE O B-GENEPROD O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SUBCELLULAR O O O O O O O O O O O B-SUBCELLULAR O B-GENEPROD O O O O O O O O O O O O O O O O O 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-CELL_LINE O B-GENEPROD O O O O O O O O O O O O O O O O O O O

H Wild-type HEK293A cells, HEK+Rbpt5 cells, Rabaptin5-knockout cells without (Rbpt5-KO) or with stable re-expression of wild-type (Rbpt5-KO+wt) or AAA-mutant Rabaptin5 (Rbpt5-KO+AAA) were stained with lysotracker and with DAPI for nuclei. Scale bar, 10 µm. I The number of lysosomes (lysotracker-positive structures) per cell was quantified from cells as in panel C (mean ± S.D. of three independent experiments). Images for at least 15 cells per sample were quantified. The value for HEK+Rbpt5 cells is an underestimate, because the density of puncta makes them difficult to distinguish as separate structures.


A HeLa cells were transfected with nontargeting siRNA ( siCtr ) or siRNAs silencing Rabaptin5 ( siRbpt5 ) or FIP200 ( siFIP200 ) for 72 h . The cells were infected with Salmonella by centrifugation at 500 × g for 5min at 37 ° C and incubation for 10 min at 37 ° C , washed three times , and incubated in fresh culture medium containing gentamicin to prevent growth of extracellular bacteria for 0 , 1 , 3 , or 6 h before lysis of the host cells and plating of the bacteria on LB agar plates at various dilutions to determine the number of live bacteria at the different time points , shown as a percentage of internalized cells after infection ( mean ± S . D . of three independent experiments ) . The fractions of internalized bacteria alive 1 h after infection are shown separately in the middle ( mean ± S . D . of three independent experiments ; ANOVA : * P < 0 . 05 ) . On the right , the fraction of infected cells was determined for HeLa cells transfected with siRNAs and infected as above with Salmonella expressing GFP , washed , and immediately fixed for fluorescence microscopy and stained with anti - transferrin receptor and anti - LC3B as cellular markers . Z - stacks for > 5 ' 000cells / sample were acquired and analyzed in Fiji to determine the fraction of infected cells ( mean ± S . D . of three independent experiments ) . The average number of bacteria per infected cell was identical ( 2 . 16 , 2 . 11 , and 2 . 12 bacteria per cell transfected with siCtr , siRbpt5 , and siFIP200 , respectively ) .

O B-CELL_LINE O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O B-ORGANISM O O O O O O O O O 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-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CELL_LINE O O O O O O O O O B-ORGANISM O B-GENEPROD O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O B-GENEPROD I-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A HeLa cells were transfected with nontargeting siRNA (siCtr) or siRNAs silencing Rabaptin5 (siRbpt5) or FIP200 (siFIP200) for 72 h. The cells were infected with Salmonella by centrifugation at 500×g for 5min at 37°C and incubation for 10 min at 37°C, washed three times, and incubated in fresh culture medium containing gentamicin to prevent growth of extracellular bacteria for 0, 1, 3, or 6 h before lysis of the host cells and plating of the bacteria on LB agar plates at various dilutions to determine the number of live bacteria at the different time points, shown as a percentage of internalized cells after infection (mean ± S.D. of three independent experiments). The fractions of internalized bacteria alive 1 h after infection are shown separately in the middle (mean ± S.D. of three independent experiments; ANOVA: *P<0.05). On the right, the fraction of infected cells was determined for HeLa cells transfected with siRNAs and infected as above with Salmonella expressing GFP, washed, and immediately fixed for fluorescence microscopy and stained with anti-transferrin receptor and anti-LC3B as cellular markers. Z-stacks for >5'000cells/sample were acquired and analyzed in Fiji to determine the fraction of infected cells (mean ± S.D. of three independent experiments). The average number of bacteria per infected cell was identical (2.16, 2.11, and 2.12 bacteria per cell transfected with siCtr, siRbpt5, and siFIP200, respectively).


B Wild - type HEK293A , HEK + Rbpt5 , Rbpt5 - KO , Rbpt5 - KO + wt , Rbpt5 - KO + AAA , and FIP200 - KO cells were infected with Salmonella and treated and analyzed as in panels A ( mean ± S . D . of three independent experiments ; ANOVA : * P < 0 . 05 , * * * * P < 0 . 0001 ) .

O O O O B-CELL_LINE O B-CELL_LINE O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O 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 Wild-type HEK293A, HEK+Rbpt5, Rbpt5-KO, Rbpt5-KO+wt, Rbpt5-KO+AAA, and FIP200-KO cells were infected with Salmonella and treated and analyzed as in panels A (mean ± S.D. of three independent experiments; ANOVA: *P<0.05, ****P<0.0001).


C Wild - type HEK293A , HEK + Rbpt5 , Rbpt5 - KO , Rbpt5 - KO + AAA , and FIP200 - KO cells were infected with Salmonella expressing GFP as in panel B , incubated in fresh culture medium containing gentamicin for 0 , 5 , 15 , 30 , and 60 min , fixed with methanol and immunostained for transferrin receptor ( TfR ) as a marker of early endosomes and for LC3B as a marker of autophagy . Salmonella were classified according to their association with a TfR - and / or LC3B - positive compartment - as illustrated on the top left ( scale bar , 2 µm ) - during the first hour after infection . In the absence of Rabaptin5 , LC3 - positive SCVs with early endosomal characteristics ( containing TfR ) were strongly reduced . ( Mean ± S . D . of three independent experiments , analyzing > 50 bacteria for each time point . )

O O O O B-CELL_LINE O B-CELL_LINE O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O B-ORGANISM O B-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-EXP_ASSAY O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O B-SUBCELLULAR I-SUBCELLULAR O O B-GENEPROD O O O O B-EXP_ASSAY O B-ORGANISM O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O 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-GENEPROD O B-GENEPROD O O B-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR O O O B-GENEPROD O O O 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 Wild-type HEK293A, HEK+Rbpt5, Rbpt5-KO, Rbpt5-KO+AAA, and FIP200-KO cells were infected with Salmonella expressing GFP as in panel B, incubated in fresh culture medium containing gentamicin for 0, 5, 15, 30, and 60 min, fixed with methanol and immunostained for transferrin receptor (TfR) as a marker of early endosomes and for LC3B as a marker of autophagy. Salmonella were classified according to their association with a TfR- and/or LC3B-positive compartment - as illustrated on the top left (scale bar, 2 µm) - during the first hour after infection. In the absence of Rabaptin5, LC3-positive SCVs with early endosomal characteristics (containing TfR) were strongly reduced. (Mean ± S.D. of three independent experiments, analyzing >50 bacteria for each time point.)


B : Consequences of ROS on the SUMO proteome vary amongst cell lines . HeLa , MCF7 , U2OS or RPE cells were treated with increasing H2O2 concentration for 15 min and lysed in Laemmli buffer supplemented with 20 mM N - Ethylmaleimide ( NEM ) . Samples were analyzed by immunoblotting with SUMO1 antibodies .

O O O O B-SMALL_MOLECULE O O O O O O O O O B-CELL_LINE O B-CELL_LINE O B-CELL_LINE O B-CELL_LINE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O B-EXP_ASSAY O B-GENEPROD O O

B: Consequences of ROS on the SUMO proteome vary amongst cell lines. HeLa, MCF7, U2OS or RPE cells were treated with increasing H2O2 concentration for 15 min and lysed in Laemmli buffer supplemented with 20 mM N-Ethylmaleimide (NEM). Samples were analyzed by immunoblotting with SUMO1 antibodies.