RNAseq data were subjected to gene set enrichment analysis ( GSEA ) . Shown are enrichment plots for the indicated set of p53 downstream genes in pairwise comparisons of Nutlin - treated MEFs with the indicated p53 genotypes .

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RNAseq data were subjected to gene set enrichment analysis (GSEA). Shown are enrichment plots for the indicated set of p53 downstream genes in pairwise comparisons of Nutlin-treated MEFs with the indicated p53 genotypes.


Reverse transcription quantitative PCR ( RTqPCR ) analysis of p53 target genes in MEFs treated for 24 hours with 1 µg / ml doxorubicin ( Doxo ) . Shown are expression values normalized to β - actin as mean ± SD ( n = 6 ) .

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Reverse transcription quantitative PCR (RTqPCR) analysis of p53 target genes in MEFs treated for 24 hours with 1 µg/ml doxorubicin (Doxo). Shown are expression values normalized to β-actin as mean ± SD (n=6).


Western blots of protein lysates prepared from MEFs treated for 24 hours with ( G ) 10 µM

Western blots of protein lysates prepared from MEFs treated for 24 hours with (G) 10 µM Nutlin


Western blots of protein lysates prepared from MEFs treated for 24 hours with 0 . 4 µg / ml Doxo .

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Western blots of protein lysates prepared from MEFs treated for 24 hours with 0.4 µg/ml Doxo.


Proliferation of primary MEFs . Cells were treated o / n with 0 . 2 µg / ml doxorubicin ( Doxo ) and pulse - labeled with 32 µM 5 - Bromo - 2 - deoxyuridine ( BrdU ) , fixed and processed for flow cytometry analysis . n = 4 .

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Proliferation of primary MEFs. Cells were treated o/n with 0.2 µg/ml doxorubicin (Doxo) and pulse-labeled with 32 µM 5-Bromo-2-deoxyuridine (BrdU), fixed and processed for flow cytometry analysis. n=4.


Long - term proliferation assay with primary MEFs of indicated genotypes . + / + and - / - : n = 3 ; EE / EE : n = 6 .

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Long-term proliferation assay with primary MEFs of indicated genotypes. +/+ and -/-: n=3; EE/EE: n=6.


MEFs were immortalized with the adenoviral oncogene E1A . 12S ( E1A MEF ) and treated with 0 . 4 µg / ml Doxo for 17 hours . Apoptosis ( Annexin V ) was quantified by flow cytometry . + / + and - / - : n = 3 ; EE / EE : n = 6 . Western blots show expression of E1A and β - actin as loading control .

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MEFs were immortalized with the adenoviral oncogene E1A.12S (E1A MEF) and treated with 0.4 µg/ml Doxo for 17 hours. Apoptosis (Annexin V) was quantified by flow cytometry. +/+ and -/-: n=3; EE/EE: n=6. Western blots show expression of E1A and β-actin as loading control.


Primary thymocytes were irradiated ex vivo with 6 Gy or treated with 1 µM dexamethasone as a control for p53 - independent apoptosis . Cell survival relative to untreated samples was analyzed using CellTiter Glow assay ( Promega ) . n = 11 for each time point and genotype .

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Primary thymocytes were irradiated ex vivo with 6 Gy or treated with 1 µM dexamethasone as a control for p53-independent apoptosis. Cell survival relative to untreated samples was analyzed using CellTiter Glow assay (Promega). n=11 for each time point and genotype.


mRNA expression analysis ( RTqPCR ) of p53 target genes in thymocytes 6 hours after 6 Gy irradiation . Shown are expression values normalized to β - actin .

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mRNA expression analysis (RTqPCR) of p53 target genes in thymocytes 6 hours after 6 Gy irradiation. Shown are expression values normalized to β-actin.


Mice of indicated genotype were subjected to 6 Gy whole - body irradiation and pulse - labeled with 120 mg / kg BrdU two hours before sacrifice at different time points . Small intestines were stained for ( F ) apoptosis ( TUNEL )

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Mice of indicated genotype were subjected to 6 Gy whole-body irradiation and pulse-labeled with 120 mg/kg BrdU two hours before sacrifice at different time points. Small intestines were stained for (F) apoptosis (TUNEL)


Mice of indicated genotype were subjected to 6 Gy whole - body irradiation and pulse - labeled with 120 mg / kg BrdU two hours before sacrifice at different time points . Small intestines were stained for proliferation ( BrdU ) ; scale bars 50 µm .

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Mice of indicated genotype were subjected to 6 Gy whole-body irradiation and pulse-labeled with 120 mg/kg BrdU two hours before sacrifice at different time points. Small intestines were stained for proliferation (BrdU); scale bars 50 µm.


Mice of indicated genotype were subjected to 6 Gy whole - body irradiation and pulse - labeled with 120 mg / kg BrdU two hours before sacrifice at different time points . Small intestines were stained for ( F ) apoptosis ( TUNEL ) Quantification for n = 3 mice / genotype ( 150 crypts / mouse ) .

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Mice of indicated genotype were subjected to 6 Gy whole-body irradiation and pulse-labeled with 120 mg/kg BrdU two hours before sacrifice at different time points. Small intestines were stained for (F) apoptosis (TUNEL) Quantification for n=3 mice/genotype (150 crypts/mouse).


Mice of indicated genotype were subjected to 6 Gy whole - body irradiation and pulse - labeled with 120 mg / kg BrdU two hours before sacrifice at different time points . Small intestines were stained for proliferation ( BrdU ) ; scale bars 50 µm . Quantification for n = 3 mice / genotype ( 150 crypts / mouse ) .

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Mice of indicated genotype were subjected to 6 Gy whole-body irradiation and pulse-labeled with 120 mg/kg BrdU two hours before sacrifice at different time points. Small intestines were stained for proliferation (BrdU); scale bars 50 µm. Quantification for n=3 mice/genotype (150 crypts/mouse).


Observed and expected genotype distribution of newborn offspring from mating Mdm2 + / - Trp53EE / + mice ( total number of pups n = 185 ; contingency test P = 0 . 0029 ) . Observed and expected genotype distribution of newborn offspring from mating Mdm2 + / - Trp53 + / - mice ( total number of pups n = 435 ; contingency test P = 0 . 912 ) .

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Observed and expected genotype distribution of newborn offspring from mating Mdm2+/-Trp53EE/+ mice (total number of pups n=185; contingency test P=0.0029). Observed and expected genotype distribution of newborn offspring from mating Mdm2+/-Trp53+/- mice (total number of pups n=435; contingency test P=0.912).


Representative picture of an E9 . 5 Mdm2 - / - Trp53EE / EE embryo ( right ) displays characteristic phenotypic abnormalities compared to an Mdm2 + / - ; Trp53EE / EE embryo ( left ) .

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Representative picture of an E9.5 Mdm2-/-Trp53EE/EE embryo (right) displays characteristic phenotypic abnormalities compared to an Mdm2+/-;Trp53EE/EE embryo (left).


IHC for p53 and cleaved caspase - 3 shows strong accumulation of p53EE protein and massive apoptosis in Mdm2 - / - Trp53EE / EE compared to Mdm2 + / - ; Trp53EE / EE control embryos .

IHC for p53 and cleaved caspase-3 shows strong accumulation of p53EE protein and massive apoptosis in Mdm2-/-Trp53EE/EE compared to Mdm2+/-;Trp53EE/EE control embryos.


Mdm2 - / - Trp53 - / - ( double knock - out , DKO ) MEFs were transduced with pInducer20 - p53EE lentivirus to enable tetracyclin ( Tet ) - inducible expression of mouse p53EE . Induction of p53EE sensitized cells to apoptosis induced by 24 hour treatment with 1 µg / ml doxorubicin ( Doxo ) as detected by flow cytometry for Annexin V .

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Mdm2-/-Trp53-/- (double knock-out, DKO) MEFs were transduced with pInducer20-p53EE lentivirus to enable tetracyclin (Tet)-inducible expression of mouse p53EE. Induction of p53EE sensitized cells to apoptosis induced by 24 hour treatment with 1 µg/ml doxorubicin (Doxo) as detected by flow cytometry for Annexin V.


mRNA expression of p53 target genes was measured in cells from ( A ) relative to β - actin by RTqPCR following treatment with Tet ± 1 µg / ml Doxo . Expression of the pro - apoptotic p53 target genes Bbc3 ( Puma ) and Bax is not significantly induced . n = 6 .

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mRNA expression of p53 target genes was measured in cells from (A) relative to β-actin by RTqPCR following treatment with Tet ± 1 µg/ml Doxo. Expression of the pro-apoptotic p53 target genes Bbc3 (Puma) and Bax is not significantly induced. n=6.


Proliferation of MEFs with indicated genotypes was analyzed by live - cell imaging in the presence of 10 µM nutlin - 3a ( Nutlin ) and / or 0 . 05 µg / ml doxorubicin ( Doxo ) . Shown is the median confluence ± SEM ( n = 12 ) .

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Proliferation of MEFs with indicated genotypes was analyzed by live-cell imaging in the presence of 10 µM nutlin-3a (Nutlin) and/or 0.05 µg/ml doxorubicin (Doxo). Shown is the median confluence ±SEM (n=12).


Cell viability assays for MEFs with indicated genotypes treated with Mdm2 inhibitors ± 0 . 4 µg / ml Doxo . n = 3 .

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Cell viability assays for MEFs with indicated genotypes treated with Mdm2 inhibitors ± 0.4 µg/ml Doxo. n=3.


Western blot of E1A - MEFs with indicated genotypes treated with 10 µM Nutlin ± 0 . 4 µg / ml Doxo for 18 hours reveals induction of apoptosis ( cCasp - 3 , cleaved caspase - 3 ; cParp , cleaved Parp ) in double - treated p53EE / EE MEFs in the absence of p53 target gene ( p21 , Mdm2 ) activation . * , 10 hours 0 . 4 µg / ml Doxo ; * * , 10 hours 0 . 2 µg / ml Doxo .

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Western blot of E1A-MEFs with indicated genotypes treated with 10 µM Nutlin ± 0.4 µg/ml Doxo for 18 hours reveals induction of apoptosis (cCasp-3, cleaved caspase-3; cParp, cleaved Parp) in double-treated p53EE/EE MEFs in the absence of p53 target gene (p21, Mdm2) activation. *, 10 hours 0.4 µg/ml Doxo; **, 10 hours 0.2 µg/ml Doxo.


mRNA expression of p53 target genes was measured in E1A MEFs of indicated genotype relative to β - actin by RTqPCR following combined treatment with 10 µM Nutlin and 1 µg / ml Doxo . n = 12 .

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mRNA expression of p53 target genes was measured in E1A MEFs of indicated genotype relative to β-actin by RTqPCR following combined treatment with 10 µM Nutlin and 1 µg/ml Doxo. n=12.


Western blot of p53EE / EE E1A - MEFs with indicated genotypes treated for 18 hours with 10 µM Nutlin , 0 . 4 µg / ml Doxo and 30 - 100 - 500 nM Ganetespib as indicated . Comb , combined treatment with 3 drugs for 18 hours . Pre , pre - treatment with ganetespib for 24 hours followed by combined treatment with Nutlin and Doxorubicin for 18 hours .

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Western blot of p53EE/EE E1A-MEFs with indicated genotypes treated for 18 hours with 10 µM Nutlin, 0.4 µg/ml Doxo and 30-100-500 nM Ganetespib as indicated. Comb, combined treatment with 3 drugs for 18 hours. Pre, pre-treatment with ganetespib for 24 hours followed by combined treatment with Nutlin and Doxorubicin for 18 hours.


Western blot of cellular fractions from unstressed and Doxo - treated MEFs of indicated genotype identifies p53EE protein in the mitochondrial fraction . PCNA , Tom20 / Bak are shown as nuclear and mitochondrial marker proteins .

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Western blot of cellular fractions from unstressed and Doxo-treated MEFs of indicated genotype identifies p53EE protein in the mitochondrial fraction. PCNA, Tom20/Bak are shown as nuclear and mitochondrial marker proteins.


Cell death measured by flow cytometry using propidium iodide exclusion in human lung adenocarcinoma H1299 cells with tet - inducible expression of human p53EE . Cells with overexpression of human p53EE mutant display a trend of increased cell death after treatment with 0 . 5 µg / ml Doxo .

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Cell death measured by flow cytometry using propidium iodide exclusion in human lung adenocarcinoma H1299 cells with tet-inducible expression of human p53EE. Cells with overexpression of human p53EE mutant display a trend of increased cell death after treatment with 0.5 µg/ml Doxo.


Cell death measured by flow cytometry using propidium iodide exclusion in human lung adenocarcinoma H1299 cells with tet - inducible expression of human p53EE . Cells with overexpression of human p53EE mutant display a trend of increased cell death after treatment with 0 . 5 µg / ml Doxo . This effect is enhanced by addition of Nutlin and blocked by the pan - caspase inhibitor QVDOph , but not the ferroptosis inhibitor Ferrostatin - 1 ( Ferr ) as control . ( A ) n = 3 ; ( B ) n = 2 .

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Cell death measured by flow cytometry using propidium iodide exclusion in human lung adenocarcinoma H1299 cells with tet-inducible expression of human p53EE. Cells with overexpression of human p53EE mutant display a trend of increased cell death after treatment with 0.5 µg/ml Doxo. This effect is enhanced by addition of Nutlin and blocked by the pan-caspase inhibitor QVDOph, but not the ferroptosis inhibitor Ferrostatin-1 (Ferr) as control. (A) n=3; (B) n=2.


Apoptosis analysis using Annexin V staining in cells from ( A ) . n = 3 .

Apoptosis analysis using Annexin V staining in cells from (A). n=3.


mRNA expression analysis ( RTqPCR ) of p53 target genes following treatment with 0 . 5 µg / ml Doxo ± 10 µM Nutlin . KO , tet - induced H1299 - pInd cells ; EE , tet - induced H1299 - pInd - p53EE cells ; WT , H460 cells . mRNA expression was normalized to β - actin ; n = 3 .

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mRNA expression analysis (RTqPCR) of p53 target genes following treatment with 0.5 µg/ml Doxo ± 10 µM Nutlin. KO, tet-induced H1299-pInd cells; EE, tet-induced H1299-pInd-p53EE cells; WT, H460 cells. mRNA expression was normalized to β-actin; n=3.


Proximity ligation assay for p53 with Tom20 in indicated untreated ( Mock ) or Doxo - treated ( 0 . 1 µg / ml , 24 hours ) H1299 cells following tet - induction . Shown are representative cells with PLA signals ( red ) counterstained with DAPI ( blue ) . Quantification of proximity ligation assays for p53EE with Tom20 , Bcl - 2 , Bcl - xL and Bak in H1299 cells . Doxo treatment ( 0 . 1 µg / ml , 24 hours ) as indicated . Plotted is the average number of PLA signals per cell for 10 fields of view .

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Proximity ligation assay for p53 with Tom20 in indicated untreated (Mock) or Doxo-treated (0.1 µg/ml, 24 hours) H1299 cells following tet-induction. Shown are representative cells with PLA signals (red) counterstained with DAPI (blue). Quantification of proximity ligation assays for p53EE with Tom20, Bcl-2, Bcl-xL and Bak in H1299 cells. Doxo treatment (0.1 µg/ml, 24 hours) as indicated. Plotted is the average number of PLA signals per cell for 10 fields of view.


Mitochondrial membrane potential of control and p53EE expressing H1299 cells in the absence and presence of increasing concentrations of BID BH3 peptide . Treatment with the mitochondrial depolarizer CCCP is shown as a positive control ; n = 3 .

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Mitochondrial membrane potential of control and p53EE expressing H1299 cells in the absence and presence of increasing concentrations of BID BH3 peptide. Treatment with the mitochondrial depolarizer CCCP is shown as a positive control; n=3.


Apoptosis measured by flow cytometry using Annexin V in H1299 cells with tet - inducible expression of the indicated human p53 mutants ; n = 3 .

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Apoptosis measured by flow cytometry using Annexin V in H1299 cells with tet-inducible expression of the indicated human p53 mutants; n=3.


mRNA expression analysis ( RTqPCR ) of p53 target genes normalized to β - actin ; n = 3 .

mRNA expression analysis (RTqPCR) of p53 target genes normalized to β-actin; n=3.


Kaplan - Meier survival plots for mice with indicated genotypes , number of mice , and median survival . Right panel , analysis of survival differences using the Log - rank ( Mantel - Cox ) test .

Kaplan-Meier survival plots for mice with indicated genotypes, number of mice, and median survival. Right panel, analysis of survival differences using the Log-rank (Mantel-Cox) test.


p53 immunohistochemistry ( IHC ) in representative spontaneous tumors ; scale bars 25 µm .

p53 immunohistochemistry (IHC) in representative spontaneous tumors; scale bars 25 µm.


Kaplan - Meier survival plots for Eµ - Myc transgenic mice with different p53 genotype . Kaplan - Meier survival plots for wild - type recipient mice transplanted with AML1 / ETO9a + NrasG12D - transduced fetal liver cells ( 5 donors / genotype ) .

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Kaplan-Meier survival plots for Eµ-Myc transgenic mice with different p53 genotype. Kaplan-Meier survival plots for wild-type recipient mice transplanted with AML1/ETO9a + NrasG12D-transduced fetal liver cells (5 donors/genotype).


p53 IHC for representative samples from ( D ) and ( E ) illustrates constitutive stabilization of p53EE in cancer cells ; scale bars 25 µm .

p53 IHC for representative samples from (D) and (E) illustrates constitutive stabilization of p53EE in cancer cells; scale bars 25 µm.


Apoptosis in Eµ - Myc lymphoma cells of indicated genotype was measured by flow cytometry ( FITC - VAD - FMK staining ) at indicated time points after treatment with 3 µg / ml MAF . Significance was calculated versus untreated . n ≥ 5 .

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Apoptosis in Eµ-Myc lymphoma cells of indicated genotype was measured by flow cytometry (FITC-VAD-FMK staining) at indicated time points after treatment with 3 µg/ml MAF. Significance was calculated versus untreated. n≥5.


Enrichment of p53 shRNA - expressing Eµ - Myc ; p53EE lymphoma cells under MAF treatment . Non - silencing control shRNA served as control . n = 3 .

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Enrichment of p53 shRNA-expressing Eµ-Myc;p53EE lymphoma cells under MAF treatment. Non-silencing control shRNA served as control. n=3.


mRNA expression of p53 target genes was measured relative to β - actin by RTqPCR following treatment of Eµ - Myc lymphoma cells of indicated genotype with 3 µg / ml MAF . n ≥ 3 .

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mRNA expression of p53 target genes was measured relative to β-actin by RTqPCR following treatment of Eµ-Myc lymphoma cells of indicated genotype with 3 µg/ml MAF. n≥3.


Kaplan - Meier survival plots for cyclophosphamide - treated versus untreated control mice transplanted with Eµ - Myc lymphoma cells of indicated genotype at day 0 . Treatment started ~ 10 days later when peripheral lymph nodes became palpable . The time course of the experiment is illustrated in a scheme .

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Kaplan-Meier survival plots for cyclophosphamide-treated versus untreated control mice transplanted with Eµ-Myc lymphoma cells of indicated genotype at day 0. Treatment started ~10 days later when peripheral lymph nodes became palpable. The time course of the experiment is illustrated in a scheme.


Residual disease in the spleen of mice from ( D ) was quantified 24 hours and 7 days after therapy using a quantitative PCR assays with primers specific to the Eµ - Myc transgene ( present only in lymphoma cells ) . Shown is the copy number of the Eµ - Myc transgene relative to a control locus present in all cells . Untreated Eµ - Myc mice ( Myc tg ) were used as positive control . Non - transgenic mice ( no Myc tg ) served as negative controls to define the detection limit . Significance was tested by t - test ( two - sided , unpaired ) .

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Residual disease in the spleen of mice from (D) was quantified 24 hours and 7 days after therapy using a quantitative PCR assays with primers specific to the Eµ-Myc transgene (present only in lymphoma cells). Shown is the copy number of the Eµ-Myc transgene relative to a control locus present in all cells. Untreated Eµ-Myc mice (Myc tg) were used as positive control. Non-transgenic mice (no Myc tg) served as negative controls to define the detection limit. Significance was tested by t-test (two-sided, unpaired).


Representative bioluminescence imaging ( BLI ) pictures of mice transplanted with AE9 + Nras AML cells . Day 0 indicates the start of combination therapy with cytarabine ( AraC ) + doxorubicin ( Doxo ) .

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Representative bioluminescence imaging (BLI) pictures of mice transplanted with AE9+Nras AML cells. Day 0 indicates the start of combination therapy with cytarabine (AraC) + doxorubicin (Doxo).


Kaplan - Meier survival plots for animals from ( G ) . Statistical significance of differences calculated by Log - rank ( Mantel - Cox ) test between treated Eµ - Myc ; p53EE / EE AML and all other groups is indicated in the table . C , untreated control ; T , therapy ( AraC + Doxo ) .

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Kaplan-Meier survival plots for animals from (G). Statistical significance of differences calculated by Log-rank (Mantel-Cox) test between treated Eµ-Myc;p53EE/EE AML and all other groups is indicated in the table. C, untreated control; T, therapy (AraC+Doxo).


( A ) Transcription at GAL3 is visualized by addition of PP7 loops at the 5 ' of GAL3 in budding yeast . Example trace of the quantified fluorescence intensity of the transcription site . Traces are binarized to determine on ( active ) and off ( inactive ) times .

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(A) Transcription at GAL3 is visualized by addition of PP7 loops at the 5' of GAL3 in budding yeast. Example trace of the quantified fluorescence intensity of the transcription site. Traces are binarized to determine on (active) and off (inactive) times.


( B ) Histogram of GAL3 on time ( burst duration ) for cells with wt and mutated UAS , respectively , reveals shorter on times for mutated UAS . Errors indicate SE of 3 experiments , n = 324 cells UASwt , n = 250 cells UASmut . ( C ) Histogram of GAL3 off times . Errors indicate SE of 3 experiments .

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(B) Histogram of GAL3 on time (burst duration) for cells with wt and mutated UAS, respectively, reveals shorter on times for mutated UAS. Errors indicate SE of 3 experiments, n = 324 cells UASwt, n = 250 cells UASmut. (C) Histogram of GAL3 off times. Errors indicate SE of 3 experiments.


( D ) cells were the UAS was mutated to a lower affinity UAS .

(D) cells were the UAS was mutated to a lower affinity UAS.


( E ) . Average on time for UASwt and UASmut from exponential fit . Errors indicate SD of fit . ( F ) Average off time for UASwt and UASmut from exponential fit . Errors indicate SD of fit .

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(E). Average on time for UASwt and UASmut from exponential fit. Errors indicate SD of fit. (F) Average off time for UASwt and UASmut from exponential fit. Errors indicate SD of fit.


( G ) Example traces of quantified fluorescence intensity at the transcription site for 5 ' PP7 and 3 ' MS2 at GAL3 .

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(G) Example traces of quantified fluorescence intensity at the transcription site for 5'PP7 and 3'MS2 at GAL3.


( H ) Cross correlation of the PP7 and MS2 signals shows a peak at 37 . 9s ± 1 . 8s delay , n = 137 cells . Shaded area and errors indicate SEM .

O O O O O O O O O O O O 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) Cross correlation of the PP7 and MS2 signals shows a peak at 37.9s±1.8s delay, n = 137 cells. Shaded area and errors indicate SEM.


( I ) Distribution of number of nascent RNAs at the transcription site determined by smFISH . The distribution of UASwt does not fit a Poisson distribution ( grey line ) , supporting that GAL3 is not constitutively transcribed , but is transcribed in bursts . Example image in shown , yellow arrows indicate TSs . Scale bars : 5 μm . n = 11 , 839 cells . ( J ) for cells with UASmut . The distribution of UASmut fits a Poisson distribution , indicating that GAL3 - UASmut is transcribed with random initiation of individual polymerases , similar to constitutive genes . n = 10 , 616 cells .

O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Distribution of number of nascent RNAs at the transcription site determined by smFISH. The distribution of UASwt does not fit a Poisson distribution (grey line), supporting that GAL3 is not constitutively transcribed, but is transcribed in bursts. Example image in shown, yellow arrows indicate TSs. Scale bars: 5 μm. n = 11,839 cells. (J) for cells with UASmut. The distribution of UASmut fits a Poisson distribution, indicating that GAL3-UASmut is transcribed with random initiation of individual polymerases, similar to constitutive genes. n = 10,616 cells.


( A ) Competitive binding experiments were performed to determine relative affinity of Gal4 to UASwt ( green ) and UASmut ( blue ) motifs . Gal4 occupancy on UASwt Cy3 / Cy5 DNA was determined by measuring protein induced fluorescence enhancement ( PIFE ) on 51 bp oligos containing either Gal4 UASwt or UASmut site 1 bp away from Cy3 fluorophore ) .

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

(A) Competitive binding experiments were performed to determine relative affinity of Gal4 to UASwt (green) and UASmut (blue) motifs. Gal4 occupancy on UASwt Cy3/Cy5 DNA was determined by measuring protein induced fluorescence enhancement (PIFE) on 51 bp oligos containing either Gal4 UASwt or UASmut site 1 bp away from Cy3 fluorophore).


( B ) Titrating unlabeled UASwt or UASmut competitor DNA reduces UASwt Cy3 / Cy5 DNA occupancy . IC50UASwt = 4 . 0 ± 0 . 6 nM , IC50UASmut = 17 . 3 ± 3 . 5 nM . n = 3 . Error bars indicate SD .

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

(B) Titrating unlabeled UASwt or UASmut competitor DNA reduces UASwt Cy3/Cy5 DNA occupancy. IC50UASwt = 4.0 ± 0.6 nM, IC50UASmut = 17.3 ± 3.5 nM. n = 3. Error bars indicate SD.


( C ) Comparison between relative affinities of Gal4 UASwt versus UASmut in naked or nucleosomal DNA shows 4 . 3 ± 1 . 1x difference in KD from naked DNA and 6 . 8 ± 1 . 7x from nucleosomal DNA . n = 3 . Error bars indicate SD .

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

(C) Comparison between relative affinities of Gal4 UASwt versus UASmut in naked or nucleosomal DNA shows 4.3 ± 1.1x difference in KD from naked DNA and 6.8 ± 1.7x from nucleosomal DNA. n = 3. Error bars indicate SD.


( D ) Experimental setup for smFRET experiments to measure Gal4 binding at nucleosomal DNA . Gal4 binds to site 8 bp into nucleosome . A FRET pair in the entry / exit region provides readout on binding events ( one fluorophore on DNA , one on histone ) . In the absence of Gal4 , nucleosomes are in the high FRET state . A Gal4 binding event traps nucleosome in low FRET state .

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

(D) Experimental setup for smFRET experiments to measure Gal4 binding at nucleosomal DNA. Gal4 binds to site 8 bp into nucleosome. A FRET pair in the entry/exit region provides readout on binding events (one fluorophore on DNA, one on histone). In the absence of Gal4, nucleosomes are in the high FRET state. A Gal4 binding event traps nucleosome in low FRET state.


( E ) Gal4 affinity to nucleosomes containing UASwt or UASmut sequences gives S1 / 2 of 7 . 2 ± 0 . 8 nM and 48 . 9 ± 10 . 8 nM , respectively . n = 3 . Error bars indicate SD .

O O O O O O O O O O O O O O O O O O O O O O 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) Gal4 affinity to nucleosomes containing UASwt or UASmut sequences gives S1/2 of 7.2 ± 0.8 nM and 48.9 ± 10.8 nM, respectively. n = 3. Error bars indicate SD.


( F ) Example smFRET traces showing Gal4 binding to UASwt in nucleosomal DNA at two different Gal4 concentrations . States are determined using HMM fit . ( G ) Same as F but for UASmut .

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

(F) Example smFRET traces showing Gal4 binding to UASwt in nucleosomal DNA at two different Gal4 concentrations. States are determined using HMM fit. (G) Same as F but for UASmut.


( H ) Binding - rates of Gal4 are concentration dependent , but are similar for UASwt and UASmut : kon UASwt = 0 . 011 ± 0 . 002 s - 1 nM - 1 , kon UASmut = 0 . 009 ± 0 . 001 s - 1 nM - 1 . n = 2 for all , except n = 4 for UASwt 10 nM Gal4 and n = 3 for UASwt 20 nM Gal4 . Error bars indicate SD .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Binding-rates of Gal4 are concentration dependent, but are similar for UASwt and UASmut: kon UASwt = 0.011 ± 0.002 s-1 nM-1, kon UASmut = 0.009 ± 0.001 s-1 nM-1. n = 2 for all, except n = 4 for UASwt 10 nM Gal4 and n = 3 for UASwt 20 nM Gal4. Error bars indicate SD.


( I ) The dissociation - rate of Gal4 at UASwt is ~ 5 - fold slower compared to the UASmut : koff UASwt = 0 . 20 ± 0 . 01 s - 1 , koff UASmut = 0 . 92 ± 0 . 05 s - 1 . n = 2 for all , except n = 4 for UASwt 10 nM Gal4 and n = 3 for UASwt 20 nM Gal4 . Error bars indicate SD .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) The dissociation-rate of Gal4 at UASwt is ~5-fold slower compared to the UASmut: koff UASwt = 0.20 ± 0.01 s-1, koff UASmut = 0.92 ± 0.05 s-1. n = 2 for all, except n = 4 for UASwt 10 nM Gal4 and n = 3 for UASwt 20 nM Gal4. Error bars indicate SD.


( J ) Histogram of Gal4 dwell time to nucleosomal DNA containing UASwt and UASmut sequences . n = 11 for UASwt , n = 8 for UASmut . Error bars indicate SE .

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

(J) Histogram of Gal4 dwell time to nucleosomal DNA containing UASwt and UASmut sequences. n = 11 for UASwt, n = 8 for UASmut. Error bars indicate SE.


( K ) Scatter plot showing the average number of nascent RNA at TS ( from smFISH ) vs affinity of Gal4 to different UAS sequences in nucleosomal DNA ( from in vitro measurements ) . In vivo transcription levels correlate with in vitro affinity of Gal4 , but transcription saturates above wildtype sequence . UAS sequences are shown in the box . For mean number of nascent RNA from smFISH , n = 2 for UASconsensus , UAS - 2C , UAS - 8T and n = 8 for UASwt , UASmut and errors indicate SEM . For in vitro affinity measurements , n = 3 and errors indicate SD .

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

(K) Scatter plot showing the average number of nascent RNA at TS (from smFISH) vs affinity of Gal4 to different UAS sequences in nucleosomal DNA (from in vitro measurements). In vivo transcription levels correlate with in vitro affinity of Gal4, but transcription saturates above wildtype sequence. UAS sequences are shown in the box. For mean number of nascent RNA from smFISH, n = 2 for UASconsensus, UAS-2C, UAS-8T and n = 8 for UASwt, UASmut and errors indicate SEM. For in vitro affinity measurements, n = 3 and errors indicate SD.


Profiles of nucleosome midpoint positions by MNase - seq experiments . Samples were digested with the indicated MNase concentrations in both raffinose ( raf ) and galactose ( gal ) containing media . Midpoints of nucleosomes are smoothed by 31 bp . In galactose the stable nucleosomes move away from the Gal4UAS , creating space for an additional fragile nucleosome ( indicated by arrow ) .

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

Profiles of nucleosome midpoint positions by MNase-seq experiments. Samples were digested with the indicated MNase concentrations in both raffinose (raf) and galactose (gal) containing media. Midpoints of nucleosomes are smoothed by 31 bp. In galactose the stable nucleosomes move away from the Gal4UAS, creating space for an additional fragile nucleosome (indicated by arrow).


( A ) Simultaneous imaging of Gal4 binding kinetics in vivo using single - molecule tracking and RNA imaging of the GAL10 target gene . Gal4 is tagged with a HALO - tag , which covalently binds to the dye JF646 . Transcription of the target gene GAL10 is visualized by PP7 - loops .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Simultaneous imaging of Gal4 binding kinetics in vivo using single-molecule tracking and RNA imaging of the GAL10 target gene. Gal4 is tagged with a HALO-tag, which covalently binds to the dye JF646. Transcription of the target gene GAL10 is visualized by PP7-loops.


( B ) Example image of a cell , showing the GAL10 TS ( arrow , left panel ) , single molecules of Gal4 ( arrows , middle panel ) and an overlay ( right panel ) . Scale bar : 2 μm .

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

(B) Example image of a cell, showing the GAL10 TS (arrow, left panel), single molecules of Gal4 (arrows, middle panel) and an overlay (right panel). Scale bar: 2 μm.


( C ) Example kymograph of a cell . Upper panel ( PP7 - GAL10 ) shows the position of the TS over time . Middle panel ( Gal4 - HALO ) show tracks of Gal4 . Data is in white , colored lines show analyzed tracks . Lower panel shows overlay , showing colocalization of some the Gal4 tracks to the GAL10 TS . Scale bar : 1 μm .

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

(C) Example kymograph of a cell. Upper panel (PP7-GAL10) shows the position of the TS over time. Middle panel (Gal4-HALO) show tracks of Gal4. Data is in white, colored lines show analyzed tracks. Lower panel shows overlay, showing colocalization of some the Gal4 tracks to the GAL10 TS. Scale bar: 1 μm.


( D ) Survival probability of the duration of Gal4 tracks ( after displacement thresholding ) at 200 ms interval from cells grown in raffinose ( n = 258 tracks in 30 cells ) or galactose ( n = 346 tracks in 25 cells ) . Lines show bi - exponential fit , indicating 2 Gal4 populations . Inset shows data in semi - logarithmic plot . ( E ) Residence time of the fast and slow component of the fits from ( D ) . The slow component changes between conditions . Error bars indicate 95 % CI .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Survival probability of the duration of Gal4 tracks (after displacement thresholding) at 200 ms interval from cells grown in raffinose (n = 258 tracks in 30 cells) or galactose (n = 346 tracks in 25 cells). Lines show bi-exponential fit, indicating 2 Gal4 populations. Inset shows data in semi-logarithmic plot. (E) Residence time of the fast and slow component of the fits from (D). The slow component changes between conditions. Error bars indicate 95% CI.


( F ) Survival probability of Gal4 residence times that colocalize with GAL10 TS ( < 250nm ) , showing that Gal4 with long residence time colocalizes with GAL10 . Data was taken at 1s interval and shows overlap between 267 Gal4 tracks and 83 GAL10 tracks , representing 458 bound molecules . Line shows exponential fit , with mean of 12 . 3s ± 0 . 83s . Errors indicate 95 % CI .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Survival probability of Gal4 residence times that colocalize with GAL10 TS (<250nm), showing that Gal4 with long residence time colocalizes with GAL10. Data was taken at 1s interval and shows overlap between 267 Gal4 tracks and 83 GAL10 tracks, representing 458 bound molecules. Line shows exponential fit, with mean of 12.3s ± 0.83s. Errors indicate 95% CI.


( A ) Schematic of 3D orbital tracking of GAL10 TS . Scale bar : 2 μm .

(A) Schematic of 3D orbital tracking of GAL10 TS. Scale bar: 2 μm.


( B ) Example trace of Gal4 binding and GAL10 transcription in the same cell .

(B) Example trace of Gal4 binding and GAL10 transcription in the same cell.


( C ) Autocorrelation of Gal4 . The exponential fit shows an average dwell time of 34 . 8 ± 0 . 5s . n = 19 traces . Shaded area and errors indicate SEM .

O O O O O O O O O O O O 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) Autocorrelation of Gal4. The exponential fit shows an average dwell time of 34.8 ± 0.5s. n = 19 traces. Shaded area and errors indicate SEM.


( D ) Autocorrelation of GAL10 . The fit of 2 linear lines reveals a burst duration of 152 . 4 ± 2 . 3s . n = 19 traces . Shaded area and errors indicate SEM .

O O O O O O O O O O O O O O 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) Autocorrelation of GAL10. The fit of 2 linear lines reveals a burst duration of 152.4 ± 2.3s. n = 19 traces. Shaded area and errors indicate SEM.


( E ) Cross correlations for Gal4 - GAL10 RNA ( blue ) and Gal4 - RNR2 RNA ( grey , negative control ) . Asymmetry and positive temporal shift of the cross correlation function is consistent with a 79 . 5 ± 0 . 2 second delay between the middle of Gal4 and GAL10 signals ( n = 19 traces ) , which is not seen in the negative control ( n = 8 traces ) . Shaded areas and errors indicate SEM .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Cross correlations for Gal4-GAL10 RNA (blue) and Gal4-RNR2 RNA (grey, negative control). Asymmetry and positive temporal shift of the cross correlation function is consistent with a 79.5 ± 0.2 second delay between the middle of Gal4 and GAL10 signals (n = 19 traces), which is not seen in the negative control (n = 8 traces). Shaded areas and errors indicate SEM.


( F ) Schematic of the different signal durations of Gal4 binding and GAL10 transcription . Gal4 binding overlaps with the GAL10 transcription for 14 . 1s .

O O O O 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) Schematic of the different signal durations of Gal4 binding and GAL10 transcription. Gal4 binding overlaps with the GAL10 transcription for 14.1s.


( A ) Example trace of PP7 - GAL10 transcription in 2 % galactose .

(A) Example trace of PP7-GAL10 transcription in 2% galactose.


( B ) Boxplot of the onset of the first transcription event in different doses of galactose . The box indicates quartiles , the horizontal line inside the box indicates the median , the whiskers indicates 1 . 5 times the interquartile range of the box . Data points outside the whiskers are indicated with circles ( 2 % : n = 86 cells , 0 . 2 % : n = 79 cells , 0 . 02 % : n = 84 cells ) .

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

(B) Boxplot of the onset of the first transcription event in different doses of galactose. The box indicates quartiles, the horizontal line inside the box indicates the median, the whiskers indicates 1.5 times the interquartile range of the box. Data points outside the whiskers are indicated with circles (2%: n = 86 cells, 0.2%: n = 79 cells, 0.02%: n = 84 cells).


( C ) The autocorrelation was used to interpret the burst duration and burst frequency changes . Burst duration is measured by the intersect of the two linear fits . When burst duration is constant , the amplitude is inversely related to the burst frequency . ( D ) Burst duration from autocorrelation is constant across 4 different galactose concentrations ( 2 % : n = 86 cells , 0 . 2 % : n = 79 cells , 0 . 02 % : n = 84 cells , 0 . 004 % : n = 53 cells ) . Error bars indicate SEM . ( E ) Amplitude of autocorrelation decreases ( burst frequency increases ) at higher doses of galactose . ( 2 % : n = 86 cells , 0 . 2 % : n = 79 cells , 0 . 02 % : n = 84 cells , 0 . 004 % : n = 53 cells ) . Error bars indicate SEM .

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

(C) The autocorrelation was used to interpret the burst duration and burst frequency changes. Burst duration is measured by the intersect of the two linear fits. When burst duration is constant, the amplitude is inversely related to the burst frequency. (D) Burst duration from autocorrelation is constant across 4 different galactose concentrations (2%: n = 86 cells, 0.2%: n = 79 cells, 0.02%: n = 84 cells, 0.004%: n = 53 cells). Error bars indicate SEM. (E) Amplitude of autocorrelation decreases (burst frequency increases) at higher doses of galactose. (2%: n = 86 cells, 0.2%: n = 79 cells, 0.02%: n = 84 cells, 0.004%: n = 53 cells). Error bars indicate SEM.


( F ) Example traces of two single cells exposed to two different doses of galactose . ( G , H ) Same - cell dose response shows same burst duration ( G ) but lower amplitude ( H ) at higher galactose concentration , indicating that galactose levels regulate burst frequency ( n = 13 cells ) . Error bars indicate SEM .

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

(F) Example traces of two single cells exposed to two different doses of galactose. (G, H) Same-cell dose response shows same burst duration (G) but lower amplitude (H) at higher galactose concentration, indicating that galactose levels regulate burst frequency (n = 13 cells). Error bars indicate SEM.


Reduced SDS - PAGE analysis of purified anti - HIV BiCEs .

Reduced SDS-PAGE analysis of purified anti-HIV BiCEs.


HIV - 1YU - 2 was preincubated with the indicated concentrations of antibodies or BiCEs and subsequently incubated with HIV reporter TZM - bl cells . 48 h later , HIV - infection was measured as luciferase activity quantified by luminescence . For each sample , a normalized HIV - infection is calculated by subtracting RLU of control ( no virus added ) , and then normalized to virus control ( no antibody or BiCE added ) set to 100 % . n = 3 independent experiments ( 10 - 1074 and 3BNC117 ) or n = 4 independent experiments ( scFv3BNC117 - Nb75 ) or n = 7 independent experiments ( scFv10 - 1074 - Nb75 ) or n = 1 experiment ( scFv10 - 1074 , Nb75 and anti - tetanus IgG ) , each experiment showing an average of triplicate wells . MW = 150 kDa ( 10 - 1074 , 3BNC117 and anti - tetanus IgG ) , MW = 43 kDa ( scFv10 - 1074 - Nb75 ) , MW = 45 kDa ( scFv3BNC117 - Nb75 ) , MW = 28 kDa ( scFv10 - 1074 ) and MW = 15 kDa ( Nb75 ) .

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

HIV-1YU-2 was preincubated with the indicated concentrations of antibodies or BiCEs and subsequently incubated with HIV reporter TZM-bl cells. 48 h later, HIV-infection was measured as luciferase activity quantified by luminescence. For each sample, a normalized HIV-infection is calculated by subtracting RLU of control (no virus added), and then normalized to virus control (no antibody or BiCE added) set to 100%. n = 3 independent experiments (10-1074 and 3BNC117) or n = 4 independent experiments (scFv3BNC117-Nb75) or n = 7 independent experiments (scFv10-1074-Nb75) or n = 1 experiment (scFv10-1074, Nb75 and anti-tetanus IgG), each experiment showing an average of triplicate wells. MW = 150 kDa (10-1074, 3BNC117 and anti-tetanus IgG), MW = 43 kDa (scFv10-1074-Nb75), MW = 45 kDa (scFv3BNC117-Nb75), MW = 28 kDa (scFv10-1074) and MW = 15 kDa (Nb75).


Raji cells expressing HIV - 1YU - 2 envelope ( Raji - Env ) or Raji control cells ( Raji - Ctr ) were incubated with 10 - 1074 or scFv10 - 1074 or with controls ( only secondary antibody or streptavidin added ) . Binding of 10 - 1074 and scFv10 - 1074 was assessed by flow cytometry using a BV421 - conjugated anti - IgG Fc antibody or streptavidin , respectively . The numbers indicate the % of cells with bound antibody . One experiment is shown .

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

Raji cells expressing HIV-1YU-2 envelope (Raji-Env) or Raji control cells (Raji-Ctr) were incubated with 10-1074 or scFv10-1074 or with controls (only secondary antibody or streptavidin added). Binding of 10-1074 and scFv10-1074 was assessed by flow cytometry using a BV421-conjugated anti-IgG Fc antibody or streptavidin, respectively. The numbers indicate the % of cells with bound antibody. One experiment is shown.


C3 deposition by the indicated antibodies or BiCEs . Results are expressed as the C3 MFI % fold change compared to no antibody treatment . n = 3 donor serum . Data information : All data are presented as mean ± SEM .

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

C3 deposition by the indicated antibodies or BiCEs. Results are expressed as the C3 MFI % fold change compared to no antibody treatment. n = 3 donor serum. Data information: All data are presented as mean ± SEM.


CDC triggered by the indicated antibodies or BiCEs . CDC was calculated as the relative % of dead cells compared to no antibody treatment . n = 3 donor serum . Data information : All data are presented as mean ± SEM .

CDC triggered by the indicated antibodies or BiCEs. CDC was calculated as the relative % of dead cells compared to no antibody treatment. n = 3 donor serum. Data information: All data are presented as mean ± SEM.


C3 deposition by the indicated antibodies or BiCEs . Results are expressed as the C3 MFI % fold change compared to no antibody treatment . n = 6 donors ( 10 - 1074 , 3BNC117 , 10 - 1074 + 3BNC17 and scFv10 - 1074 - Nb75 + scFv3BNC117 - Nb75 ) or n = 8 donors ( no antibody treatment , scFv10 - 1074 - Nb75 and scFv3BNC117 - Nb75 ) or n = 2 donors ( scFv10 - 1074 , Nb75 and anti - tetanus IgG ) . Data information : Data are presented as median with interquartile range . Significance was determined by comparing each antibody or BiCE to no antibody treatment . Only significant differences are shown ; * P < 0 . 05 and * * P < 0 . 01 , Wilcoxon signed - rank test . When n ≤ 2 , no statistical analysis was performed .

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

C3 deposition by the indicated antibodies or BiCEs. Results are expressed as the C3 MFI % fold change compared to no antibody treatment. n = 6 donors (10-1074, 3BNC117, 10-1074+3BNC17 and scFv10-1074-Nb75+scFv3BNC117-Nb75) or n = 8 donors (no antibody treatment, scFv10-1074-Nb75 and scFv3BNC117-Nb75) or n = 2 donors (scFv10-1074, Nb75 and anti-tetanus IgG). Data information: Data are presented as median with interquartile range. Significance was determined by comparing each antibody or BiCE to no antibody treatment. Only significant differences are shown; *P < 0.05 and **P < 0.01, Wilcoxon signed-rank test. When n ≤ 2, no statistical analysis was performed.


Primary CD4 T cells infected with HIV - 1NL4 - 3 - eGFP and uninfected CD4 T cells were incubated with NHS and the indicated antibodies or BiCEs in presence of an HIV - inhibitor . The frequency of eGFP + cells was analyzed by flow cytometry at the indicated time points . The numbers indicate the % of eGFP + cells . One representative donor is shown .

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Primary CD4 T cells infected with HIV-1NL4-3-eGFP and uninfected CD4 T cells were incubated with NHS and the indicated antibodies or BiCEs in presence of an HIV-inhibitor. The frequency of eGFP+ cells was analyzed by flow cytometry at the indicated time points. The numbers indicate the % of eGFP+ cells. One representative donor is shown.


Induction of eGFP + elimination over time by the indicated antibodies or BiCEs . Results are expressed as the relative % of eGFP + cells compared to no antibody treatment . n = 6 donors ( 10 - 1074 , 3BNC117 , 10 - 1074 + 3BNC17 and scFv10 - 1074 - Nb75 + scFv3BNC117 - Nb75 ) or n = 8 donors ( no antibody treatment , scFv10 - 1074 - Nb75 and scFv3BNC117 - Nb75 ) or n = 2 donors ( scFv10 - 1074 , Nb75 and anti - tetanus IgG ) . Data information : Data are presented as median with interquartile range . Significance was determined by comparing each antibody or BiCE to no antibody treatment . Only significant differences are shown ; * P < 0 . 05 and * * P < 0 . 01 , Wilcoxon signed - rank test . When n ≤ 2 , no statistical analysis was performed .

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Induction of eGFP+ elimination over time by the indicated antibodies or BiCEs. Results are expressed as the relative % of eGFP+ cells compared to no antibody treatment. n = 6 donors (10-1074, 3BNC117, 10-1074+3BNC17 and scFv10-1074-Nb75+scFv3BNC117-Nb75) or n = 8 donors (no antibody treatment, scFv10-1074-Nb75 and scFv3BNC117-Nb75) or n = 2 donors (scFv10-1074, Nb75 and anti-tetanus IgG). Data information: Data are presented as median with interquartile range. Significance was determined by comparing each antibody or BiCE to no antibody treatment. Only significant differences are shown; *P < 0.05 and **P < 0.01, Wilcoxon signed-rank test. When n ≤ 2, no statistical analysis was performed.


( D and E ) qRT - PCR analysis of codon tag reporter mRNA levels at 6 hpf relative to 2 hpf in the absence ( D ) or presence ( E ) of translation - blocking GFP MO . Bar charts show the average of three independent biological replicates . Error bars show standard deviation ( SD ) . Individual data points are shown as dots . P - values were calculated using two - sided Student ' s t - test .

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(D and E) qRT-PCR analysis of codon tag reporter mRNA levels at 6 hpf relative to 2 hpf in the absence (D) or presence (E) of translation-blocking GFP MO. Bar charts show the average of three independent biological replicates. Error bars show standard deviation (SD). Individual data points are shown as dots. P-values were calculated using two-sided Student's t-test.


( B - E ) Scatter plots showing a correlation between relative ribosome density ( 28 - 30 - nt footprints ; x - axis ) and the codon effects measured by PACE ( y - axis ) . Each dot represents a single codon . Graphs for all codons ( B ) , codons for polar amino acids ( C ) , nonpolar amino acids ( D ) , and charged amino acids ( E ) are shown . Data information : the regression line is shown in blue and the 95 % confidence interval is shown in gray . P - values were calculated using Student ' s t - test .

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(B-E) Scatter plots showing a correlation between relative ribosome density (28-30-nt footprints; x-axis) and the codon effects measured by PACE (y-axis). Each dot represents a single codon. Graphs for all codons (B), codons for polar amino acids (C), nonpolar amino acids (D), and charged amino acids (E) are shown. Data information: the regression line is shown in blue and the 95% confidence interval is shown in gray. P-values were calculated using Student's t-test.


( F - I ) Scatter plots showing a correlation between relative ribosome density ( 28 - 30 - nt footprints ; x - axis ) and the tRNA levels ( y - axis , Each dot represents a single codon . Graphs for all codons ( F ) , codons for polar amino acids ( G ) , nonpolar amino acids ( H ) , and charged amino acids ( I ) are shown . Data information : the regression line is shown in blue and the 95 % confidence interval is shown in gray . P - values were calculated using Student ' s t - test .

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(F-I) Scatter plots showing a correlation between relative ribosome density (28-30-nt footprints; x-axis) and the tRNA levels (y-axis, Each dot represents a single codon. Graphs for all codons (F), codons for polar amino acids (G), nonpolar amino acids (H), and charged amino acids (I) are shown. Data information: the regression line is shown in blue and the 95% confidence interval is shown in gray. P-values were calculated using Student's t-test.


( B ) Charged tRNA levels in mock - injected and AnsB - overexpressing embryos at 6 hpf . Total and charged tRNAs were measured by qRT - PCR coupled to the OXOPAP assay . The relative charged tRNA level in mock - injected embryos were set to one . Data information bar charts show the average of three independent biological replicates . Error bars show SD . Individual data points are shown as dots . P - values were calculated using two - sided Student ' s t - test .

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(B) Charged tRNA levels in mock-injected and AnsB-overexpressing embryos at 6 hpf. Total and charged tRNAs were measured by qRT-PCR coupled to the OXOPAP assay. The relative charged tRNA level in mock-injected embryos were set to one. Data information bar charts show the average of three independent biological replicates. Error bars show SD. Individual data points are shown as dots. P-values were calculated using two-sided Student's t-test.


( D ) Poly ( A ) tail analysis of Asn AAC codon tag reporter mRNA at 2 and 6 hpf . The developmental stages are shown above as hpf . The lane labeled A0 shows the 3 ´ UTR fragment without a poly ( A ) tail . ( E ) Quantification of the PAT assay at 6 hpf in ( D ) .

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(D) Poly(A) tail analysis of Asn AAC codon tag reporter mRNA at 2 and 6 hpf. The developmental stages are shown above as hpf. The lane labeled A0 shows the 3´ UTR fragment without a poly(A) tail. (E) Quantification of the PAT assay at 6 hpf in (D).


( F ) Poly ( A ) tail analysis of Leu CUG codon tag reporter mRNA at 2 and 6 hpf . ( G ) Quantification of the PAT assay at 6 hpf in ( F ) .

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(F) Poly(A) tail analysis of Leu CUG codon tag reporter mRNA at 2 and 6 hpf. (G) Quantification of the PAT assay at 6 hpf in (F).


( B ) Western blotting to detect the sfGFP levels at 6 hpf . Tubulin ( Tub ) was detected as a loading control .

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(B) Western blotting to detect the sfGFP levels at 6 hpf. Tubulin (Tub) was detected as a loading control.


( C ) qRT - PCR analysis of hCMV uORF2 reporter mRNA levels in wild - type embryos at 6 hpf relative to 2 hpf . Data information : bar charts show the average of three independent biological replicates . Error bars show SD . Individual data points are shown as dots . P - values were calculated using two - sided Student ' s t - test .

(C) qRT-PCR analysis of hCMV uORF2 reporter mRNA levels in wild-type embryos at 6 hpf relative to 2 hpf. Data information: bar charts show the average of three independent biological replicates. Error bars show SD. Individual data points are shown as dots. P-values were calculated using two-sided Student's t-test.


( D ) Poly ( A ) tail analysis of hCMV uORF2 reporter mRNAs at 2 and 6 hpf . The developmental stages are shown above as hpf . The lane labeled A0 shows the 3 ´ UTR fragment without a poly ( A ) tail . ( E ) Quantification of the PAT assay at 6 hpf in ( D ) .

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(D) Poly(A) tail analysis of hCMV uORF2 reporter mRNAs at 2 and 6 hpf. The developmental stages are shown above as hpf. The lane labeled A0 shows the 3´ UTR fragment without a poly(A) tail. (E) Quantification of the PAT assay at 6 hpf in (D).


( G ) qRT - PCR analysis of hCMV uORF2 reporter mRNA levels in MZznf598 embryos at 6 hpf relative to 2 hpf . ( H ) qRT - PCR analysis of hCMV uORF2 reporter mRNA levels in wild - type embryos , MZznf598 embryos and MZznf598 embryos injected with a mRNA encoding Myc - tagged full - length ( full ) or RING domain deleted ( ∆ RING ) Znf598 . mRNA levels at 6 hpf relative to 2 hpf are shown . Data information : bar charts show the average of three independent biological replicates . Error bars show SD . Individual data points are shown as dots . P - values were calculated using two - sided Student ' s t - test .

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(G) qRT-PCR analysis of hCMV uORF2 reporter mRNA levels in MZznf598 embryos at 6 hpf relative to 2 hpf. (H) qRT-PCR analysis of hCMV uORF2 reporter mRNA levels in wild-type embryos, MZznf598 embryos and MZznf598 embryos injected with a mRNA encoding Myc-tagged full-length (full) or RING domain deleted (∆RING) Znf598. mRNA levels at 6 hpf relative to 2 hpf are shown. Data information: bar charts show the average of three independent biological replicates. Error bars show SD. Individual data points are shown as dots. P-values were calculated using two-sided Student's t-test.


( J ) Results of PACE in MZznf598 zebrafish embryos . Black circles show the relative stability of the reporter mRNAs ( averages of two biological replicates ) in MZznf598 embryos . Gray circles show the relative stability of reporter mRNAs in wild - type embryos The stability of a codon - tag reporter with a UGA stop codon was set to one . Error bars represent maximum and minimum data points . The relative effect of each codon on mRNA stability in wild - type embryos is shown as a color gradient from red ( destabilizing ) to blue ( stabilizing ) .

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(J) Results of PACE in MZznf598 zebrafish embryos. Black circles show the relative stability of the reporter mRNAs (averages of two biological replicates) in MZznf598 embryos. Gray circles show the relative stability of reporter mRNAs in wild-type embryos The stability of a codon-tag reporter with a UGA stop codon was set to one. Error bars represent maximum and minimum data points. The relative effect of each codon on mRNA stability in wild-type embryos is shown as a color gradient from red (destabilizing) to blue (stabilizing).


( B ) Results of the tandem ORF assay with Lys AAG × 8 and Lys AAA × 8 sequences . ( C ) Results of the tandem ORF assay with Gly × 8 and Pro × 8 sequences . ( D ) Results of the tandem ORF assay with Asn AAC and Asn AAU codon tags . ( E ) Results of the tandem ORF assay using EGFP ORFs with different codon optimalities . Data information : normalized Fluc activity with no insert was set to one . All experiments were repeated three times , and the average Fluc activity is shown as bar charts . Error bars show SD . Individual data points are shown as dots . P - values were calculated using two - sided Student ' s t - test .

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(B) Results of the tandem ORF assay with Lys AAG×8 and Lys AAA×8 sequences. (C) Results of the tandem ORF assay with Gly×8 and Pro×8 sequences. (D) Results of the tandem ORF assay with Asn AAC and Asn AAU codon tags. (E) Results of the tandem ORF assay using EGFP ORFs with different codon optimalities. Data information: normalized Fluc activity with no insert was set to one. All experiments were repeated three times, and the average Fluc activity is shown as bar charts. Error bars show SD. Individual data points are shown as dots. P-values were calculated using two-sided Student's t-test.


X - ray analysis of 6 - months - old H2 - c - fosLTR / Egfrwa / + and H2 - c - fosLTR / Egfrwa / wa littermates . Scale bars :

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X-ray analysis of 6-months-old H2-c-fosLTR/Egfrwa/+ and H2-c-fosLTR/Egfrwa/wa littermates. Scale bars: 1cm


Bone tumor number per mouse at 5 - 6 months of age ( n = 23 wa / + , 15 wa / wa )

Bone tumor number per mouse at 5-6 months of age (n=23 wa/+, 15 wa/wa)


Quantification of tumor size in tibiae at 5 - 6 months of age ( n = 23 wa / + , 15 wa / wa )

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Quantification of tumor size in tibiae at 5-6 months of age (n=23 wa/+, 15 wa/wa)


Alkaline Phosphatase ( ALP ) levels in the serum at endpoint ( age = 5 - 6 months ; n = 16 wa / + , 11 wa / wa )

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Alkaline Phosphatase (ALP) levels in the serum at endpoint (age = 5-6 months; n=16 wa/+, 11 wa/wa)


X - ray analysis before ( age = 2 months ) and after ( age = 7 months ) vehicle or Erlotinib

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X-ray analysis before (age = 2 months) and after (age = 7 months) vehicle or Erlotinib treatment


Quantification of tumor number during treatment ( n = 6 Vehicle , 5 Erlotinib )

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Quantification of tumor number during treatment (n=6 Vehicle, 5 Erlotinib)


Tumor size during treatment ( n = 6 Vehicle , 5 Erlotinib )

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Tumor size during treatment (n=6 Vehicle, 5 Erlotinib)


Analysis of serum ALP levels during treatment ( n = 5 )

Analysis of serum ALP levels during treatment (n=5)


PET summation images ( 0 - 90 min ) in horizontal ( upper panel ) and coronal view ( lower panel ) depicting [ 11C ] Erlotinib distribution in one H2 - c - fosLTR / Egfrwt mouse ( M125 ) . Anatomical structures are labelled with arrows ( T , tumor ; L , liver ; H , heart ; B , brain ) . Scale bars : 1cm Concentration - time curves of [ 11C ] Erlotinib in bone tumors in right scapula of three H2 - c - fosLTR / Egfrwt mice measured with PET . Broken line indicates threshold for in vitro effect of Erlotinib ( 1 µM )

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PET summation images (0-90 min) in horizontal (upper panel) and coronal view (lower panel) depicting [11C]Erlotinib distribution in one H2-c-fosLTR/Egfrwt mouse (M125). Anatomical structures are labelled with arrows (T, tumor; L, liver; H, heart; B, brain). Scale bars: 1cm Concentration-time curves of [11C]Erlotinib in bone tumors in right scapula of three H2-c-fosLTR/Egfrwt mice measured with PET. Broken line indicates threshold for in vitro effect of Erlotinib (1 µM)


X - ray analysis of 7 - months - old H2 - c - fosLTR / Egfrwt and H2 - c - fosLTR / Egfr ∆ Ob littermates . Scale bars :

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X-ray analysis of 7-months-old H2-c-fosLTR/Egfrwt and H2-c-fosLTR/Egfr∆Ob littermates. Scale bars: 1cm


Bone tumor number per mouse at 6 - 7 months of age ( n = 22 wt , 11 ∆ Ob ) Quantification of tumor size in tibiae ( n = 22 wt , 11 ∆ Ob )

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Bone tumor number per mouse at 6-7 months of age (n=22 wt, 11 ∆Ob) Quantification of tumor size in tibiae (n=22 wt, 11 ∆Ob)


ALP levels in the serum at 7 - month endpoint ( n = 17 wt , 8 ∆ Ob )

ALP levels in the serum at 7-month endpoint (n=17 wt, 8 ∆Ob)


µPET / CT analysis of 7 - months - old H2 - c - fosLTR / Egfrwt and H2 - c - fosLTR / Egfr ∆ Ob littermates Standardized uptake values ( SUV ) of the µPET - tracer Na [ 18F ] F in the pelvic osteosarcoma of 4 - and 7 - months - old H2 - c - fosLTR / Egfrwt and H2 - c - fosLTR / Egfr ∆ Ob mice . n = 6 wt , 4 ∆ Ob for 4 month time - point , n = 6 wt , 3 ∆ Ob for 7 month time -

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µPET/CT analysis of 7-months-old H2-c-fosLTR/Egfrwt and H2-c-fosLTR/Egfr∆Ob littermates Standardized uptake values (SUV) of the µPET-tracer Na[18F]F in the pelvic osteosarcoma of 4- and 7-months-old H2-c-fosLTR/Egfrwt and H2-c-fosLTR/Egfr∆Ob mice. n=6 wt, 4 ∆Ob for 4 month time-point, n=6 wt, 3 ∆Ob for 7 month time-point


PCNA and cleaved Caspase 3 IHC staining and quantification shown as % positive cells ( for PCNA ) and as positive cells per mm² ( for cleaved Caspase 3 ) in OS from H2 - c - fosLTR / Egfrwt ( n = 6 ) and H2 - c - fosLTR / Egfr ∆ Ob ( n = 5 ) mice . Scale bars :

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PCNA and cleaved Caspase 3 IHC staining and quantification shown as % positive cells (for PCNA) and as positive cells per mm² (for cleaved Caspase 3) in OS from H2-c-fosLTR/Egfrwt (n=6) and H2-c-fosLTR/Egfr∆Ob (n=5) mice. Scale bars: 100µm


Egfr , Ccnd1 , c - fos and transgenic c - fos ( c - fostg ) mRNA expression levels in tumors of H2 - c - fosLTR / Egfrwt and H2 - c - fosLTR / Egfr ∆ Ob mice normalized to Tbp . n = 17 wt , 14 ∆ Ob ( Egfr , Ccnd1 ) , n = 16 wt , 13 ∆ Ob ( c - fos ) , n = 16 wt , 14 ∆ Ob ( c -

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Egfr, Ccnd1, c-fos and transgenic c-fos (c-fostg) mRNA expression levels in tumors of H2-c-fosLTR/Egfrwt and H2-c-fosLTR/Egfr∆Ob mice normalized to Tbp. n=17 wt, 14 ∆Ob (Egfr, Ccnd1), n=16 wt, 13 ∆Ob (c-fos), n=16 wt, 14 ∆Ob (c-fostg


IHC staining and quantification showing pRSK2 ( n = 4 ) , pCREB ( n = 7 wt , 6 ∆ Ob ) and c - Fos ( n = 5 ) positive cells ( % ) in OS from H2 - c - fosLTR / Egfrwt and H2 - c - fosLTR / Egfr ∆ Ob littermates . Scale bars :

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IHC staining and quantification showing pRSK2 (n=4), pCREB (n=7 wt, 6∆Ob) and c-Fos (n=5) positive cells (%) in OS from H2-c-fosLTR/Egfrwt and H2-c-fosLTR/Egfr∆Ob littermates. Scale bars: 100µm


Western Blot analysis of primary OS cells isolated from H2 ‑ c ‑ fosLTR / Egfrwt and H2 - c - fosLTR / Egfr ∆ Ob

Western Blot analysis of primary OS cells isolated from H2‑c‑fosLTR/Egfrwt and H2-c-fosLTR/Egfr∆Ob mice


c - fos and transgenic c - fos ( c fostg ) mRNA expression levels in primary H2 - c - fosLTR OS cells after 4x in vitro passages , cultured under standard conditions ( n = 3 independent cell lines )

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c-fos and transgenic c-fos (c fostg) mRNA expression levels in primary H2-c-fosLTR OS cells after 4x in vitro passages, cultured under standard conditions (n=3 independent cell lines)


Western Blot analysis of H2 - c - fosLTR / Egfrwt OS cells treated for 24h with

Western Blot analysis of H2-c-fosLTR/Egfrwt OS cells treated for 24h with Erlotinib


c - fos and c - fostg mRNA expression levels in H2 - c - fosLTR / Egfrwt OS cells treated for 24h with Erlotinib ( 10µM ) or DMSO as control ( n = 4 independent cell lines )

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c-fos and c-fostg mRNA expression levels in H2-c-fosLTR/Egfrwt OS cells treated for 24h with Erlotinib (10µM) or DMSO as control (n=4 independent cell lines)


Western Blot analysis of starved H2 - c - fosLTR / Egfrwt OS cells , pre - treated with DMSO ( 1 : 1000 ) , Afatinib ( 5µM ) , GSK2233470 ( 10µM ) , Rapamycin ( 10nM ) or U0126 ( 10µM ) for 30 minutes and stimulated with EGF ( 50ng / ml ) as

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Western Blot analysis of starved H2-c-fosLTR/Egfrwt OS cells, pre-treated with DMSO (1:1000), Afatinib (5µM), GSK2233470 (10µM), Rapamycin (10nM) or U0126 (10µM) for 30 minutes and stimulated with EGF (50ng/ml) as indicated


Western Blot analysis of primary OS cells isolated from a p53f / f Rb1f / f Osx - Cre mouse after 24h Erlotinib

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Western Blot analysis of primary OS cells isolated from a p53f/f Rb1f/f Osx-Cre mouse after 24h Erlotinib treatment


c - fos mRNA expression levels in p53f / f Rb1f / f Osx - Cre OS cells treated for 24h with Erlotinib ( 10µM ) ( n = 3 )

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c-fos mRNA expression levels in p53f/f Rb1f/f Osx-Cre OS cells treated for 24h with Erlotinib (10µM) (n=3)


Western Blot analysis of starved p53f / f Rb1f / f Osx - Cre OS cells , pre - treated with DMSO ( 1 : 1000 ) or Afatinib ( 5µM ) for 30 minutes and stimulated with EGF ( 50ng / ml ) as

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Western Blot analysis of starved p53f/f Rb1f/f Osx-Cre OS cells, pre-treated with DMSO (1:1000) or Afatinib (5µM) for 30 minutes and stimulated with EGF (50ng/ml) as indicated


X - ray analysis of 6 - months - old H2 - c - fosLTR and H2 - c - fosLTR / ColAREG littermates . Scale bars :

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X-ray analysis of 6-months-old H2-c-fosLTR and H2-c-fosLTR/ColAREG littermates. Scale bars: 1cm


Bone tumor number per mouse at 5 - 6 months of age ( n = 22 wt , 13 ColAREG )

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Bone tumor number per mouse at 5-6 months of age (n=22 wt, 13 ColAREG)


Quantification of tumor size in tibiae at 5 - 6 months of age ( n = 22 wt , 13 ColAREG )

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Quantification of tumor size in tibiae at 5-6 months of age (n=22 wt, 13 ColAREG)


Alkaline Phosphatase ( ALP ) levels in the serum at endpoint ( age = 5 - 6 months ; n = 29 wt , 19 ColAREG )

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Alkaline Phosphatase (ALP) levels in the serum at endpoint (age = 5-6 months; n=29 wt, 19 ColAREG)


c - fos mRNA expression levels in osteosarcomas of H2 - c - fosLTR ( n = 11 ) and H2 - c - fosLTR / ColAREG mice ( n = 14 )

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c-fos mRNA expression levels in osteosarcomas of H2-c-fosLTR (n=11) and H2-c-fosLTR/ColAREG mice (n=14)


Western Blot analysis of bone tumor protein lysates from H2 - c - fosLTR and H2 - c - fosLTR / ColAREG

Western Blot analysis of bone tumor protein lysates from H2-c-fosLTR and H2-c-fosLTR/ColAREG mice


Representative images of OS biopsies stained with antibodies against EGFR and c ‑ Fos . Scale bars :

Representative images of OS biopsies stained with antibodies against EGFR and c‑Fos. Scale bars: 50µm


Kaplan - Meier survival curve comparing the survival of patients with EGFR and c - Fos double positive osteosarcomas against patients without co - expression of both proteins . ( n = 52 )

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Kaplan-Meier survival curve comparing the survival of patients with EGFR and c-Fos double positive osteosarcomas against patients without co-expression of both proteins. (n=52)


Western Blot analysis showing EGFR and c - Fos protein levels in human OS cell lines cultured under standard

Western Blot analysis showing EGFR and c-Fos protein levels in human OS cell lines cultured under standard conditions


Cell viability of 143b or LM7 cells cultured for 24h in medium ( + 10 % FCS ) with DMSO ( 1 : 2000 ) or Erlotinib ( 10µM ) ( n = 3 , representative result from 3 independent experiments , shown as fold change , normalized to DMSO )

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Cell viability of 143b or LM7 cells cultured for 24h in medium (+10% FCS) with DMSO (1:2000) or Erlotinib (10µM) (n=3, representative result from 3 independent experiments, shown as fold change, normalized to DMSO)


Cell viability of 143b or LM7 cells cultured for 24h in medium ( + 0 . 5 % FCS ) containing 1xPBS ( contr . , 1 : 200 ) or EGF ( 50ng / ml ) ( n = 3 , representative result from 3 independent experiments , shown as fold change , normalized to control )

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Cell viability of 143b or LM7 cells cultured for 24h in medium (+0.5% FCS) containing 1xPBS (contr., 1:200) or EGF (50ng/ml) (n=3, representative result from 3 independent experiments, shown as fold change, normalized to control)


143b xenograft growth curve during therapeutic regime ( n = 8 vehicle , 6 Erlotinib ; two independent experiments ) 143b tumor weight at endpoint ( n = 8 Vehicle , 6 Erlotinib ; two independent experiments )

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143b xenograft growth curve during therapeutic regime (n=8 vehicle, 6 Erlotinib; two independent experiments) 143b tumor weight at endpoint (n=8 Vehicle, 6 Erlotinib; two independent experiments)


LM7 xenograft growth curve during therapeutic regime ( n = 7 Vehicle , 5 Erlotinib ; two independent experiments ) LM7 tumor weight at endpoint . n = 7 vehicle , 5 Erlotinib , two independent

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LM7 xenograft growth curve during therapeutic regime (n=7 Vehicle, 5 Erlotinib; two independent experiments) LM7 tumor weight at endpoint. n=7 vehicle, 5 Erlotinib, two independent experiment


IHC analysis of PCNA and cleaved Caspase 3 in 143b - derived tumors ( n = 4 ) . Scale bars : 50µm IHC analysis of pCREB and c - Fos in 143b - derived tumors . ( n = 5 for pCREB and n = 4 for c - Fos analysis ) . Scale bars :

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IHC analysis of PCNA and cleaved Caspase 3 in 143b-derived tumors (n=4). Scale bars: 50µm IHC analysis of pCREB and c-Fos in 143b-derived tumors. (n=5 for pCREB and n=4 for c-Fos analysis). Scale bars: 50µm


Western Blot analysis of pCREB / CREB and p - c - Fos / c - Fos protein expression in lysates directly isolated from 143b xenografts at endpoint , after 14 days of vehicle or Erlotinib

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Western Blot analysis of pCREB/CREB and p-c-Fos/c-Fos protein expression in lysates directly isolated from 143b xenografts at endpoint, after 14 days of vehicle or Erlotinib treatment


( a ) Western blot analysis of HeLa cells treated with siRNA for 84 h .

(a) Western blot analysis of HeLa cells treated with siRNA for 84 h.


( b ) Western blot analysis of HeLa cells treated with a second independent siRNA targeting ATG5 , ATG7 or NDP52 . GAPDH and total protein stained with Coomassie blue serve as loading controls .

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(b) Western blot analysis of HeLa cells treated with a second independent siRNA targeting ATG5, ATG7 or NDP52. GAPDH and total protein stained with Coomassie blue serve as loading controls.


( c ) Western blot analysis of HeLa cells cultured in Hank ' s buffered salt solution ( Starved ) or DMEM + 10 % FBS ( Serum ) .

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(c) Western blot analysis of HeLa cells cultured in Hank's buffered salt solution (Starved) or DMEM+ 10% FBS (Serum).


( d ) Western blot analysis of HeLa cells treated ( 48 h ) with an inhibitor of mTORC1 ( RAP , 20 nM ) or control ( DMSO ) .

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(d) Western blot analysis of HeLa cells treated (48 h) with an inhibitor of mTORC1 (RAP, 20 nM) or control (DMSO).


( e ) Western blot analysis comparing the levels of DICER in MDA - 231 , T47D and MDA - 435 cells .

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(e) Western blot analysis comparing the levels of DICER in MDA-231, T47D and MDA-435 cells.


( f ) Western blot analysis of MDA - 231 , T47D and MDA - 435 cells treated ( 48 h ) with inhibitors of mTORC1 ( RAP , 20 nM ) , mTORC1 and 2 ( PP242 , 100 nM ) , or DMSO ( control ) .

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(f) Western blot analysis of MDA-231, T47D and MDA-435 cells treated (48 h) with inhibitors of mTORC1 (RAP, 20 nM), mTORC1 and 2 (PP242, 100 nM), or DMSO (control).


( g ) Western blot analysis of HeLa cells treated with CQ or DMSO for 12 h .

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(g) Western blot analysis of HeLa cells treated with CQ or DMSO for 12 h.


( h ) Western blot analysis of HeLa cells treated with BAF ( 200 nM , 12 h ) , RAP ( 20 nM , 24 h ) , or RAP and BAF . The results in a - h are representative of three experiments and use α - tubulin ( TUBA ) as a loading control .

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(h) Western blot analysis of HeLa cells treated with BAF (200 nM, 12 h), RAP (20 nM, 24 h), or RAP and BAF. The results in a-h are representative of three experiments and use α-tubulin (TUBA) as a loading control.


( i ) Western blot analysis of DICER ( monoclonal antibody 13D6 ) or control ( EEF1A1 ) in HeLa cells treated with control siRNA or siRNA targeting DICER .

(i) Western blot analysis of DICER (monoclonal antibody 13D6) or control (EEF1A1) in HeLa cells treated with control siRNA or siRNA targeting DICER.


( j ) Confocal analysis of the signal from an anti - DICER monoclonal antibody ( clone 13D6 ) or an isotype control monoclonal antibody in HeLa cells . Scale bar , 10 μm .

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(j) Confocal analysis of the signal from an anti-DICER monoclonal antibody (clone 13D6) or an isotype control monoclonal antibody in HeLa cells. Scale bar, 10 μm.


( k , l ) RT - qPCR analysis of AGO1 , AGO2 and DICER mRNA in HeLa cells treated for 12 h with DMSO , BAF or CQ ( k ) or siRNA for 84 h ( l ) . Error bars show s . e . m . Uncropped images of blots are shown in Supplementary Fig . S3 .

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(k,l) RT-qPCR analysis of AGO1, AGO2 and DICER mRNA in HeLa cells treated for 12 h with DMSO, BAF or CQ (k) or siRNA for 84 h (l). Error bars show s.e.m. Uncropped images of blots are shown in Supplementary Fig. S3.


( a ) Localization using confocal microscopy of endogenous DICER ( monoclonal antibody 13D6 ) and NDP52 in HeLa cells treated with RAP or control . The arrows highlight some examples of co - localization . The lower left insets show higher magnifications of the areas outlined in the main panel . Scale bars , 5 μm . ( b ) Quantification of DICER co - localization with NDP52 . Total fluorescence intensity of DICER co - localized with punctae ( > 0 . 2 μm3 ) of NDP52 was quantified with Volocity software in RAP - treated cells / control - treated cells over several Z - stacked microscope fields per experiment ( n = 4 , error bars s . e . m . ) .

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

(a) Localization using confocal microscopy of endogenous DICER (monoclonal antibody 13D6) and NDP52 in HeLa cells treated with RAP or control. The arrows highlight some examples of co-localization. The lower left insets show higher magnifications of the areas outlined in the main panel. Scale bars, 5 μm. (b) Quantification of DICER co-localization with NDP52. Total fluorescence intensity of DICER co-localized with punctae (>0.2 μm3) of NDP52 was quantified with Volocity software in RAP-treated cells/control-treated cells over several Z-stacked microscope fields per experiment (n = 4, error bars s.e.m.).


( c ) Localization using confocal microscopy of endogenous DICER ( monoclonal antibody 13D6 ) and HcRed - LC3 in HeLa cells treated with BAF or control . The arrows highlight some examples of co - localization . Insets as in a . Scale bar , 5 μm . ( d ) Quantification of DICER co - localization with HcRed - LC3 was performed as for NDP52 in b .

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(c) Localization using confocal microscopy of endogenous DICER (monoclonal antibody 13D6) and HcRed-LC3 in HeLa cells treated with BAF or control. The arrows highlight some examples of co-localization. Insets as in a. Scale bar, 5 μm. (d) Quantification of DICER co-localization with HcRed-LC3 was performed as for NDP52 in b.


( e ) Confocal microscopy analysis of GFP - DCP1A and HcRed - LC3wt in HeLa cells . Scale bar , 10 μm .

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(e) Confocal microscopy analysis of GFP-DCP1A and HcRed-LC3wt in HeLa cells. Scale bar, 10 μm.


( f - i ) Localization of DICER ( monoclonal antibody 13D6 ) detected with anti - mouse IgG ( 10 nm gold beads ) by electron microscopy in CQ - ( 20 μM , 12 h ) treated HeLa cells . Mito , mitochondria ; MVB , multivesicular body . The black arrows highlight all gold beads .

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(f-i) Localization of DICER (monoclonal antibody 13D6) detected with anti-mouse IgG (10 nm gold beads) by electron microscopy in CQ- (20 μM, 12 h) treated HeLa cells. Mito, mitochondria; MVB, multivesicular body. The black arrows highlight all gold beads.


( j ) Western blot analysis of fractions from a discontinuous gradient of Histodenz ( 15 % , 20 % , 24 % , 26 % ) described to enrich autophagosomes ( light AV ) and autophagolysosomes ( heavy AV ; ref . ) . HEK293T cells were treated with CQ ( 20 μM , 16 h ) . Fractions enriched in multivesicular body and lysosomal markers are indicated . Material that was pelleted by centrifugation at 100 , 000g ( 100 K pellet ) or that remained in solution after the 100 , 000g spin ( soluble ) was not added to the gradient .

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(j) Western blot analysis of fractions from a discontinuous gradient of Histodenz (15%, 20%, 24%, 26%) described to enrich autophagosomes (light AV) and autophagolysosomes (heavy AV; ref. ). HEK293T cells were treated with CQ (20 μM, 16 h). Fractions enriched in multivesicular body and lysosomal markers are indicated. Material that was pelleted by centrifugation at 100,000g (100 K pellet) or that remained in solution after the 100,000g spin (soluble) was not added to the gradient.


( k ) Western blot analysis of UB and p62 in discontinuous Histodenz gradients ( as in j ) of HEK293T cells treated with control ( DMSO ) or CQ . Uncropped images of blots are shown in Supplementary Fig . S3 .

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(k) Western blot analysis of UB and p62 in discontinuous Histodenz gradients (as in j) of HEK293T cells treated with control (DMSO) or CQ. Uncropped images of blots are shown in Supplementary Fig. S3.


( a ) Western blot analysis of GEMIN4 in fractions from a discontinuous gradient of Histodenz ( 15 % , 20 % , 24 % , 26 % ) described to enrich autophagosomes ( light AV ) and autophagolysosomes ( heavy AV ) . HEK293T cells were treated with CQ ( 20 μM , 16 h ) . Fractions enriched in multivesicular body ( MVB ) and lysosomal markers are indicated .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Western blot analysis of GEMIN4 in fractions from a discontinuous gradient of Histodenz (15%, 20%, 24%, 26%) described to enrich autophagosomes (light AV) and autophagolysosomes (heavy AV). HEK293T cells were treated with CQ (20 μM, 16 h). Fractions enriched in multivesicular body (MVB) and lysosomal markers are indicated.


( b ) Western blot of GEMIN4 or loading control ( TUBA ) in HeLa cells treated with control siRNA or siRNA targeting ATG5 , ATG6 or ATG7 .

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(b) Western blot of GEMIN4 or loading control (TUBA) in HeLa cells treated with control siRNA or siRNA targeting ATG5, ATG6 or ATG7.


( c ) Western blot analysis of GEMIN4 or control ( TUBA ) in HeLa cells cultured in DMEM 10 % FBS ( Serum ) , Hank ' s buffered salt solution ( Starve ) for 2 h , or treated for 16 h with control ( DMSO ) , BAF ( 200 nM ) , RAP ( 20 nM ) , RAP + BAF , or PP242 ( 100 nM ) .

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(c) Western blot analysis of GEMIN4 or control (TUBA) in HeLa cells cultured in DMEM 10% FBS (Serum), Hank's buffered salt solution (Starve) for 2 h, or treated for 16 h with control (DMSO), BAF (200 nM), RAP (20 nM), RAP+BAF, or PP242 (100 nM).


( d ) Top , western blot of GEMIN4 immunoprecipitates ( IP ) from HeLa cells . Bottom , western blot analysis of GEMIN4 in immunoprecipitates of anti - NDP52 , - p62 or control antibody from HeLa cells .

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(d) Top, western blot of GEMIN4 immunoprecipitates (IP) from HeLa cells. Bottom, western blot analysis of GEMIN4 in immunoprecipitates of anti-NDP52, -p62 or control antibody from HeLa cells.


( e ) Western blot of DICER immunoprecipitates from HeLa cells .

(e) Western blot of DICER immunoprecipitates from HeLa cells.


( f ) Western blot of AGO2 immunoprecipitates from HeLa cells .

(f) Western blot of AGO2 immunoprecipitates from HeLa cells.


( g ) Western blot analysis of GEMIN4 , compared with loading controls ( DCP1A , CLN3 ) in HeLa cells treated with control siRNA or siRNA targeting GEMIN3 and GEMIN4 .

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(g) Western blot analysis of GEMIN4, compared with loading controls (DCP1A, CLN3) in HeLa cells treated with control siRNA or siRNA targeting GEMIN3 and GEMIN4.


( h ) Western blot analysis of HeLa cells treated with indicated combinations of control siRNA or siRNA targeting GEMIN3 , GEMIN4 and ATG5 . Western blot analysis of CLN3 and DCP1A are used to demonstrate equal loading of wells .

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(h) Western blot analysis of HeLa cells treated with indicated combinations of control siRNA or siRNA targeting GEMIN3, GEMIN4 and ATG5. Western blot analysis of CLN3 and DCP1A are used to demonstrate equal loading of wells.


( i ) Western blot analysis with anti - UB monoclonal antibody ( mono - and polyubiquitylated proteins , FK2 clone ) in Flag immunoprecipitates performed under stringent denaturing conditions . Flag - AGO2 was induced for 24 h with tetracycline in HEK293T cells with Tet - inducible Flag - AGO2 stably integrated that had been treated 24 h before with the indicated siRNAs .

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(i) Western blot analysis with anti-UB monoclonal antibody (mono- and polyubiquitylated proteins, FK2 clone) in Flag immunoprecipitates performed under stringent denaturing conditions. Flag-AGO2 was induced for 24 h with tetracycline in HEK293T cells with Tet-inducible Flag-AGO2 stably integrated that had been treated 24 h before with the indicated siRNAs.


( j ) Western blot analysis of HeLa cells treated with control siRNA or siRNA targeting ATG5 for 24 h . TUBA , α - tubulin loading control .

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(j) Western blot analysis of HeLa cells treated with control siRNA or siRNA targeting ATG5 for 24 h. TUBA, α-tubulin loading control.


( k ) Top , RT - qPCR analysis of miR - 16 or let - 7a levels in AGO2 immunoprecipitates from DMSO - and BAF - treated HeLa cells . Error bars represent s . e . m . Bottom , a representative western blot of AGO2 immunoprecipitates used in one replicate of these experiments . Uncropped images of blots are shown in Supplementary Fig . S3 .

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(k) Top, RT-qPCR analysis of miR-16 or let-7a levels in AGO2 immunoprecipitates from DMSO- and BAF-treated HeLa cells. Error bars represent s.e.m. Bottom, a representative western blot of AGO2 immunoprecipitates used in one replicate of these experiments. Uncropped images of blots are shown in Supplementary Fig. S3.


( a ) Northern blot of miR - 16 and let - 7a in HeLa cells treated with siRNA for 84 h . Levels of U6 , ethidium - bromide - stained total RNA on northern membrane ( loading ) and 28S and 18S ribosomal RNA on 1 % agarose gel are shown as controls for equal loading .

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(a) Northern blot of miR-16 and let-7a in HeLa cells treated with siRNA for 84 h. Levels of U6, ethidium-bromide-stained total RNA on northern membrane (loading) and 28S and 18S ribosomal RNA on 1% agarose gel are shown as controls for equal loading.


( b - d ) RT - qPCR analysis of let - 7a and miR - 16 ( b ) , pre - let - 7a and pre - miR - 16 ( c ) and let - 7a * and miR - 16 * ( d ) in HeLa cells depleted of NDP52 or ATG5 with siRNA ( 84 h ) . n = 3 independent experiments ; error bars represent s . e . m .

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(b-d) RT-qPCR analysis of let-7a and miR-16 (b), pre-let-7a and pre-miR-16 (c) and let-7a* and miR-16* (d) in HeLa cells depleted of NDP52 or ATG5 with siRNA (84 h). n = 3 independent experiments; error bars represent s.e.m.


( e ) Quantification of fluorescence signal in AGO2 immunoprecipitates from HeLa cell extracts incubated with duplexes of let - 7a - let - 7a * labelled on the 3 ′ end of either let - 7 or let - 7a * with FITC . Cells were treated 16 h before with DMSO ( control ) , BAF ( 200 nm ) or geldanamycin ( 10 μM ) . Representative results of one experiment are shown .

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(e) Quantification of fluorescence signal in AGO2 immunoprecipitates from HeLa cell extracts incubated with duplexes of let-7a-let-7a* labelled on the 3′ end of either let-7 or let-7a* with FITC. Cells were treated 16 h before with DMSO (control), BAF (200 nm) or geldanamycin (10 μM). Representative results of one experiment are shown.


( f ) Western blot analysis of AGO2 immunoprecipitates ( IP ) used in e .

(f) Western blot analysis of AGO2 immunoprecipitates (IP) used in e.


( g ) Normalized results of four independent experiments ( n = 4 ) identical to e . Error bars represent s . e . m . Uncropped images of blots are shown in Supplementary Fig . S3 .

(g) Normalized results of four independent experiments (n = 4) identical to e. Error bars represent s.e.m. Uncropped images of blots are shown in Supplementary Fig. S3.