Figure 4 ( A ) BI ‐ 1 WT and KO MEFs were treated with EBSS for 2 h , and the levels of phospho ‐ p70S6k were determined by western blot analysis . Total p70S6k is also shown . ( B ) LC3 was monitored by immunofluorescence in cells treated with EBSS for 3 h in the presence or absence of 10 μM JNK inhibitor SP600125 . Mean and standard deviation are presented ( N = 3 ) . Student ' s t ‐ test was used to analyse statistical significance , * P0 . 001 and * * P0 . 0001 . ( C ) BI ‐ 1 WT and KO cells were treated with EBSS for indicated time points , in the presence or absence of 10 μM of the JNK inhibitor SP600125 . Levels of phosphorylation of JNK ( pJNK ) and LC3 ‐ II were determined by western blot . The levels of total JNK and Hsp90 are shown as control ( N = 4 ) . Image was assembled from cropped lanes of the same western blot analysis of the same gel . ( D ) BI ‐ 1 WT and KO cells were treated with EBSS for indicated time points in the presence or absence of 10 μM SP600125 , and the electrophoretic shift associated with BCL ‐ 2 phosphorylation was monitored by western blot . ( E ) BI ‐ 1 WT and KO cells were co ‐ transfected with expression vectors for Beclin ‐ 1 - MYC , BI ‐ 1 - HA , and BCL ‐ XL - FLAG . After 24 h , cells were treated with EBSS for 2 h or left untreated . The association of MYC ‐ tagged expressed Beclin ‐ 1 and BCL ‐ XL - FLAG was assessed by immunoprecipitation of Beclin ‐ 1 followed by western blot analysis . ( F ) 293T cells were co ‐ transfected with expression vectors for Beclin ‐ 1 - MYC , BI ‐ 1 - HA , and BCL ‐ XL - FLAG . Cell extracts were prepared in CHAPS buffer and Beclin ‐ 1 - MYC immunoprecipitated , and the possible co ‐ precipitation of BI ‐ 1 - HA , and BCL ‐ XL - FLAG was assessed by western blot analysis ( N = 3 ) . Asterisks indicate BI ‐ 1 oligomers . ( G ) ( G ) 293T cells were co ‐ transfected as described in ( F ) and BI ‐ 1 - HA was immunoprecipitated , and the possible co ‐ precipitation of Beclin ‐ 1 - MYC , and BCL ‐ XL - FLAG determined by western blot analysis ( N = 3 ) . Figure source data can be found with the Supplementary Information .

O O B-PANEL_START O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(A) BI‐1 WT and KO MEFs were treated with EBSS for 2 h, and the levels of phospho‐p70S6k were determined by western blot analysis. Total p70S6k is also shown.(B) LC3 was monitored by immunofluorescence in cells treated with EBSS for 3 h in the presence or absence of 10 μM JNK inhibitor SP600125. Mean and standard deviation are presented (N=3). Student's t‐test was used to analyse statistical significance, *P0.001 and **P0.0001.(C) BI‐1 WT and KO cells were treated with EBSS for indicated time points, in the presence or absence of 10 μM of the JNK inhibitor SP600125. Levels of phosphorylation of JNK (pJNK) and LC3‐II were determined by western blot. The levels of total JNK and Hsp90 are shown as control (N=4). Image was assembled from cropped lanes of the same western blot analysis of the same gel.(D) BI‐1 WT and KO cells were treated with EBSS for indicated time points in the presence or absence of 10 μM SP600125, and the electrophoretic shift associated with BCL‐2 phosphorylation was monitored by western blot.(E) BI‐1 WT and KO cells were co‐transfected with expression vectors for Beclin‐1-MYC, BI‐1-HA, and BCL‐XL-FLAG. After 24 h, cells were treated with EBSS for 2 h or left untreated. The association of MYC‐tagged expressed Beclin‐1 and BCL‐XL-FLAG was assessed by immunoprecipitation of Beclin‐1 followed by western blot analysis.(F) 293T cells were co‐transfected with expression vectors for Beclin‐1-MYC, BI‐1-HA, and BCL‐XL-FLAG. Cell extracts were prepared in CHAPS buffer and Beclin‐1-MYC immunoprecipitated, and the possible co‐precipitation of BI‐1-HA, and BCL‐XL-FLAG was assessed by western blot analysis (N=3). Asterisks indicate BI‐1 oligomers.(G) (G) 293T cells were co‐transfected as described in (F) and BI‐1-HA was immunoprecipitated, and the possible co‐precipitation of Beclin‐1-MYC, and BCL‐XL-FLAG determined by western blot analysis (N=3). Figure source data can be found with the Supplementary Information.


Figure 5 ( A ) BI ‐ 1 KO MEFs were stably transduced with lentiviral vectors expressing an shRNA against beclin ‐ 1 ( shBeclin ‐ 1 ) or ire1α ( shIRE1α ) mRNA or luciferase ( shLuc ) as control . The levels of LC3 , Beclin ‐ 1 , IRE1α and Hsp90 were monitored by western blot . ( B ) BI ‐ 1 KO MEFs were stably transduced with lentiviral vectors as described in A . Cells were exposed to EBSS for 3 h , and then LC3 levels were analysed by western blot . Image was assembled from cropped lanes of the same western blot analysis . ( C ) Endogenous LC3 distribution was visualized using immunofluorescence and confocal microscopy in BI ‐ 1 KO / shLuc and BI ‐ 1 KO / shBeclin ‐ 1 cells . Quantification represents the visualization of at least 180 cells . Student ' s t ‐ test was used to analyse statistical significance . Mean and standard deviation are presented , * P0 . 001 , NS : non ‐ significant . ( D ) LC3 was visualized and quantified in BI ‐ 1 KO / shLuc and BI ‐ 1 KO / shIRE1α cells described in ( B ) by immunofluorescence and confocal microscopy analysis . ( E ) BI ‐ 1 KO cells were transiently transfected with a TRAF2 dominant ‐ negative ( TRAF2 ‐ DN ) construct or empty vector ( mock ) , and after 48 h cells were stimulated with EBSS and the levels of LC3 ‐ II and Hsp90 were monitored by western blot . Right panel : quantification of relative LC3 ‐ II levels normalized with Hsp90 and non ‐ treated cells . Mean and standard deviation are presented . ( F ) 293T cells were co ‐ transfected with expression vectors for HA ‐ tagged IRE1α ( IRE1 - HA ) , TRAF2 - FLAG , and MYC ‐ tagged BI ‐ 1 ( BI1 - MYC ) . After 48 h of transfection , HA ‐ tagged proteins were immunoprecipitated and the possible interaction with TRAF2 was analysed by western blot . Right panel : the percentage of TRAF2 dissociation from IRE1α by the presence or absence of BI ‐ 1 was quantified and normalized with the expression levels observed in the inputs . For comparison , the co ‐ IP signal observed in the absence of BI ‐ 1 was normalized as 100 % co ‐ IP in each independent experiment ( N = 3 ) . Mean and standard deviation are presented , * P0 . 05 . Figure source data can be found with the Supplementary Information .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(A) BI‐1 KO MEFs were stably transduced with lentiviral vectors expressing an shRNA against beclin‐1 (shBeclin‐1) or ire1α (shIRE1α) mRNA or luciferase (shLuc) as control. The levels of LC3, Beclin‐1, IRE1α and Hsp90 were monitored by western blot.(B) BI‐1 KO MEFs were stably transduced with lentiviral vectors as described in A. Cells were exposed to EBSS for 3 h, and then LC3 levels were analysed by western blot. Image was assembled from cropped lanes of the same western blot analysis.(C) Endogenous LC3 distribution was visualized using immunofluorescence and confocal microscopy in BI‐1 KO/shLuc and BI‐1 KO/shBeclin‐1 cells. Quantification represents the visualization of at least 180 cells. Student's t‐test was used to analyse statistical significance. Mean and standard deviation are presented, *P0.001, NS: non‐significant.(D) LC3 was visualized and quantified in BI‐1 KO/shLuc and BI‐1 KO/shIRE1α cells described in (B) by immunofluorescence and confocal microscopy analysis.(E) BI‐1 KO cells were transiently transfected with a TRAF2 dominant‐negative (TRAF2‐DN) construct or empty vector (mock), and after 48 h cells were stimulated with EBSS and the levels of LC3‐II and Hsp90 were monitored by western blot. Right panel: quantification of relative LC3‐II levels normalized with Hsp90 and non‐treated cells. Mean and standard deviation are presented.(F) 293T cells were co‐transfected with expression vectors for HA‐tagged IRE1α (IRE1-HA), TRAF2-FLAG, and MYC‐tagged BI‐1 (BI1-MYC). After 48 h of transfection, HA‐tagged proteins were immunoprecipitated and the possible interaction with TRAF2 was analysed by western blot. Right panel: the percentage of TRAF2 dissociation from IRE1α by the presence or absence of BI‐1 was quantified and normalized with the expression levels observed in the inputs. For comparison, the co‐IP signal observed in the absence of BI‐1 was normalized as 100% co‐IP in each independent experiment (N=3). Mean and standard deviation are presented, *P0.05. Figure source data can be found with the Supplementary Information.


Figure 6 ( A ) The expression of dBI ‐ 1 was knocked down in Drosophila melanogaster . Then , relative expression levels of dBI ‐ 1 mRNA were monitored by semiquantitative PCR . Actin levels were monitored as control . ( B ) ( B ) LC3 levels were monitored in control ( Da ‐ Gal4 > huLC3 : GFP ) or dBI ‐ 1 RNAi larvae ( Da ‐ Gal4 > huLC3 : GFP , Dcr2 , dBI ‐ 1i ) under basal or fasting conditions . Then , huLC3 - GFP levels were analysed by western blot . In addition , dBI ‐ 1i larvae were treated with 100 μM SP600125 ( added in the growing media ) . ( C ) Left panel : the presence of LC3 ‐ positive vesicles ( white arrowheads ) was monitored by confocal microscopy in control ( Da ‐ Gal4 > huLC3 > GFP ) and dBI ‐ 1 knockdown pupae ( Da ‐ Gal4 > huLC3 : GFP , Dcr2 , dBI ‐ 1i ) after 6 h of puparium formation . The organization of actin cytoskeleton was monitored by staining with phalloidin ( Ph , red ) . Nucleus was stained with Topro ( blue ) . Scale bar : left 20 μm and right 11 μm . Right panel : overexpression of dBI ‐ 1 ( dBI ‐ 1EY03662 ) delays salivary gland degradation . Overexpression of dBI ‐ 1 was confirmed by semiquantitative RT - PCR . Actin levels were monitored as loading control . Right panel : wild ‐ type control and dBI ‐ 1EY03662 pupae were analysed at 14 h after puparium formation . Superficial and internal confocal planes of the cells are presented . Scale bar : 40 μm . ( D ) Wild ‐ type control and dBI ‐ 1EY03662 larvae were cultured in fasting conditions for different periods of time and fat body stained with lysotracker ( red ) and Hoechst ( blue ) . Then , lysosomal content in the fat body was visualized by epifluorescence microscopy . The percentage of cells presenting lysotracker ‐ positive stain is indicated . Scale bar : 50 μm . ( E ) Control or dBI ‐ 1 knockdown adult flies were exposed to nutrient starvation and then animal viability was monitored over time for several days . In all , 100 individuals were monitored in each condition . Data represent mean and standard error ( N = 3 ) . Two ‐ way ANOVA was used to analyse statistical significance between groups . ( F ) Second instar dBI ‐ 1 RNAi or control larvae were grown in food supplemented with 25 μg / ml Tm dissolved in DMSO or 0 . 5 % DMSO as control . The number of individual reaching the adult fly stage was evaluated . Mean and standard error are presented ( N = 3 ) , * * P0 . 01 .

O O B-PANEL_START O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(A) The expression of dBI‐1 was knocked down in Drosophila melanogaster. Then, relative expression levels of dBI‐1 mRNA were monitored by semiquantitative PCR. Actin levels were monitored as control.(B) (B) LC3 levels were monitored in control (Da‐Gal4>huLC3:GFP) or dBI‐1 RNAi larvae (Da‐Gal4>huLC3:GFP, Dcr2, dBI‐1i) under basal or fasting conditions. Then, huLC3-GFP levels were analysed by western blot. In addition, dBI‐1i larvae were treated with 100 μM SP600125 (added in the growing media).(C) Left panel: the presence of LC3‐positive vesicles (white arrowheads) was monitored by confocal microscopy in control (Da‐Gal4>huLC3>GFP) and dBI‐1 knockdown pupae (Da‐Gal4>huLC3:GFP, Dcr2, dBI‐1i) after 6 h of puparium formation. The organization of actin cytoskeleton was monitored by staining with phalloidin (Ph, red). Nucleus was stained with Topro (blue). Scale bar: left 20 μm and right 11 μm. Right panel: overexpression of dBI‐1 (dBI‐1EY03662) delays salivary gland degradation. Overexpression of dBI‐1 was confirmed by semiquantitative RT-PCR. Actin levels were monitored as loading control. Right panel: wild‐type control and dBI‐1EY03662 pupae were analysed at 14 h after puparium formation. Superficial and internal confocal planes of the cells are presented. Scale bar: 40 μm.(D) Wild‐type control and dBI‐1EY03662 larvae were cultured in fasting conditions for different periods of time and fat body stained with lysotracker (red) and Hoechst (blue). Then, lysosomal content in the fat body was visualized by epifluorescence microscopy. The percentage of cells presenting lysotracker‐positive stain is indicated. Scale bar: 50 μm.(E) Control or dBI‐1 knockdown adult flies were exposed to nutrient starvation and then animal viability was monitored over time for several days. In all, 100 individuals were monitored in each condition. Data represent mean and standard error (N=3). Two‐way ANOVA was used to analyse statistical significance between groups.(F) Second instar dBI‐1 RNAi or control larvae were grown in food supplemented with 25 μg/ml Tm dissolved in DMSO or 0.5% DMSO as control. The number of individual reaching the adult fly stage was evaluated. Mean and standard error are presented (N=3), **P0.01.


Figure 7 ( A ) The levels of LC3 , Atg5 / Atg12 complex , Beclin ‐ 1 , Bcl ‐ 2 , Bcl ‐ XL , IRE1α and Hsp90 were monitored in liver protein extracts of bi ‐ 1 + / + and bi ‐ 1 − / − at 6 ‐ month ‐ old mice . Each well represents independent mice . ( B ) A similar analysis was performed for indicated markers in kidney protein extracts as described in liver . ( C ) bi ‐ 1 + / + and bi ‐ 1 − / − mice were injected with 50 ng / ml of tunicamycin by intraperitoneal injection ( N = 3 ) and then LC3 levels were monitored in liver protein extracts by western blot analysis . Right panel : quantification of relative LC3 ‐ II levels , * * P0 . 005 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7(A) The levels of LC3, Atg5/Atg12 complex, Beclin‐1, Bcl‐2, Bcl‐XL, IRE1α and Hsp90 were monitored in liver protein extracts of bi‐1+/+ and bi‐1−/− at 6‐month‐old mice. Each well represents independent mice.(B) A similar analysis was performed for indicated markers in kidney protein extracts as described in liver.(C) bi‐1+/+ and bi‐1−/− mice were injected with 50 ng/ml of tunicamycin by intraperitoneal injection (N=3) and then LC3 levels were monitored in liver protein extracts by western blot analysis. Right panel: quantification of relative LC3‐II levels, **P0.005.


Figure 1A Heatmap showing the unsupervised consensus clustering of TCGA melanomas ( n = 445 : 356 metastases and 89 primary melanomas ) . The identified clusters are indicated below the heatmap : cluster 1 ( n = 55 , red ) , cluster 2 ( n = 304 , blue ) and cluster 3 ( n = 86 , green ) . Type of melanoma samples ( primary or metastatic ) and of metastatic site ( subcutaneous , lymph node or distant ) within each cluster are indicated below and above the heatmap , respectively . B Heatmap showing the unsupervised consensus clustering of TCGA primary melanomas ( n = 89 ) using the 1 , 000 most variable expressed genes in the same dataset . The identified signature clearly divides the TCGA dataset in two groups : Primary - A ( n = 34 , red ) and Primary - B ( n = 55 , pink ) , as indicated . C Boxplots showing the distributions of the normalized rank - sums of the up - regulated ( left panel ) and down - regulated ( right panel ) genes in the identified signature . Down - regulated genes in Primary - B melanomas better discriminate the two groups of primary melanoma samples ( p = 5 . 9e - 15 , Wilcoxon rank - sum test ) . Median , minimum , maximum , 25th , 75th whiskers of ranksum distribution are shown .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A Heatmap showing the unsupervised consensus clustering of TCGA melanomas (n=445: 356 metastases and 89 primary melanomas). The identified clusters are indicated below the heatmap: cluster 1 (n=55, red), cluster 2 (n=304, blue) and cluster 3 (n=86, green). Type of melanoma samples (primary or metastatic) and of metastatic site (subcutaneous, lymph node or distant) within each cluster are indicated below and above the heatmap, respectively. B Heatmap showing the unsupervised consensus clustering of TCGA primary melanomas (n=89) using the 1,000 most variable expressed genes in the same dataset. The identified signature clearly divides the TCGA dataset in two groups: Primary-A (n=34, red) and Primary-B (n=55, pink), as indicated. C Boxplots showing the distributions of the normalized rank-sums of the up-regulated (left panel) and down-regulated (right panel) genes in the identified signature. Down-regulated genes in Primary-B melanomas better discriminate the two groups of primary melanoma samples (p=5.9e-15, Wilcoxon rank-sum test). Median, minimum, maximum, 25th, 75th whiskers of ranksum distribution are shown.


Figure 2A Box and whiskers ( from 5th to 95th percentile ) plots depicting TINCR RNA levels in the TCGA SKCM RNA - seq dataset of melanoma samples . TINCR expression is higher in Primary A melanomas , and its reduction in Primary - B melanomas and all groups of metastatic samples is statistically significant ( Mann - Whitney test , * * p < 0 . 01 ) . Median , minimum , maximum , 25th , 75th whiskers of expression values are shown . B Scatter - plot showing correlation between the expression of TINCR RNA and normalized rank - sums s of the down - regulated ( upper panel ) and up - regulated ( lower panel ) genes in the primary melanoma signature . TINCR expression strongly correlates ( Spearman ' s correlation coefficient of 0 . 79 , Spearman rank p - value : * * p < 0 . 01 ) with the expression of the down - regulated genes . C Dot plot showing qRT - PCR analysis of TINCR expression in primary ( 8 ) and metastatic ( 8 ) melanoma samples from surgical specimens or patient - derived xenografts ( 4 primary and 8 metastatic PDXs ) . TINCR RNA levels relative to the L32P housekeeping gene are shown . Paired primary and metastasis PDXs are marked with an asterisk . The difference between primary and metastatic samples is significant ( Mann - Whitney test , * * p < 0 . 01 ) . Median , minimum , maximum , 25th , 75th whiskers of expression values are shown .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A Box and whiskers (from 5th to 95th percentile) plots depicting TINCR RNA levels in the TCGA SKCM RNA-seq dataset of melanoma samples. TINCR expression is higher in Primary A melanomas, and its reduction in Primary-B melanomas and all groups of metastatic samples is statistically significant (Mann-Whitney test, **p< 0.01). Median, minimum, maximum, 25th, 75th whiskers of expression values are shown.B Scatter-plot showing correlation between the expression of TINCR RNA and normalized rank-sums s of the down-regulated (upper panel) and up-regulated (lower panel) genes in the primary melanoma signature. TINCR expression strongly correlates (Spearman's correlation coefficient of 0.79, Spearman rank p-value: **p<0.01) with the expression of the down-regulated genes.C Dot plot showing qRT-PCR analysis of TINCR expression in primary (8) and metastatic (8) melanoma samples from surgical specimens or patient-derived xenografts (4 primary and 8 metastatic PDXs). TINCR RNA levels relative to the L32P housekeeping gene are shown. Paired primary and metastasis PDXs are marked with an asterisk. The difference between primary and metastatic samples is significant (Mann-Whitney test, **p< 0.01). Median, minimum, maximum, 25th, 75th whiskers of expression values are shown.


Figure 3A qRT - PCR quantification of TINCR RNA expression in MMC70 and WM902B cell lines , independently infected with two shRNA targeting TINCR expression ( shTINCR - 1 and - 2 ) . Cells transduced with shScamble ( shSCR ) were used as control . TINCR RNA levels relative to L32P housekeeping gene and control ( mean ± sd ) measured in three independent biological experiments are shown . In both cell lines TINCR knockdown efficiency is more than 70 % with the two shRNAs . Statistical analyses were performed using Student t - test ( * * : p < 0 . 01 ) . B In vitro migration assay of MMC70 and WM902B cells transduced with shTINCR - 1 , shTINCR - 2 or shSCR . Relative migration ( mean ± sd ) from three independent experiments is indicated . Statistical analyses were performed using Student t - test ( * * * : p < 0 . 001 ) . C In vitro cell proliferation of shSCR or shTINCR infected MMC70 and WM902B cells assayed 72h after infection at the indicated time points using CellTiter - Glo luminescent kit ( Promega ) . Cell growth from three biological replicates is indicated as proliferation rate relative to control cells ( mean ± sd ) . The difference between control and TINCR - silenced samples at all time points is not significant ( Student t - test ) . D Upper panel : in vivo growth of tumors derived from intradermal injection of shTINCR - 2 or shSCR WM902B cells in NSG mice . 600 , 000 WM902B cells per mouse were inoculated . Tumor volumes ( mean ± sd ) were measured 8 weeks after transplantation . Results from two independent experiments ( four animals per group ) are shown . Statistical analysis between control and TINCR - silenced groups was performed using the Student t - test ( * * = p < 0 . 01 ) . Lower panel : qRT - PCR analysis of TINCR RNA expression in tumors derived from in vivo transplant of shSCR and shTINCR WM902B cells ( 8 weeks after injection ) . TINCR RNA levels relative to L32P housekeeping gene and control ( mean ± sd ) from four biological replicates ( animals ) from both experiments are shown . E Drug - response curves showing increased resistance of shTINCR - WM902B cells after treatment with BRAF ( Vemurafenib ; from 10 to 10 , 000 nM ) and MEK ( Trametinib ; from 4 to 2 , 000 nM ) inhibitors . The shSCR - ( control ) and shTINCR - WM902B cells were treated with increasing concentrations of drugs and their viability assayed after 72 hours using CellTiter - Glo luminescent kit ( Promega ) . Error bars represent s . e . m . of three biological replicates . The dose response curve was fit with nonlinear regression ( GraphPad Prism ) . G Western blot analysis of a panel of proteins that are specifically associated to the proliferative or invasive phenotype . Total protein lysates were extracted from shSCR - and shTINCR - WM902B cells ( lane 1 and 2 ) or shSCR - and shTINCR - MMC70 cells ( lane 3 and 4 ) 10 days after infection and analyzed by immunoblot assay using the indicated antibodies . Vinculin was used as loading control .

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Figure 3A qRT-PCR quantification of TINCR RNA expression in MMC70 and WM902B cell lines, independently infected with two shRNA targeting TINCR expression (shTINCR-1 and -2). Cells transduced with shScamble (shSCR) were used as control. TINCR RNA levels relative to L32P housekeeping gene and control (mean±sd) measured in three independent biological experiments are shown. In both cell lines TINCR knockdown efficiency is more than 70% with the two shRNAs. Statistical analyses were performed using Student t -test (**: p<0.01).B In vitro migration assay of MMC70 and WM902B cells transduced with shTINCR-1, shTINCR-2 or shSCR. Relative migration (mean±sd) from three independent experiments is indicated. Statistical analyses were performed using Student t -test (***: p<0.001).C In vitro cell proliferation of shSCR or shTINCR infected MMC70 and WM902B cells assayed 72h after infection at the indicated time points using CellTiter-Glo luminescent kit (Promega). Cell growth from three biological replicates is indicated as proliferation rate relative to control cells (mean±sd). The difference between control and TINCR-silenced samples at all time points is not significant (Student t-test).D Upper panel: in vivo growth of tumors derived from intradermal injection of shTINCR-2 or shSCR WM902B cells in NSG mice. 600,000 WM902B cells per mouse were inoculated. Tumor volumes (mean±sd) were measured 8 weeks after transplantation. Results from two independent experiments (four animals per group) are shown. Statistical analysis between control and TINCR-silenced groups was performed using the Student t -test (**=p<0.01). Lower panel: qRT-PCR analysis of TINCR RNA expression in tumors derived from in vivo transplant of shSCR and shTINCR WM902B cells (8 weeks after injection). TINCR RNA levels relative to L32P housekeeping gene and control (mean±sd) from four biological replicates (animals) from both experiments are shown.E Drug-response curves showing increased resistance of shTINCR-WM902B cells after treatment with BRAF (Vemurafenib; from 10 to 10,000 nM) and MEK (Trametinib; from 4 to 2,000 nM) inhibitors. The shSCR- (control) and shTINCR-WM902B cells were treated with increasing concentrations of drugs and their viability assayed after 72 hours using CellTiter-Glo luminescent kit (Promega). Error bars represent s.e.m. of three biological replicates. The dose response curve was fit with nonlinear regression (GraphPad Prism).G Western blot analysis of a panel of proteins that are specifically associated to the proliferative or invasive phenotype. Total protein lysates were extracted from shSCR- and shTINCR-WM902B cells (lane 1 and 2) or shSCR- and shTINCR-MMC70 cells (lane 3 and 4) 10 days after infection and analyzed by immunoblot assay using the indicated antibodies. Vinculin was used as loading control.


Figure 4A qRT - PCR analysis of TINCR expression ( left ) of MM13 and MM2 PDX cells infected with lentiviral vectors expressing full - length TINCR cDNA . PDX cells infected with empty vector ( Empty ) were used as control . TINCR RNA levels relative to L32P housekeeping gene and control ( mean ± sd ) from three biological replicates are shown . Statistical analyses were performed using Student t - test ( * * * : p < 0 . 001 ) . B In vitro relative migration ( mean ± sd ) from three independent experiments ( biological replicates ) is shown in MM13 and MM2 TINCR overexpressing ( TINCR ) compared to control ( Empty ) cells . Statistical analyses were performed using Student t - test ( * * * : p < 0 . 001 ) . C In vitro cell proliferation was assayed 72 hours post infection in Empty and TINCR MM13 and MM2 PDX cells at the indicated time points ( 1 - 5 days ) using CellTiter - Glo viability assay . Relative proliferation rate ( mean ± sd ) of three independent experiments are shown . D Drug - response curves showing sensitization to MEK inhibitor Trametinib of MM13 ( NRAS - mutant PDX ) and to Trametinib and BRAF inhibitor Vemurafenib of TINCR MM2 cells ( BRAF - mutant PDX ) . The Empty ( control ) and TINCR expressing MM13 cells were treated with increasing concentrations of drug ( ranging from 5 to 5000 nM ) and cell viability was assayed after 72 hours by CellTiter - Glo viability assay . Error bars represent s . e . m . of three biological replicates . The dose response curve was fit with nonlinear regression ( GraphPad Prism ) . E Western blot analysis of the expression of the invasive and proliferative phenotype markers EPHA2 , EGFR , AXL , PDGFRβ , SOX10 , and MITF in TINCR - overexpressing MM13 and MM2 cells . Total protein lysates were extracted from PDX cells 7 days after infection and analyzed by immunoblot assay using the indicated antibodies . Vinculin was used as loading control . This experiment is representative of three independent experiments showing comparable levels of TINCR overexpression ( 8 - 10 folds ) . F In vivo growth of Empty vector and TINCR cDNA expressing MM13 cells following intradermal injection into NSG mice . Four animals ( 200 , 000 cells / mouse ) per group were used in each experiment . Tumor growth ( mean ± sd ) was monitored weekly and tumor volume ( mean ± sd ) measured at 4 weeks for primary tumors ( originated in the site of injection , left panel ) and at 8 weeks for spontaneous metastases which colonized lymph nodes ( right panel ) . The results of two independent experiments are shown . Statistical analyses of control and TINCR - overexpressing groups from both experiments were performed using Student t - test ( * * : p < 0 . 01 ) . G Representative pictures of lymph node metastases derived from Empty and TINCR expressing MM13 PDX cells are shown . Hematoxylin - eosin staining of sections of liver and lung metastases derived from Empty and TINCR MM13 PDX cells . Metastasis formation was evaluated 8 weeks after transplantation into NSG mice . Pictures of representative lungs and livers infiltrated with metastases are shown in the inset . Scale bars : 200μm .

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Figure 4A qRT-PCR analysis of TINCR expression (left) of MM13 and MM2 PDX cells infected with lentiviral vectors expressing full-length TINCR cDNA. PDX cells infected with empty vector (Empty) were used as control. TINCR RNA levels relative to L32P housekeeping gene and control (mean±sd) from three biological replicates are shown. Statistical analyses were performed using Student t -test (***: p<0.001).B In vitro relative migration (mean±sd) from three independent experiments (biological replicates) is shown in MM13 and MM2 TINCR overexpressing (TINCR) compared to control (Empty) cells. Statistical analyses were performed using Student t -test (***: p<0.001).C In vitro cell proliferation was assayed 72 hours post infection in Empty and TINCR MM13 and MM2 PDX cells at the indicated time points (1-5 days) using CellTiter-Glo viability assay. Relative proliferation rate (mean±sd) of three independent experiments are shown.D Drug-response curves showing sensitization to MEK inhibitor Trametinib of MM13 (NRAS-mutant PDX) and to Trametinib and BRAF inhibitor Vemurafenib of TINCR MM2 cells (BRAF-mutant PDX). The Empty (control) and TINCR expressing MM13 cells were treated with increasing concentrations of drug (ranging from 5 to 5000 nM) and cell viability was assayed after 72 hours by CellTiter-Glo viability assay. Error bars represent s.e.m. of three biological replicates. The dose response curve was fit with nonlinear regression (GraphPad Prism).E Western blot analysis of the expression of the invasive and proliferative phenotype markers EPHA2, EGFR, AXL, PDGFRβ, SOX10, and MITF in TINCR-overexpressing MM13 and MM2 cells. Total protein lysates were extracted from PDX cells 7 days after infection and analyzed by immunoblot assay using the indicated antibodies. Vinculin was used as loading control. This experiment is representative of three independent experiments showing comparable levels of TINCR overexpression (8-10 folds).F In vivo growth of Empty vector and TINCR cDNA expressing MM13 cells following intradermal injection into NSG mice. Four animals (200,000 cells/mouse) per group were used in each experiment. Tumor growth (mean±sd) was monitored weekly and tumor volume (mean±sd) measured at 4 weeks for primary tumors (originated in the site of injection, left panel) and at 8 weeks for spontaneous metastases which colonized lymph nodes (right panel). The results of two independent experiments are shown. Statistical analyses of control and TINCR-overexpressing groups from both experiments were performed using Student t -test (**: p<0.01).G Representative pictures of lymph node metastases derived from Empty and TINCR expressing MM13 PDX cells are shown. Hematoxylin-eosin staining of sections of liver and lung metastases derived from Empty and TINCR MM13 PDX cells. Metastasis formation was evaluated 8 weeks after transplantation into NSG mice. Pictures of representative lungs and livers infiltrated with metastases are shown in the inset. Scale bars: 200μm.


Figure 5A Western blot analysis of integrated stress response activation in WM902B and MMC70 cells . Cells were harvested 96 h after shRNA infection and western blot performed using the indicated antibodies . B Representative comparison of translational efficiency ( TE ) in control ( shSCR ) and shTINCR WM902B cells 72h post infection . The genes with statistically significant TE up - and downregulation ( FDR < 0 . 1 ) are highlighted in red and blue respectively . ATF4 is circled among the upregulated genes . C Heatmap representing top translationally upregulated and down regulated genes in TINCR WM902B cells . The plotted values are the log2 fold TE change values in each biological replicate ( rep1 and rep2 ) relative to control ( shSCR ) . D Bar - graph representing ribosome protected fragments ( RPF ) normalized coverage ( mean ± sd ) mapping to ATF4 CDS in control ( shSCR ) and TINCR ( shTINCR ) depleted WM902B cells from two biological replicates is shown . E UCSC genome browser tracks of ribosome footprint data illustrating RPF density distribution across ATF4 mRNA in shSCR and shTINCR WM902B cells .

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Figure 5A Western blot analysis of integrated stress response activation in WM902B and MMC70 cells. Cells were harvested 96 h after shRNA infection and western blot performed using the indicated antibodies.B Representative comparison of translational efficiency (TE) in control (shSCR) and shTINCR WM902B cells 72h post infection. The genes with statistically significant TE up- and downregulation (FDR< 0.1) are highlighted in red and blue respectively. ATF4 is circled among the upregulated genes.C Heatmap representing top translationally upregulated and down regulated genes in TINCR WM902B cells. The plotted values are the log2 fold TE change values in each biological replicate (rep1 and rep2) relative to control (shSCR).D Bar-graph representing ribosome protected fragments (RPF) normalized coverage (mean±sd) mapping to ATF4 CDS in control (shSCR) and TINCR (shTINCR) depleted WM902B cells from two biological replicates is shown.E UCSC genome browser tracks of ribosome footprint data illustrating RPF density distribution across ATF4 mRNA in shSCR and shTINCR WM902B cells.


Figure 6B Q - PCR validation of TINCR and ATF4 RNA recovery in TINCR antisense pulldown . Enrichment relative to input ( mean ± sd ) from three independent pulldown experiments is shown . Statistical analyses were performed using Student t - test ( * * : p < 0 . 01 , * * * : p < 0 . 001 ) . C UCSC genome browser tracks of TINCR - RIA - seq sequencing reads illustrating coverage and enrichment across ATF4 transcript in WM902B and MMC70 cells .

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Figure 6B Q-PCR validation of TINCR and ATF4 RNA recovery in TINCR antisense pulldown. Enrichment relative to input (mean±sd) from three independent pulldown experiments is shown. Statistical analyses were performed using Student t -test (**: p<0.01, ***: p<0.001).C UCSC genome browser tracks of TINCR-RIA-seq sequencing reads illustrating coverage and enrichment across ATF4 transcript in WM902B and MMC70 cells.


Figure 1p53 ChIPseq in MEFs of the indicated genotype treated with or without 10 µM nutlin - 3a ( Nutlin ) for 16 hours . Shown are 2 kb regions surrounding the summit of the 468 p53 binding peaks called in Nutlin - treated p53 + / + , but not p53 - / - or p53EE / EE MEFs . For p53EE / EE MEFs three independent replicates are shown . RNAseq was performed with MEFs of the indicated genotype untreated or treated with 10 µM Nutlin for 16 hours . Shown are all Nutlin - regulated genes from the MSigDB gene set Hallmarks _ P53 _ Pathway with a mean log2FC ≥ 1 in p53 + / + cells . Shown are the z - transformed RNA expression values ( FPKM ) . 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 ) . Western blots of protein lysates prepared from MEFs treated for 24 hours with ( G ) 10 µM NutlinWestern blots of protein lysates prepared from MEFs treated for 24 hours with 0 . 4 µg / ml Doxo .

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Figure 1p53 ChIPseq in MEFs of the indicated genotype treated with or without 10 µM nutlin-3a (Nutlin) for 16 hours. Shown are 2 kb regions surrounding the summit of the 468 p53 binding peaks called in Nutlin-treated p53+/+, but not p53-/- or p53EE/EE MEFs. For p53EE/EE MEFs three independent replicates are shown.RNAseq was performed with MEFs of the indicated genotype untreated or treated with 10 µM Nutlin for 16 hours. Shown are all Nutlin-regulated genes from the MSigDB gene set Hallmarks_P53_Pathway with a mean log2FC≥1 in p53+/+ cells. Shown are the z-transformed RNA expression values (FPKM).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).Western blots of protein lysates prepared from MEFs treated for 24 hours with (G) 10 µM NutlinWestern blots of protein lysates prepared from MEFs treated for 24 hours with 0.4 µg/ml Doxo.


Figure 2Proliferation 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 . 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 . 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 ) 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 ) . 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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Figure 2Proliferation 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.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.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)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).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).


Figure 3Observed 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 ) . 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 .

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Figure 3Observed 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).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.


Figure 4Mdm2 - / - 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 . 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 . 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 . 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 . 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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Figure 4Mdm2-/-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.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.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.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.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.


Figure 5Cell 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 . 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 . 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 . 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 .

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Figure 5Cell 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.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.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.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.


Figure 6Kaplan - 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 . 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 .

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Figure 6Kaplan-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.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.


Figure 7Apoptosis 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 . 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 . 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 ) . 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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Figure 7Apoptosis 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.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.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).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).


Figure 1 ( 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 . ( 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 . ( 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 . ( 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 .

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Figure 1(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. (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. (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.(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.


Figure 2 ( 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 . ( 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 . ( 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 . ( 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 . ( 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 .

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Figure 2(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.(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.(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. (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.(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.


Figure 3Profiles 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 ) .

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Figure 3Profiles 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).


Figure 4 ( 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 . ( 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 . ( 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 .

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Figure 4(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.(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. (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.


Figure 5 ( 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 . ( 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 . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(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.(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.(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.


Figure 6 ( 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 ) . ( 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 B-PANEL_START O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(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).(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.


Figure 1Reduced SDS - PAGE analysis of purified anti - HIV BiCEs .

Figure 1Reduced SDS-PAGE analysis of purified anti-HIV BiCEs.


Figure 2HIV - 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 .

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Figure 2HIV-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.


Figure 3C3 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 .

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Figure 3C3 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.


Figure 4C3 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 . 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4C3 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.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.