A - H . Base pairing patterns for sRNA - mRNA pairs identified by CLASH were calculated using IntaRNA software ( Busch et al . 2008 ) are shown ( left ) hdeA - GadY ( D ) . Point mutations predicted sRNA seed sequences ( blue shading ) are indicated . ( Histograms , right ) Median ( MFI ) was assessed by FACS for mRNA - sfGFP fusions with compensatory base changes . In each case , introduction of a point mutation into an sRNA or mRNA construct ( M1 ) is expected to reduce sRNA repression , which should be restored by combining complementary point mutants ( last bar , M1 - M1 ) . A two - t - was used to calculate significance from biological triplicate cultures . * p < 0 . 05 .

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A-H. Base pairing patterns for the sRNA-mRNA pairs identified by CLASH were calculated using IntaRNA software (Busch et al. 2008) and are shown (left) hdeA-GadY (D). Point mutations and predicted sRNA seed sequences (blue shading) are indicated. (Histograms, right) Median fluorescence intensity (MFI) was assessed by FACS for mRNA-sfGFP fusions with compensatory base changes. In each case, introduction of a point mutation into an sRNA or mRNA construct (M1) is expected to reduce sRNA repression, which should be restored by combining complementary point mutants (last bar, M1-M1). A two-tailed t-test was used to calculate significance from biological triplicate cultures. *p<0.05.


A - H . Base pairing patterns for sRNA - mRNA pairs identified by CLASH were calculated using IntaRNA software ( Busch et al . 2008 ) are shown ( left ) for rssA - RyeB ( E ) . Point mutations predicted sRNA seed sequences ( blue shading ) are indicated . ( Histograms , right ) Median ( MFI ) was assessed by FACS for mRNA - sfGFP fusions with compensatory base changes . In each case , introduction of a point mutation into an sRNA or mRNA construct ( M1 ) is expected to reduce sRNA repression , which should be restored by combining complementary point mutants ( last bar , M1 - M1 ) . A two - t - was used to calculate significance from biological triplicate cultures . * p < 0 . 05 .

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A-H. Base pairing patterns for the sRNA-mRNA pairs identified by CLASH were calculated using IntaRNA software (Busch et al. 2008) and are shown (left) for rssA-RyeB (E). Point mutations and predicted sRNA seed sequences (blue shading) are indicated. (Histograms, right) Median fluorescence intensity (MFI) was assessed by FACS for mRNA-sfGFP fusions with compensatory base changes. In each case, introduction of a point mutation into an sRNA or mRNA construct (M1) is expected to reduce sRNA repression, which should be restored by combining complementary point mutants (last bar, M1-M1). A two-tailed t-test was used to calculate significance from biological triplicate cultures. *p<0.05.


A - H . Base pairing patterns for sRNA - mRNA pairs identified by CLASH were calculated using IntaRNA software ( Busch et al . 2008 ) are shown ( left ) for chuA - Esr41 ( F ) . Point mutations predicted sRNA seed sequences ( blue shading ) are indicated . ( Histograms , right ) Median ( MFI ) was assessed by FACS for mRNA - sfGFP fusions with compensatory base changes . In each case , introduction of a point mutation into an sRNA or mRNA construct ( M1 ) is expected to reduce sRNA repression , which should be restored by combining complementary point mutants ( last bar , M1 - M1 ) . A two - t - was used to calculate significance from biological triplicate cultures . * p < 0 . 05 .

A-H. Base pairing patterns for the sRNA-mRNA pairs identified by CLASH were calculated using IntaRNA software (Busch et al. 2008) and are shown (left) for chuA-Esr41 (F). Point mutations and predicted sRNA seed sequences (blue shading) are indicated. (Histograms, right) Median fluorescence intensity (MFI) was assessed by FACS for mRNA-sfGFP fusions with compensatory base changes. In each case, introduction of a point mutation into an sRNA or mRNA construct (M1) is expected to reduce sRNA repression, which should be restored by combining complementary point mutants (last bar, M1-M1). A two-tailed t-test was used to calculate significance from biological triplicate cultures. *p<0.05.


A - H . Base pairing patterns for sRNA - mRNA pairs identified by CLASH were calculated using IntaRNA software ( Busch et al . 2008 ) are shown ( left ) for cirA - Esr41 ( G ) . Point mutations predicted sRNA seed sequences ( blue shading ) are indicated . ( Histograms , right ) Median ( MFI ) was assessed by FACS for mRNA - sfGFP fusions with compensatory base changes . In each case , introduction of a point mutation into an sRNA or mRNA construct ( M1 ) is expected to reduce sRNA repression , which should be restored by combining complementary point mutants ( last bar , M1 - M1 ) . A two - t - was used to calculate significance from biological triplicate cultures . * p < 0 . 05 .

A-H. Base pairing patterns for the sRNA-mRNA pairs identified by CLASH were calculated using IntaRNA software (Busch et al. 2008) and are shown (left) for cirA-Esr41 (G). Point mutations and predicted sRNA seed sequences (blue shading) are indicated. (Histograms, right) Median fluorescence intensity (MFI) was assessed by FACS for mRNA-sfGFP fusions with compensatory base changes. In each case, introduction of a point mutation into an sRNA or mRNA construct (M1) is expected to reduce sRNA repression, which should be restored by combining complementary point mutants (last bar, M1-M1). A two-tailed t-test was used to calculate significance from biological triplicate cultures. *p<0.05.


A - H . Base pairing patterns for sRNA - mRNA pairs identified by CLASH were calculated using IntaRNA software ( Busch et al . 2008 ) are shown ( left ) for bfr - Esr41 ( H ) . Point mutations predicted sRNA seed sequences ( blue shading ) are indicated . ( Histograms , right ) Median ( MFI ) was assessed by FACS for mRNA - sfGFP fusions with compensatory base changes . In each case , introduction of a point mutation into an sRNA or mRNA construct ( M1 ) is expected to reduce sRNA repression , which should be restored by combining complementary point mutants ( last bar , M1 - M1 ) . A two - t - was used to calculate significance from biological triplicate cultures . * p < 0 . 05 .

A-H. Base pairing patterns for the sRNA-mRNA pairs identified by CLASH were calculated using IntaRNA software (Busch et al. 2008) and are shown (left) for bfr-Esr41 (H). Point mutations and predicted sRNA seed sequences (blue shading) are indicated. (Histograms, right) Median fluorescence intensity (MFI) was assessed by FACS for mRNA-sfGFP fusions with compensatory base changes. In each case, introduction of a point mutation into an sRNA or mRNA construct (M1) is expected to reduce sRNA repression, which should be restored by combining complementary point mutants (last bar, M1-M1). A two-tailed t-test was used to calculate significance from biological triplicate cultures. *p<0.05.


A - C . FACS of constitutively expressed sfGFP fusions to chuA ( A ) , cirA ( B ) , or bfr ( C ) in presence of Esr41 ( orange ) , RyhB ( blue ) , or a scrambled control ( red ) . histogram shows GFP for each sRNA - mRNA fusion .

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A-C. FACS analysis of constitutively expressed sfGFP fusions to chuA (A), cirA (B), or bfr (C) in the presence of Esr41 (orange), RyhB (blue), or a scrambled RNA control (red). The histogram shows GFP fluorescence for each sRNA-mRNA fusion.


D . Esr41 confers resistance to Colicin 1A 1B in sensitive background , E . coli DH5alpha . Top agar lawns of DH5alpha expressing a control scrambled ( pJV300 ) , Esr41 ( pZE12 : : esr41 ) , or RyhB ( pZE12 : : ryhB ) were spotted with colicins indicated ( top ) . Zones of clearing indicate sensitivity to tested colicin .

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D. Esr41 confers resistance to Colicin 1A and 1B in the sensitive background, E. coli DH5alpha. Top agar lawns of DH5alpha expressing a control scrambled RNA (pJV300), Esr41 (pZE12::esr41), or RyhB (pZE12::ryhB) were spotted with colicins indicated (top). Zones of clearing indicate sensitivity to the tested colicin.


E . Esr41 confers a competitive disadvantage on EHEC under iron - limiting conditions . Wild type EHEC isogenic ∆ esr41 ( black ) , or EHEC ∆ esr41and chromosomally repaired EHEC ∆ esr41 : : esr41 were inoculated at equal densities cultured in MEM - HEPES media for 3 days . proportion of each was determined at each time point ( days ) is expressed as a relative to starting inoculum where 1 is an equal fitness ( see Appended Supplementary Methods ) .

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E. Esr41 confers a competitive disadvantage on EHEC under iron-limiting conditions. Wild type EHEC and isogenic ∆esr41 strain (black), or EHEC ∆esr41and the chromosomally repaired strain EHEC ∆esr41::esr41 were inoculated at equal densities and cultured in MEM-HEPES media for 3 days. The proportion of each strain was determined at each time point (days) and is expressed as a ratio relative to the starting inoculum where 1 is an equal fitness (see Appended Supplementary Methods).


A - D of uninjected , FLT3 / WT mRNA FLT3 / ITD mRNA injected ( 150 ng per ) on day 2 post fertilization ( dpf ) . Data information : ov : otic vesicles Scale bar = 500 μm .

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A-D The morphology of uninjected, FLT3/WT mRNA and FLT3/ITD mRNA injected (150 ng per embryo) embryos on day 2 post fertilization (dpf). Data information: ov: otic vesicles Scale bar = 500 μm.


E - H Whole mount in situ hybridization ( WISH ) of notochord - specific marker col9a2 in uninjected , FLT3 / WT mRNA FLT3 / ITD mRNA injected on 2 dpf . Data information : ov : otic vesicles ; cm : cephalic mesoderm ; nc : notochord . Scale bar = 500 μm .

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E-H Whole mount in situ hybridization (WISH) of notochord-specific marker col9a2 in uninjected, FLT3/WT mRNA and FLT3/ITD mRNA injected embryos on 2 dpf. Data information: ov: otic vesicles; cm: cephalic mesoderm; nc: notochord. Scale bar = 500 μm.


FLT3 signaling was detected by Western blotting in 293FT - transfected with FLT3 / ITD mRNA ( I )

FLT3 signaling was detected by Western blotting in 293FT cells-transfected with FLT3/ITD mRNA (I)


FLT3 signaling was detected by Western blotting in zebrafish - injected with FLT3 / ITD mRNA ( J ) .

FLT3 signaling was detected by Western blotting in zebrafish embryos-injected with FLT3/ITD mRNA (J).


K effect of FLT3 inhibitor quizartinib ( Qui ) on dorsalization axis - duplication phenotype - induced by FLT3 / ITD mRNA injection in zebrafish . Data information : experiments were performed in triplicates data are presented as mean ± . * P < 0 . 05 , * * P < 0 . 01 ( Student ' s t - ) . NS , not significant .

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K The effect of FLT3 inhibitor quizartinib (Qui) on the dorsalization and axis-duplication phenotype-induced by FLT3/ITD mRNA injection in zebrafish. Data information: the experiments were performed in triplicates and the data are presented as mean ± SEM. *P<0.05, **P<0.01 (Student's t-test). NS, not significant.


of fst expression by RT - qPCR ( L ) after FLT3 / ITD overexpression in zebrafish at 6 hpf . Data information : experiments were performed in triplicates data are presented as mean ± . * P < 0 . 05 , * * P < 0 . 01 ( Student ' s t - ) . NS , not significant .

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Quantification of fst expression by RT-qPCR (L) after FLT3/ITD overexpression in zebrafish embryos at 6 hpf. Data information: the experiments were performed in triplicates and the data are presented as mean ± SEM. *P<0.05, **P<0.01 (Student's t-test). NS, not significant.


of fst expression by Western Blotting ( M ) , after FLT3 / ITD overexpression in zebrafish at 6 hpf .

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Quantification of fst expression by Western Blotting (M), after FLT3/ITD overexpression in zebrafish embryos at 6 hpf.


of fst expression by WISH ( N ) after FLT3 / ITD overexpression in zebrafish at 6 hpf .

Quantification of fst expression by WISH (N) after FLT3/ITD overexpression in zebrafish embryos at 6 hpf.


A - C WISH of fst in FLT3 / WT - ( A ) , FLT3 / ITD - plasmid injected zebrafish without ( B ) or with ( C ) quizartinib treatment ( 2 . 5 μM ) from 6 to 36 hpf . fst expression was expanded by FLT3 / ITD in 86 % of ( B , arrow , 32 / 37 ) which could be effectively blocked by treating with FLT3 inhibitor quizartinib in 83 % of ( C , 29 / 35 ) . Data information : Scale bar = 500 μm .

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A-C WISH of fst in FLT3/WT- (A), FLT3/ITD- plasmid DNA injected zebrafish embryos without (B) or with (C) quizartinib treatment (2.5 μM) from 6 to 36 hpf. fst expression was expanded by FLT3/ITD DNA in 86% of embryos (B, arrow, 32/37) which could be effectively blocked by treating with FLT3 inhibitor quizartinib in 83% of embryos (C, 29/35). Data information: Scale bar = 500 μm.


Generation characterization of FLT3 / ITD - transgenic zebrafish . GFP expression was detected by florescent microscopy ( F - H ) in blood circulation thymus by WISH ( I J , blue arrow ) in WT sibling Runx1 - FLT3 / ITD transgenic zebrafish ( F1 ) at 4 dpf . FLT3 / ITD - positive zebrafish ( F1 ) were confirmed by PCR genotyping of GFP FLT3 / ITD using genomic from fin clip of WT siblings Runx1 - FLT3 / ITD transgenic zebrafish ( F1 ) at two months old . Fish 4 , 5 6 showed germline transmission of FLT3 / ITD transgene ( K ) . Data information : Scale bar = 500 μm .

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Generation and characterization of FLT3/ITD-transgenic zebrafish. GFP expression was detected by florescent microscopy (F-H) and in blood circulation and thymus by WISH (I and J, blue arrow) in WT sibling and Runx1-FLT3/ITD transgenic zebrafish (F1) embryos at 4 dpf. FLT3/ITD-positive zebrafish (F1) were confirmed by PCR genotyping of GFP and FLT3/ITD using genomic DNA from fin clip of WT siblings and Runx1-FLT3/ITD transgenic zebrafish (F1) at two months old. Fish 4, 5 and 6 showed germline transmission of FLT3/ITD transgene (K). Data information: Scale bar = 500 μm.


Kidney marrow ( KM ) was collected from Runx1 - FLT3 / ITD transgenic zebrafish ( F1 ) at 18 months old . hematopoietic composition of KM from WT siblings ( n = 6 ) Runx1 - FLT3 / ITD transgenic ( n = 6 ) zebrafish were examined by Giemsa ( L ) flow cytometry ( abbreviation for panel M : M , Myeloid ; P , Progenitor ; L , Lymphoid ; E , Erythroid ) . Data are presented in box plot . * * P < 0 . 01 ( Student ' s t - ) .

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Kidney marrow (KM) was collected from Runx1-FLT3/ITD transgenic zebrafish (F1) at 18 months old. The morphology and hematopoietic composition of KM from WT siblings (n = 6) and Runx1-FLT3/ITD transgenic (n = 6) zebrafish were examined by Giemsa staining (L) and flow cytometry (abbreviation for panel M: M, Myeloid cells; P, Progenitor cells; L, Lymphoid cells; E, Erythroid cells). Data are presented in box plot. **P<0.01 (Student's t-test).


Kidney marrow ( KM ) was collected from Runx1 - FLT3 / ITD transgenic zebrafish ( F1 ) at 18 months old . hematopoietic composition of KM from WT siblings ( n = 6 ) Runx1 - FLT3 / ITD transgenic ( n = 6 ) zebrafish were examined by flow cytometry Data are presented in box plot . whiskers , boxes , central lines in panel N represented minimum - to - maximum values , 25th - to - 75th percentile , 50th percentile ( median ) , respectively . * * P < 0 . 01 ( Student ' s t - ) .

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Kidney marrow (KM) was collected from Runx1-FLT3/ITD transgenic zebrafish (F1) at 18 months old. The morphology and hematopoietic composition of KM from WT siblings (n = 6) and Runx1-FLT3/ITD transgenic (n = 6) zebrafish were examined by flow cytometry Data are presented in box plot. The whiskers, boxes, and central lines in panel N represented the minimum-to-maximum values, 25th-to-75th percentile, and the 50th percentile (median), respectively. **P<0.01 (Student's t-test).


O Expression of fst was detected by RT - qPCR in KM from WT sibling Runx1 - FLT3 / ITD transgenic zebrafish at 18 months old . RT - qPCR experiments were performed in triplicates data were presented as mean ± . * * P < 0 . 01 .

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

O Expression of fst was detected by RT-qPCR in KM from WT sibling and Runx1-FLT3/ITD transgenic zebrafish at 18 months old. The RT-qPCR experiments were performed in triplicates and data were presented as mean ± SEM. **P<0.01.


P Detection of FST expression , p - ERK1 / 2 , p - CREB in mononuclear from normal peripheral blood ( PBSC ) FLT3 / ITD AML ( diagnostic samples with leukemia blasts > 80 % ) by Western blotting . ^ : non - specific of p - ATF1 due to conserved motif .

P Detection of FST expression, p-ERK1/2, and p-CREB in mononuclear cells from normal peripheral blood stem cell (PBSC) and FLT3/ITD AML patients (diagnostic samples with leukemia blasts > 80%) by Western blotting. ^: non-specific staining of p-ATF1 protein due to the conserved motif.


B - C direct binding of p - CREB to human FST promoter was detected by ChIP - PCR ( B ) ChIP - qPCR ( C ) . c - Fos was used as positive control of p - CREB target gene . Normal IgG was used as negative control of ChIP . Data information experiments were performed in triplicates , data were presented as mean ± . * * P < 0 . 01 , * * * P < 0 . 001 ( Student ' s t - ) .

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B-C The direct binding of p-CREB to human FST promoter was detected by ChIP-PCR (B) and ChIP-qPCR (C). c-Fos was used as positive control of p-CREB target gene. Normal IgG was used as negative control of ChIP. Data information the experiments were performed in triplicates, and the data were presented as mean ± SEM. **P<0.01, ***P<0.001 (Student's t-test).


D Dual luciferase demonstrating direct binding of pCREB on human FST promoter . pRL - CMV , Renilla luciferase vector ; pGL - CRE - pGL - CRE + , firefly luciferase expression driven by human FST promoter with deleted CRE site ( CRE - ) or wildtype ( CRE + ) ; p - GFPSpark , GFP expressing vector ; p - CREBY134F , CREBY134F - GFP expressing vector . Data information : experiments were performed in triplicates , data were presented as mean ± . * * P < 0 . 01 , * * * P < 0 . 001 ( Student ' s t - ) .

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D Dual luciferase assay demonstrating the direct binding of pCREB on human FST promoter. pRL-CMV, Renilla luciferase vector; pGL-CRE- and pGL-CRE+, firefly luciferase expression driven by human FST promoter with deleted CRE site (CRE-) or wildtype (CRE+); p-GFPSpark, GFP expressing vector; p-CREBY134F, CREBY134F-GFP expressing vector. Data information: the experiments were performed in triplicates, and the data were presented as mean ± SEM. **P<0.01, ***P<0.001 (Student's t-test).


E FST expression FLT3 / ITD signaling were detected by Western blotting in Ba / F3 - parental ( P in short ) Ba / F3 - FLT3 / ITD ( ITD in short ) .

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E FST expression and FLT3/ITD signaling were detected by Western blotting in Ba/F3-parental (P in short) and Ba/F3-FLT3/ITD (ITD in short) cells.


F - H Phosphor - flow of p - CREB in Ba / F3 - parental , Ba / F3 - FLT3 / ITD , Ba / F3 - FLT3 / ITD - treated with FLT3 inhibitor Quizartinib ( Qui in short ) . Isotype antibody were used as control to calculate mean ( MFI ) ( F - G ) . transcription expression of Fst was detected by RT - qPCR after Quizartinib treatment ( 10 nM ) in Ba / F3 - FLT3 / ITD for one day ( H ) . Data information : experiments were performed in triplicates , data were presented as mean ± . * * P < 0 . 01 , * * * P < 0 . 001 ( Student ' s t - ) .

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

F-H Phosphor-flow analysis of p-CREB in Ba/F3-parental, Ba/F3-FLT3/ITD, and Ba/F3-FLT3/ITD cells-treated with FLT3 inhibitor Quizartinib (Qui in short). Isotype antibody were used as control to calculate the mean fluorescence intensity (MFI) ratio (F-G). The transcription and expression of Fst was detected by RT-qPCR after Quizartinib treatment (10 nM) in Ba/F3-FLT3/ITD cells for one day (H). Data information: the experiments were performed in triplicates, and the data were presented as mean ± SEM. **P<0.01, ***P<0.001 (Student's t-test).


I - K expression of FST phosphorylation of CREB were detected by Western Blotting ( I K ) phospho - flow ( J ) in MOLM - 13 ( I ) Ba / F3 - FLT3 / ITD ( K ) treated with Quizartinib BRD7389 for one day , respectively . Data information : experiments were performed in triplicates , data were presented as mean ± . * * P < 0 . 01 , * * * P < 0 . 001 ( Student ' s t - ) .

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I-K The expression of FST and phosphorylation of CREB were detected by Western Blotting (I and K) and phospho-flow analysis (J) in MOLM-13 (I) and Ba/F3-FLT3/ITD (K) cells treated with Quizartinib and BRD7389 for one day, respectively. Data information: the experiments were performed in triplicates, and the data were presented as mean ± SEM. **P<0.01, ***P<0.001 (Student's t-test).


L RSK FST expression were detected by Western Blotting after p90RSK knockout by CRISPR / Cas9 in MOLM - 13 .

L RSK and FST expression were detected by Western Blotting after p90RSK knockout by CRISPR/Cas9 in MOLM-13 cells.


M phosphorylation of CREB FST expression were detected by Western Blotting in Ba / F3 - FLT3 / ITD treated with CREB inhibitor 666 - 15 for one day . ^ : non - specific of p - ATF1 due to conserved motif .

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M The phosphorylation of CREB and FST expression were detected by Western Blotting in Ba/F3-FLT3/ITD cells treated with CREB inhibitor 666-15 for one day. ^: non-specific staining of p-ATF1 protein due to the conserved motif.


N CREB FST expression were detected by Western Blotting after CREB knockout by CRISPR / Cas9 in MOLM - 13 .

N CREB and FST expression were detected by Western Blotting after CREB knockout by CRISPR/Cas9 in MOLM-13 cells.


O growth of Ba / F3 - parental ( with IL - 3 ) , Ba / F3 - FLT3 / ITD ( without IL - 3 ) Ba / F3 - FLT3 / ITD ( with IL - 3 ) was measured after three days treatment of CREB inhibitor 666 - 15 in vitro . Data information : experiments were performed in triplicates , data were presented as mean ± . * * P < 0 . 01 , * * * P < 0 . 001 ( Student ' s t - ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O 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 I-CONTROLLED_VAR I-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 The growth of Ba/F3-parental (with IL-3), Ba/F3-FLT3/ITD (without IL-3) and Ba/F3-FLT3/ITD (with IL-3) cells was measured after three days treatment of CREB inhibitor 666-15 in vitro. Data information: the experiments were performed in triplicates, and the data were presented as mean ± SEM. **P<0.01, ***P<0.001 (Student's t-test).


P rescue effect of CREB inhibitor 666 - 15 on FLT3 / ITD - induced dorsalization axis duplication in zebrafish at 1 dpf .

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

P The rescue effect of CREB inhibitor 666-15 on FLT3/ITD-induced dorsalization and axis duplication in zebrafish embryos at 1 dpf.


A FST expression in different AML lines was detected by Western Blotting .

A FST expression in different AML cell lines was detected by Western Blotting.


FST317 FST344 overexpression resulted in significant increases of FST transcription by RT - qPCR by Western Blot ( B ) Green , ML - 2 - GFP ; Blue , ML - 2 - FST317 ; Red , ML - 2 - FST344 . RT - qPCR experiments were performed in triplicates ( B ) . Data information : data were presented as mean ± . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( Student ' s t - ) . ns : Not significant .

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

FST317 and FST344 overexpression resulted in significant increases of FST transcription by RT-qPCR and protein by Western Blot (B) Green, ML-2-GFP; Blue, ML-2-FST317; Red, ML-2-FST344. The RT-qPCR experiments were performed in triplicates (B). Data information: data were presented as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001 (Student's t-test). ns: Not significant.


FST317 FST344 overexpression promoted ML - 2 growth in vitro ( C ) . Green , ML - 2 - GFP ; Blue , ML - 2 - FST317 ; Red , ML - 2 - FST344 . Data information : data were presented as mean ± . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( Student ' s t - ) . ns : Not significant .

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

FST317 and FST344 overexpression promoted ML-2 cell growth in vitro (C). Green, ML-2-GFP; Blue, ML-2-FST317; Red, ML-2-FST344. Data information: data were presented as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001 (Student's t-test). ns: Not significant.


D - E clonogenicity of ML - 2 overexpressing GFP , FST317 , FST344 in vitro for 14 days . CFU experiments were performed in triplicates ( E ) . Data information : data were presented as mean ± . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( Student ' s t - ) . ns : Not significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-E The clonogenicity of ML-2 overexpressing GFP, FST317, and FST344 in vitro for 14 days. The CFU experiments were performed in triplicates (E). Data information: data were presented as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001 (Student's t-test). ns: Not significant.


engraftment of ML - 2 ( with luciferase gene ) overexpressing GFP , FST317 , FST344 was quantified by bioluminescence imaging ( F G ) Data information : data were presented as mean ± . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( Student ' s t - ) . ns : Not significant .

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

The engraftment of ML-2 (with luciferase gene) overexpressing GFP, FST317, and FST344 was quantified by bioluminescence imaging (F and G) Data information: data were presented as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001 (Student's t-test). ns: Not significant.


engraftment of ML - 2 ( with luciferase gene ) overexpressing GFP , FST317 , FST344 survival of ML - 2 - engrafted NSG mice in vivo was recorded ( H ) . Survival curve in panel H was analyzed by Log - rank . * P < 0 . 05 , * * P < 0 . 01 .

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

The engraftment of ML-2 (with luciferase gene) overexpressing GFP, FST317, and FST344 and the survival of ML-2-engrafted NSG mice in vivo was recorded (H). Survival curve in panel H was analyzed by Log-rank test. *P<0.05, **P<0.01.


- seq of upregulation of RET , IL2RA CCL5 after FST344 overexpression in ML - 2 .

RNA-seq of upregulation of RET, IL2RA and CCL5 after FST344 overexpression in ML-2 cells.


RT - qPCR validation of upregulation of RET , IL2RA CCL5 after FST344 overexpression in ML - 2 . RT - qPCR experiments were performed in triplicates ( J ) . Data information : data were presented as mean ± . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( Student ' s t - ) . ns : Not significant .

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

RT-qPCR validation of upregulation of RET, IL2RA and CCL5 after FST344 overexpression in ML-2 cells. RT-qPCR experiments were performed in triplicates (J). Data information: data were presented as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001 (Student's t-test). ns: Not significant.


Overall survival of from TCGA - AML on differential expression of

Overall survival analysis of patients from TCGA-AML based on the differential expression of IL2RA


Overall survival of from TCGA - AML on differential expression of CCL5 .

Overall survival analysis of patients from TCGA-AML based on the differential expression of CCL5.


M Activation of MAPK / ERK pathway in ML - 2 by FST344 overexpression were validated by Western Blotting . Scale bar = 1 mm .

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M Activation of MAPK/ERK pathway in ML-2 by FST344 overexpression were validated by Western Blotting. Scale bar = 1 mm.


A FST knockdown in MOLM - 13 by shRNA effectively reduced FST expression .

A FST knockdown in MOLM-13 by shRNA effectively reduced FST expression.


were measured after FST knockdown in vitro . Scale bar = 10 μm .

The morphology were measured after FST knockdown in vitro. Scale bar = 10 μm.


apoptosis ( C ) measured after FST knockdown in vitro . Scale bar = 10 μm . apoptosis assays ( C ) were performed in triplicates . Data information : data were presented as mean ± . * P < 0 . 05 , * * P < 0 . 01 ( Student ' s t - ) .

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The apoptosis (C) measured after FST knockdown in vitro. Scale bar = 10 μm. The apoptosis assays (C) were performed in triplicates. Data information: data were presented as mean ± SEM. *P<0.05, **P<0.01 (Student's t-test).


clonogenicity of MOLM - 13 ( D E ) were measured after FST knockdown in vitro . Scale bar = 10 μm . Data information : data were presented as mean ± . * P < 0 . 05 , * * P < 0 . 01 ( Student ' s t - ) .

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The clonogenicity of MOLM-13 (D and E) were measured after FST knockdown in vitro. Scale bar = 10 μm. Data information: data were presented as mean ± SEM. *P<0.05, **P<0.01 (Student's t-test).


engraftment of MOLM - 13 after FST knockdown was detected by flow cytometry of human CD45 mouse CD45 . 1 positive in recipient mouse BM aspiration at week 2 post transplantation .

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

The engraftment of MOLM-13 after FST knockdown was detected by flow cytometry of human CD45 and mouse CD45.1 positive cells in recipient mouse BM aspiration at week 2 post transplantation.


engraftment of MOLM - 13 after FST knockdown was detected by flow cytometry of human CD45 mouse CD45 . 1 positive in recipient mouse BM aspiration at week 2 post transplantation . Data information : data were presented as mean ± . * P < 0 . 05 , * * P < 0 . 01 ( Student ' s t - ) .

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

The engraftment of MOLM-13 after FST knockdown was detected by flow cytometry of human CD45 and mouse CD45.1 positive cells in recipient mouse BM aspiration at week 2 post transplantation. Data information: data were presented as mean ± SEM. *P<0.05, **P<0.01 (Student's t-test).


effect of FST knockdown on survival of NSG mice - engrafted with MOLM - 13 . scr : scrambled control ( 7 mice ) ; sh : short hairpin ( 8 mice for sh1 sh2 respectively ) . survival curve was analyzed by Log - rank . * * P < 0 . 01 .

The effect of FST knockdown on the survival of NSG mice-engrafted with MOLM-13. scr: scrambled sequence control (7 mice); sh: short hairpin RNA (8 mice for sh1 and sh2 respectively). The survival curve was analyzed by Log-rank test. **P<0.01.


of MOLM - 13 after FST knockout by CRISPR / Cas9 in vitro . Scale bar = 10 μm .

The morphology of MOLM-13 after FST knockout by CRISPR/Cas9 in vitro. Scale bar = 10 μm.


clonogenicity of MOLM - 13 after FST knockout by CRISPR / Cas9 in vitro . Scale bar = 10 μm . Data information : data were presented as mean ± . * P < 0 . 05 , * * P < 0 . 01 ( Student ' s t - ) .

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

The clonogenicity of MOLM-13 after FST knockout by CRISPR/Cas9 in vitro. Scale bar = 10 μm. Data information: data were presented as mean ± SEM. *P<0.05, **P<0.01 (Student's t-test).


engraftment of MOLM - 13 after FST knockout was detected by flow cytometry of human CD45 mouse CD45 . 1 positive in recipient mouse BM aspiration at week 2 post transplantation .

The engraftment of MOLM-13 after FST knockout was detected by flow cytometry of human CD45 and mouse CD45.1 positive cells in recipient mouse BM aspiration at week 2 post transplantation.


engraftment of MOLM - 13 after FST knockout was detected by flow cytometry of human CD45 mouse CD45 . 1 positive in recipient mouse BM aspiration at week 2 post transplantation . Data information : data were presented as mean ± . * P < 0 . 05 , * * P < 0 . 01 ( Student ' s t - ) .

The engraftment of MOLM-13 after FST knockout was detected by flow cytometry of human CD45 and mouse CD45.1 positive cells in recipient mouse BM aspiration at week 2 post transplantation. Data information: data were presented as mean ± SEM. *P<0.05, **P<0.01 (Student's t-test).


E effect of FST knockout on survival of NSG mice - engrafted with MOLM - 13 . Cas 9 : Cas 9 only ( 10 mice ) ; sgRNA # 3 : Cas 9 + sgRNA # 3 ( 10 mice ) ; sgRNA # 4 : Cas 9 + sgRNA # 4 ( 8 mice ) . Data information : survival curves were analyzed by Log - rank . * P < 0 . 05 .

E The effect of FST knockout on the survival of NSG mice-engrafted with MOLM-13 cells. Cas 9: Cas 9 only (10 mice); sgRNA#3: Cas 9 + sgRNA#3 (10 mice); sgRNA#4: Cas 9 + sgRNA#4 (8 mice). Data information: survival curves were analyzed by Log-rank test. *P<0.05.


F knockdown efficiency of different FST - specific antisense oligoes ( ASOs ) in MOLM - 13 was detected by RT - qPCR after three days treatment in vitro . knockdown RT - qPCR experiments were performed in triplicates . Data information : data were presented as mean ± . * P < 0 . 05 , * * P < 0 . 01 ( Student ' s t - ) .

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

F The knockdown efficiency of different FST-specific antisense oligoes (ASOs) in MOLM-13 cells was detected by RT-qPCR after three days treatment in vitro. The knockdown and RT-qPCR experiments were performed in triplicates. Data information: data were presented as mean ± SEM. *P<0.05, **P<0.01 (Student's t-test).


G MOLM - 13 growth was measured after 3 days treatment of FST - ASO in vitro . ASO treatment experiments were performed in triplicates . Data information : data were presented as mean ± . * P < 0 . 05 , * * P < 0 . 01 ( Student ' s t - ) .

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

G MOLM-13 cell growth was measured after 3 days treatment of FST-ASO in vitro. The ASO treatment experiments were performed in triplicates. Data information: data were presented as mean ± SEM. *P<0.05, **P<0.01 (Student's t-test).


H Intraperitoneal injection of FST - ASO ( 10mg / kg weekly , 6 mice ) significantly prolonged survival of MOLM - 13 - engrafted NSG mice . random was used for negative control ( Neg - ASO , 6 mice ) . Data information : survival curves were analyzed by Log - rank . * P < 0 . 05 .

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

H Intraperitoneal injection of FST-ASO (10mg/kg weekly, 6 mice) significantly prolonged the survival of MOLM-13-engrafted NSG mice. The random sequence was used for negative control (Neg-ASO, 6 mice). Data information: survival curves were analyzed by Log-rank test. *P<0.05.


Flt3 / ITD knock - in mouse were genotyped ( A ) .

Flt3/ITD knock-in mouse were genotyped (A).


spleen ( B C , 4 mice each ) were measured in WT siblings Flt3 / ITD - knock - in mice ( 8 mice each ) . data were presented in box plot . whiskers , boxes , central lines represented minimum - to - maximum values , 25th - to - 75th percentile , 50th percentile ( median ) , respectively . * * P < 0 . 01 ( Student ' s t - ) .

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

The spleen weight (B and C, 4 mice each) were measured in WT siblings and Flt3/ITD-knock-in mice (8 mice each). data were presented in box plot. The whiskers, boxes, and central lines represented the minimum-to-maximum values, 25th-to-75th percentile, and the 50th percentile (median), respectively. **P<0.01 (Student's t-test).


serum Fst level ( D ) were measured in WT siblings Flt3 / ITD - knock - in mice ( 8 mice each ) . data were presented in box plot . whiskers , boxes , central lines represented minimum - to - maximum values , 25th - to - 75th percentile , 50th percentile ( median ) , respectively . * * P < 0 . 01 ( Student ' s t - ) .

The serum Fst level (D) were measured in WT siblings and Flt3/ITD-knock-in mice (8 mice each). data were presented in box plot. The whiskers, boxes, and central lines represented the minimum-to-maximum values, 25th-to-75th percentile, and the 50th percentile (median), respectively. **P<0.01 (Student's t-test).


engraftment of MOLM - 13 in NSG mice was confirmed at week 1 post injection ( E - F ) .

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

The engraftment of MOLM-13 in NSG mice was confirmed at week 1 post injection (E-F).


Serum FST level was measured in MOLM - 13 - engrafted NSG mice at pre - injection week 2 post injection ( G , PBS group , 3 mice ; MOLM - 13 group , 4 mice ) . Data information : data were presented as scatter dot plot . n . s : not significant , * P < 0 . 05 , * * P < 0 . 01 ( Student ' s t - ) .

Serum FST level was measured in MOLM-13-engrafted NSG mice at pre-injection and week 2 post injection (G, PBS group, 3 mice; MOLM-13 group, 4 mice). Data information: data were presented as scatter dot plot. n.s: not significant, *P<0.05, **P<0.01 (Student's t-test). In


After FST knockdown , serum FST level was also measured in MOLM - 13 - engrafted NSG mice at week 2 post injection ( H , 3 mice for each group ) . Data information : data were presented as mean ± . * P < 0 . 05 , * * P < 0 . 01 ( Student ' s t - ) .

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

After FST knockdown, serum FST level was also measured in MOLM-13-engrafted NSG mice at week 2 post injection (H, 3 mice for each group). Data information: data were presented as mean ± SEM. *P<0.05, **P<0.01 (Student's t-test).


Serum FST level was significantly increased in AML - derived xenografted mouse at week 6 post injection . Human leukemic engraftments were confirmed by flow cytometry of human CD45 mouse CD45 . 1 in recipient mouse BM aspiration ( J ) .

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

Serum FST level was significantly increased in primary AML-derived xenografted mouse at week 6 post injection. Human leukemic engraftments were confirmed by flow cytometry of human CD45 and mouse CD45.1 cells in recipient mouse BM aspiration (J).


Serum FST level was significantly increased in AML - derived xenografted mouse at week 6 post injection . Serum FST from pre - injection post - engraftment mouse were measured ( K , one mouse for each AML sample ) . Data information : data were presented as scatter dot plot . n . s : not significant , * P < 0 . 05 , * * P < 0 . 01 ( Student ' s t - ) .

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

Serum FST level was significantly increased in primary AML-derived xenografted mouse at week 6 post injection. Serum FST from pre-injection and post-engraftment mouse were measured (K, one mouse for each primary AML sample). Data information: data were presented as scatter dot plot. n.s: not significant, *P<0.05, **P<0.01 (Student's t-test).


L Correlation between serum FST leukemia blast from FLT3 / ITD - mutated AML at diagnosis . Correlation ( Pearson correlation coefficient ) was performed by Graphpad Prism 6 .

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L Correlation between serum FST levels and leukemia blast percentage from FLT3/ITD-mutated AML at diagnosis. Correlation analysis (Pearson correlation coefficient) was performed by Graphpad Prism 6.


Serum FST decreased in CR increased after relapse in 4 AML receiving Quizartinib monotherapy . Data information : data were presented as mean ± . * P < 0 . 05 , * * P < 0 . 01 ( Student ' s t - ) .

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Serum FST decreased in CR and increased after relapse in 4 AML patients receiving Quizartinib monotherapy. Data information: data were presented as mean ± SEM. *P<0.05, **P<0.01 (Student's t-test).


N Serum FST continued to rise during disease progression from a who did not respond to Quizartinib . in N were recruited in QUANTUM - R accrual has been completed . data were presented as mean ± . * P < 0 . 05 , * * P < 0 . 01 ( Student ' s t - ) .

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

N Serum FST continued to rise during disease progression from a patient who did not respond to Quizartinib. Patients in N were recruited in the QUANTUM-R and patient accrual has been completed. data were presented as mean ± SEM. *P<0.05, **P<0.01 (Student's t-test).


A . SF - 1 , FATE1 β - tubulin shown in basal condition after Dox treatment in H295R / TR , H295R / TRSF - 1 , H295R / TRFATE1 H295R / TR N - FlagFATE1cells .

O O O O O O O O O O O O O O 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. SF-1, FATE1 and β-tubulin protein levels shown in basal condition and after Dox treatment in H295R/TR, H295R/TRSF-1, H295R/TRFATE1 and H295R/TR N-FlagFATE1cells.


B . FATE1 mRNA expression is increased in H295R / TRSF - 1 by Dox treatment . efficiency of FATE1 knockdown by specific ( siFATE1 ) vs . control ( siC ) siRNA nucleofection is shown ( mean ± ; n = 4 with 3 replicates / experiment ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O 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. FATE1 mRNA expression is increased in H295R/TRSF-1 cells by Dox treatment. The efficiency of FATE1 knockdown by specific (siFATE1) vs. control (siC) siRNA nucleofection is shown (mean ± SEM; n=4 with 3 replicates/experiment).


C . Immunofluorescence microscopy showing endogenous FATE1 ( red ) induction of expression by Dox - treatment of H295R / TRSF - 1 . SF - 1 ( green ) , DAPI ( blue ) . Scale bars , 10 μm .

C. Immunofluorescence microscopy showing endogenous FATE1 protein (red) induction of expression by Dox-treatment of H295R/TRSF-1 cells. SF-1 (green), DAPI (blue). Scale bars, 10 μm.


D . Subcellular localization of endogenous FATE1 ( red ) transfected markers for Golgi , ER mitochondria , respectively ( green ) in Dox - treated H295R / TRSF - 1 . DAPI ( blue ) . Scale bars , 10 μm .

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D. Subcellular localization of endogenous FATE1 (red) and transfected fluorescent markers for Golgi, ER and mitochondria, respectively (green) in Dox-treated H295R/TRSF-1 cells. DAPI (blue). Scale bars, 10 μm.


E . Subcellular localization of endogenous FATE1 ( green ) in Dox - treated H295R / TRSF - 1 costained with an antibody against mitochondrial marker HSP60 ( red ) . SF - 1 ( blue ) . Scale bar , 10 μm .

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E. Subcellular localization of the endogenous FATE1 protein (green) in Dox-treated H295R/TRSF-1 cells costained with an antibody against the mitochondrial marker HSP60 (red). SF-1 (blue). Scale bar, 10 μm.


F . Immunogold electron microscopy showing association of FATE1 with mitochondrial outer surface in Dox - treated H295R / TR N - FlagFATE1 . Scale bar , 500 nm .

F. Immunogold electron microscopy showing association of FATE1 with the mitochondrial outer surface in Dox-treated H295R/TR N-FlagFATE1 cells. Scale bar, 500 nm.


G . Dox - treated H295R / TR N - FlagFATE1 were fractioned into ( N ) , heavy membranes ( HM ) , membranes ( LM ) ( C ) fractions localization of SF - 1 , FATE1 , ribosomal RPL7 , VDAC1 GAPDH was revealed by immunoblot .

G. Dox-treated H295R/TR N-FlagFATE1 cells were fractioned into nuclear (N), heavy membranes (HM), light membranes (LM) and cytosolic (C) fractions and localization of SF-1, FATE1, ribosomal protein RPL7, VDAC1 and GAPDH was revealed by immunoblot.


H . Effect of increasing concentrations ( 0 , 1 , 10 100 μg / ml ) of proteinase K 0 . 1 % Triton X - 100 ( TX - 100 ) treatment of mitochondrial fraction from Dox - treated H295R / TR N - FlagFATE1 on VDAC1 , FATE1 , TOM20 cytochrome c .

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

H. Effect of increasing concentrations (0, 1, 10 and 100 μg/ml) of proteinase K and 0.1% Triton X-100 (TX-100) treatment of the mitochondrial fraction from Dox-treated H295R/TR N-FlagFATE1 cells on VDAC1, FATE1, TOM20 and cytochrome c.


I . FATE1 VDAC1 are associated with pellet ( ; P ) fraction after high - speed centrifugation of alkaline - extracted mitochondrial fraction of Dox - treated H295R / TR SF - 1 , while cytochrome c is found in supernatant ( S ) .

O O O O O O O O O O O O O O O O O O O O O O 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. FATE1 and VDAC1 are associated with the pellet (membrane; P) fraction after high-speed centrifugation of the alkaline-extracted mitochondrial fraction of Dox-treated H295R/TR SF-1 cells, while cytochrome c is found in the supernatant (S).


B . Subcellular localization of EGFP - fusions of full - ( FL ) FATE1 its mutants : N - terminal ( aa . 1 - 124 ) , C - terminal ( aa . 125 - 183 ) , 1 - 162 , transmembrane domain ( aa . 155 - 183 ) L151D ( green ) transfected in H295R / TRSF - 1 . Mitochondria were stained by anti - TOM20 antibody ( red ) by DAPI ( blue ) . Scale bars , 10 μm .

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B. Subcellular localization of EGFP-fusions of full-length (FL) FATE1 and its mutants: N-terminal (aa. 1-124), C-terminal (aa.125-183), 1-162, transmembrane domain (aa. 155-183) and L151D (green) transfected in H295R/TRSF-1 cells. Mitochondria were stained by anti-TOM20 antibody (red) and DNA by DAPI (blue). Scale bars, 10 μm.


C . EMD Flag - tagged FATE1 , but not other OMM TOM20 , were co - immunoprecipitated from Dox - treated H295R / TR N - FlagFATE1 . Control immunoprecipitations were performed with anti - myc ( α - myc ) non - immune rabbit IgG ( IgG - R ) antibodies .

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

C. EMD and Flag-tagged FATE1, but not the other OMM protein TOM20, were co-immunoprecipitated from Dox-treated H295R/TR N-FlagFATE1 cells. Control immunoprecipitations were performed with anti-myc (α-myc) and non-immune rabbit IgG (IgG-R) antibodies.


D . Confocal microscopy showed colocalization of EMD , as revealed by immunofluorescence microscopy ( red ) , with ER , stained by ER - GFP BacMam ( green ) . Scale bars , 10 μm .

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D. Confocal fluorescence microscopy showed colocalization of cytoplasmic EMD, as revealed by immunofluorescence microscopy (red), with ER, stained by ER-GFP BacMam (green). Scale bars, 10 μm.


E . Mic60 / mitofilin Flag - tagged FATE1 were coimmunoprecipitated from Dox - treated H295R / TR N - FlagFATE1 . Control immunoprecipitations were performed with anti - myc ( α - myc ) antibody .

E. Mic60/mitofilin and Flag-tagged FATE1 were coimmunoprecipitated from Dox-treated H295R/TR N-FlagFATE1 cells. Control immunoprecipitations were performed with anti-myc (α-myc) antibody.


F . Mitochondrial localization of Mic60 / mitofilin ( green ) FATE1 ( red ) in Dox - treated H295R / TRSF - 1 . Scale bar , 10 μm .

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F. Mitochondrial localization of Mic60/mitofilin (green) and FATE1 (red) in Dox-treated H295R/TRSF-1 cells. Scale bar, 10 μm.


G . Left , triple immunofluorescence microscopy labelling of endogenous FATE1 ( green ) , ER labelled by calreticulin ( red ) mitochondria labelled by HSP60 ( blue ) in Dox - treated H295R / TRSF - 1 . One showing close apposition of red , green blue ( signals ) is shown at higher magnification . Scale bar , 10 μm . Right , graph showing of FATE1 signal in total mitochondria ( histogram ) vs . ER - mitochondria contact sites ( red histogram ) ( mean ± ; n = 13 ) . * * p < 0 . 01 .

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G. Left, triple immunofluorescence microscopy labelling of endogenous FATE1 (green), ER labelled by calreticulin (red) and mitochondria labelled by HSP60 staining (blue) in Dox-treated H295R/TRSF-1 cells. One area showing close apposition of red, green and blue staining (white signals) is shown at higher magnification. Scale bar, 10 μm. Right, graph showing quantification of FATE1 signal in total mitochondria (white histogram) vs. ER-mitochondria contact sites (red histogram) (mean ± SEM; n=13). **p<0.01.


H . H295R / TRSF - 1 were fractioned into crude mitochondria ( CM ) , endoplasmic reticulum ( ER ) , purified mitochondria ( PM ) mitochondria - associated membranes ( MAM ) fractions . H , total homogenate . localization of SERCA2 , SOAT1 , VDAC1 , GRP75 , S1R , EMD , Mic60 / mitofilin , FATE1 TOM20 in different fractions was revealed by immunoblot .

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H. H295R/TRSF-1 cells were fractioned into crude mitochondria (CM), endoplasmic reticulum (ER), purified mitochondria (PM) and mitochondria-associated membranes (MAM) fractions. H, total homogenate. The localization of SERCA2, SOAT1, VDAC1, GRP75, S1R, EMD, Mic60/mitofilin, FATE1 and TOM20 proteins in the different fractions was revealed by immunoblot.


A . H295R / TR N - FlagFATE1 were transfected with D1 ER marker for ER ( green ) mtRFP marker for mitochondria ( red ) . Manders ' coefficient for green - red signals colocalization in basal ( histograms ) Dox - treated ( black histograms ) is shown on right ( mean ± ; n = 12 ) . * * * p < 0 . 001 . Scale bars , 10 μm .

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A. H295R/TR N-FlagFATE1 cells were transfected with D1 ER marker for ER (green) and mtRFP marker for mitochondria (red). Manders' coefficient for green-red signals colocalization in basal (white histograms) and Dox-treated (black histograms) cells is shown on the right (mean ± SEM; n=12). ***p<0.001. Scale bars, 10 μm.


B . Effect of FATE1 expression on ER - mitochondria in H295R / TR N - FlagFATE1 measured using a split - GFP probe [ 29 ] . is shown in graph . histogram , number of objects / in basal conditions ; black histogram , number of objects / in Dox - treated ( mean ± ; n = 6 with 90 analyzed in total ) . * * p < 0 . 01 . Scale bars , 5 μm .

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B. Effect of FATE1 expression on ER-mitochondria distance in H295R/TR N-FlagFATE1 cells measured using a split-GFP probe [29]. Quantification is shown in the graph. White histogram, number of fluorescent objects/cell in basal conditions; black histogram, number of fluorescent objects/cell in Dox-treated cells (mean ± SEM; n=6 with 90 cells analyzed in total). **p<0.01. Scale bars, 5 μm.


C . Transmission electron microscopy of H295R / TR N - FlagFATE1 cultured in basal conditions or treated with Dox . ER - mitochondria contacts are indicated by arrowheads . Scale bars , 200 nm . Right , of number of contacts normalized by number of mitochondria of mitochondria with ER contact sites is shown in basal conditions ( histograms ) after Dox treatment ( black histograms ) of ( mean ± ; n = 38 for basal 47 for Dox - treated ) . * * * p < 0 . 001 .

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C. Transmission electron microscopy images of H295R/TR N-FlagFATE1 cells cultured in basal conditions or treated with Dox. ER-mitochondria contacts are indicated by white arrowheads. Scale bars, 200 nm. Right, quantification of the number of contacts normalized by the number of mitochondria and the percentage of mitochondria with ER contact sites is shown in basal conditions (white histograms) and after Dox treatment (black histograms) of cells (mean ± SEM; n=38 for basal and 47 for Dox-treated cells). ***p<0.001.


D . Transmission electron microscopy of H295R / TRSF - 1 cultured in basal conditions or treated with Dox . ER - mitochondria contacts are indicated by arrowheads . Scale bars , 200 nm . Right , of number of contacts normalized by number of mitochondria of mitochondria with ER contact sites is shown in basal conditions ( histograms ) after Dox treatment ( black histograms ) of ( mean ± ; n = 41 for basal 42 for Dox - treated ) . * * * p < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Transmission electron microscopy images of H295R/TRSF-1 cells cultured in basal conditions or treated with Dox. ER-mitochondria contacts are indicated by white arrowheads. Scale bars, 200 nm. Right, quantification of the number of contacts normalized by the number of mitochondria and the percentage of mitochondria with ER contact sites is shown in basal conditions (white histograms) and after Dox treatment (black histograms) of cells (mean ± SEM; n=41 for basal and 42 for Dox-treated cells). ***p<0.001.


E . Mitochondrial shape ( BacMam Mitochondria - RFP ; in red ) in H295R / TR N - FlagFATE1 shown by confocal microscopy in basal conditions after Dox treatment . FATE1 ( green ) , DAPI ( blue ) . A higher magnification of merged green red signal is shown in insets . Scale bars , 10 μm . F . Mitochondrial fragmentation index in H295R / TR N - FlagFATE1 after Dox treatment ( red histogram ) compared to cultured in basal conditions ( histogram ) ( mean ± ; n = 111 for basal 101 for Dox - treated ) . * * * p < 0 . 001 .

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E. Mitochondrial shape (BacMam Mitochondria-RFP; in red) in H295R/TR N-FlagFATE1 cells shown by fluorescence confocal microscopy in basal conditions and after Dox treatment. FATE1 (green), DAPI (blue). A higher magnification of merged green and red signal is shown in the insets. Scale bars, 10 μm.F. Mitochondrial fragmentation index in H295R/TR N-FlagFATE1 cells after Dox treatment (red histogram) compared to cells cultured in basal conditions (white histogram) (mean ± SEM; n=111 for basal and 101 for Dox-treated cells). ***p<0.001.


G . Mitochondrial fragmentation index in H295R / TRSF - 1 after Dox treatment ( green histogram ) compared to cultured in basal conditions ( histogram ) . ( mean ± ; n = 37 for basal 49 for Dox - treated ) . * * * p < 0 . 001 .

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G. Mitochondrial fragmentation index in H295R/TRSF-1 cells after Dox treatment (green histogram) compared to cells cultured in basal conditions (white histogram). (mean ± SEM; n=37 for basal and 49 for Dox-treated cells). ***p<0.001.


H . Mitochondrial potential ( ∆ Ψ ) measured by TMRM after Dox treatment of H295R / TR N - FlagFATE1 ( red histogram ) compared to cultured in basal conditions ( histogram ) . Mean ± is shown . ( mean ± ; n = 3 with 12 replicates / experiment ) . ns , not significant .

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H. Mitochondrial membrane potential (∆Ψ) measured by TMRM fluorescence after Dox treatment of H295R/TR N-FlagFATE1 cells (red histogram) compared to cells cultured in basal conditions (white histogram). Mean ± SEM is shown. (mean ± SEM; n=3 with 12 replicates/experiment). ns, not significant.


A . MitochondrialCa2 + uptake . H295R / TR parental clone ( / grey histograms ) H295R / TRSF - 1 ( / green histograms ) , H295R / TR N - FlagFATE1 ( / red histograms ) H295R / TRFATE1 ( / violet histograms ) ( mean ± ; n = 3 - 5 with 12 replicates / condition ) . * * p < 0 . 01 ; * * * p < 0 . 001 ; ns , not significant . B . CytosolicCa2 + . Histograms as in A ( mean ± ; n = 3 - 5 with 12 replicates / condition ) . ns , not significant .

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A. MitochondrialCa2+ uptake. H295R/TR parental clone (white/grey histograms) H295R/TRSF-1 (white/green histograms), H295R/TR N-FlagFATE1 (white/red histograms) and H295R/TRFATE1 cells (white/violet histograms) (mean ± SEM; n=3-5 with 12 replicates/condition). **p<0.01; ***p<0.001; ns, not significant.B. CytosolicCa2+ concentration. Histograms as in A (mean ± SEM; n=3-5 with 12 replicates/condition). ns, not significant.


C . Mitochondrial ( left ) ( right ) Ca2 + concentrations in Dox - treated H295R / TRSF - 1 transfected with control ( siC ; histograms ) or FATE1 - specific ( siFATE1 ; green histograms ) siRNAs ( mean ± ; n = 3 - 5 with 12 replicates / condition ) . * * * p < 0 . 001 ; ns , not significant .

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C. Mitochondrial (left) and cytosolic (right) Ca2+ concentrations in Dox-treated H295R/TRSF-1 cells transfected with control (siC; white histograms) or FATE1-specific (siFATE1; green histograms) siRNAs (mean ± SEM; n=3-5 with 12 replicates/condition). ***p<0.001; ns, not significant.


D . Caspase 3 / 7 activity in H295R / TR N - FlagFATE1 cultured in basal conditions ( histograms ) or in presence of Dox ( red histograms ) treated with H2O2 ( 500 μM ) C2 - ceramide ( 50 μM ) or staurosporine ( STS ) ( 1 μM ) . ( mean ± ; n = 5 - 8 with 3 replicates / condition ) . * p < 0 . 05 ; * * p < 0 . 01 ; ns , not significant .

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D. Caspase 3/7 activity in H295R/TR N-FlagFATE1 cells cultured in basal conditions (white histograms) or in the presence of Dox (red histograms) treated with H2O2 (500 μM) C2-ceramide (50 μM) or staurosporine (STS) (1 μM). (mean ± SEM; n=5-8 with 3 replicates/condition). *p<0.05; **p<0.01; ns, not significant.


E . Caspase 3 / 7 activity in Dox - treated H295R / TRSF - 1 transfected with control ( siC ; histograms ) or FATE1 - specific ( siFATE1 ; green histograms ) siRNAs treated with H2O2 or STS ( mean ± ; n = 5 - 8 with 3 replicates / condition ) . * p < 0 . 05 ; ns , not significant .

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E. Caspase 3/7 activity in Dox-treated H295R/TRSF-1 cells transfected with control (siC; white histograms) or FATE1-specific (siFATE1; green histograms) siRNAs and treated with H2O2 or STS (mean ± SEM; n=5-8 with 3 replicates/condition). *p<0.05; ns, not significant.


F . Flow cytometry of TUNEL in H295R / TR N - Flag FATE1 cultured in basal conditions or in presence of Dox treated with vehicle ( control ) H2O2 ( 500 μM ) , C2 - ceramide ( 50 μM ) or staurosporine ( STS ) ( 1 μM ) .

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F. Flow cytometry analysis of TUNEL staining in H295R/TR N-Flag FATE1 cells cultured in basal conditions or in the presence of Dox treated with vehicle (control) H2O2 (500 μM), C2-ceramide (50 μM) or staurosporine (STS) (1 μM).


G . Caspase 3 / 7 activity in H295R / TR N - FlagFATE1 cultured in basal conditions ( histograms ) or in presence of Dox ( red histograms ) treated with mitotane ( 50 μM ) ( mean ± ; n = 5 - 8 with 3 replicates / condition ) . * * p < 0 . 01 .

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G. Caspase 3/7 activity in H295R/TR N-FlagFATE1 cells cultured in basal conditions (white histograms) or in the presence of Dox (red histograms) and treated with mitotane (50 μM) (mean ± SEM; n=5-8 with 3 replicates/condition). **p<0.01.


H . Caspase 3 / 7 activity in Dox - treated H295R / TRSF - 1 transfected with control ( siC ; histograms ) or FATE1 - specific ( siFATE1 ; green histograms ) siRNAs treated with mitotane ( mean ± ; n = 5 - 8 with 3 replicates / condition ) . * p < 0 . 05 .

H. Caspase 3/7 activity in Dox-treated H295R/TRSF-1 cells transfected with control (siC; white histograms) or FATE1-specific (siFATE1; green histograms) siRNAs and treated with mitotane (mean ± SEM; n=5-8 with 3 replicates/condition). *p<0.05.


A . Immunohistochemistry of FATE1 expression in normal adrenal cortex adrenal hyperplasia , adenoma carcinoma ( ACC ) . Testis tissue was used as positive control . Scale bars , 200 µm ( low magnification ) ; 50 μ m ( high magnification ) . B . Table summarizing relative of FATE1IHC ( H - score ) in groups of normal , benign malignant tissues . highest FATE1 expression were found in ACC . n , number of samples studied for each category of tissues . NBST , normal benign steroidogenic tissues .

A. Immunohistochemistry analysis of FATE1 expression in normal adrenal cortex and adrenal hyperplasia, adenoma and carcinoma (ACC). Testis tissue was used as positive control. Scale bars, 200 µm (low magnification); 50 μ m (high magnification).B. Table summarizing the relative intensity of FATE1IHC staining (H-score) in groups of normal, benign and malignant tissues. The highest FATE1 expression levels were found in ACC. n, number of samples studied for each category of tissues. NBST, normal and benign steroidogenic tissues.


C . Prognostic parameters in ACC . Results are presented as hazard ratios ( HR ) including 95 % confidence interval ( CI ) . significance level was set at α = 5 % for comparisons . results for SF - 1 , FATE1 combined SF - 1 / FATE1 expression are shown both in univariate mutivariate ( adjusted for , sex ENSAT tumour stage ) .

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C. Prognostic parameters in ACC patients. Results are presented as hazard ratios (HR) including 95% confidence interval (CI). The significance level was set at α=5% for all comparisons. The results for SF-1, FATE1 and combined SF-1/FATE1 expression levels are shown both in univariate and mutivariate (adjusted for age, sex and ENSAT tumour stage) analysis.


D . Overall survival of ACC according to FATE1 expression in their tumours , as measured by quantitative immunohistochemistry ( low , green curve ; high , red curve ) in mutivariate . p = 0 . 008 .

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D. Overall survival of ACC patients according to FATE1 expression in their tumours, as measured by quantitative immunohistochemistry (low, green curve; high, red curve) in mutivariate analysis. p=0.008.


A ) of effect of BCL - 2 - family on HCT AKO , measured as of showing released cyt c or pyknotic nuclei . Data correspond to at least three independent experiments , n > 30 per condition per experiment . * * * p < 0 , 001 * p < 0 , 05 with respect to GFP condition .

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A) Quantification of the effect of individual BCL-2-family proteins on HCT AKO cell death, measured as percentage of cells showing released cyt c or pyknotic nuclei. Data correspond to at least three independent experiments, n>30 cells per condition per experiment. *** p<0,001 and * p<0,05 with respect to GFP condition.


C ) Western blot of SMAC cyt c release in GFP - BAX , tBID - GFP GFP - BCLXL expressing at 12 h , 16 h 24 h after transfection in HCT AKO . SN ( supernatant , fraction ) , P ( pellet , fraction ) PS ( Ponceau ) .

C) Western blot analysis of SMAC and cyt c release in GFP-BAX, tBID-GFP and GFP-BCLXL expressing cells at 12 h, 16 h and 24 h after transfection in HCT AKO. SN (supernatant, cytosolic fraction), P (pellet, membrane fraction) and PS (Ponceau Staining).


F ) Kinetics of expression of tBID - GFP ( blue ) PI intake upon tBID expression ( purple ) unstransfected HCT116 AKO ( in grey ) . Plots show average ( lines ) its standard deviation ( shaded areas ) from 3 technical replicates 3 independent experiments . was obtained by total per well using incucyte system every 60 min .

F) Kinetics of the expression of tBID-GFP (blue) and PI intake upon tBID expression (purple) and unstransfected HCT116 AKO cells (in grey). Plots show the average fluorescence intensity (dark lines) and its standard deviation (shaded areas) from 3 technical replicates and 3 independent experiments. Fluorescence intensity was obtained by analysis the total fluorescence per well using incucyte system every 60 min.


G ) of PI + from total transfected with GFP - BAX , tBID - GFP , GFP - BCLXL GFP . n = 3 independent experiments with > 10000 per condition per experiment . * * * p < 0 , 001 with respect to untransfected condition .

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G) Quantification of PI+ cells from total transfected cells with GFP-BAX, tBID-GFP, GFP-BCLXL and GFP. n=3 independent experiments with >10000 cells per condition per experiment. *** p<0,001 with respect to untransfected condition.


H ) induced by tBID - GFP in HCT AKO in absence presence of ZVAD , NSA Ferrostatin - 1 . Data correspond to three independent experiments , n > 30 per condition per experiment . * * * p < 0 , 001 with respect to MOCK condition .

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H) Cell death induced by tBID-GFP in HCT AKO cells in the absence and presence of ZVAD, NSA and Ferrostatin-1. Data correspond to three independent experiments, n>30 cells per condition per experiment. *** p<0,001 with respect to MOCK condition.


A ) Representative confocal immunofluorescence bright field ( BF ) of HCT DKO expressing tBID - GFP ( in green ) treated or not with COMBO ( ABT737 + S63586 ) . Cyt c shown in magenta nuclei in cyan . Scale bar 10 µm . B ) Effect of tBID - GFP mCherry - BCLXL on , measured as of showing released cyt c or pyknotic nuclei . Data correspond to at least three independent experiments , n > 30 per condition per experiment . * * * p < 0 , 001 , * * p < 0 , 025 * p < 0 , 05 with respect to tBID - GFP in HCT AKO condition .

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A) Representative confocal immunofluorescence and bright field (BF) images of HCT DKO cells expressing tBID-GFP (in green) treated or not with COMBO (ABT737 + S63586). Cyt c shown in magenta and nuclei in cyan. Scale bar 10 µm. B) Effect of tBID-GFP and mCherry-BCLXL on cell death, measured as percentage of cells showing released cyt c or pyknotic nuclei. Data correspond to at least three independent experiments, n> 30 cells per condition per experiment. *** p<0,001, ** p<0,025 and * p<0,05 with respect to tBID-GFP in HCT AKO condition.


C ) Representative confocal immunofluorescence of HCT DKO upon indicated treatments . Nuclei ( cyan ) cyt c ( magenta ) are shown . Scale bar 10µm . Arrowheads represent showing released cytochrome c pyknotic nuclei .

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C) Representative confocal immunofluorescence images of HCT DKO cells upon indicated treatments. Nuclei (cyan) and cyt c (magenta) are shown. Scale bar 10µm. Arrowheads represent cells showing released cytochrome c and pyknotic nuclei.


E ) % of induced by TRAIL ( 1µg / ml ) , COMBO their combination at different treatment durations on HCT DKO measured as in panel B . Paired Student ' s t - * * p < 0 , 025 * p < 0 , 05 with respect to MOCK condition .

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E) % of cell death induced by TRAIL (1µg/ml), COMBO and their combination at different treatment durations on HCT DKO measured as in panel B. Paired Student's t-test ** p<0,025 and * p<0,05 with respect to MOCK condition.


I ) Effect of TRAIL ( 1µg / ml ) , COMBO their combination on HCT DKO TKO at different time points normalized to control . Each dot corresponds to average of 3 technical replicates from 4 independent experiments . * * * p < 0 , 001 .

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I) Effect of TRAIL (1µg/ml), COMBO and their combination on HCT DKO and TKO cell death at different time points normalized to control cells. Each dot corresponds to the average of 3 technical replicates from 4 independent experiments. *** p<0,001.


J ) Representative HyVolution - superresolution of TFAM - RFP expressing U2OS BAX / BAK DKO in in absence ( top ) presence of tBID - GFP ( bottom ) . TFAM appears in red , TOM20 / mitochondria in grey tBID - GFP in green .

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J) Representative HyVolution - superresolution images of TFAM-RFP expressing U2OS BAX/BAK DKO cells in the in the absence (top) and presence of tBID-GFP (bottom). TFAM appears in red, TOM20/mitochondria in grey and tBID-GFP in green.


K ) of with TFAM release on U2OS BAX / BAK DKO transfected with GFP - BAX / tBID - GFP TFAM - RFP in presence / absence of COMBO . Dots correspond to experiments ( n = 3 experiments with more than 10 per condition per experiment ) . Unpaired Student ' s t - * * * p < 0 , 001 with respect to non - treated conditions .

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K) Quantification of cells with TFAM release on U2OS BAX/BAK DKO cells transfected with GFP-BAX/tBID-GFP and TFAM-RFP in the presence/ absence of COMBO. Dots correspond to individual experiments (n=3 individual experiments with more than 10 cells per condition per experiment). Unpaired Student's t-test *** p<0,001 with respect to non-treated conditions.


B ) induced by tBID - GFP its variants on HCT AKO , measured as of showing released cyt c or pyknotic nuclei . Data correspond to three independent experiments , with n > 30 per condition per experiment . * * * p < 0 , 001 * p < 0 , 05 with respect to tBID - GFP WT condition .

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B) Cell death induced by tBID-GFP and its variants on HCT AKO, measured as percentage of cells showing released cyt c or pyknotic nuclei. Data correspond to three independent experiments, with n> 30 cells per condition per experiment. *** p<0,001 and * p<0,05 with respect to tBID-GFP WT condition.


D ) Effect of tBID wt α6 - mutants overexpression on HCT AKO , measured as DRAQ7 + / GFP + normalized to tBID wt condition . n = 3 independent experiments , * * p < 0 , 025 * p < 0 , 05 with respect to tBID - GFP wt condition . E ) Effect of tBID wt α6 - mutants overexpression on MEF BID KO , measured as DRAQ7 + / GFP + normalized to tBID wt condition . Effect of GFP overexpression on BID KO relative to tBID wt was assessed by pyknotic nuclei formation . n = 3 independent experiments * * * p < 0 , 001 with respect to tBID - GFP wt condition .

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D) Effect of tBID wt and α6-mutants overexpression on HCT AKO cells, measured as DRAQ7+/GFP+ cells and normalized to tBID wt condition. n=3 independent experiments, ** p<0,025 and * p<0,05 with respect to tBID-GFP wt condition. E) Effect of tBID wt and α6-mutants overexpression on MEF BID KO cells, measured as DRAQ7+/GFP+ cells and normalized to tBID wt condition. Effect of GFP overexpression on BID KO cells relative to tBID wt was assessed by pyknotic nuclei formation quantification. n=3 independent experiments *** p<0,001 with respect to tBID-GFP wt condition.


A ) Representative CLEM of mitochondria in HCT AKO ( control ) HCT AKO expressing GFP - BAX or tBID - GFP . Black arrows indicate MOM disruptions . B - D ) Effect of GFP - BAX tBID - GFP on HCT AKO mitochondrial structure . ( B ) Number of disruptions at MOM per mitochondrial , each dot represents an . ( C ) Mean per mitochondrion at condition tested , each dot represents an mitochondrion . ( D ) of cristae , each dot represents an crista . Cristae shape calculated as / width . Data represent n > 50 mitochondria from 3 - 5 independent . In B - D ) , boxes represent 96 % confidence interval , average is represented by inside box whiskers correspond to S . D . statistical significance was assessed by unpaired Student ' s t - . * * * p < 0 , 001 * * p < 0 , 025 with respect to HCT AKO or untransfected condition .

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A) Representative CLEM images of mitochondria in HCT AKO cells (control) and HCT AKO cells expressing GFP-BAX or tBID-GFP. Black arrows indicate MOM disruptions. B-D) Effect of GFP -BAX and tBID-GFP on HCT AKO mitochondrial structure. (B) Number of disruptions at the MOM per mitochondrial area, each dot represents an individual cell. (C) Mean area per mitochondrion at the condition tested, each dot represents an individual mitochondrion. (D) Analysis of cristae morphology, each dot represents an individual crista. Cristae shape calculated as length/width. Data represent n>50 mitochondria from 3-5 independent cells. In B-D), boxes represent 96% confidence interval, the average is represented by the line inside the box and whiskers correspond to S.D. The statistical significance was assessed by unpaired Student's t-test. *** p<0,001 and ** p<0,025 with respect to HCT AKO or untransfected condition.


E ) Representative confocal STED of GFP - BAX tBID - GFP expressed in HCT AKO . Scale bar 1 µm for BAX , 5 µm for tBID . F ) Zoomed - in structures of GFP - BAX supramolecular assemblies with shape of lines ( * double lines ) , arcs rings in HCT AKO . Scale bar 100 nm .

E) Representative confocal and STED images of GFP-BAX and tBID-GFP expressed in HCT AKO. Scale bar 1 µm for BAX images, 5 µm for tBID images. F) Zoomed-in structures of GFP-BAX supramolecular assemblies with the shape of lines (*double lines), arcs and rings in HCT AKO cells. Scale bar 100 nm.


A ) representative western blot of cyt c SMAC in HeLa . B , C ) Western Blot of active BAX ( B ) BAK ( C ) , in HeLa treated with STS or infected with Shigella flexneri ( MOI 30 ) at different time points after infection .

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A) representative western blot of cytosolic cyt c and SMAC in HeLa cells. B,C) Western Blot analysis of active BAX (B) and BAK (C), in HeLa cells treated with STS or infected with Shigella flexneri (MOI 30) at different time points after infection.


E ) Representative Western Blot of total ( lysate ) cyt c SMAC in HCT wt , HCT DKO HCT TKO infected with Shigella flexneri at indicated time points after treatment / infection .

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E) Representative Western Blot analysis of cytosolic and total (lysate) cyt c and SMAC in HCT wt, HCT DKO and HCT TKO cells infected with Shigella flexneri at the indicated time points after treatment/infection.


A ) Caspase - 3 / 7 activity induced by ABT199 etoposide on Nalm6 ( venetoclax - sensitive ) Nalm6 199R ( venetoclax - resistant ) , normalized to untreated . Unpaired Student ' s t - * * * p < 0 , 001 * * p < 0 , 025 ( Nalm6 with respect to Nalm6 199R at different conditions ) . Dots correspond to technical replicates of n = 3 independent experiments .

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A) Caspase-3/7 activity induced by ABT199 and etoposide on Nalm6 (venetoclax-sensitive) and Nalm6 199R (venetoclax-resistant) cells, normalized to untreated cells. Unpaired Student's t-test *** p<0,001 and ** p<0,025 (Nalm6 with respect to Nalm6 199R at different conditions). Dots correspond to technical replicates of n=3 independent experiments.


D ) Representative kinetics of , represented as DRAQ7 + in Nalm6 199R ( grey ) , Nalm6 199R BID KO # 1 ( blue ) Nalm6 199R BID KO # 5 ( purple ) upon 250 ng / ml TRAIL treatment . Plots show average ( lines ) its standard deviation ( shaded areas ) from 5 technical replicates . was obtained by total per well using incucyte system every 60 min .

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D) Representative kinetics of cell death, represented as DRAQ7+ cells in Nalm6 199R cells (grey), Nalm6 199R BID KO #1 cells (blue) and Nalm6 199R BID KO #5 cells (purple) upon 250 ng/ml TRAIL treatment. Plots show the average fluorescence intensity (dark lines) and its standard deviation (shaded areas) from 5 technical replicates. Fluorescence intensity was obtained by analysis the total fluorescence per well using incucyte system every 60 min.E


H ) induced by GFP - BAK GFP - BAK R127H on HCT AKO , measured as DRAQ7 + / GFP + . Unpaired Student ' s t - . * * * p < 0 , 001 with respect to GFP BAK wt .

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H) Cell death induced by GFP-BAK and GFP-BAK R127H on HCT AKO, measured as DRAQ7+/GFP+ cells. Unpaired Student's t-test. *** p<0,001 with respect to GFP BAK wt.


I ) Effect of 24 h incubation with 250 ng / ml TRAIL on Nalm6 199R , Nalm6 199R BAK KO Nalm6 199R BOK KO , measured as DRAQ7 + with respect of total surface normalized to untreated conditions . Each dot corresponds to average normalized value of 3 - 4 technical replicates in n = 3 independent experiments .

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I) Effect of 24 h incubation with 250 ng/ml TRAIL on Nalm6 199R, Nalm6 199R BAK KO and Nalm6 199R BOK KO, measured as DRAQ7+ cells with respect of total cell surface and normalized to untreated conditions. Each dot corresponds to the average normalized value of 3-4 technical replicates in n=3 independent experiments.


( I ) Representative image showing changes in colon following DSS - induced colitis with concurrent treatment with MBELNs . ( J ) Column graph showing changes in colon . Data are mean ± from seven biological replicates , * * P < 0 . 01 using one - way ANOVA . (

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(I) Representative image showing changes in colon morphology and length following DSS-induced colitis with concurrent treatment with MBELNs. (J) Column graph showing changes in colon length. Data are mean ± SEM from seven biological replicates, **P<0.01 using one-way ANOVA. (


Hematoxylin eosin ( HE ) to show histological changes in colon tissue Scale bar 200 μm .

Hematoxylin and eosin (HE) staining to show histological changes in colon tissue Scale bar 200 μm.


( M ) Enzyme - linked immunosorbent ( ELISA ) for interleukin ( IL ) - 6 IL - 1β from colon tissue . Data are mean ± from seven biological replicates , * * P < 0 . 01 using one - way ANOVA .

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(M) Enzyme-linked immunosorbent assay (ELISA) for interleukin (IL)-6 and IL-1β from colon tissue. Data are mean ± SEM from seven biological replicates, **P<0.01 using one-way ANOVA.


( A ) Mass spectrometry ( MS ) of aryl hydrocarbon receptor ( AhR ) pulldown from Mulberry bark derived exosome - like nanoparticles ( MBELNs ) 2 , 3 , 7 , 8 - tetrachlorodibenzo - p - dioxin ( TCDD ) treated MC38 lysate . Data from three technical replicates are shown .

(A) Mass spectrometry (MS) analysis of aryl hydrocarbon receptor (AhR) pulldown protein from Mulberry bark derived exosome-like nanoparticles (MBELNs) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) treated MC38 cell lysate. Data from three technical replicates are shown.


( D ) Representative showing effect of MBELNs with without TCDD ( 1 nm ) / carbobenzoxy - Leu - Leu - leucinal ( MG132 ; 20 µM ) on co - expression of AhR COPS8 using immunofluorescence . Scale bar 10 μm , are from three biological replicates .

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(D) Representative images showing effect of MBELNs with and without TCDD (1 nm)/ carbobenzoxy-Leu-Leu-leucinal (MG132; 20 µM) on co-expression of AhR and COPS8 using immunofluorescence. Scale bar 10 μm, images are from three biological replicates.


( E ) Western blot showing MBELNs , TCDD MG132 effect on co - expression of AhR COPS8 . data represent as mean ± from three biological replicates * * P < 0 . 01 , * * * P < 0 . 001 using one - way ANOVA .

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(E) Western blot showing MBELNs, TCDD and MG132 effect on co-expression of AhR and COPS8. The data represent as mean ± SEM from three biological replicates **P<0.01, ***P<0.001 using one-way ANOVA.


( F ) Heat map showing MS of AhR pulldown from MC38 treated with MBELNs in combination with proteasome inhibitor , MG132 . Data are represented from three technical replicates .

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(F) Heat map showing MS analysis of AhR pulldown protein from MC38 cells treated with MBELNs in combination with proteasome inhibitor, MG132. Data are represented from three technical replicates.


( C ) Transmission electron microscopy ( ) of crypts of COPS8fl / fl COPS8ΔIEC mice . base of crypt in COPS8ΔIEC mice is occupied by poorly differentiated columnar epithelial that lack secretory granules , rudimentary electron - dense granules ( black arrows ) , granules in lumen ( blue arrows ) have a contracted endoplasmic reticulum ( ER ) ( arrows ) . Scale bar 5μm , data are from seven biological replicates per genotype .

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(C) Transmission electron microscopy (TEM) of crypts of COPS8fl/fl and COPS8ΔIEC mice. The base of the crypt in COPS8ΔIEC mice is occupied by poorly differentiated columnar epithelial cells that lack secretory granules, rudimentary electron-dense granules (black arrows), granules in the lumen (blue arrows) and have a contracted endoplasmic reticulum (ER) (white arrows). Scale bar 5μm, data are from seven biological replicates per genotype.


( G - H ) Real - time quantitative reverse transcription Polymerase chain reaction ( PCR ) ( qRT - PCR ) of expression of genes encoding AMPs in ileum ( G ) colon ( H ) in COPS8fl / fl COPS8ΔIEC mice . Error bars indicate mean ± SD from three biological replicates . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 using Student ' s t - .

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(G-H) Real-time quantitative reverse transcription Polymerase chain reaction (PCR) (qRT-PCR) analysis of the expression of genes encoding AMPs in the ileum (G) and colon (H) in COPS8fl/fl and COPS8ΔIEC mice. Error bars indicate the mean ± SD from three biological replicates. *P<0.05, **P<0.01, ***P<0.001 using Student's t-test.


( A ) Real - time quantitative reverse transcription polymerase chain reaction ( PCR ) ( qRT - PCR ) of differential abundance of selected taxa in stool samples of ileum colon of Villin - Cre COPS8 - lox alleles expressing ( COPS8fl / fl ) COPS8 knockout ( KO ) ( COPS8ΔIEC ) mice . Error bars indicate mean ± SD from three biological replicates per genotype . * * P < 0 . 01 , NS - nonsignificant using Student ' s t - . (

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(A) Real-time quantitative reverse transcription polymerase chain reaction (PCR) (qRT-PCR) analysis of the differential abundance of selected bacterial taxa in stool samples of ileum and colon of Villin-Cre and COPS8-lox alleles expressing (COPS8fl/fl ) and COPS8 knockout (KO) (COPS8ΔIEC) mice. Error bars indicate the mean ± SD from three biological replicates per genotype. **P<0.01, NS- nonsignificant using Student's t-test. (


Mice were treated by 2 % dextran sodium sulfate ( DSS ) in drinking water for 7 days . ( F ) Representative hematoxylin eosin ( HE ) stained sections of colon cecum from COPS8fl / fl COPS8ΔIEC mice after DSS - indcued colitis ( day 9 ) . Scale bar 100μm , representative data from five biological replicates per genotype . ( G ) Histopathologically scored sections of distal colon ; cecum colon were analyzed from COPS8fl / fl COPS8ΔIEC mice after DSS - indcued colitis ( day 7 ) . Data are represented as mean ± from five biological replicates per genotype . * * P < 0 . 01 using Student ' s t - . (

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Mice were treated by 2% dextran sodium sulfate (DSS) in drinking water for 7 days. (F) Representative hematoxylin and eosin (HE) stained sections of colon and cecum from COPS8fl/fl and COPS8ΔIEC mice after DSS-indcued colitis (day 9). Scale bar 100μm, representative data from five biological replicates per genotype. (G) Histopathologically scored sections of distal colon; cecum and colon length were analyzed from COPS8fl/fl and COPS8ΔIEC mice after DSS-indcued colitis (day 7). Data are represented as mean ± SEM from five biological replicates per genotype. **P<0.01 using Student's t-test. (


Mice were treated by 2 % dextran sodium sulfate ( DSS ) in drinking water for 7 days . ( H ) Cytokine ( interleukin ( IL ) - 1β , IL - 6 necrosis factor - α ( TNF - α ) ) in colon collected on day 7 in colitis induced COPS8fl / fl COPS8ΔIEC mice . Data are represented as mean ± from five biological replicates per genotype . * P < 0 . 05 , * * P < 0 . 01 , NS - non - significant using Student ' s t - .

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Mice were treated by 2% dextran sodium sulfate (DSS) in drinking water for 7 days. (H) Cytokine levels (interleukin (IL)-1β, IL-6 and tumor necrosis factor-α (TNF-α)) in the colon collected on day 7 in colitis induced COPS8fl/fl and COPS8ΔIEC mice. Data are represented as mean ± SEM from five biological replicates per genotype. *P<0.05, **P<0.01, NS- non-significant using Student's t-test.


Mice were treated by 2 % dextran sodium sulfate ( DSS ) in drinking water for 7 days . ( I ) frequency of CD11b + Ly6C + CD11b + Ly6G + in colonic lamina propria ( cLP ) of COPS8fl / fl or COPS8ΔIEC mice with DSS - induced colitis . column graph represents of Ly6C + Ly6G + , presented as mean ± from three biological replicates per genotype . * P < 0 . 05 , NS - non - significant using Student ' s t - . ( J ) Representative FACS plots of intracellular of Forkhead box P3 ( FOXP3 ) , Interferon gamma ( IFN - γ ) IL - 17A in CD3 + CD4 + T from colonic lamina propria of COPS8fl / fl or COPS8ΔIEC mice with DSS - induced colitis . column graph represents of Treg + of Th1 / Th17 , represented as mean ± from three biological replicates per genotype . * P < 0 . 05 using Student ' s t - .

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Mice were treated by 2% dextran sodium sulfate (DSS) in drinking water for 7 days. (I) The frequency of CD11b+Ly6C+ and CD11b+Ly6G+ cells in colonic lamina propria (cLP) of COPS8fl/fl and or COPS8ΔIEC mice with DSS-induced colitis. The column graph represents percentage of Ly6C+ and Ly6G+ cells, presented as mean ± SEM from three biological replicates per genotype. *P<0.05, NS- non-significant using Student's t-test. (J) Representative FACS plots and percentage of intracellular staining of Forkhead box protein P3 (FOXP3), Interferon gamma (IFN-γ) and IL-17A in CD3+CD4+ T cells from colonic lamina propria of COPS8fl/fl or COPS8ΔIEC mice with DSS-induced colitis. The column graph represents percentage of Treg+ and ratio of Th1/Th17 cells, represented as mean ± SEM from three biological replicates per genotype. *P<0.05 using Student's t-test.


( H ) Flow cytometric of CD45 + infiltrating immune in lamina propria of ileum . Data are represented as mean ± from three biological replicates per genotype . * * P < 0 . 01 , NS - non - significant using one - way ANOVA .

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(H) Flow cytometric analysis of CD45+ infiltrating immune cells in the lamina propria of the ileum. Data are represented as mean ± SEM from three biological replicates per genotype. **P<0.01, NS- non-significant using one-way ANOVA.


( I ) Real - time quantitative reverse transcription polymerase chain reaction ( qRT - PCR ) of expression of genes encoding inflammatory cytokines in ileum . COPS8fl / fl COPS8ΔIEC mice were either untreated ( phosphate buffered saline ( PBS ) ) or treated with antibiotic cocktail ( Abx ) for 3 months . Data are mean ± from three biological replicates . * * P < 0 . 01 , NS - non - significant using one - way ANOVA .

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(I) Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis of the expression of genes encoding inflammatory cytokines in the ileum. COPS8fl/fl and COPS8ΔIEC mice were either untreated (phosphate buffered saline (PBS)) or treated with the antibiotic cocktail (Abx) for 3 months. Data are mean ± SEM from three biological replicates. **P<0.01, NS- non-significant using one-way ANOVA.


( B ) Representative image showing changes in colon following DSS - induced colitis with concurrent treatment with MBELNs COPS8fl / fl COPS8ΔIEC mice . Representative data from seven biological replicates per genotype .

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(B) Representative image showing changes in colon morphology and length following DSS-induced colitis with concurrent treatment with MBELNs COPS8fl/fl and COPS8ΔIEC mice. Representative data from seven biological replicates per genotype.


Hematoxylin eosin ( HE ) to show histological changes Scale bar

Hematoxylin and eosin staining (HE) to show histological changes Scale bar 200μm


( F ) Expression of anti - microbial peptide ( AMPs ) in colon of COPS8fl / fl COPS8ΔIEC mice while being treated with MBELNs by oral administration . Data are mean ± from three biological replicates per group . * P < 0 . 05 , * * P < 0 . 01 , NS - non - significant using one - way ANOVA .

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(F) Expression of anti-microbial peptide (AMPs) in the colon of COPS8fl/fl and COPS8ΔIEC mice while being treated with MBELNs by oral administration. Data are mean ± SEM from three biological replicates per group. *P<0.05, **P<0.01, NS- non-significant using one-way ANOVA.


( I - J ) Transcriptional expression of factor kappa B ( NF - κB ) ( I ) inhibitor of factor kappa - β kinase ( IKK - β ) ( J ) mRNA using Real - time quantitative reverse transcription Polymerase chain reaction ( qRT - PCR ) in colonic epithelial upon treatment with MBELNs in addition to DSS . Data are mean ± from three biological replicates per group . * * P < 0 . 01 , NS - non - significant using one - way ANOVA .

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(I-J) Transcriptional expression of nuclear factor kappa B (NF-κB) (I) and inhibitor of nuclear factor kappa-β kinase (IKK-β) (J) mRNA using Real-time quantitative reverse transcription Polymerase chain reaction (qRT-PCR) in colonic epithelial cells upon treatment with MBELNs in addition to DSS. Data are mean ± SEM from three biological replicates per group. **P<0.01, NS- non-significant using one-way ANOVA.