G . Inhibition of ER stress impairs the ability of Carfilzomib , Bortezomib , MLN9708 and MG132 to reprogram M2 towards M1 - like macrophages . BMDMs were induced by IL - 4 ( 20 ng / mL ) for 24 hours , then pretreated with 4 - PBA ( 5 mM ) for 1 hour and stimulated by Carfilzomib ( 1μM ) , Bortezomib ( 1μM ) , MLN9708 ( 2 μM ) , MG132 ( 5 μM ) . RNA was extracted from cells 6 hours after stimulation and the expression of Il - 1β , Il - 6 and Inos were detected through RT - qPCR . The data are means ± SD of three independent experiments . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( student ' s t - test ) .

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G. Inhibition of ER stress impairs the ability of Carfilzomib, Bortezomib, MLN9708 and MG132 to reprogram M2 towards M1-like macrophages. BMDMs were induced by IL-4 (20 ng/mL) for 24 hours, then pretreated with 4-PBA (5 mM) for 1 hour and stimulated by Carfilzomib (1μM), Bortezomib (1μM), MLN9708 (2 μM), MG132 (5 μM). RNA was extracted from cells 6 hours after stimulation and the expression of Il-1β, Il-6 and Inos were detected through RT-qPCR. The data are means ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 (student's t-test).


A , B Deficiency of Ern1 reduces the expression of inflammatory related genes . Wild - type and Ern1 - / - Raw264 . 7 cells ( A ) or BMDMs ( B ) were pretreated by IL - 4 ( 20 ng / mL ) for 24 hours , then stimulated by DMSO or Carfilzomib ( 1 μM ) . The mRNA of Il - 1β and Il - 6 were quantified through RT - qPCR 6 hours after stimulation . The data are means ± SD of three independent experiments . * * p < 0 . 01 , * * * p < 0 . 001 ( student ' s t - test ) .

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A, B Deficiency of Ern1 reduces the expression of inflammatory related genes. Wild-type and Ern1-/- Raw264.7 cells (A) or BMDMs (B) were pretreated by IL-4 (20 ng/mL) for 24 hours, then stimulated by DMSO or Carfilzomib (1 μM). The mRNA of Il-1β and Il-6 were quantified through RT-qPCR 6 hours after stimulation. The data are means ± SD of three independent experiments. **p < 0.01, ***p < 0.001 (student's t-test).


E . Kira6 inhibits the expression of ER stress related genes and inflammatory related genes . BMDMs was treated by IL - 4 ( 20 ng / mL ) for 24 hours , treated with Kira6 ( 100 nM ) for 1 hour , and then stimulated by Carfilzomib ( 1 μM ) for 6 hours . RNA was extracted from cells and the expression of Bip , Chop , Il - 1β and Il - 6 were quantified through RT - qPCR . The data are means ± SD of three independent experiments . * p < 0 . 05 , * * p < 0 . 01 ( student ' s t - test ) . F , G . Carfilzomib has no effect on splicing of XBP1 in M2 macrophages . BMDMs ( F ) or Raw264 . 7 cells ( G ) were pretreated by IL - 4 ( 20 ng / mL ) for 24 hours , then stimulated with DMSO , Carfilzomib ( 1 μM ) or TUN ( 500 nM ) . The mRNA expression of sXBP1 and unXBP1 were detected through RT - qPCR 6 hours after stimulation . The data are means ± SD of three independent experiments . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( student ' s t - test ) .

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E. Kira6 inhibits the expression of ER stress related genes and inflammatory related genes. BMDMs was treated by IL-4 (20 ng/mL) for 24 hours, treated with Kira6 (100 nM) for 1 hour, and then stimulated by Carfilzomib (1 μM) for 6 hours. RNA was extracted from cells and the expression of Bip, Chop, Il-1β and Il-6 were quantified through RT-qPCR. The data are means ± SD of three independent experiments. *p < 0.05, **p < 0.01 (student's t-test). F, G. Carfilzomib has no effect on splicing of XBP1 in M2 macrophages. BMDMs (F) or Raw264.7 cells (G) were pretreated by IL-4 (20 ng/mL) for 24 hours, then stimulated with DMSO, Carfilzomib (1 μM) or TUN (500 nM). The mRNA expression of sXBP1 and unXBP1 were detected through RT-qPCR 6 hours after stimulation. The data are means ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 (student's t-test).


J . Carfilzomib promotes the association between IRE1α and TRAF2 in M2 macrophages . Raw264 . 7 cells were pretreated by IL - 4 ( 20ng / mL ) for 24 hours , then stimulated by Carfilzomib ( 1 μM ) for 2 or 4 hours . Coimmunoprecipitation ( Co - IP ) and IB were performed with indicated antibodies .

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J. Carfilzomib promotes the association between IRE1α and TRAF2 in M2 macrophages. Raw264.7 cells were pretreated by IL-4 (20ng/mL) for 24 hours, then stimulated by Carfilzomib (1 μM) for 2 or 4 hours. Coimmunoprecipitation (Co-IP) and IB were performed with indicated antibodies.


L . Carfilzomib activates NF - κB signaling pathway through IRE1α in M2 macrophages . Wild - type and Ern1 - / - Raw264 . 7 cells were pretreated by IL - 4 ( 20 ng / mL ) for 24 hours , then stimulated by Carfilzomib ( 1 μM ) for indicated time points . WCL were analyzed by IB with indicated antibodies .

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L. Carfilzomib activates NF-κB signaling pathway through IRE1α in M2 macrophages. Wild-type and Ern1-/- Raw264.7 cells were pretreated by IL-4 (20 ng/mL) for 24 hours, then stimulated by Carfilzomib (1 μM) for indicated time points. WCL were analyzed by IB with indicated antibodies.


C . Representative images of Hematoxylin and eosin ( H & E ) staining of the lung tissue obtained from different treatment groups . Expression of Ki - 67 in lung tissues was detected by immunohistochemistry method . D . Statistics of the tumor area and the number of Ki67 positive cells in sections of lung tissue . Data are means ± SD of three independent experiments . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( student ' s t - test ) .

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C. Representative images of Hematoxylin and eosin (H&E) staining of the lung tissue obtained from different treatment groups. Expression of Ki-67 in lung tissues was detected by immunohistochemistry method. D. Statistics of the tumor area and the number of Ki67 positive cells in sections of lung tissue. Data are means ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 (student's t-test).


E . Carfilzomib treatment promotes the infiltration of M1 macrophages . Tumor bearing TD mice were treated as indicated in ( A ) and euthanized . Lung tissues were dissected for evaluating M1 / M2 populations through analyze the expression of CD80 and CD206 by flow cytometry . F . Statistics represent the proportion of CD80 or CD206 positive cells in lung tissue . Data are means ± SD of three independent experiments . * * p < 0 . 01 ( student ' s t - test ) . G . Carfilzomib treatment promotes the infiltration and activation of CD8 + T cells . Lung tissues of ( E ) were used for evaluating the infiltration of CD8 + T cells and their expression of CD69 by flow cytometry . H . Statistics about the proportion of CD8 + or CD69 + positive cells in lung tissue . Data are means ± SD of three independent experiments . * * p < 0 . 01 ( student ' s t - test ) .

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E. Carfilzomib treatment promotes the infiltration of M1 macrophages. Tumor bearing TD mice were treated as indicated in (A) and euthanized. Lung tissues were dissected for evaluating M1/M2 populations through analyze the expression of CD80 and CD206 by flow cytometry. F. Statistics represent the proportion of CD80 or CD206 positive cells in lung tissue. Data are means ± SD of three independent experiments. **p < 0.01 (student's t-test). G. Carfilzomib treatment promotes the infiltration and activation of CD8+ T cells. Lung tissues of (E) were used for evaluating the infiltration of CD8+ T cells and their expression of CD69 by flow cytometry. H. Statistics about the proportion of CD8+ or CD69+ positive cells in lung tissue. Data are means ± SD of three independent experiments. **p < 0.01 (student's t-test).


K , L . Carfilzomib shrinks tumor through modulating tumor microenvironment . EG7 cells were inoculated in wild - type or LyzM - cre ; lsl - dTA to allow tumor xenograft to reach a volume of around 80 mm3 . Mice were randomized for treatment ( n = 5 ) by saline or Carfilzomib for two weeks . The xenografts were dissected to photograph ( K ) or weigh ( L ) after treatment . Data are shown as mean ± SD and n indicates the number of biological replicates . * p < 0 . 05 , * * * p < 0 . 001 ( student ' s t - test ) .

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K, L. Carfilzomib shrinks tumor through modulating tumor microenvironment. EG7 cells were inoculated in wild-type or LyzM-cre;lsl-dTA to allow tumor xenograft to reach a volume of around 80 mm3. Mice were randomized for treatment (n=5) by saline or Carfilzomib for two weeks. The xenografts were dissected to photograph (K) or weigh (L) after treatment. Data are shown as mean ±SD and n indicates the number of biological replicates. *p < 0.05, ***p < 0.001 (student's t-test).


D . CD4 + and CD8 + T cells play a role in mediating cancer - shrinking effect of Carfilzomib . Lung - cancer - bearing TD mice were treated by saline , Carfilzomib , anti - mouse CD4 antibody ( ip . ) , anti - mouse CD8 antibody ( ip . ) , or Carfilzomib plus anti - mouse CD4 / 8 antibody . CT scanning was performed after two - week treatment . E . Representative images of H & E staining of the treated lung tissues . Expression of Ki - 67 in lung tissues was detected by immunohistochemistry method . F . Carfilzomib synergized with PD1 antibody in shrinking lung cancers . Lung cancer bearing TD mice were treated by saline , anti - PD1 antibody ( ip ) , Carfilzomib , or Carfilzomib plus anti - PD1 ( ip ) . CT scanning was performed after two - week treatment . G . Representative images of H & E staining of the lung tissues . Expression of Ki - 67 in lung tissue was detected by immunohistochemistry method .

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

D. CD4+ and CD8+ T cells play a role in mediating cancer-shrinking effect of Carfilzomib. Lung-cancer-bearing TD mice were treated by saline, Carfilzomib, anti-mouse CD4 antibody (ip.), anti-mouse CD8 antibody (ip.), or Carfilzomib plus anti-mouse CD4/8 antibody. CT scanning was performed after two-week treatment. E. Representative images of H&E staining of the treated lung tissues. Expression of Ki-67 in lung tissues was detected by immunohistochemistry method. F. Carfilzomib synergized with PD1 antibody in shrinking lung cancers. Lung cancer bearing TD mice were treated by saline, anti-PD1 antibody (ip), Carfilzomib, or Carfilzomib plus anti-PD1 (ip). CT scanning was performed after two-week treatment. G. Representative images of H&E staining of the lung tissues. Expression of Ki-67 in lung tissue was detected by immunohistochemistry method.


A . Carfilzomib , Bortezomib and MLN9708 promote the expression of M1 macrophage markers ( Il - 1β , Il - 6 , Tnfα ) and reduce the expression of M2 macrophage markers ( Il - 10 , Tgfβ ) in macrophages derived from PBMCs . After induced by IL - 4 ( 20 ng / ml ) for 24 hours to differentiate into mature M2 macrophages , PBMCs derived macrophages were stimulated by Carfilzomib ( 1 μM ) , Bortezomib ( 1 μM ) or MLN9708 ( 2 μM ) . RNA was extracted from cells 6 hours after stimulation and the expression of mRNA were quantified through RT - qPCR . Data from three experiments are presented as the mean ± SD . T test was used for statistical analysis of differences between groups . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( student ' s t - test ) .

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A. Carfilzomib, Bortezomib and MLN9708 promote the expression of M1 macrophage markers (Il-1β, Il-6, Tnfα) and reduce the expression of M2 macrophage markers (Il-10, Tgfβ) in macrophages derived from PBMCs. After induced by IL-4 (20 ng/ml) for 24 hours to differentiate into mature M2 macrophages, PBMCs derived macrophages were stimulated by Carfilzomib (1 μM), Bortezomib (1 μM) or MLN9708 (2 μM). RNA was extracted from cells 6 hours after stimulation and the expression of mRNA were quantified through RT-qPCR. Data from three experiments are presented as the mean ± SD. T test was used for statistical analysis of differences between groups. *p < 0.05, **p < 0.01, ***p < 0.001 (student's t-test).


C - F . Carfilzomib , Bortezomib and MLN9708 increase the expression of CD80 ( C ) and reduce the expression of CD206 ( E ) in M2 macrophages derived from PBMCs . After activated by IL - 4 ( 20 ng / ml ) for 24 hours to differentiate into mature M2 macrophages , PBMCs derived macrophages were stimulated by Carfilzomib ( 500 nM ) , Bortezomib ( 500 nM ) or MLN9708 ( 500 nM ) . The representative histogram of CD80 and CD206 expression was analyzed by flow cytometry 12 hours after stimulation . ( D , F ) Statistics represent the proportion of CD80 ( D ) or CD206 ( F ) positive cells in macrophages derived from PBMCs . Data from three experiments are presented as the mean ± SD . T test was used for statistical analysis of differences between groups . * p < 0 . 05 , * * p < 0 . 01 ( student ' s t - test ) .

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

C-F. Carfilzomib, Bortezomib and MLN9708 increase the expression of CD80 (C) and reduce the expression of CD206 (E) in M2 macrophages derived from PBMCs. After activated by IL-4 (20 ng/ml) for 24 hours to differentiate into mature M2 macrophages, PBMCs derived macrophages were stimulated by Carfilzomib (500 nM), Bortezomib (500 nM) or MLN9708 (500 nM). The representative histogram of CD80 and CD206 expression was analyzed by flow cytometry 12 hours after stimulation. (D, F) Statistics represent the proportion of CD80 (D) or CD206 (F) positive cells in macrophages derived from PBMCs. Data from three experiments are presented as the mean ± SD. T test was used for statistical analysis of differences between groups. *p < 0.05, **p < 0.01 (student's t-test).


C , AltFUS ( arrow ) endogenous expression in Human tissues ( brain , muscles and kidney - 100 μg ) , in HEK293 and HeLa cultured cells ( 100 μg ) and using the over - expression of altFUS CDS in HEK293 cells ( 50 μg ) as positive control ( representative image from n = 3 ) . The asterisk indicates a protein species detected with the anti - altFUS antibody specifically in the brain .

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C, AltFUS (arrow) endogenous expression in Human tissues (brain, muscles and kidney - 100 μg), in HEK293 and HeLa cultured cells (100 μg) and using the over-expression of altFUS CDS in HEK293 cells (50 μg) as positive control (representative image from n=3). The asterisk indicates a protein species detected with the anti-altFUS antibody specifically in the brain.


AltFUS ( arrow ) endogenous expression in the motor cortex of three C9orf72 and three sporadic ALS patients ( D ) ( representative image from n = 3 ) . The asterisk indicates a protein species detected with the anti - altFUS antibody specifically in the brain .

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AltFUS (arrow) endogenous expression in the motor cortex of three C9orf72 and three sporadic ALS patients (D) (representative image from n=3). The asterisk indicates a protein species detected with the anti-altFUS antibody specifically in the brain.


AltFUS ( arrow ) endogenous expression in iPSC - derived motor neurons of three lines from controls and from ALS patients ( E ) ( representative image from n = 3 ) . The asterisk indicates a protein species detected with the anti - altFUS antibody specifically in the brain .

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AltFUS (arrow) endogenous expression in iPSC-derived motor neurons of three lines from controls and from ALS patients (E) (representative image from n=3). The asterisk indicates a protein species detected with the anti-altFUS antibody specifically in the brain.


I , Endogenous altFUS mitochondrial expression in HEK293 cells following fractionation ( representative image from n = 3 ) , with Tubulin as a marker of the cytosolic fraction and VDAC as a marker of the mitochondrial fraction . We used siFUS transfected cells as a negative control and altFUS transfected cells as a positive control for altFUS expression ( WCL = Whole Cell Lysate , Cyto = Cytosol fraction , Mito = mitochondrial fraction ) .

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I, Endogenous altFUS mitochondrial expression in HEK293 cells following fractionation (representative image from n=3), with Tubulin as a marker of the cytosolic fraction and VDAC as a marker of the mitochondrial fraction. We used siFUS transfected cells as a negative control and altFUS transfected cells as a positive control for altFUS expression (WCL = Whole Cell Lysate, Cyto = Cytosol fraction, Mito = mitochondrial fraction).


J , Representative images of the mitochondrial network ( Tomm20 in red ) in mock and altFUS - Flag ( green ) transfected HeLa cells ( n = 3 ) . The white scale bar corresponds to 10μm . K , Proportion of tubules and globules in the mitochondrial network of mock HeLa cells and HeLa cells transfected with altFUS - Flag Quantification was done over a minimum of 100 cells across a technical duplicate per independent experiments ( n = 3 , i . e . a minimum of 300 cells per biological conditions , p - value < 0 . 001 , Mann - Whitney U test ) . The boxes extend to the 25th and 75th percentiles , with the median marked . The whiskers correspond to the 5th and 95th percentiles .

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J, Representative images of the mitochondrial network (Tomm20 in red) in mock and altFUS-Flag (green) transfected HeLa cells (n=3). The white scale bar corresponds to 10μm. K, Proportion of tubules and globules in the mitochondrial network of mock HeLa cells and HeLa cells transfected with altFUS-Flag Quantification was done over a minimum of 100 cells across a technical duplicate per independent experiments (n=3, i.e. a minimum of 300 cells per biological conditions, p-value < 0.001, Mann-Whitney U test). The boxes extend to the 25th and 75th percentiles, with the median marked. The whiskers correspond to the 5th and 95th percentiles.


E , Scatter plot of the proteins identified by AP - MS indicating their enrichment ( Fold - change over control ) and their SAINT probability score . Proteins above the 0 . 8 threshold ( grey line ) are indicated in black , others in grey . AltFUS is indicated in red ( bait ) and preys known to regulate the autophagy or the cellular stress response are indicated in green .

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E, Scatter plot of the proteins identified by AP-MS indicating their enrichment (Fold-change over control) and their SAINT probability score. Proteins above the 0.8 threshold (grey line) are indicated in black, others in grey. AltFUS is indicated in red (bait) and preys known to regulate the autophagy or the cellular stress response are indicated in green.


B , LC3 - II accumulation after bafilomycin treatment from mock , altFUS , FUS , FUS ( Ø ) , FUS - R495x , FUS ( Ø ) - R495x transfected cells and FUS ( Ø ) - R495x and altFUS co - transfected cells across 3 independent experiments ( n = 3 , mean ± SD ) . The quantification corresponds to the treated / untreated ratio of LC3 - II abundance Statistical significance is relative to the mock condition unless otherwise indicated ( * * * * = p value < 0 . 0001 , * * * = p value < 0 . 001 , * * = p value < 0 . 01 , n . s . = non - significant , two - way ANOVA with Tukey ' s multiple comparison correction ) .

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

B, LC3-II accumulation after bafilomycin treatment from mock, altFUS, FUS, FUS(Ø), FUS-R495x, FUS(Ø)-R495x transfected cells and FUS(Ø)-R495x and altFUS co-transfected cells across 3 independent experiments (n=3, mean ± SD). The quantification corresponds to the treated/untreated ratio of LC3-II abundance Statistical significance is relative to the mock condition unless otherwise indicated (**** = p value < 0.0001, *** = p value < 0.001, ** = p value < 0.01, n.s. = non-significant, two-way ANOVA with Tukey's multiple comparison correction).


D - E , Quantification of FUS cytoplasmic granules , number ( D ) and area ( μm2 ) ( E ) in cells over - expressing the bicistronic ( + ) or monocistronic ( - ) construct for FUS , FUS - G156E , FUS - R495x , FUS - K510E , FUS - Q519x , FUS - Q519I - fs527x , FUS - R521C , and FUS - P525L . Statistical comparisons are made between bicistronic and monocistronic versions of each construct ( n = 3 - biological replicates , mean ± SD , * * * * = p value < 0 . 0001 , * * * = p value < 0 . 001 , * * = p value < 0 . 01 , * = p value < 0 . 05 , n . s . = non - significant , one - way ANOVA test with Sidak ' s multiple comparison ) .

O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Quantification of FUS cytoplasmic granules, number (D) and area (μm2) (E) in cells over-expressing the bicistronic (+) or monocistronic (-) construct for FUS, FUS-G156E, FUS-R495x, FUS-K510E, FUS-Q519x, FUS-Q519I-fs527x, FUS-R521C, and FUS-P525L. Statistical comparisons are made between bicistronic and monocistronic versions of each construct (n=3 - biological replicates, mean ± SD, **** = p value < 0.0001, *** = p value < 0.001, ** = p value < 0.01, * = p value < 0.05, n.s. = non-significant, one-way ANOVA test with Sidak's multiple comparison).


C - E , Locomotion assay represented by the percentage of climbing success in control and RU - 486 - treated transgenic Drosophila expressing mCherry or altFUS ( C ) , the bicistronic FUS or the monocistronic FUS ( Ø ) ( D ) , and the bicistronic FUS - R495x or the monocistronic FUS ( Ø ) - R495x ( E ) at day 1 , 10 and 20 post - induction . Statistical comparisons were made between each population ( n = 4 - biological replicates ) . Indicated significance is between the monocistronic and the bicistronic transgenic flies of the RU - 486 - treated population ( mean ± SD , n . s . = non - significant , * = p value < 0 . 05 , * * * = p value < 0 . 001 , two - way ANOVA with Tukey ' s multiple comparison correction ) .

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C-E, Locomotion assay represented by the percentage of climbing success in control and RU-486-treated transgenic Drosophila expressing mCherry or altFUS (C), the bicistronic FUS or the monocistronic FUS(Ø) (D), and the bicistronic FUS-R495x or the monocistronic FUS(Ø)-R495x (E) at day 1, 10 and 20 post-induction. Statistical comparisons were made between each population (n=4 - biological replicates). Indicated significance is between the monocistronic and the bicistronic transgenic flies of the RU-486-treated population (mean ± SD, n.s. = non-significant, * = p value < 0.05, *** = p value < 0.001, two-way ANOVA with Tukey's multiple comparison correction).


A , Graphical representation of FUS synonymous mutations ( yellow ) found in ALS patients . The canonical FUS mRNA is represented in dark blue ( ENST00000254108 or NM _ 004960 ) . The FUS protein coding sequence is indicated in light blue , and the altFUS protein coding sequence is indicated in green . B , Images by confocal microscopy of TDP - 43 ( white ) , FUS ( GFP tagged - green ) and altFUS ( Flag tagged - red ) in HeLa cells over - expressing GFP - FUS ( Flag ) , GFP - FUS ( P31L - Flag ) - S44 = , GFP - FUS ( A38V - Flag ) - G51 = , GFP - FUS ( A46V - Flag ) - G59 = , GFP - FUS ( R64P - Flag ) - S77 = ( representative images from n = 3 ) . White arrows indicate some TDP - 43 aggregates . The white scale bar corresponds to 10 μm . C , Quantification of cells with TDP - 43 aggregates in HeLa cells ( see panel B ) . The data are represented as the fold - change compared to the GFP - FUS ( Flag ) expressing cells . Statistical significance is indicated above the bars ( n = 3 - biological replicates , mean ± SD , * * * * = p value < 0 . 0001 , two - way ANOVA with Tukey ' s multiple comparison correction ) .

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

A, Graphical representation of FUS synonymous mutations (yellow) found in ALS patients. The canonical FUS mRNA is represented in dark blue (ENST00000254108 or NM_004960). The FUS protein coding sequence is indicated in light blue, and the altFUS protein coding sequence is indicated in green. B, Images by confocal microscopy of TDP-43 (white), FUS (GFP tagged - green) and altFUS (Flag tagged - red) in HeLa cells over-expressing GFP-FUS(Flag), GFP-FUS(P31L-Flag)-S44=, GFP-FUS(A38V-Flag)-G51=, GFP-FUS(A46V-Flag)-G59=, GFP-FUS(R64P-Flag)-S77= (representative images from n=3). White arrows indicate some TDP-43 aggregates. The white scale bar corresponds to 10 μm. C, Quantification of cells with TDP-43 aggregates in HeLa cells (see panel B). The data are represented as the fold-change compared to the GFP-FUS(Flag) expressing cells. Statistical significance is indicated above the bars (n=3 - biological replicates, mean ± SD, **** = p value < 0.0001, two-way ANOVA with Tukey's multiple comparison correction).


Comparison of SWATH - MS - based quantification to DDA - MS - based strategies ( MS1 XIC and spectral counting ) with regard to consistency ( as judged based upon protein - level SEC chromatogram robustness towards increasing requirements on the number of consecutive detections ) and precision ( judged based on the correlation between sibling peptide SEC chromatograms ) of quantification and overall performance in error - controlled complex - centric query of CORUM complexes in the respective protein level chromatogram sets ( also see Appendix Figure S3C ) . TP , true positive assignments according to error model , P , all positives .

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Comparison of SWATH-MS-based quantification to DDA-MS-based strategies (MS1 XIC and spectral counting) with regard to consistency (as judged based upon protein-level SEC chromatogram robustness towards increasing requirements on the number of consecutive detections) and precision (judged based on the correlation between sibling peptide SEC chromatograms) of quantification and overall performance in error-controlled complex-centric query of CORUM complexes in the respective protein level chromatogram sets (also see Appendix Figure S3C). TP, true positive assignments according to error model, P, all positives.


Peptide - level SEC chromatograms are grouped by UniprotKB identifier to detect co - elution signals indicative of protein elution ranges / peaks . Based on external size calibration of the apparent analyte molecular weight per SEC fraction , signals can be attributed to likely assembled or monomeric state ( also see Appendix Figure S4 ) . For TCPE , three distinct elution signals , numbered 1 - 3 , are detected , two in the assembled and one in the monomer elution range .

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Peptide-level SEC chromatograms are grouped by UniprotKB identifier to detect co-elution signals indicative of protein elution ranges/peaks. Based on external size calibration of the apparent analyte molecular weight per SEC fraction, signals can be attributed to likely assembled or monomeric state (also see Appendix Figure S4). For TCPE, three distinct elution signals, numbered 1-3, are detected, two in the assembled and one in the monomer elution range.


Proteins are observed eluting in 1 - 6 distinct peaks and with a wide range of apparent vs . monomeric molecular weight ratios ( distributions , left panels ) . The molecular weight ratios of the three peaks detected for TCPE ( displayed in A ) are indicated . Many of the proteins eluting in a single peak ( top panel and bar ) appear assembled . Proteins eluting multiple times ( 27 % of the proteins ) do so preferentially in the assembled range , suggesting frequent participation in multiple differently sized macromolecular assemblies ( lower panels and pie chart ) . For a list of all detected protein peaks , see Dataset EV3 .

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Proteins are observed eluting in 1-6 distinct peaks and with a wide range of apparent vs. monomeric molecular weight ratios (distributions, left panels). The molecular weight ratios of the three peaks detected for TCPE (displayed in A) are indicated. Many of the proteins eluting in a single peak (top panel and bar) appear assembled. Proteins eluting multiple times (27 % of the proteins) do so preferentially in the assembled range, suggesting frequent participation in multiple differently sized macromolecular assemblies (lower panels and pie chart). For a list of all detected protein peaks, see Dataset EV3.


Proteins observed in multiple assembled peaks ( n = 659 ) are enriched in components of the proteasome and other known large complex assemblies .

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Proteins observed in multiple assembled peaks (n = 659) are enriched in components of the proteasome and other known large complex assemblies.


For nearly half the CORUM complex hypotheses queried , two or more distinct subunit co - elution signals were detected ( See methods and Appendix information on CCprofiler ) .

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For nearly half the CORUM complex hypotheses queried, two or more distinct subunit co-elution signals were detected (See methods and Appendix information on CCprofiler).


Among the four distinct co - elution signals detected from the eight canonical CSN subunits ' chromatograms ( here with CSN7A , not CSN7B ) are two distinct signals indicating distinct co - elution of two different complex variants .

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Among the four distinct co-elution signals detected from the eight canonical CSN subunits' chromatograms (here with CSN7A, not CSN7B) are two distinct signals indicating distinct co-elution of two different complex variants.


Distinct co - elution of holo - CSN ( observed at the expected fraction 35 ) and Mini - CSN CSN1 / 3 / 8 ( observed eluting offset only one fraction late , F45 , of the expected fraction , F44 ) . Expected fractions are estimated from the cumulative sum of one copy per component and external size calibration . Coloring adapted to highlight subversion components and their partitioning across holo - and sub - complex . CSN1 / 3 / 8 interact and form a submodule within the CSN holo - complex structure ( PDB accession 4D10 ) . The observations are consistent between the two whole workflow replicates ( see Appendix Figure S6A ) . Lower right panel , validation of distinct elution behavior of holo - CSN exclusive ( CSN7A ) and shared subunit ( CSN8 ) by immunoblotting . For full immunoblotting data ( CSN1 , CSN3 , CSN4 , CSN5 , CSN7A and CSN8 ) see Appendix Figure S7C ) .

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

Distinct co-elution of holo-CSN (observed at the expected fraction 35) and Mini-CSN CSN1/3/8 (observed eluting offset only one fraction late, F45, of the expected fraction, F44). Expected fractions are estimated from the cumulative sum of one copy per component and external size calibration. Coloring adapted to highlight subversion components and their partitioning across holo- and sub-complex. CSN1/3/8 interact and form a submodule within the CSN holo-complex structure (PDB accession 4D10). The observations are consistent between the two whole workflow replicates (see Appendix Figure S6A). Lower right panel, validation of distinct elution behavior of holo-CSN exclusive (CSN7A) and shared subunit (CSN8) by immunoblotting. For full immunoblotting data (CSN1, CSN3, CSN4, CSN5, CSN7A and CSN8) see Appendix Figure S7C).


CSN1 / 3 / 8 display distinct fractionation patterns in co - fractionation experiments performed in other laboratories , specifically in orthogonal ion exchange fractionation of HEK293 lysates ( Wan et al . 2015 , upper panels ) and size exclusion chromatographic fractionation of U2OS lysates ( Kirkwood et al . 2013 , lower panel ) , in line with the CSN1 / 3 / 8 as distinct entity .

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CSN1/3/8 display distinct fractionation patterns in co-fractionation experiments performed in other laboratories, specifically in orthogonal ion exchange fractionation of HEK293 lysates (Wan et al. 2015, upper panels) and size exclusion chromatographic fractionation of U2OS lysates (Kirkwood et al. 2013, lower panel), in line with the CSN1/3/8 as distinct entity.


Baculoviral co - expression of human CSN1 , CSN3 and CSN8 in Sf21VM insect cells , with CSN8 N - terminally Strep ( II ) - and CSN1 & CSN3 N - terminally His6 - tagged , followed by affinity purification and SDS - PAGE displays banding pattern in line with the formation of a stable trimer CSN1 / 3 / 8 .

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Baculoviral co-expression of human CSN1, CSN3 and CSN8 in Sf21VM insect cells, with CSN8 N-terminally Strep(II)- and CSN1 & CSN3 N-terminally His6-tagged, followed by affinity purification and SDS-PAGE displays banding pattern in line with the formation of a stable trimer CSN1/3/8.


Protein - level SEC chromatograms of the 22 canonical 26S proteasome subunits . Vertical black lines indicate the apexes of six distinct co - elution signals detected in complex - centric scoring ; two of which represent well - known co - occurring variants , the full 26S ( i ) and the 20S ( ii ) particle devoid the 19S lid and ATPase ( Indicated by structural models , PDB accession 5GJR ) and four of which , composed of predominantly 20S α and β subunits , appear at reduced size ( 222 - 107 kDa , fractions 39 , 40 , 42 and 46 ) . The observations are consistent between the two whole workflow replicates ( see Appendix Figure S6B ) .

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Protein-level SEC chromatograms of the 22 canonical 26S proteasome subunits. Vertical black lines indicate the apexes of six distinct co-elution signals detected in complex-centric scoring; two of which represent well-known co-occurring variants, the full 26S (i) and the 20S (ii) particle devoid the 19S lid and ATPase (Indicated by structural models, PDB accession 5GJR) and four of which, composed of predominantly 20S α and β subunits, appear at reduced size (222 - 107 kDa, fractions 39, 40, 42 and 46). The observations are consistent between the two whole workflow replicates (see Appendix Figure S6B).


Subunit mass distribution across early and late assembly intermediate elution ranges suggests predominant components of the intermediary species accumulating in HEK293 cells .

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Subunit mass distribution across early and late assembly intermediate elution ranges suggests predominant components of the intermediary species accumulating in HEK293 cells.


SECexplorer web - interface for querying custom protein sets for co - elution behavior in the SEC - SWATH - MS data , viewing chromatograms for interpretation and with algorithmic assistance .

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SECexplorer web-interface for querying custom protein sets for co-elution behavior in the SEC-SWATH-MS data, viewing chromatograms for interpretation and with algorithmic assistance.


Zoom in to high MW peak shoulder of holo - COP9 Signalosome ( compare Figure 5 ) , where defined co - elution signals of CSN substrate components CUL4A and DDB2 suggest the partial resolution of substrate - bound and free pools of CSN holo - complex .

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Zoom in to high MW peak shoulder of holo-COP9 Signalosome (compare Figure 5), where defined co-elution signals of CSN substrate components CUL4A and DDB2 suggest the partial resolution of substrate-bound and free pools of CSN holo-complex.


Estimation of the fraction of holo - CSN in the likely substrate - bound pool versus the free pool , with eight measurements along the eight subunits and based on Gaussian deconvolution of two signals underlying the observed peak and shoulder ( also see Appendix Figure S7 ) .

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Estimation of the fraction of holo-CSN in the likely substrate-bound pool versus the free pool, with eight measurements along the eight subunits and based on Gaussian deconvolution of two signals underlying the observed peak and shoulder (also see Appendix Figure S7).


A Statistical analysis of NFATc1 expression with respect to tumor grading in KPCmice tissues .

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A Statistical analysis of NFATc1 expression with respect to tumor grading in KPCmice tissues.


B Immunohistochemistry detection of NFATc1 and p53 , illustrating G1 ‐ G2 and G3 in human PDAC tissues . Scale bars , 100 μm .

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B Immunohistochemistry detection of NFATc1 and p53, illustrating G1‐G2 and G3 in human PDAC tissues. Scale bars, 100 μm.


C Statistical illustration of TMA analysis demonstrating NFATc1 strong nuclear positive tumors ( n = 161 patients ) with respect to functional status of p53 expression in human PDAC tissues .

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C Statistical illustration of TMA analysis demonstrating NFATc1 strong nuclear positive tumors (n = 161 patients) with respect to functional status of p53 expression in human PDAC tissues.


D Representative H & amp ; amp ; E ‐ stained sections as well as immunohistochemistry detection of HA ‐ NFATc1 and p53 in well ‐ and poorly differentiated pancreatic tissues of KNCmice . Scale bars , 100 μm . E Quantification of p53 expression with respect to the tumor grading by evaluating both the intensity of immunostaining and the number of cells with overexpression by means of an immunoreactivity score ( IRS ) in KNCmice ( every dot represents one mouse ) .

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D Representative H&amp;E‐stained sections as well as immunohistochemistry detection of HA‐NFATc1 and p53 in well‐ and poorly differentiated pancreatic tissues of KNCmice. Scale bars, 100 μm.E Quantification of p53 expression with respect to the tumor grading by evaluating both the intensity of immunostaining and the number of cells with overexpression by means of an immunoreactivity score (IRS) in KNCmice (every dot represents one mouse).


F Incidence of livermetastases in KNCmice ( mean values ± SD ) .

F Incidence of livermetastases in KNCmice (mean values ± SD).


A Genome ‐ wide expression and GSEA analysis show p53 ‐ enriched signatures with " EMT " and " stem cell transcript " in KNCtumor cells .

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A Genome‐wide expression and GSEA analysis show p53‐enriched signatures with "EMT" and "stem cell transcript" in KNCtumor cells.


B Heat map showing p53 ‐ dependent regulation of " EMT " ‐ and " stemness " ‐ related genes in KNCtumor cells . Fold change relative to control cells is displayed in a green red color scheme for selected genes with FClog2 1 . 0 or FClog2 > − 1 . 0 .

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B Heat map showing p53‐dependent regulation of "EMT"‐ and "stemness"‐related genes in KNCtumor cells. Fold change relative to control cells is displayed in a green red color scheme for selected genes with FClog2 1.0 or FClog2 > −1.0.


C Statistical analysis of wound healing assay performed in primary KNCtumor cells following p53 depletion . Time ‐ lapse analyzer software was used to quantify the wound closure rate . Means ± SD are shown from one out of three independent experiments .

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C Statistical analysis of wound healing assay performed in primary KNCtumor cells following p53 depletion. Time‐lapse analyzer software was used to quantify the wound closure rate. Means ± SD are shown from one out of three independent experiments.


D Western blot analysis revealing protein expression levels upon p53 depletion in KNCtumor cells .

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D Western blot analysis revealing protein expression levels upon p53 depletion in KNCtumor cells.


E X ‐ fold mRNA expression of EMT ‐ related marker genes after p53 depletion in KNCtumor cells . Means ± SD from at least three independent experiments are shown . Asterisks display significance ( * P 0 . 05 ) .

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E X‐fold mRNA expression of EMT‐related marker genes after p53 depletion in KNCtumor cells. Means ± SD from at least three independent experiments are shown. Asterisks display significance (*P 0.05).


F Representative protein expression of EMT inducers in pancreatic lysates prepared from KNCmice with different tumor gradings .

F Representative protein expression of EMT inducers in pancreatic lysates prepared from KNCmice with different tumor gradings.


A Kaplan - Meier curves showing survival of KNC , KPNC , and KPΔNC mice under the control of p48 ‐ Cre pancreas ‐ specific promoters ( P 0 . 0001 for KPNC or KPΔNC cohorts versus KNC , log ‐ rank test , for pairwise combination ) .

A Kaplan-Meier curves showing survival of KNC, KPNC, and KPΔNC mice under the control of p48‐Cre pancreas‐specific promoters (P 0.0001 for KPNC or KPΔNC cohorts versus KNC, log‐rank test, for pairwise combination).


B Pathological features of KPNC and KP ∆ NC mice before tumor extraction . Pancreatic tissue is encircled .

B Pathological features of KPNC and KP∆NC mice before tumor extraction. Pancreatic tissue is encircled.


C , D HE and corresponding immunohistochemistry stainings of ( C ) KPNC and ( D ) KP ∆ NCmouse tumors . Scale bars , 100 μm .

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C, D HE and corresponding immunohistochemistry stainings of (C) KPNC and (D) KP∆NCmouse tumors. Scale bars, 100 μm.


E Tumor grading of PDAC from KPNC mice ( n = 10 ) .

E Tumor grading of PDAC from KPNC mice (n = 10).


A Quantitative RT ‐ PCR analysis of EMT ‐ related markers obtained from tumors derived from mice of the indicated genotypes ( n = 3 , means ± SD ) .

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A Quantitative RT‐PCR analysis of EMT‐related markers obtained from tumors derived from mice of the indicated genotypes (n = 3, means ± SD).


B Relative mRNA expression of NFATc1 ‐ and EMT ‐ related marker genes on administration of CsA ( 1 μM ) in KPNC cells . Data represents means ± SD from at least three independent experiments . Asterisks show significance ( * P 0 . 05 ) .

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B Relative mRNA expression of NFATc1‐ and EMT‐related marker genes on administration of CsA (1 μM) in KPNC cells. Data represents means ± SD from at least three independent experiments. Asterisks show significance (*P 0.05).


C Western blot analysis revealing protein expression levels in KPNC cells from the indicated proteins following transfection of siRNA control or siRNA toward NFATc1 , respectively .

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C Western blot analysis revealing protein expression levels in KPNC cells from the indicated proteins following transfection of siRNA control or siRNA toward NFATc1, respectively.


A Sox2 mRNA expression of pancreatic lysates from KNC , KPNC , and KPC mice , analyzed by real ‐ time PCR in three independent experiments ( means ± SD ) .

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A Sox2 mRNA expression of pancreatic lysates from KNC, KPNC, and KPC mice, analyzed by real‐time PCR in three independent experiments (means ± SD).


B , C Western blot analysis shows Sox2 expression in pancreatic lysates of ( B ) KNC mice with indicated tumor grading and in ( C ) KPNC and KPC mice .

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B, C Western blot analysis shows Sox2 expression in pancreatic lysates of (B) KNC mice with indicated tumor grading and in (C) KPNC and KPC mice.


D Protein expression levels after siRNA ‐ mediated p53 depletion in KNC tumor cells .

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D Protein expression levels after siRNA‐mediated p53 depletion in KNC tumor cells.


E , F NFATc1 and Sox2 protein expression after ( E ) NFATc1 depletion or ( F ) upon CsA ( 1 μM , 24 h ) treatment in Panc1 cells .

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E, F NFATc1 and Sox2 protein expression after (E) NFATc1 depletion or (F) upon CsA (1 μM, 24 h) treatment in Panc1 cells.


G Sox2 and E ‐ cadherin mRNA expression after NFATc1 depletion in L3 . 6 cells .

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G Sox2 and E‐cadherin mRNA expression after NFATc1 depletion in L3.6 cells.


I , J ChIP assays show NFATc1 binding to Sox2 enhancer and promoter in KNC tumor cells ( I )

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I, J ChIP assays show NFATc1 binding to Sox2 enhancer and promoter in KNC tumor cells (I)


I , J ChIP assays show RNA polymerase II binding to Sox2 promoter upon CsA ( 1 μM , 24 h ) treatment in L3 . 6 cells ( J ) .

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I, J ChIP assays show RNA polymerase II binding to Sox2 promoter upon CsA (1 μM, 24 h) treatment in L3.6 cells (J).


K Co ‐ immunoprecipitation of endogenous HA ‐ NFATc1 and Sox2 was performed in KPNC cells .

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K Co‐immunoprecipitation of endogenous HA‐NFATc1 and Sox2 was performed in KPNC cells.


L ChIP experiments show NFATc1 binding along with H3K27 acetylation ( H3K27ac ) mark at the selected Snai1 enhancer region and RNA polymerase II binding at Snai1 promoter in the presence or absence of Sox2 in KPNC tumor cells . Representative results from at least three independent experiments are shown ( means ± SD ; P 0 . 05 ) .

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L ChIP experiments show NFATc1 binding along with H3K27 acetylation (H3K27ac) mark at the selected Snai1 enhancer region and RNA polymerase II binding at Snai1 promoter in the presence or absence of Sox2 in KPNC tumor cells. Representative results from at least three independent experiments are shown (means ± SD; P 0.05).


M Relative mRNA expression levels of EMT ‐ related marker genes upon Sox2 depletion in KPNC tumor cells ( P 0 . 05 ) .

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M Relative mRNA expression levels of EMT‐related marker genes upon Sox2 depletion in KPNC tumor cells (P 0.05).


N Zeb1 and Snail mRNA levels in L3 . 6 cells after shRNA ‐ mediated depletion of Sox2 and transient overexpression of NFATc1 shows that loss of Sox2 significantly rescues NFATc1 ‐ mediated induction of EMT genes ( * P 0 . 05 ) .

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N Zeb1 and Snail mRNA levels in L3.6 cells after shRNA‐mediated depletion of Sox2 and transient overexpression of NFATc1 shows that loss of Sox2 significantly rescues NFATc1‐mediated induction of EMT genes (*P 0.05).


A NFATc1 and Sox2 protein expression in L3 . 6 cells ( adherent versus spheres ) .

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A NFATc1 and Sox2 protein expression in L3.6 cells (adherent versus spheres).


B Relative mRNA expression of NFATc1 and Sox2 in L3 . 6 cells as well as in KPNCmicetumor cells ( adherent versus spheres ) . Graph represents data from three independent experiments ( means ± SD ) . Asterisks display significance ( * P 0 . 05 ) .

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B Relative mRNA expression of NFATc1 and Sox2 in L3.6 cells as well as in KPNCmicetumor cells (adherent versus spheres). Graph represents data from three independent experiments (means ± SD). Asterisks display significance (*P 0.05).


C Cell sorting was performed to collect CD44 high and low populations in KPNC spheres ( left panel ) , which were analyzed for NFATc1 and Sox2 mRNA expression ( right panel ) .

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C Cell sorting was performed to collect CD44 high and low populations in KPNC spheres (left panel), which were analyzed for NFATc1 and Sox2 mRNA expression (right panel).


D Sox2 mRNA expression upon CsA ( 1 μM ) treatment in L3 . 6 cells ( adherent versus spheres ) ( * P 0 . 05 ) .

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D Sox2 mRNA expression upon CsA (1 μM) treatment in L3.6 cells (adherent versus spheres) (*P 0.05).


E Bright ‐ field light microscopy images as well as quantification of KPNC spheres after transient NFATc1 depletion .

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E Bright‐field light microscopy images as well as quantification of KPNC spheres after transient NFATc1 depletion.


A Expression analysis of indicated miRNAs upon adenoviral re ‐ expression of p53 in KPΔNC tumor cells , detected by real ‐ time PCR ( P 0 . 05 ) .

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A Expression analysis of indicated miRNAs upon adenoviral re‐expression of p53 in KPΔNC tumor cells, detected by real‐time PCR (P 0.05).


B Protein expression of EMT marker genes after adenoviral re ‐ expression of GFP ‐ tagged p53 in KPΔNC tumor cells .

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B Protein expression of EMT marker genes after adenoviral re‐expression of GFP‐tagged p53 in KPΔNC tumor cells.


C , D Protein ( C ) expression levels of EMT and stemness marker genes upon miRNA ‐ 34a and miRNA ‐ 200c overexpression using specific mimics in KPNC tumor cells . Representative results from at least three independent experiments are shown ( means ± SD ; P 0 . 05 ) .

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C, D Protein (C) expression levels of EMT and stemness marker genes upon miRNA‐34a and miRNA‐200c overexpression using specific mimics in KPNC tumor cells. Representative results from at least three independent experiments are shown (means ± SD; P 0.05).


C , D mRNA ( D ) expression levels of EMT and stemness marker genes upon miRNA ‐ 34a and miRNA ‐ 200c overexpression using specific mimics in KPNC tumor cells . Representative results from at least three independent experiments are shown ( means ± SD ; P 0 . 05 ) .

C, D mRNA (D) expression levels of EMT and stemness marker genes upon miRNA‐34a and miRNA‐200c overexpression using specific mimics in KPNC tumor cells. Representative results from at least three independent experiments are shown (means ± SD; P 0.05).


E Western blot analysis of EMT marker expression in KNC cells following depletion of miR ‐ 200c .

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E Western blot analysis of EMT marker expression in KNC cells following depletion of miR‐200c.


F ChIP experiments showing NFATc1 binding along with H3K27 acetylation mark at the selected Snai1 enhancer region as well as Sox2 and RNA polymerase II binding at Snai1 promoter after miR ‐ 200c overexpression in KPNC tumor cells . Means ± SD are shown from one out of three independent experiments .

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F ChIP experiments showing NFATc1 binding along with H3K27 acetylation mark at the selected Snai1 enhancer region as well as Sox2 and RNA polymerase II binding at Snai1 promoter after miR‐200c overexpression in KPNC tumor cells. Means ± SD are shown from one out of three independent experiments.


G ChIP analysis in KP ∆ NC tumor cells after adenoviral GFP ‐ tagged p53 overexpression . NFATc1 and H3K27ac binding are illustrated on Snai1 enhancer , and Sox2 and RNA polymerase II binding are demonstrated on Snai1 promoter . Means ± SD are shown from one out of three independent experiments .

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G ChIP analysis in KP∆NC tumor cells after adenoviral GFP‐tagged p53 overexpression. NFATc1 and H3K27ac binding are illustrated on Snai1 enhancer, and Sox2 and RNA polymerase II binding are demonstrated on Snai1 promoter. Means ± SD are shown from one out of three independent experiments.


H Quantification of KP ∆ NC tumour cell - derived spheres after adenoviralGFP ‐ tagged p53 overexpression .

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H Quantification of KP∆NC tumour cell-derived spheres after adenoviralGFP‐tagged p53 overexpression.


( A ) Top panel : Scheme illustrating the tetracycline inducible Flp - In 293 system that controls the expression of HA - IKKε or HA - GFP . Bottom panel : Representative western blot showing induced expression of HA - IKKε in Flp - In 293 cells treated with doxycycline ( Dox , 50 ng / ml ) for 16 hours compared to endogenous IKKε in T47D , MDA - MB - 231 and MDA - MB - 468 breast cancer cell lines .

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(A) Top panel: Scheme illustrating the tetracycline inducible Flp-In 293 system that controls the expression of HA-IKKε or HA-GFP. Bottom panel: Representative western blot showing induced expression of HA-IKKε in Flp-In 293 cells treated with doxycycline (Dox, 50 ng/ml) for 16 hours compared to endogenous IKKε in T47D, MDA-MB-231 and MDA-MB-468 breast cancer cell lines.


( B ) Heatmap and hierarchical clustering of metabolite concentrations in Flp - In 293 HA - GFP and Flp - In 293 HA - IKKε cells treated with doxycycline ( Dox , 50 ng / ml , 16 hours ) ( n = 5 technical replicates ) .

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(B) Heatmap and hierarchical clustering of metabolite concentrations in Flp-In 293 HA-GFP and Flp-In 293 HA-IKKε cells treated with doxycycline (Dox, 50 ng/ml, 16 hours) (n=5 technical replicates).


( C ) Serine production from glucose ( serine m + 3 , 13C6 - glucose labelling , left panel ) and glutamine ( serine m + 1 , 15N2 - glutamine labelling , right panel ) in Flp - In 293 HA - GFP or Flp - In 293 HA - IKKε cells treated with doxycycline ( 50 ng / ml , 16 hours ) metabolite levels were normalised to the internal standard HEPES .

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(C) Serine production from glucose (serine m+3, 13C6-glucose labelling, left panel) and glutamine (serine m+1, 15N2-glutamine labelling, right panel) in Flp-In 293 HA-GFP or Flp-In 293 HA-IKKε cells treated with doxycycline (50 ng/ml, 16 hours) metabolite levels were normalised to the internal standard HEPES.


( E ) Contribution of pyruvate and glucose - derived carbon to TCA cyle metabolites in Flp - In 293 HA - GFP or Flp - In 293 HA - IKKε cells treated with doxycycline ( 50 ng / ml , 16 hours ) . ( n = 5 technical replicates ) . metabolite levels were normalised to the internal standard HEPES .

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(E) Contribution of pyruvate and glucose-derived carbon to TCA cyle metabolites in Flp-In 293 HA-GFP or Flp-In 293 HA-IKKε cells treated with doxycycline (50 ng/ml, 16 hours). (n=5 technical replicates). metabolite levels were normalised to the internal standard HEPES.


( F ) Fractional enrichment of serine , malate and citrate 13C - isotopologues in Flp - In 293 HA - GFP and Flp - In 293 HA - IKKε cells treated with doxycycline ( 50 ng / ml , 16 hours ) ( n = 5 technical replicates ) .

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(F) Fractional enrichment of serine, malate and citrate 13C-isotopologues in Flp-In 293 HA-GFP and Flp-In 293 HA-IKKε cells treated with doxycycline (50 ng/ml, 16 hours) (n=5 technical replicates).


( G ) Fractional enrichment of the serine 15 - N - isotopologue in Flp - In 293 HA - GFP and Flp - In 293 HA - IKKε cells treated with doxycycline ( 50 ng / ml , 16 hours ) . m + 1 shows the naturally occurring 13C isotopologue ( n = 5 technical replicates ) . metabolite levels were normalised to the internal standard HEPES .

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(G) Fractional enrichment of the serine 15-N-isotopologue in Flp-In 293 HA-GFP and Flp-In 293 HA-IKKε cells treated with doxycycline (50 ng/ml, 16 hours). m+1 shows the naturally occurring 13C isotopologue (n=5 technical replicates). metabolite levels were normalised to the internal standard HEPES.


Representative western blot showing level of IKKε in IKBKE ( IKKε ) - silenced ( A ) T47D breast cancer cell lines .

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Representative western blot showing level of IKKε in IKBKE (IKKε)-silenced (A) T47D breast cancer cell lines.


Representative western blot showing level of IKKε in IKBKE ( IKKε ) - silenced ( B ) MDA - MB - 468 breast cancer cell lines .

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Representative western blot showing level of IKKε in IKBKE (IKKε)-silenced (B) MDA-MB-468 breast cancer cell lines.


( C - D ) Heatmap and hierarchical clustering of metabolite concentrations in ( C ) IKBKE ( IKKε ) - silenced T47D cells and ( D ) IKBKE ( IKKε ) - silenced MDA - MB - 468 cells ( n = 5 technical replicates ) . metabolite levels were normalised to the internal standard HEPES .

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(C-D) Heatmap and hierarchical clustering of metabolite concentrations in (C) IKBKE (IKKε)-silenced T47D cells and (D) IKBKE (IKKε)-silenced MDA-MB-468 cells (n=5 technical replicates). metabolite levels were normalised to the internal standard HEPES.


( E ) Glycine production , representative of serine production , from glutamine ( glycine m + 1 , 15N - glutamine labelling ) and glucose ( glycine m + 2 , 13C6 - glucose labelling ) , and contribution of pyruvate and glucose - derived carbon to TCA cycle metabolites ( citrate m + 2 , malate m + 2 , 13C6 - glucose labelling ) in IKBKE ( IKKε ) - silenced T47D cells . ( n = 5 technical replicates ) . metabolite levels were normalised to the internal standard HEPES .

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(E) Glycine production, representative of serine production, from glutamine (glycine m+1, 15N-glutamine labelling) and glucose (glycine m+2, 13C6-glucose labelling), and contribution of pyruvate and glucose-derived carbon to TCA cycle metabolites (citrate m+2, malate m+2, 13C6-glucose labelling) in IKBKE (IKKε)-silenced T47D cells. (n=5 technical replicates). metabolite levels were normalised to the internal standard HEPES.


( F ) Serine production from glutamine ( serine m + 1 , 15N - glutamine labelling ) , and serine and glycine production from glucose ( glycine m + 2 , serine m + 3 , 13C6 - glucose labelling ) as well as contribution of pyruvate and glucose - derived carbon to TCA cycle metabolites ( malate m + 2 , 13C6 - glucose labelling ) in IKBKE ( IKKε ) - silenced MDA - MB - 468 cells .

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(F) Serine production from glutamine (serine m+1, 15N-glutamine labelling), and serine and glycine production from glucose (glycine m+2, serine m+3, 13C6-glucose labelling) as well as contribution of pyruvate and glucose-derived carbon to TCA cycle metabolites (malate m+2, 13C6-glucose labelling) in IKBKE (IKKε)-silenced MDA-MB-468 cells.


( A ) Basal oxygen consumption in Flp - In 293 HA - IKKε wt , Flp - In 293 HA - IKKε KD - m and Flp - In 293 HA - IKKε UbLD - m cells following treatment with doxycycline ( Dox , 16 hours ) , measured using Oroboros high resolution respirometry . Data are normalised to non - treated control cells .

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(A) Basal oxygen consumption in Flp-In 293 HA-IKKε wt, Flp-In 293 HA-IKKε KD-m and Flp-In 293 HA-IKKε UbLD-m cells following treatment with doxycycline (Dox, 16 hours), measured using Oroboros high resolution respirometry. Data are normalised to non-treated control cells.


( B ) Average TMRM staining intensity in Flp - In 293 HA - IKKε wt , Flp - In 293 HA - IKKε KD - m and Flp - In 293 HA - IKKε UbLD - m expressing cells induced by doxycycline ( Dox , 16 hours ) . Data are normalised to non - treated control cells .

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(B) Average TMRM staining intensity in Flp-In 293 HA-IKKε wt, Flp-In 293 HA-IKKε KD-m and Flp-In 293 HA-IKKε UbLD-m expressing cells induced by doxycycline (Dox, 16 hours). Data are normalised to non-treated control cells.


( C ) Basal mitochondrial oxygen consumption rate ( OCR ) in a panel of IKBKE ( IKKε ) - silenced breast cancer cell lines , measured using Seahorse XF96e or XF24 analysis .

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(C) Basal mitochondrial oxygen consumption rate (OCR) in a panel of IKBKE (IKKε)-silenced breast cancer cell lines, measured using Seahorse XF96e or XF24 analysis.


( D ) OCR in mitochondria isolated from Flp - In 293 HA - GFP or HA - IKKε cells treated with doxycycline ( 50 ng / ml , 16 hours ) , measured using Oroboros high resolution respirometry .

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(D) OCR in mitochondria isolated from Flp-In 293 HA-GFP or HA-IKKε cells treated with doxycycline (50 ng/ml, 16 hours), measured using Oroboros high resolution respirometry.


( F ) Representative western blot showing l evel of IKKε in three independent single cell clones of Flp - In 293 HA - GFP or Flp - In 293 HA - IKKε cells following treatment with doxycycline ( 100 ng / ml , 16 hours ) .

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(F) Representative western blot showing level of IKKε in three independent single cell clones of Flp-In 293 HA-GFP or Flp-In 293 HA-IKKε cells following treatment with doxycycline (100 ng/ml, 16 hours).


( G ) Relative pyruvate dehydrogenase ( PDH ) activity in Flp - In 293 HA - GFP or Flp - In 293 HA - IKKε cells treated with doxycycline ( 50 ng / ml , 16 hours ) .

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(G) Relative pyruvate dehydrogenase (PDH) activity in Flp-In 293 HA-GFP or Flp-In 293 HA-IKKε cells treated with doxycycline (50 ng/ml, 16 hours).


( H ) Average TMRM staining intensity in Flp - In 293 HA - GFP or Flp - In 293 HA - IKKε cells treated with doxycycline ( Dox ) and DCA ( both for 16 hours ) . Data are normalised to non - treated Flp - In 293 HA - IKKε cells .

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(H) Average TMRM staining intensity in Flp-In 293 HA-GFP or Flp-In 293 HA-IKKε cells treated with doxycycline (Dox) and DCA (both for 16 hours). Data are normalised to non-treated Flp-In 293 HA-IKKε cells.


( I ) Basal OCR in Flp - In 293 HA - GFP or Flp - In 293 HA - IKKε cells treated with doxycycline ( 50 ng / ml ) in combination with dichloroacetate ( DCA ) for 16 hours , measured using Oroboros high resolution respirometry .

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(I) Basal OCR in Flp-In 293 HA-GFP or Flp-In 293 HA-IKKε cells treated with doxycycline (50 ng/ml) in combination with dichloroacetate (DCA) for 16 hours, measured using Oroboros high resolution respirometry.


( J ) Basal OCR in Flp - In 293 HA - GFP or Flp - In 293 HA - IKKε cells treated with doxycycline ( 50 ng / ml ) in combination with pyruvate deprivation for 16 hours , measured using Oroboros high resolution respirometry .

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(J) Basal OCR in Flp-In 293 HA-GFP or Flp-In 293 HA-IKKε cells treated with doxycycline (50 ng/ml) in combination with pyruvate deprivation for 16 hours, measured using Oroboros high resolution respirometry.


( A ) Representative western blot showing level of ATF4 in ATF4 - silenced Flp - In 293 HA - IKKε cells treated with doxycycline ( 16 hours ) .

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(A) Representative western blot showing level of ATF4 in ATF4-silenced Flp-In 293 HA-IKKε cells treated with doxycycline (16 hours).


( B ) qRT - PCR analysis of PHGDH , PSAT1 and PSPH mRNA levels in Flp - In 293 HA - GFP or Flp - In 293 HA - IKKε cells treated with doxycycline ( Dox ) for 16 hours . Data are expressed as fold changes , relative to levels in non - treated Flp - In 293 HA - GFP cells and normalised to β - Actin ( n = 4 biological replicates )

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(B) qRT-PCR analysis of PHGDH, PSAT1 and PSPH mRNA levels in Flp-In 293 HA-GFP or Flp-In 293 HA-IKKε cells treated with doxycycline (Dox) for 16 hours. Data are expressed as fold changes, relative to levels in non-treated Flp-In 293 HA-GFP cells and normalised to β-Actin (n=4 biological replicates)


( C ) Representative western blot showing levels of IKKε , IRF3 , phosphorylated IRF3 ( S396 ) and SBP enzymes in Flp - In 293 HA - GFP or Flp - In 293 HA - IKKε cells treated with doxycycline ( Dox ) for 16 hours .

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(C) Representative western blot showing levels of IKKε, IRF3, phosphorylated IRF3 (S396) and SBP enzymes in Flp-In 293 HA-GFP or Flp-In 293 HA-IKKε cells treated with doxycycline (Dox) for 16 hours.


( D ) qRT - PCR analysis of PHGDH , PSAT1 and PSPH mRNA levels in ATF4 - silenced Flp - In 293 HA - IKKε cells treated with doxycycline ( Dox ) for 16 hours . Data are expressed as fold changes , relative to levels in a non - silenced , non - treated control , and normalised to β - Actin ( n = 5 biological replicates ) .

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

(D) qRT-PCR analysis of PHGDH, PSAT1 and PSPH mRNA levels in ATF4-silenced Flp-In 293 HA-IKKε cells treated with doxycycline (Dox) for 16 hours. Data are expressed as fold changes, relative to levels in a non-silenced, non-treated control, and normalised to β-Actin (n=5 biological replicates).


( E ) Representative western blot showing levels of IKKε , PHGDH , PSAT1 and PSPH in ATF4 - silenced Flp - In 293 HA - IKKε cells treated with doxycycline ( Dox ) for 16 hours .

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

(E) Representative western blot showing levels of IKKε, PHGDH, PSAT1 and PSPH in ATF4-silenced Flp-In 293 HA-IKKε cells treated with doxycycline (Dox) for 16 hours.


( A ) qRT - PCR analysis of PHGDH , PSAT1 and PSPH mRNA levels in a panel of IKBKE ( IKKε ) - silenced breast cancer cell lines . Data are expressed as fold changes , relative to levels in a non - silenced control of each cell line and normalised to β - Actin ( n ≥ 3 biological replicates ) .

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

(A) qRT-PCR analysis of PHGDH, PSAT1 and PSPH mRNA levels in a panel of IKBKE (IKKε)-silenced breast cancer cell lines. Data are expressed as fold changes, relative to levels in a non-silenced control of each cell line and normalised to β-Actin (n≥3 biological replicates).


( B ) Representative western blot showing levels of IKKε and the SBP enzymes in IKBKE ( IKKε ) - silenced ZR - 75 - 1 , T47D , MDA - MB - 468 and MCF7 breast cancer cell lines .

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

(B) Representative western blot showing levels of IKKε and the SBP enzymes in IKBKE (IKKε)-silenced ZR-75-1, T47D, MDA-MB-468 and MCF7 breast cancer cell lines.


( C - E ) Levels of the SBP enzymes in a panel of IKBKE ( IKKε ) - silenced breast cancer cell lines . ( C ) PHGDH , ( D ) PSAT1 and ( E ) PSPH levels in indicated cell lines normalised to Vinculin . Densitometry analysis quantified single sample density as a percentage of total blot density per cell line prior to vinculin normalisation ( n ≥ 3 biological replicates ) .

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

(C-E) Levels of the SBP enzymes in a panel of IKBKE (IKKε)-silenced breast cancer cell lines. (C) PHGDH, (D) PSAT1 and (E) PSPH levels in indicated cell lines normalised to Vinculin. Densitometry analysis quantified single sample density as a percentage of total blot density per cell line prior to vinculin normalisation (n≥3 biological replicates).


( F ) qRT - PCR analysis of PHGDH , PSAT1 and PSPH mRNA levels in ATF4 - silenced ZR - 75 - 1 , T47D and MDA - MB - 468 breast cancer cell lines . Data are expressed as fold changes , relative to levels in non - silenced control cells and normalised to β - Actin ( n = 3 biological replicates ) .

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

(F) qRT-PCR analysis of PHGDH, PSAT1 and PSPH mRNA levels in ATF4-silenced ZR-75-1, T47D and MDA-MB-468 breast cancer cell lines. Data are expressed as fold changes, relative to levels in non-silenced control cells and normalised to β-Actin (n=3 biological replicates).


( G ) Representative western blot showing levels of PHGDH , PSAT1 and PSPH in ATF4 - silenced ZR - 75 - 1 , MDA - MB - 468 , MDA - MB - 231 , T47D and HCC1143 breast cancer cell lines .

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

(G) Representative western blot showing levels of PHGDH, PSAT1 and PSPH in ATF4-silenced ZR-75-1, MDA-MB-468, MDA-MB-231, T47D and HCC1143 breast cancer cell lines.


( A ) Correlation of change in OCR ( ΔOCR ) in a panel of IKBKE ( IKKε ) - silenced breast cancer cell lines ( from Fig . 3C ) and the change in cell confluency ( Δconfluency ) upon treatment of the panel of cell lines with NCT502 ( from Fig . EV5A ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Correlation of change in OCR (ΔOCR) in a panel of IKBKE (IKKε)-silenced breast cancer cell lines (from Fig. 3C) and the change in cell confluency (Δconfluency) upon treatment of the panel of cell lines with NCT502 (from Fig. EV5A).


( B ) Correlation of ΔOCR in a panel of IKBKE ( IKKε ) - silenced breast cancer cell lines ( from Fig . 3C ) and the Δconfluency upon treatment of the panel of cell lines with 6 - Diazo - 5 - oxo - L - norleucine ( DON ) ( from Fig . EV5C ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Correlation of ΔOCR in a panel of IKBKE (IKKε)-silenced breast cancer cell lines (from Fig. 3C) and the Δconfluency upon treatment of the panel of cell lines with 6-Diazo-5-oxo-L-norleucine (DON) (from Fig. EV5C).


( C ) Association between IKBKE ( IKKε ) and PSAT1 mRNA overexpression evaluated by a chi - squared independence test . The + sign indicates samples with significant ( p < 0 . 05 ) overexpression of IKBKE or PSAT1 . Number and percentage of samples , as well as the Chi - square values are shown .

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

(C) Association between IKBKE (IKKε) and PSAT1 mRNA overexpression evaluated by a chi-squared independence test. The + sign indicates samples with significant (p<0.05) overexpression of IKBKE or PSAT1. Number and percentage of samples, as well as the Chi-square values are shown.


Expression of IKBKE ( IKKε ) and the SBP enzymes PSAT1 , in the METABRIC dataset . Samples were stratified by Pam50 intrinsic subtypes and ER status . Brown - Forsythe and Welch ANOVA test with unpaired t - test with Welch correction were applied . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 IKBKE (IKKε) and the SBP enzymes PSAT1, in the METABRIC dataset. Samples were stratified by Pam50 intrinsic subtypes and ER status. Brown-Forsythe and Welch ANOVA test with unpaired t-test with Welch correction were applied. *p<0.05, **p< 0.01, ***p<0.001, ****p<0.0001.


Expression of SBP enzymes PHGDH and PSPH in the METABRIC dataset . The expression of a proliferation related gene set and ATF4 are also shown . Samples were stratified by Pam50 intrinsic subtypes and ER status . Brown - Forsythe and Welch ANOVA test with unpaired t - test with Welch correction were applied . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 SBP enzymes PHGDH and PSPH in the METABRIC dataset. The expression of a proliferation related gene set and ATF4 are also shown. Samples were stratified by Pam50 intrinsic subtypes and ER status. Brown-Forsythe and Welch ANOVA test with unpaired t-test with Welch correction were applied. *p<0.05, **p< 0.01, ***p<0.001, ****p<0.0001.


( J ) Association between IKBKE ( IKKε ) and ATF4 mRNA overexpression evaluated by chi - squared independence test . The + sign indicates samples with significant ( p < 0 . 05 ) overexpression of IKBKE or PSAT1 . Number and percentage of samples , as well as the Chi - square values are shown .

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

(J) Association between IKBKE (IKKε) and ATF4 mRNA overexpression evaluated by chi-squared independence test. The + sign indicates samples with significant (p<0.05) overexpression of IKBKE or PSAT1. Number and percentage of samples, as well as the Chi-square values are shown.


( K , L ) Correlation of IKBKE ( IKKε ) and PSAT1 ( K ) or IKBKE ( IKKε ) and ATF4 ( L ) expression in the ER negative sample subset .

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

(K, L) Correlation of IKBKE (IKKε) and PSAT1 (K) or IKBKE (IKKε) and ATF4 (L) expression in the ER negative sample subset.


( C ) Adult phenotypes of the ash1 and NSD mutants . In ash122 / ash121 mutants ( designated as ash1 - ) the loss of Abd - B expression results in partial transformation of abdominal segments 6 and 5 toward segments 5 and 4 , which is visible from the partial loss of pigmentation on tergites 5 and 6 ( t5 and t6 ) and appearance of bristles on sternite 6 ( s6 , marked with black arrows ) . The loss of Ubx expression causes transformation of the third thoracic to the second thoracic segment visible as partial haltere ( H ) to wing and third leg ( 3L ) to second leg ( 2L ) transformations . The former is evident from the change in the haltere shape and the appearance of multiple bristles ( black arrowheads ) . The latter is indicated by the apical and pre - apical bristles ( red arrows ) on the tibia of the third leg of ash1 mutants . These are normally present on 2L but absent on 3L ( compare to wild - type ) . Also note the appearance of additional hypopleural bristles on the third thoracic segment of the ash1 - flies ( red arrowheads ) , which indicate its transformation towards the second thoracic segment . Phenotype of the NSDds46 / NSDds46 ( NSD - ) flies is indistinguishable from wild - type and the phenotype of the double ash122 , NSDds46 / ash122 , NSDds46 ( ash1 - , NSD - ) flies is no more severe than that of the single ash122 / ash121 ( ash1 - ) mutants .

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

(C) Adult phenotypes of the ash1 and NSD mutants. In ash122/ash121 mutants (designated as ash1-) the loss of Abd-B expression results in partial transformation of abdominal segments 6 and 5 toward segments 5 and 4, which is visible from the partial loss of pigmentation on tergites 5 and 6 (t5 and t6) and appearance of bristles on sternite 6 (s6, marked with black arrows). The loss of Ubx expression causes transformation of the third thoracic to the second thoracic segment visible as partial haltere (H) to wing and third leg (3L) to second leg (2L) transformations. The former is evident from the change in the haltere shape and the appearance of multiple bristles (black arrowheads). The latter is indicated by the apical and pre-apical bristles (red arrows) on the tibia of the third leg of ash1 mutants. These are normally present on 2L but absent on 3L (compare to wild-type). Also note the appearance of additional hypopleural bristles on the third thoracic segment of the ash1- flies (red arrowheads), which indicate its transformation towards the second thoracic segment. Phenotype of the NSDds46/NSDds46 (NSD-) flies is indistinguishable from wild-type and the phenotype of the double ash122,NSDds46/ ash122,NSDds46 (ash1-,NSD-) flies is no more severe than that of the single ash122/ash121 (ash1-) mutants.


( D ) Ubx expression in the haltere imaginal discs . The expression was assayed by immunostaining with antibodies against Ubx ( red ) and acetylated H3K18 ( green , positive control ) . While ash122 / ash121 ( ash1 - ) larvae show stochastic clonal loss of the Ubx immunostaining in haltere discs ( yellow dashed lines ) , Set2 - larvae have uniform expression of Ubx throughout the haltere disc , resembling that in the wild - type larvae . Scale bars indicate 100μm .

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

(D) Ubx expression in the haltere imaginal discs. The expression was assayed by immunostaining with antibodies against Ubx (red) and acetylated H3K18 (green, positive control). While ash122/ash121 (ash1-) larvae show stochastic clonal loss of the Ubx immunostaining in haltere discs (yellow dashed lines), Set2- larvae have uniform expression of Ubx throughout the haltere disc, resembling that in the wild-type larvae. Scale bars indicate 100μm.


( A ) Adult phenotypes of the ash122 / ash19011 flies supplemented with transgenic constructs expressing either the full - length Ash1 ( Ash1FL ) or the truncated variants lacking the PHD ( Ash1 ∆ PHD ) or the SET ( Ash1 ∆ SET ) domains . Note the third leg ( L3 ) to second leg ( L2 ) , haltere ( H ) to wing ( black arrows ) , t5 to t4 and t6 to t5 transformations in the Ash1 ∆ PHD and the Ash1 ∆ SET but not in the Ash1FL flies . The latter are evident from the partial loss of pigmentation in t6 and t5 , or the appearance of small bristles ( trichomas ) on t6 of the Ash1 ∆ PHD and the Ash1 ∆ SET flies in the area that is normally naked ( Ash1FL , yellow dashed line ) .

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

(A) Adult phenotypes of the ash122/ash19011 flies supplemented with transgenic constructs expressing either the full-length Ash1 (Ash1FL) or the truncated variants lacking the PHD (Ash1∆PHD) or the SET (Ash1∆SET) domains. Note the third leg (L3) to second leg (L2), haltere (H) to wing (black arrows), t5 to t4 and t6 to t5 transformations in the Ash1∆PHD and the Ash1∆SET but not in the Ash1FL flies. The latter are evident from the partial loss of pigmentation in t6 and t5, or the appearance of small bristles (trichomas) on t6 of the Ash1∆PHD and the Ash1∆SET flies in the area that is normally naked (Ash1FL, yellow dashed line).


( B ) Two - fold dilutions of total nuclear protein extracts from the third instar larvae of the ash122 / ash19011 mutants supplemented with various transgenic constructs and wild type flies were analyzed by western blot with anti - Ash1 antibodies . Arrowhead indicates the position of Ash1 protein . Note that transgenic proteins are expressed at comparable level .

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

(B) Two-fold dilutions of total nuclear protein extracts from the third instar larvae of the ash122/ash19011 mutants supplemented with various transgenic constructs and wild type flies were analyzed by western blot with anti-Ash1 antibodies. Arrowhead indicates the position of Ash1 protein. Note that transgenic proteins are expressed at comparable level.


( C ) Coomassie staining of SDS - PAGE separated protein extracts from ( B ) was used to control the loading .

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

(C) Coomassie staining of SDS-PAGE separated protein extracts from (B) was used to control the loading.


Two - fold serial dilutions of the total protein extracts from the wild - type , ash122 / ash19011 ( ash1 - ) , ash122 , NSDds46 / ash19011 , NSDds46 ( ash1 - , NSD - ) and Set21 ( Set2 - ) larval brains , imaginal discs and salivary glands were analyzed by western blot with antibodies against H3K36me1 ( A ) , H3K36me2 ( B ) and H3K36me3 ( C ) . Note the strong ( > 10 - fold ) reduction of H3K36me3 signal in the Set2 - extract and the slight ( ~ 2 - fold ) reduction of H3K36me1 signal in the ash1 - and ash1 - , NSD - extracts . The protein extracts from the wild - type , double ash1 - , NSD - and single NSD - and Set2 - mutants ( right panels ) were analyzed together on the same membrane , however the images of the H3K36me1 and H3K36me3 western blots were modified to splice out the marker lane between the ash1 - , NSD - and the Set2 - extracts . Western blots with constitutively expressed BEAF - 32 protein were used as loading controls .

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

Two-fold serial dilutions of the total protein extracts from the wild-type, ash122/ash19011 (ash1-), ash122,NSDds46/ash19011,NSDds46 (ash1-, NSD-) and Set21 (Set2-) larval brains, imaginal discs and salivary glands were analyzed by western blot with antibodies against H3K36me1 (A), H3K36me2 (B) and H3K36me3 (C). Note the strong (>10-fold) reduction of H3K36me3 signal in the Set2- extract and the slight (~2-fold) reduction of H3K36me1 signal in the ash1- and ash1-, NSD- extracts. The protein extracts from the wild-type, double ash1-, NSD- and single NSD- and Set2- mutants (right panels) were analyzed together on the same membrane, however the images of the H3K36me1 and H3K36me3 western blots were modified to splice out the marker lane between the ash1-,NSD- and the Set2- extracts. Western blots with constitutively expressed BEAF-32 protein were used as loading controls.


Chromatin from the wild - type ( dark blue bars ) , ash122 / ash19011 ( ash1 - , red bars ) and transgenic ash122 / ash19011 ( Ash1FL , light blue bars ; Ash1 ∆ PHD , green bars ; Ash1 ∆ SET , orange bars ) larvae was subjected to immunoprecipitation with the antibodies against H3K36me1 , H3K36me2 Histograms show the mean of the two independent experiments ( n = 2 ) with dots indicating individual experimental results . An intergenic region on chromosome 3R ( intergenic ) and the constitutively active TBP - associated factor 4 ( Taf4 ) gene serve as controls . The loss of Ash1 ChIP signal in the ash1 - larvae indicates that the selected genes are the genuine Ash1 binding sites .

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

Chromatin from the wild-type (dark blue bars), ash122/ash19011 (ash1-, red bars) and transgenic ash122/ash19011 (Ash1FL, light blue bars; Ash1∆PHD, green bars; Ash1∆SET, orange bars) larvae was subjected to immunoprecipitation with the antibodies against H3K36me1 , H3K36me2 Histograms show the mean of the two independent experiments (n=2) with dots indicating individual experimental results. An intergenic region on chromosome 3R (intergenic) and the constitutively active TBP-associated factor 4 (Taf4) gene serve as controls. The loss of Ash1 ChIP signal in the ash1- larvae indicates that the selected genes are the genuine Ash1 binding sites.