C Activation of NF - κB pathway by RIPK2 pocket mutants . RIPK2 - deficient THP - 1 were reconstituted with wild - type RIPK2 or RIPK2 mutants , stimulated with L18 - MDP , harvested at indicated time points analyzed by immunoblotting .

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

C Activation of the NF-κB pathway by RIPK2 pocket mutants. RIPK2-deficient THP-1 cells were reconstituted with wild-type RIPK2 or RIPK2 mutants, stimulated with L18-MDP, harvested at indicated time points and analyzed by immunoblotting.


D IL - 8 production of RIPK2 - deficient THP - 1cells reconstituted with wild - type RIPK2 or RIPK2 mutants stimulated with L18 - MDP was assessed by ELISA . N = 3 - 8 experiments . Shown is average ± . ns = P > 0 . 05 ; * = P ≤ 0 . 05 ; * * * = P ≤ 0 . 001 ; * * * * = P ≤ 0 . 0001 ; two - way ANOVA .

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

D IL-8 production of RIPK2-deficient THP-1cells reconstituted with wild-type RIPK2 or RIPK2 mutants and stimulated with L18-MDP was assessed by ELISA. N = 3-8 experiments. Shown is average ± SEM. ns = P > 0.05; * = P ≤ 0.05; *** = P ≤ 0.001; **** = P ≤ 0.0001; two-way ANOVA.


E Ubiquitination of RIPK2 pocket mutants . RIPK2 - deficient THP - 1 were reconstituted with wild - type or mutant RIPK2 , left unstimulated or stimulated with L18 - MDP subjected to UBA pulldown immunoblotting .

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

E Ubiquitination of RIPK2 pocket mutants. RIPK2-deficient THP-1 cells were reconstituted with wild-type or mutant RIPK2, left unstimulated or stimulated with L18-MDP and subjected to UBA pulldown and immunoblotting.


A : Thermal stability using selected RIPK2 mutants . THP - 1 expressing RIPK2 were subjected to heat treatment non - denatured fraction was analysed by immunoblotting .

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

A: Thermal stability assay using selected RIPK2 mutants. THP-1 cells expressing RIPK2 were subjected to heat treatment and the non-denatured fraction was analysed by immunoblotting.


B : Binding of RIPK2 to XIAP - BIR2 . Lysates from RIPK2 - deficient THP - 1 reconstituted with WT or mutant RIPK2 were subjected to pulldown experiments with recombinantly expressed XIAP - BIR2 analysed by immunoblotting .

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

B: Binding of RIPK2 to XIAP-BIR2. Lysates from RIPK2-deficient THP-1 cells reconstituted with WT or mutant RIPK2 were subjected to pulldown experiments with recombinantly expressed XIAP-BIR2 and analysed by immunoblotting.


( A ) Illustration of crossing ageing scheme used to obtain total extracts from fly heads for transcriptional profiling successive qPCR assays . Expression of different Atrophin forms with GMR driver was induced , owing to a ‐ sensitive mutant Gal80 repressor . F1 flies were allowed to develop at 18 ° C ; at this Gal80 repressor keeps transgenes silent . Newly eclosed flies ( 0 - 48 h ) were collected killed immediately ( 0d ) or aged for 2 or 14 days at 29 ° C . This inactivates Gal80 transgenes are switched on by GMR ‐ Gal4 . This protocol allows comparing siblings that differ exclusively in their transgenes expression . Control flies crossed to GMR ‐ Gal4 ; UbiGal80ts in experiments are from w1118 stock in which UAS transgenes have been generated .

O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Illustration of the crossing and ageing scheme used to obtain total RNA extracts from fly heads for the transcriptional profiling and all successive qPCR assays. Expression of different Atrophin forms with the GMR driver was induced, owing to a temperature‐sensitive mutant Gal80 repressor. F1 flies were allowed to develop at 18°C; at this temperature the Gal80 repressor keeps transgenes silent. Newly eclosed flies (0-48 h) were collected and killed immediately (0d) or aged for 2 or 14 days at 29°C. This inactivates Gal80 and transgenes are switched on by GMR‐Gal4. This protocol allows comparing siblings that differ exclusively in their age and transgenes expression. Control flies crossed to GMR‐Gal4; UbiGal80ts in all experiments are from the w1118 stock in which all UAS transgenes have been generated.


( B ) Tangential eye sections of flies representative of different populations used in microarray at different time points . Weak degeneration is only visible after 14 days with polyQ Atro ; in particular with Atro75QN there is an initial loss of photoreceptors ( PR , arrow ) , 30 . 7 % of ommatidia has lost at least 1 PR , that is only 5 . 1 % of neuronal PR have been lost at this stage ( N = 333 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O 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) Tangential eye sections of flies representative of all the different populations used in the microarray analysis at all different time points. Weak degeneration is only visible after 14 days with polyQ Atro; in particular with Atro75QN there is an initial loss of photoreceptors (PR, arrow), 30.7% of the ommatidia has lost at least 1 PR, that is only 5.1% of all neuronal PR have been lost at this stage (N=333).


( A ) Heat maps for three subsets of pattern clustering generated using Euclidean metric . Upregulated genes are in red downregulated genes are in green . downregulating transcriptional activity increases with time . main branch in clustering of different Atro mutants is along time points . See also Supplementary Figure 4 .

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

(A) Heat maps for three subsets of the pattern clustering analysis generated using the Euclidean Distance metric. Upregulated genes are in red and downregulated genes are in green. The downregulating transcriptional activity increases with time. The main branch in clustering of the different Atro mutants is along time points. See also Supplementary Figure 4.


( A ) qPCR of fold changes ± s . d . of ft transcription . Downregulation starts at 2 days progresses at 14 days .

O O 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) qPCR analysis of the fold changes±s.d. of ft transcription. Downregulation starts at 2 days and progresses at 14 days.


( B ) qPCR of enrichment ± s . d . of different regions of ft regulatory elements in ChIP for Atro from BG3neuronal extracts . Enrichment is calculated as of DNAimmunoprecipitated from extracts of in which Atro expression has been induced , with respect to amount immunoprecipitated from uninduced extracts according to ( Atro - No Ab ) induced / ( Atro − No Ab ) uninduced formula . region from IV chromosome has been previously shown not to be immunoprecipitated in ChIP for Atro ( Haecker et al , 2007 ) has been used as a negative control .

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

(B) qPCR analysis of the enrichment±s.d. of different regions of the ft regulatory elements in ChIP for Atro from BG3neuronal cell extracts. Enrichment is calculated as the percentage of DNAimmunoprecipitated from extracts of cells in which Atro expression has been induced, with respect to the amount immunoprecipitated from uninduced cell extracts according to the (Atro-No Ab)induced/(Atro−No Ab)uninduced formula. The DNA region from IV chromosome has been previously shown not to be immunoprecipitated in ChIP for Atro ( Haecker et al, 2007) and has been used as a negative control.


( C ) Histograms showing number of ommatidia with a full complement ( 7 ) of PR in flies expressing Atro75QN with Rhodopsin1 driver in either a control ( w1118 ) or different mutant backgrounds aged at 29 ° C for 28 days . Heterozygosis for two independent ft alleles a sav allele significantly enhances loss of PR . Mild overexpression of Wts via a GMR - wts transgene , which does not display any strong phenotype per se , significantly suppresses loss of PR . No interaction was detected in this with wtsx1 ykiB5 alleles in heterozygosity . N = 430 - 963 from at least four eyes .

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

(C) Histograms showing the number of ommatidia with a full complement (7) of PR in flies expressing Atro75QN with the Rhodopsin1 driver in either a control (w1118) or different mutant backgrounds and aged at 29°C for 28 days. Heterozygosis for two independent ft alleles and a sav allele significantly enhances the loss of PR. Mild overexpression of Wts via a GMR-wts transgene, which does not display any strong phenotype per se, significantly suppresses the loss of PR. No interaction was detected in this assay with wtsx1 and ykiB5 alleles in heterozygosity. N=430-963 from at least four eyes.


( D ) Tangential eye sections histograms showing degeneration of GMR > Atro75QN , Ubi - Gal80ts flies in combination with different UAS transgenes aged 14 days as in Figure 1A . UAS - EGFP is used as a negative control . Either Ft or Wts overexpression strongly suppresses loss of PR caused by Atro75QN . Lower panels are provided as examples of . A more modest but still significant rescue is observed by overexpressing a Yki RNAi contruct or Hpo ; however , in this case it is to be considered that overexpression of Hpoper se brings about loss of at least one PR in ∼ 5 % of ommatidia ( data not shown ) . No effect is detected with overexpression of Sav . Two ‐ t ‐ : * P0 . 05 ; * * P0 . 01 . wtsEPG4808 is a previously uncharacterised EP element insertion at 5 ′ of wts gene . Rare homozygous escapers display larger eyes , whereas in combination with GMR - GMR this gives rise to smaller rough eyes , , finally , GMR of GMR > wtsEPG4808 indicates a 10 ‐ fold increase in head content of wts mRNA ( data not shown ) .

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

(D) Tangential eye sections and histograms showing the degeneration of GMR>Atro75QN, Ubi-Gal80ts flies in combination with different UAS transgenes and aged 14 days as in Figure 1A. UAS-EGFP is used as a negative control. Either Ft or Wts overexpression strongly suppresses loss of PR caused by Atro75QN. Lower panels are provided as examples of quantification. A more modest but still significant rescue is observed by overexpressing a Yki RNAi contruct or Hpo; however, in this case it is to be considered that the overexpression of Hpoper se brings about the loss of at least one PR in ∼5% of ommatidia (data not shown). No effect is detected with overexpression of Sav. Two‐tailed t‐test: *P0.05; **P0.01. The wtsEPG4808 is a previously uncharacterised EP element insertion at the 5′ of the wts gene. Rare homozygous escapers display larger eyes, whereas in combination with GMR-GMR this line gives rise to smaller rough eyes, and, finally, GMR analysis of GMR>wtsEPG4808 indicates a 10‐fold increase in the head content of wts mRNA (data not shown).


( A ) Tangential eye sections through ftfd clones aged 1 , 7 14 days , of ftfd , dDGC13 double mutant clones of ftfd clones in a ykiB5 heterozygous background aged 14 days . Clones are marked by absence of yellow pigment . Arrows point at degenerating photoreceptors , arrowheads point at intact wt photoreceptors in mosaic ommatidia . circled ommatidium is genetically wt but non ‐ autonomously flipped in its polarity has not degenerated . Lower panels are masks that show clonal borders of PR number per ommatidia . After 14 days , almost ftfd have degenerated , whereas almost ftfd , dDGC13 some ftfd , ykiB5 / + neuronalphotoreceptors have not .

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

(A) Tangential eye sections through ftfd clones aged 1, 7 and 14 days, of ftfd,dDGC13 double mutant clones and of ftfd clones in a ykiB5 heterozygous background aged 14 days. Clones are marked by the absence of yellow pigment. Arrows point at degenerating photoreceptors, arrowheads point at intact wt photoreceptors in mosaic ommatidia. The circled ommatidium is genetically wt but non‐autonomously flipped in its polarity and has not degenerated. Lower panels are masks that show clonal borders and quantification of PR number per ommatidia. After 14 days, almost all ftfd cells have degenerated, whereas almost all ftfd,dDGC13 and some ftfd,ykiB5/+ neuronalphotoreceptors have not.


( B ) Histograms showing of degeneration in ftfd , ftfddDGC13 ftfdykiB5 / + clones . Loss of ft leads to statistically significant loss of neurons this is suppressed at 14 days by ykiB5and much more dramatically by dDGC13 . * * * P0 . 001 , * * P0 . 01 ; * P0 . 05 in two ‐ t ‐ .

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

(B) Histograms showing the quantification of degeneration in ftfd, ftfddDGC13 and ftfdykiB5/+ clones. Loss of ft leads to statistically significant loss of neurons and this is suppressed at 14 days by ykiB5and much more dramatically by dDGC13. ***P0.001, **P0.01; *P0.05 in two‐tailed t‐test.


( C ) Degeneration in mosaic ommatidia in ftfd clones . At this stage of degeneration approximately half of non ‐ pigmented ( genotypically mutant ) PR display a degenerative phenotype , whereas virtually pigmented ( genotypically wt ) PR are normal . χ2 ‐ : 82 . 38 ; P0 . 001 . Total PR N = 310 ( pigmented ) 327 ( not pigmented ) .

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

(C) Degeneration in mosaic ommatidia in ftfd clones. At this stage of degeneration approximately half of the non‐pigmented (genotypically mutant) PR cells display a degenerative phenotype, whereas virtually all pigmented (genotypically wt) PR cells are normal. χ2‐test: 82.38; P0.001. Total PR N=310 (pigmented) and 327 (not pigmented).


( D ) Tangential eye sections through MARCM clone mutants for ftfd that express either no transgene or an RNAi construct against yki ( UAS - ykiIR ) with Tub - Gal4 aged 14 days at 29 ° C . Because transgene UAS - ykiIR is on chromosomal arm 2 L , same as ft , clones generated with this system carry two copies of UAS - ykiIR therefore downregulate yki very effectively . No other outside clones expresses construct is therefore wt for yki . Mask panel is presented on right of each section . A of photoreceptor numbers ( far right ) displays a very significant increase in number of wt ommatidia ( N = 210 219 from four eyes ) . * * * P0 . 001 in two ‐ t ‐ . If classes of ommatidia are considered , χ2 ‐ = 232 . 30 ; P0 . 001 for 3 degrees of freedom .

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

(D) Tangential eye sections through MARCM clone mutants for ftfd that express either no transgene or an RNAi construct against yki (UAS-ykiIR) with Tub-Gal4 and aged 14 days at 29°C. Because the transgene UAS-ykiIR is on chromosomal arm 2 L, the same as ft, clones generated with this system carry two copies of UAS-ykiIR and therefore downregulate yki very effectively. No other cell outside the clones expresses the construct and is therefore wt for yki. Mask panel is presented on the right of each section. A quantification of photoreceptor numbers (far right) displays a very significant increase in the number of wt ommatidia (N=210 and 219 from four eyes). ***P0.001 in two‐tailed t‐test. If all classes of ommatidia are considered, χ2‐test=232.30; P0.001 for 3 degrees of freedom.


( A ) Tangential eye sections through wtsX1 mutant fly eyes . In clones for null wtsX1 allele , degeneration inside clones is at severe stages at 14 days .

O O O O O O 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) Tangential eye sections through wtsX1 mutant fly eyes. In clones for the null wtsX1 allele, degeneration inside the clones is at severe stages at 14 days.


( B ) Tangential eye sections through sav3 clones . In sav3 after enclosure many photoreceptors are intact but after 14 days at 29 ° C most sav mutant photoreceptors have degenerated .

O O O O O O O O O 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) Tangential eye sections through sav3 clones. In sav3 after enclosure many photoreceptors are intact but after 14 days at 29°C most sav mutant photoreceptors have degenerated.


( C ) Histograms showing of ommatidia with full complement of photoreceptors in hpo , wts sav mutant clones . progression of loss of photoreceptors is evident statistically significant in cases . Mutants for hpoBF33 do not survive for 14 days at 29oC corresponding sections are shown in Supplementary Figure 12 . N = 195 - 268 . * * * P0 . 001 , * * P0 . 01 ; * P0 . 05 in two ‐ t ‐ .

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

(C) Histograms showing the quantification of ommatidia with the full complement of photoreceptors in hpo, wts and sav mutant clones. The progression of loss of photoreceptors is evident and statistically significant in all cases. Mutants for hpoBF33 do not survive for 14 days at 29oC and corresponding sections are shown in Supplementary Figure 12. N=195-268. ***P0.001, **P0.01; *P0.05 in two‐tailed t‐test.


( A ) Regression of overgrowth versus neurodegeneration observed in loss of function mutants ( left ) overexpression of transgenes ( right ) . Overgrowth is shown in Supplementary Figure 14 . For loss of function mutant , neurodegeneration was quantified at 14 days in clones ( as shown in Figures 4 5 ) . For UAS ‐ overexpression transgenes neurodegeneration was quantified at 14 days when expressed with GMRGal4 ; UbiGal80ts . For CycD + Cdk4 , these specific data are missing have not been plotted ; however , Supplementary Figure 13 shows that these mutants do not cause neurodegeneration in a different setup in which they are expressed during both development adult life . In both cases regression coefficient r2 is extremely low ( 0 . 21 0 . 03 ) , indicating an absence of correlation between two processes .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Regression analysis of the overgrowth versus the neurodegeneration observed in loss of function mutants (left) and overexpression of transgenes (right). Overgrowth quantification is shown in Supplementary Figure 14. For loss of function mutant, the neurodegeneration was quantified at 14 days in clones (as shown in Figures 4 and 5). For UAS‐overexpression transgenes neurodegeneration was quantified at 14 days when expressed with GMRGal4; UbiGal80ts. For CycD+Cdk4, these specific data are missing and have not been plotted; however, Supplementary Figure 13 shows that these mutants do not cause neurodegeneration in a different setup in which they are expressed during both development and adult life. In both cases the regression coefficient r2 is extremely low (0.21 and 0.03), indicating an absence of correlation between the two processes.


( B ) Tangential eye sections through eyes of flies expressing with GMR - Gal4 , Ubi - Gal80ts either UAS - EGFP or UAS - Yki or UAS - YkiS111A , S168A , S250A aged 1 or 28 days . Arrows point at missing or degenerated photoreceptors . UAS - YkiS111A , S168A , S250A is so effective that even very low expression leaking out with this system is enough to affect development generate mild eye roughness overproliferation of lattice . Histograms showing of loss in eyes shown in A also eyes from flies aged at intermediate 14 ‐ day stage . Mild but significant degeneration is observed for both Yki forms , with respect to negative control ; however , no statistical difference is observed between two Yki that have dramatically different effects on overgrowth . * * * P0 . 001 , * * P0 . 01 ; * P0 . 05 in two ‐ 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 B-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 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 B-CONTROLLED_VAR 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

(B) Tangential eye sections through the eyes of flies expressing with GMR-Gal4, Ubi-Gal80ts either UAS-EGFP or UAS-Yki or UAS-YkiS111A,S168A,S250A and aged 1 or 28 days. Arrows point at missing or degenerated photoreceptors. The UAS-YkiS111A,S168A,S250A is so effective that even the very low expression leaking out with this system is enough to affect development and generate mild eye roughness and overproliferation of lattice cells. Histograms showing the quantification of cell loss in eyes shown in A and also eyes from flies aged at the intermediate 14‐day stage. Mild but significant degeneration is observed for both Yki forms, with respect to the negative control; however, no statistical difference is observed between the two Yki proteins that have dramatically different effects on overgrowth. ***P0.001, **P0.01; *P0.05 in two‐tailed t‐test.


Neurodegeneration by polyQ Atro partially requires dachs . Tangential eye sections through dGC13 clones ( marked by absence of yellow pigment ) either in control flies or those expressing Atro75QN with Rhodopsin1 driver aged 28 days . Mask panels below each section exemplify clonal boundaries PR no . for ommatidia . Early signs of degeneration indicated by reduced complement of photoreceptors are detected specifically in pigmented ( arrowhead ) . At this stage , inside d mutant clones ommatidia are unaffected display a wt arrangement ( arrows ) . ( B ) Histograms showing of loss in eyes shown in ( A ) . A statistically significant rescue is obtained both for number of wt ommatidia ( * P0 . 05 in two ‐ t ‐ ) for other categories of ommatidia ( χ2 = 44 . 76 ; P0 . 001 for 2 degrees of freedom ) .

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

Neurodegeneration by polyQ Atro partially requires dachs. Tangential eye sections through dGC13 clones (marked by the absence of yellow pigment) either in control flies or those expressing Atro75QN with the Rhodopsin1 driver and aged 28 days. Mask panels below each section exemplify clonal boundaries and PR no. for the ommatidia. Early signs of degeneration indicated by reduced complement of photoreceptors are detected specifically in the pigmented area (arrowhead). At this stage, inside d mutant clones all ommatidia are unaffected and display a wt arrangement (arrows). (B) Histograms showing the quantification of cell loss in eyes shown in (A). A statistically significant rescue is obtained both for the number of wt ommatidia (*P0.05 in two‐tailed t‐test) and for all other categories of ommatidia (χ2=44.76; P0.001 for 2 degrees of freedom).


( A ) EM scan of a wt ( left ) ftfd mutant ommatidium ( right ) of a ftfd clone after 14 days . Scale bar : 1 μm . High ‐ magnification panels ( far right , top to bottom ) display autophagosomes with undigested debris , damaged mitochondria forming phagophores ( arrowheads ) , found in ftfd mutant . Scale bar : 0 . 2 μm for zoom ‐ in panels .

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

(A) EM scan of a wt (left) and ftfd mutant ommatidium (right) of a ftfd clone after 14 days. Scale bar: 1 μm. High‐magnification panels (far right, top to bottom) display autophagosomes with undigested debris, damaged mitochondria and forming phagophores (arrowheads), found in ftfd mutant cells. Scale bar: 0.2 μm for zoom‐in panels.


( B ) Graphs of of autophagic vesicles ( AV ) per photoreceptor found in EM sections of 7 ‐ day ( left ) ‐ 14 ‐ day ( right ) ‐ old ftfd clones . Significant accumulation of AV is found in mutant ( not pigmented ) with respect to genotypically wt ( pigmented ) . * * P0 . 01 * * * P0 . 001 in one ‐ t ‐ . N = 12 pigmented versus 20 non ‐ pigmented for 7 ‐ day graph 13 pigmented versus 21 non ‐ pigmented for 14 ‐ day graph .

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

(B) Graphs of the quantification of autophagic vesicles (AV) per photoreceptor found in EM sections of 7‐day (left)‐ and 14‐day (right)‐old ftfd clones. Significant accumulation of AV is found in mutant cells (not pigmented) with respect to genotypically wt (pigmented) cells. **P0.01 and ***P0.001 in one‐tailed t‐test. N=12 pigmented cells versus 20 non‐pigmented cells for the 7‐day graph and 13 pigmented cells versus 21 non‐pigmented cells for the 14‐day graph.


( C ) Confocal pictures of whole ‐ mount retinae of a ftfd clone aged 7 days expressing GFP ∷ Atg8a ubiquitously with Tub - Gal4 . Red is phalloidin marking rhabdomeres , green is GFP clone is marked by absence of β ‐ gal ( blue ) . Small GFP ∷ Atg8a dots accumulate specifically inside ft mutant ( arrow ) .

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

(C) Confocal pictures of whole‐mount retinae of a ftfd clone aged 7 days and expressing GFP∷Atg8a ubiquitously with Tub-Gal4. Red is phalloidin marking rhabdomeres, green is GFP and the clone is marked by the absence of β‐gal staining (blue). Small GFP∷Atg8a dots accumulate specifically inside ft mutant cells (arrow).


( D ) Confocal pictures of whole ‐ mount retinae of a ftfd clone aged 7 days ( left ) 14 days ( right ) . Red is phalloidin marking rhabdomeres , blue is p62 clone is marked by absence of GFP ( green ) . p62 starts to gather in small dots specifically inside ft mutant ( arrow ) then accumulates massively ( arrow ) in ft mutant clones as many degenerate .

O O O O O O O O O O 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 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

(D) Confocal pictures of whole‐mount retinae of a ftfd clone aged 7 days (left) and 14 days (right). Red is phalloidin marking rhabdomeres, blue is p62 and the clone is marked by the absence of GFP staining (green). p62 starts to gather in small dots specifically inside ft mutant cells (arrow) and then accumulates massively (arrow) in the ft mutant clones as many cells degenerate.


( A ) EM scan of a 7 ‐ day ‐ old sav3 mutant ommatidium zoom ‐ in on autophagosomes containing undigested debris ( arrowhead ) . Scale bar : 1 μm for panel on left 0 . 2 μm for zoom ‐ in panel on right .

O O O O O O O O O O O O O O O O O O O O O O O O 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) EM scan of a 7‐day‐old sav3 mutant ommatidium and zoom‐in on autophagosomes containing undigested debris (arrowhead). Scale bar: 1 μm for panel on the left and 0.2 μm for zoom‐in panel on the right.


( B ) Graph of of autophagic vesicles ( AV ) per photoreceptor found in EM sections of 1 ‐ week ‐ old sav3 clones . Significant accumulation of AV is found in mutant . * * * P0 . 001 in one ‐ t ‐ . N = 45 pigmented versus 42 non ‐ pigmented .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Graph of the quantification of autophagic vesicles (AV) per photoreceptor found in EM sections of 1‐week‐old sav3 clones. Significant accumulation of AV is found in mutant cells. ***P0.001 in one‐tailed t‐test. N=45 pigmented cells versus 42 non‐pigmented cells.


A . Rab25 expression decreases nutrient withdrawal ( 24 h ) induced in ovarian A2780 IOSE29 measured using ELISA plus ( left ) ; were cultured in complete media ( containing 5 % FBS ) , serum free media ( SF ) , complete media plus 2DG , serum glucose free media ( SF - glu ) , or in acid , glucose SF Earls buffer salt solution ( EBSS ) . ( a ) p < 0 . 01 versus control ( b ) p < 0 . 05 versus pcDNA . ( Right ) of apoptotic ( sub - G0 population ) by flow cytometry in A2780 following glucose serum withdrawal ( Glu / SF ) . ( a ) p < 0 . 001 versus A2780pcDNA control ( b ) p < 0 . 05 A2780Rab25 versus A2780pcDNA control .

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

A. Rab25 expression decreases nutrient withdrawal (24 h) induced cell death in ovarian A2780 and IOSE29 cells measured using the Cell death ELISA plus assay (left); cells were cultured in complete media (containing 5% FBS), serum free media (SF), complete media plus 2DG, serum and glucose free media (SF-glu), or in amino acid, glucose and SF Earls buffer salt solution (EBSS). (a) p < 0.01 versus control and (b) p < 0.05 versus pcDNA. (Right) Percentage of apoptotic cells (sub-G0 population) by flow cytometry in A2780 cells following glucose and serum withdrawal (Glu/SF). (a) p < 0.001 versus A2780pcDNA control and (b) p < 0.05 A2780Rab25 versus A2780pcDNA control.


B . Decreasing Rab25 expression increases sensitivity to nutrient stress induced . Expression of Rab25 was down - regulated by siRNA or shRNA specific to Rab25 .

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

B. Decreasing Rab25 expression increases sensitivity to nutrient stress induced cell death. Expression of Rab25 was down-regulated by siRNA or shRNA specific to Rab25.


C . Rab25 regulates autophagy activity in ovarian cancer under serum glucose deprivation conditions . Western blot for 16kD LC3 - II fragment , indicative of autophagy activity , in A2780 after 4 6 h of serum glucose withdrawal .

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

C. Rab25 regulates autophagy activity in ovarian cancer cells under serum and glucose deprivation conditions. Western blot for the 16kD LC3-II fragment, indicative of autophagy activity, in A2780 cells after 4 and 6 h of serum and glucose withdrawal.


D . Electron microscopy of autophagy in ovarian A2780 after 4 24 h of serum glucose withdrawal ( upper panel , low magnification ) . High magnification of boxed areas with arrowheads depicting autophagic vacuoles ( lower panel , inset ; N : nucleus ) . Average number of autophagosomes per was calculated by counting number of autophagosomes in 16 from two - independent experiments .

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

D. Electron microscopy of autophagy in ovarian A2780 cells after 4 and 24 h of serum and glucose withdrawal (upper panel, low magnification). High magnification images of boxed areas with arrowheads depicting autophagic vacuoles (lower panel, inset; N: nucleus). Average number of autophagosomes per cell was calculated by counting the number of autophagosomes in 16 individual cells from two-independent experiments.


E - H . Expression of Rab25 decreases glucose serum deprivation induced signalling activation . expression was measured by RPPA or WB . E . RPPA detection of time - dependent activation of AMPK after nutrient withdrawal .

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 B-MEASURED_VAR O B-CONTROLLED_VAR O O

E-H. Expression of Rab25 decreases glucose and serum deprivation induced signalling activation. Protein expression was measured by RPPA or WB analysis.E. RPPA detection of time-dependent activation of AMPK after nutrient withdrawal.


E - H . Expression of Rab25 decreases glucose serum deprivation induced signalling activation . expression was measured by RPPA or WB . F . WB of AMPK acetyl - CoA carboxylase ( ACC ) phosphorylation in HEY ovarian cancer ( upper panel ) . WB of phospho - ACC in A2780 , IOSE80ht SKOV3 ovarian after 1 h of nutrient withdrawal ( lower panel ) .

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

E-H. Expression of Rab25 decreases glucose and serum deprivation induced signalling activation. Protein expression was measured by RPPA or WB analysis.F. WB analysis of AMPK and acetyl-CoA carboxylase (ACC) phosphorylation in HEY ovarian cancer cells (upper panel). WB of phospho-ACC levels in A2780, IOSE80ht and SKOV3 ovarian cells after 1 h of nutrient withdrawal (lower panel).


E - H . Expression of Rab25 decreases glucose serum deprivation induced signalling activation . expression was measured by RPPA or WB . G . RPPA detection of phosphorylation of ACC after withdrawal of serum glucose .

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 B-MEASURED_VAR O O O O B-CONTROLLED_VAR O

E-H. Expression of Rab25 decreases glucose and serum deprivation induced signalling activation. Protein expression was measured by RPPA or WB analysis.G. RPPA detection of phosphorylation of ACC after withdrawal of serum and glucose.


E - H . Expression of Rab25 decreases glucose serum deprivation induced signalling activation . expression was measured by RPPA or WB . H . Effect of Rab25 down - regulation on AMPK ( left panel ) ACC ( right panel ) phosphorylation . Total , isolated from A2780pcDNA Rab25 expressing 72 h post - transfection with either non - target ( NT ) RNAi or Rab25 specific RNAi , was separated by polyacrylamide gel electrophoresis ( PAGE ) followed by WB . ( a ) p < 0 . 001 versus NT RNAi control ( b ) p < 0 . 001 , pcDNA versus Rab25 .

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-CONTROLLED_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 B-CONTROLLED_VAR 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 B-CONTROLLED_VAR O

E-H. Expression of Rab25 decreases glucose and serum deprivation induced signalling activation. Protein expression was measured by RPPA or WB analysis.H. Effect of Rab25 down-regulation on AMPK (left panel) and ACC (right panel) phosphorylation. Total cellular protein, isolated from A2780pcDNA and Rab25 expressing cells 72 h post-transfection with either non-target (NT) RNAi or Rab25 specific RNAi, was separated by polyacrylamide gel electrophoresis (PAGE) followed by WB analysis. (a) p < 0.001 versus NT RNAi control and (b) p < 0.001, pcDNA versus Rab25.


Expression of Rab25 increases endogenous ATP level in ovarian cancer . * p < 0 . 001 Rab25 versus pcDNA . mean of ATP per mg of ( on standard curve generated using a known amount of ATP ) is 2 . 36 − 10 moles / mg for IOSE29htpcDNA , 6 . 21 − 10 moles / mg for IOSE29htRab25 , 8 . 14 − 11 moles / mg for IOSE80htpcDNA , 1 . 05 − 10 moles / mg for IOSE80htRab25 , 2 . 85 − 10 moles / mg for A2780pcDNA , 3 . 81 − 10 moles / mg for A2780Rab25 , 7 . 23 − 10 moles / mg for HEYpcDNA 9 . 82 − 10 moles / mg for HEYRab25 .

O O B-CONTROLLED_VAR O O B-MEASURED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Rab25 increases endogenous ATP level in ovarian cancer cells. *p < 0.001 Rab25 versus pcDNA. The mean cellular concentration of ATP per mg of protein (based on the standard curve generated using a known amount of ATP) is 2.36−10 moles/mg for IOSE29htpcDNA cells, 6.21−10 moles/mg for IOSE29htRab25 cells, 8.14−11 moles/mg for IOSE80htpcDNA cells, 1.05−10 moles/mg for IOSE80htRab25 cells, 2.85−10 moles/mg for A2780pcDNA cells, 3.81−10 moles/mg for A2780Rab25 cells, 7.23−10 moles/mg for HEYpcDNA cells and 9.82−10 moles/mg for HEYRab25 cells.


Rab25 expression increases ATP synthesis delays fall in total ATP after glucose serum withdrawal , p < 0 . 001 Rab25 versus control pcDNA .

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

Rab25 expression increases ATP synthesis and delays fall in total cellular ATP levels after glucose and serum withdrawal, p < 0.001 Rab25 versus control pcDNA.


Down - regulation of Rab25 expression decreases ability to maintain ATP after glucose serum withdrawal ( a ) p < 0 . 05 SF - Glu versus FBS ( b ) p < 0 . 05 versus control .

O O O O B-CONTROLLED_VAR O O O O 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

Down-regulation of Rab25 expression decreases ability to maintain ATP after glucose and serum withdrawal (a) p < 0.05 SF-Glu versus FBS and (b) p < 0.05 versus control.


Expression of Rab25 increases total glycogen content while down - regulation of Rab25 expression decreases total glycogen content ( µg of glycogen / mg ) . were cultured in complete media for 24 h before glycogen measurement . ( a ) p < 0 . 01 Rab25 versus pcDNA , ( b ) p < 0 . 05 Rab25 RNAi versus NT RNAi ( c ) p < 0 . 05 shRAb25 versus shRNA control .

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

Expression of Rab25 increases total cellular glycogen content while down-regulation of Rab25 expression decreases total cellular glycogen content (µg of glycogen/mg protein). Cells were cultured in complete media for 24 h before glycogen measurement. (a) p < 0.01 Rab25 versus pcDNA, (b) p < 0.05 Rab25 RNAi versus NT RNAi and (c) p < 0.05 shRAb25 versus shRNA control.


Decrease in glycogen content after glucose serum withdrawal * p < 0 . 001 Rab25 versus pcDNA .

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

Decrease in cellular glycogen content after glucose and serum withdrawal *p < 0.001 Rab25 versus pcDNA.


Effect of GPi ( Bay U6571 ) oligomycin on ATP production . A2780pcDNA transfected Rab25 expressing were pretreated with vehicle ( control ) , 30 µM of Bay U6571 ( GPi ) or 25 µg / ml of oligomycin for 2 h . were then cultured in serum - glucose free RPMI 1640 in presence or absence of Bay U6571 or oligomycin for indicated times . ( a ) p < 0 . 01 oligomycin time 0 versus control time 0 ( b ) p < 0 . 01 versus time 0 at each treatment group .

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

Effect of GPi (Bay U6571) and oligomycin on ATP production. A2780pcDNA transfected and Rab25 expressing cells were pretreated with vehicle (control), 30 µM of Bay U6571 (GPi) or 25 µg/ml of oligomycin for 2 h. Cells were then cultured in serum- and glucose free RPMI 1640 in the presence or absence of Bay U6571 or oligomycin for the indicated times. (a) p < 0.01 oligomycin time 0 versus control time 0 and (b) p < 0.01 versus time 0 at each treatment group.


Effect of Bay U6571 on glycogen ( left panel ) ATP ( right panel ) in HEY . ( a ) p < 0 . 01 versus pcDNA control time 0 at 5 % FBS ( b ) p < 0 . 01 GPi versus control at each corresponding treatment .

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

Effect of Bay U6571 on cellular glycogen (left panel) and ATP (right panel) levels in HEY cells. (a) p < 0.01 versus pcDNA control time 0 at 5% FBS and (b) p < 0.01 GPi versus control at each corresponding treatment.


Down regulation of Rab25 expression inhibition of glycogen breakdown by GPi administration increases 2DG induced * p < 0 . 05 versus shRNA control .

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

Down regulation of Rab25 expression and inhibition of glycogen breakdown by GPi administration increases 2DG induced cell death *p < 0.05 versus shRNA control.


A . Rab25 expression increases phospho but not total AKT GSK3 in A2780 leading to decreased phosphorylation of GS ( pGS ) .

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

A. Rab25 expression increases phospho but not total AKT and GSK3 levels in A2780 cells leading to decreased phosphorylation of GS (pGS).


B . WB of AKT GSK3 phosphorylation confirming inhibitory effect of PI3K pathways inhibitors PI103 , GPi synthase kinase 3 inhibitor ( GSK3i ) in HEY .

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

B. WB analysis of AKT and GSK3 phosphorylation confirming the inhibitory effect of PI3K pathways inhibitors PI103, GPi and synthase kinase 3 inhibitor (GSK3i) in HEY cells.


C . Inhibition of GSK3 activity increases total glycogen . pcDNA transfected Rab25 expressing ovarian cancer were cultured in complete media in presence of 10 µM GSK3i for indicated times . Total glycogen content was measured normalized with total content . ( a ) p < 0 . 001 versus no GSK3i treated pcDNA .

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

C. Inhibition of GSK3 activity increases total cellular glycogen levels. pcDNA transfected and Rab25 expressing ovarian cancer cells were cultured in complete media in the presence of 10 µM GSK3i for the indicated times. Total cellular glycogen content was measured and normalized with total protein content. (a) p < 0.001 versus no GSK3i treated pcDNA cells.


D - E . Effect of PI3K inhibitor PI103 on glycogen content ATP production . Ovarian cancer were pretreated with PI103 for at least 2 h in complete media . were then subjected to 2 hglucose FBS withdrawal to deplete endogenous glycogen followed by culturing in RPMI - glucose media in presence of PI103 for 2 h ( 2 hSF ) . After 2 h of nutrient stress , complete media containing PI103 was added to for 30 min ( 5 % FBS ) for recovery . A second glucose FBS withdrawal ( 15 min SF ) followed immediately to examine effect on glycogen ATP . a , p < 0 . 001 5 % FBS versus 2 hSF , b , p < 0 . 01 15 min SF versus 5 % FBS . D . There is an increase in glycogen content during recovery phase ( 5 % FBS ) a decrease in glycogen when are subject to a second nutrient stress ( 15 min SF ) .

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

D-E. Effect of PI3K inhibitor PI103 on cellular glycogen content and ATP production. Ovarian cancer cells were pretreated with PI103 for at least 2 h in complete media. Cells were then subjected to 2 hglucose and FBS withdrawal to deplete endogenous glycogen followed by culturing cells in RPMI-glucose media in the presence of PI103 for 2 h (2 hSF). After 2 h of nutrient stress, complete media containing PI103 was added to the cells for 30 min (5% FBS) for recovery. A second glucose and FBS withdrawal (15 min SF) followed immediately to examine the effect on glycogen and ATP levels. a, p < 0.001 5% FBS versus 2 hSF, b, p < 0.01 15 min SF versus 5% FBS.D. There is an increase in glycogen content during recovery phase (5% FBS) and a decrease in glycogen when cells are subject to a second nutrient stress (15 min SF).


D - E . Effect of PI3K inhibitor PI103 on glycogen content ATP production . Ovarian cancer were pretreated with PI103 for at least 2 h in complete media . were then subjected to 2 hglucose FBS withdrawal to deplete endogenous glycogen followed by culturing in RPMI - glucose media in presence of PI103 for 2 h ( 2 hSF ) . After 2 h of nutrient stress , complete media containing PI103 was added to for 30 min ( 5 % FBS ) for recovery . A second glucose FBS withdrawal ( 15 min SF ) followed immediately to examine effect on glycogen ATP . a , p < 0 . 001 5 % FBS versus 2 hSF , b , p < 0 . 01 15 min SF versus 5 % FBS . E . Utilization of glycogen to produce ATP .

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

D-E. Effect of PI3K inhibitor PI103 on cellular glycogen content and ATP production. Ovarian cancer cells were pretreated with PI103 for at least 2 h in complete media. Cells were then subjected to 2 hglucose and FBS withdrawal to deplete endogenous glycogen followed by culturing cells in RPMI-glucose media in the presence of PI103 for 2 h (2 hSF). After 2 h of nutrient stress, complete media containing PI103 was added to the cells for 30 min (5% FBS) for recovery. A second glucose and FBS withdrawal (15 min SF) followed immediately to examine the effect on glycogen and ATP levels. a, p < 0.001 5% FBS versus 2 hSF, b, p < 0.01 15 min SF versus 5% FBS.E. Utilization of glycogen to produce ATP.


F . Addition of AKT inhibitor MK2206 ( AKTi ) abolishes Rab25 - dependent glycogen storage . ( a ) p < 0 . 05 versus shRNA control ( b ) p < 0 . 01 SF + AKTi versus SF .

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

F. Addition of AKT inhibitor MK2206 (AKTi) abolishes Rab25-dependent glycogen storage. (a) p < 0.05 versus shRNA control and (b) p < 0.01 SF + AKTi versus SF.


G . Inhibiting AKT pathway glucose metabolism decreases viability . were pretreated with 10 µM of PI103 or AKTi , 100 µM LND or 3BrPy for 2 h before switching to SF media in presence of inhibitors for an addition16 h before titre blue viability . * p < 0 . 001 shRNA Rab25 versus shRNA control .

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 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-CONTROLLED_VAR O O O O

G. Inhibiting AKT pathway and glucose metabolism decreases cell viability. Cells were pretreated with 10 µM of PI103 or AKTi, 100 µM LND or 3BrPy for 2 h before switching to SF media in the presence of inhibitors for an addition16 h before cell titre blue viability assay. *p < 0.001 shRNA Rab25 versus shRNA control.


A . Rab25 over - expression increases H3 - labelled 2DG uptake . ( a ) p < 0 . 001 Rab25 versus pcDNA . Results are mean ± s . d . of a triplicate in one of representative experiment .

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

A. Rab25 over-expression increases H3-labelled 2DG uptake. (a) p < 0.001 Rab25 versus pcDNA. Results are mean ± s.d. of a triplicate in one of representative experiment.


B . Down - regulation of Rab25 or AKT expression , or inhibition AKT activity by MK2206 decreases glucose uptake in HEY ( a ) p < 0 . 001 versus NT siRNA control , ( b ) p < 0 . 05 versus siRab25 ( c ) p < 0 . 05 versus siAKT .

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

B. Down-regulation of Rab25 or AKT expression, or inhibition AKT activity by MK2206 decreases glucose uptake in HEY cells (a) p < 0.001 versus NT siRNA control, (b) p < 0.05 versus siRab25 and (c) p < 0.05 versus siAKT.


C . Effect of down regulation of Rab25 AKT inhibitor MK2206 ( AKTi ) on glucose uptake . were either pretreated for 2 h with AKTi before assessing glucose uptake . ( a ) p < 0 . 05 versus shRNA control ( b ) p < 0 . 05 versus shRab25 .

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

C. Effect of down regulation of Rab25 and AKT inhibitor MK2206 (AKTi) on glucose uptake. Cells were either pretreated for 2 h with AKTi before assessing glucose uptake. (a) p < 0.05 versus shRNA control and (b) p < 0.05 versus shRab25.


D . Rab25 co - localization with GLUT1 in A2780 . Immunofluorescence microscopy of GLUT1 ( red ) Rab25 ( green ) . Co - localization of GLUT1 Rab25 ( i . e . yellow ) is indicated by arrows . Single scale bar , 10 µM .

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

D. Rab25 co-localization with GLUT1 in A2780 cells. Immunofluorescence microscopy staining of GLUT1 (red) and Rab25 (green). Co-localization of GLUT1 and Rab25 (i.e. yellow) is indicated by arrows. Single scale bar, 10 µM.


E - G . Resistance to 2DG induced in Rab25 is mediated through PI3K / AKT pathway . Inhibition of PI3K / AKT pathway was achieved by addition of 10 µM of PI103 or MK2206 . E . AKT inhibition increases sensitivity to 2DG induced . * p < 0 . 001 versus HEYpcDNA .

O O O 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 O B-CONTROLLED_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-G. Resistance to 2DG induced cell death in Rab25 cells is mediated through the PI3K/AKT pathway. Inhibition of the PI3K/AKT pathway was achieved by addition of 10 µM of PI103 or MK2206.E. AKT inhibition increases sensitivity to 2DG induced cell death. *p < 0.001 versus HEYpcDNA cells.


E - G . Resistance to 2DG induced in Rab25 is mediated through PI3K / AKT pathway . Inhibition of PI3K / AKT pathway was achieved by addition of 10 µM of PI103 or MK2206 . F . Down - regulation of AKT by siRNA specific to AKT .

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

E-G. Resistance to 2DG induced cell death in Rab25 cells is mediated through the PI3K/AKT pathway. Inhibition of the PI3K/AKT pathway was achieved by addition of 10 µM of PI103 or MK2206.F. Down-regulation of AKT by siRNA specific to AKT.


E - G . Resistance to 2DG induced in Rab25 is mediated through PI3K / AKT pathway . Inhibition of PI3K / AKT pathway was achieved by addition of 10 µM of PI103 or MK2206 . G . Or down - regulation of Rab25 by shRNA specific to Rab25 enhances 2DG induced . * p < 0 . 001 versus RNAi control .

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

E-G. Resistance to 2DG induced cell death in Rab25 cells is mediated through the PI3K/AKT pathway. Inhibition of the PI3K/AKT pathway was achieved by addition of 10 µM of PI103 or MK2206.G. Or down-regulation of Rab25 by shRNA specific to Rab25 enhances 2DG induced cell death. *p < 0.001 versus RNAi control cells.


Ovarian cancer HEY ( upper panel ) were co - transfected with AKT - IFPN Rab25 - IFPC or with AKT - IFPC Rab25 - IFPN . become due to an interaction between AKT Rab25 bringing two halves of into proximity creating a stable restoring . Hela stably expressing AKT - IFPN ( lower panel ) were transfected with Rab25 - IFPC or PDK - IFPC as control . A brightfield image corresponding to image is shown transfected indicated by an arrow .

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

Ovarian cancer HEY cells (upper panel) were co-transfected with AKT-IFPN and Rab25-IFPC or with AKT-IFPC and Rab25-IFPN. Cells become fluorescent due to an interaction between AKT and Rab25 bringing the two halves of the fluorescent protein into proximity creating a stable complex and restoring fluorescence. Hela cells stably expressing AKT-IFPN (lower panel) were transfected with Rab25-IFPC or PDK-IFPC as control. A brightfield image corresponding to the fluorescent image is shown and transfected cells indicated by an arrow.


Detection of Rab25 AKT interaction by immunoprecipitation . Ovarian expressing HA - tagged Rab25 were IP with anti - HA antibody . resultant was separated by gel electrophoresis detected by WB using anti - AKT antibody .

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

Detection of Rab25 and AKT interaction by immunoprecipitation. Ovarian cells expressing HA-tagged Rab25 were IP with anti-HA antibody. The resultant complex was separated by gel electrophoresis and detected by WB using anti-AKT antibody.


Generation of Rab25 deletion mutants . Expression of deletion mutants was detected by immunofluorescence ( upper panel ) WB ( middle panel ) using anti - GFP antibody which interacts with YFP . Diagrammatic representation of structure of mutants is shown in lower panel .

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

Generation of Rab25 deletion mutants. Expression of the deletion mutants was detected by immunofluorescence staining (upper panel) and WB (middle panel) using anti-GFP antibody which interacts with YFP. Diagrammatic representation of the structure of the mutants is shown in the lower panel.


Rab25 deletion mutants do not alter glycogen ATP . Ovarian cancer lysates were collected 24 h post transfection . ( a ) p < 0 . 05 versus empty vector transfected .

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

Rab25 deletion mutants do not alter cellular glycogen and ATP levels. Ovarian cancer cell lysates were collected 24 h post transfection. (a) p < 0.05 versus empty vector transfected cells.


A . Rab25 expression correlates with glycogen in patienttumours . Total total extracts , isolated from 31 ovarian cancer specimens , were subjected to Rab25 gene expression glycogen content using qPCR glycogen , respectively .

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

A. Rab25 expression correlates with glycogen levels in patienttumours. Total RNA and total cellular extracts, isolated from 31 ovarian cancer patient specimens, were subjected to Rab25 gene expression and glycogen content analysis using qPCR and glycogen assay, respectively.


B - C . Rab25 expression signature identifies with a poor prognosis . Ovarian cancer were classified as either mirroring Rab25 - associated gene expression signature ( Rab25 signature ) or not ( non - Rab25 signature ) using linear discriminant in BRB tools , in two independent published ovarian datasets . Progression free survival curvesfor with advanced disease ( Stage II to IV ) are shown . Univariate analyses were plotted using Kaplan - Meier method Coxplots used for multivariate analyses ( co - variates included stage , grade , histology residual disease ) . B . Tothill et al , 2008 . C . Dressman et al , 2007 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-C. The Rab25 expression signature identifies patients with a poor prognosis. Ovarian cancer patients were classified as either mirroring the Rab25-associated gene expression signature (Rab25 signature) or not (non-Rab25 signature) using linear discriminant analysis in BRB tools, in two independent published ovarian datasets. Progression free survival curvesfor patients with advanced disease (Stage II to IV) are shown. Univariate analyses were plotted using Kaplan-Meier method and Coxplots used for multivariate analyses (co-variates included stage, grade, histology and residual disease).B. Tothill et al, 2008.C. Dressman et al, 2007.


D . Prediction of breast cancer overall survival in two independent published breast datasets ( upper panel ) Pawitan et al , 2005 ( lower panel ) Chin et al , 2006 , on Rab25 - associated gene signature . Breast classification was determined by BRB tool class prediction function to identify with Rab25 - associated gene expression signature ( Rab25 - signature ) or without ( non - Rab25 signature ) . from datasets were included in class prediction .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Prediction of breast cancer overall survival in two independent published breast datasets (upper panel) Pawitan et al, 2005 and (lower panel) Chin et al, 2006, based on Rab25-associated gene signature. Breast patient classification was determined by BRB tool class prediction function to identify patient with Rab25-associated gene expression signature (Rab25-signature) or without (non-Rab25 signature). All patients from the datasets were included in class prediction.


A Determination of a time window for 2a2iL treatment needed to induce a naïve - like pluripotency state in primed hPSCs . One day treatment with 2a followed by addition of 2a2iL for up to 14 days . Scale bars : 200 µm .

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

A Determination of a time window for 2a2iL treatment needed to induce a naïve-like pluripotency state in primed hPSCs. One day treatment with 2a followed by the addition of 2a2iL for up to 14 days. Scale bars: 200 µm.


C Characteristics of 2a2iL - RH6 represented by dome - shaped colonies , ALP activity , a normal karyotype ( 46 , XY ) . 2a2iL - RH6 expressed common pluripotency markers OCT4 , NANOG , SOX2 , TRA - 1 - 81 , but not SSEA1 . Nuclei were stained with 4 ′ , 6 - diamidino - 2 - phenylindole ( DAPI ) Propidium iodide ( PI ) . Scale bars : 200 , 100 , 50 µm in inset .

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

C Characteristics of 2a2iL-RH6 cells represented by dome-shaped colonies, ALP activity, and a normal male karyotype (46, XY). 2a2iL-RH6 expressed the common pluripotency markers OCT4, NANOG, SOX2, and TRA-1-81, but not SSEA1. Nuclei were stained with 4′, 6-diamidino-2-phenylindole (DAPI) and Propidium iodide (PI). Scale bars: 200, 100, and 50 µm in the inset.


A Doubling time in pRH6 versus 2a2iL - RH6 . * * P < 0 . 01 ( t - ) . Data presented as mean ± SD ( n = 3 ) .

O O O O O O O O O O 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 Doubling time in pRH6 versus 2a2iL-RH6. **P<0.01 (t-test). Data presented as mean ± SD (n=3).


B Cloning efficiency in presence ( + ) absence ( − ) of ROCK inhibitor ( ROCKi ) in pRH6 versus 2a2iL - RH6 . ROCKi ( − ) * P < 0 . 05 , ROCKi ( + ) * P < 0 . 01 ( t - ) , data presented as mean ± SD ( n = 3 ) .

O O O O O O O O O O O O O B-CONTROLLED_VAR 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 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 Cloning efficiency in the presence (+) and absence (−) of ROCK inhibitor (ROCKi) in pRH6 versus 2a2iL-RH6. ROCKi (−)*P<0.05, ROCKi (+) *P<0.01 (t-test), data presented as mean ± SD (n=3).


C Firefly luciferase expression of constructs that contain distal proximal enhancers of OCT4 after transfection of pRh6 2a2iL - RH6 . of firefly luciferase expression to Renilla luciferase expression ( expressed from co - transfected plasmid ) measured 48 h after transfection showed proximal enhancer activity in pRH6 distal enhancer activation in 2a2iL - RH6 . OCT4 PE - Luc * * * P < 0 . 001 , OCT4 DE - Luc * P < 0 . 05 ( t - ) . Data presented as mean ± SD ( n = 3 ) .

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 B-MEASURED_VAR 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

C Firefly luciferase expression of constructs that contain distal and proximal enhancers of OCT4 after transfection of pRh6 and 2a2iL-RH6. The ratio of firefly luciferase expression to Renilla luciferase expression (expressed from co-transfected plasmid) measured 48 h after transfection showed proximal enhancer activity in pRH6 and distal enhancer activation in 2a2iL-RH6. OCT4 PE-Luc ***P<0.001, OCT4 DE-Luc *P<0.05 (t-test). Data presented as mean ± SD (n=3).


D qRT - PCR of pluripotency - related genes in pRH6 2a2iL - RH6 . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( t - ) . Data presented as mean ± SD ( n = 3 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 pluripotency-related genes in pRH6 and 2a2iL-RH6. *P<0.05, **P<0.01, ***P<0.001 (t-test). Data presented as mean ± SD (n=3).


E Immunofluorescence for several naïve specific markers in 2a2iL - RH6 . Scale bar : 100 µm .

E Immunofluorescence staining for several naïve specific markers in 2a2iL-RH6. Scale bar: 100 µm.


F Western blot of naïve - related pluripotency markers in pRH6 2a2iL - RH6 . * * P < 0 . 01 , * * * P < 0 . 001 ( t - ) . Data presented as mean ± SD ( n = 3 ) .

O O O O O O O O O O O O O O O O O O O O O O O O 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 Western blot analysis of naïve-related pluripotency markers in pRH6 and 2a2iL-RH6. **P<0.01, ***P<0.001 (t-test). Data presented as mean ± SD (n=3).


G Immunofluorescence against pRH5 2a2iL - RH5 ( 46 , XX ) for H3K27me3 . Nuclei were stained with 4 ′ , 6 - diamidino - 2 - phenylindole ( DAPI ) . Reactivation of inactive X - chromosome in 2a2iL - RH5 was confirmed by lack of H3K27me3 foci . Scale bar : 50 µm .

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 B-MEASURED_VAR O O O O O O O O

G Immunofluorescence staining against pRH5 and 2a2iL-RH5 (46, XX) for H3K27me3. Nuclei were stained with 4′, 6-diamidino-2-phenylindole (DAPI). Reactivation of inactive X-chromosome in female 2a2iL-RH5 was confirmed by the lack of H3K27me3 nuclear foci. Scale bar: 50 µm.


A Phase contrast of embryoid bodies ( EBs ) from 2a2iL - RH6 that differentiated into neurons . Scale bars : 200 100 µm in inset .

O O O 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 Phase contrast images of embryoid bodies (EBs) from 2a2iL-RH6 cells that differentiated into neurons. Scale bars: 200 and 100 µm in the inset.


B qRT - PCR of 20 day - EBs from pRH6 2a2iL - RH6 for markers of different embryonic germ layer derivatives . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( t - ) . Data presented as mean ± SD ( n = 3 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 qRT-PCR analysis of 20 day-EBs from pRH6 and 2a2iL-RH6 for markers of different embryonic germ layer derivatives. *P<0.05, **P<0.01, ***P<0.001 (t-test). Data presented as mean ± SD (n=3).


C Immunofluorescence of differentiated 2a2iL - RH6 that depict expression of markers for neuron - like ( TUJ - 1 ) , cardiac - muscle - like ( CTNT ) , hepatocyte - like ( SOX17 ) . Nuclei were stained with 4 ′ , 6 - diamidino - 2 - phenylindole ( DAPI ) . Scale bar : 100 µm .

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 B-MEASURED_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 O O O O O O O

C Immunofluorescence images of differentiated 2a2iL-RH6 cells that depict expression of markers for neuron-like cells (TUJ-1), cardiac-muscle-like cells (CTNT), and hepatocyte-like cells (SOX17). Nuclei were stained with 4′, 6-diamidino-2-phenylindole (DAPI). Scale bar: 100 µm.


D 2a2iL - RH6 form teratomas that consist of three embryonic germ layer - derived tissues . ( i ) Rosette structures ( ectoderm ) , ( ii ) cartilage ( mesoderm ) , ( iii ) primitive gut ( endoderm ; scale bar 50 µm ) .

D 2a2iL-RH6 cells form teratomas that consist of three embryonic germ layer-derived tissues. (i) Rosette structures (ectoderm), (ii) cartilage (mesoderm), and (iii) primitive gut (endoderm; scale bar 50 µm).


E From left to right : Phase contrast GFP representation in 2a2iL - RH6 - eGFP ; presence of injected 2a2iL - RH6 - eGFP in mouse blastocyst integration into inner mass ( ICM ) after 3 , 24 48 h incubation periods . Scale bars : 200 , 100 , 50 µm in inset .

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

E From left to right: Phase contrast and GFP representation in 2a2iL-RH6-eGFP; presence of injected 2a2iL-RH6-eGFP in the mouse blastocyst and integration into the inner cell mass (ICM) after 3, 24 and 48 h incubation periods. Scale bars: 200, 100, and 50 µm in the inset.


F of tissue - sections from human - mouse chimeras in E10 . 5 showed positive for GFP ( top ) human antigen ( HNA ) ( bottom ) in different parts of , including head ( i ) , trunk ( ii ) , tail ( iii ) with higher magnification in right panels . Scale bars : 200 100 µm in inset .

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

F Fluorescence images of tissue-sections from human-mouse chimeras in E10.5 showed cells positive for GFP (top) and human nuclear antigen (HNA) (bottom) in different parts of the embryos, including the head (i), trunk (ii), and tail (iii) with the higher magnification in the right panels. Scale bars: 200 and 100 µm in the inset.


G Genomic PCR for a human specific mitochondrial ( 391 bp ) in human - mouse chimeras .

G Genomic PCR analysis for a human specific mitochondrial sequence (391 bp) in human-mouse chimeras.


H Immunofluorescence against NANOG shows lack of expression in E10 . 5 human - mouse chimera tissue sections . Scale bars : 200 100 µm in inset .

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

H Immunofluorescence staining against NANOG shows lack of expression in E10.5 human-mouse chimera tissue sections. Scale bars: 200 and 100 µm in the inset.


I Immunofluorescence against TUJ1 , BRACHYURY ( BRA ) , SOX17 in E10 . 5 human - mouse chimera tissue sections represent differentiation - derivatives of 2a2iL - RH6 . Scale bars : 200 100 µm in inset .

O O O B-MEASURED_VAR O B-MEASURED_VAR 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

I Immunofluorescence staining against TUJ1, BRACHYURY (BRA), and SOX17 in E10.5 human-mouse chimera tissue sections represent differentiation-derivatives of 2a2iL-RH6. Scale bars: 200 and 100 µm in the inset.


B of ICM - outgrowths by protocols 1 2 during first 4 days after seeding . nRH11 - 14 ( naïve Royan H11 , 12 , 13 , 14 ) : naïve hESC lines produced from human using 2a2iL condition . Scale bar : 100 µm .

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

B Morphology of ICM-outgrowths by protocols 1 and 2 during the first 4 days after seeding. nRH11-14 (naïve Royan H11, 12, 13, and 14): naïve hESC lines produced from human embryos using 2a2iL condition. Scale bar: 100 µm.


C Comparison of efficiency of naïve - like hESCs derivation from human blastocysts by two protocols .

C Comparison of the efficiency of naïve-like hESCs derivation from human blastocysts by two protocols.


D Representative example of naïve - like hESCs ( nRH13 ) at passage 33 . Dome - shaped colonies were stained for ALP activity karyotyped . Scale bars : 200 100 µm in inset .

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

D Representative example of naïve-like hESCs (nRH13) at passage 33. Dome-shaped colonies were stained for ALP activity and karyotyped. Scale bars: 200 and 100 µm in the inset.


E Expression of common pluripotency markers OCT4 , SOX2 , TRA - 1 - 81 , naïve - related pluripotency markers NANOG , KLF17 , TFCP2L1 in nRH13 . Nuclei were stained with 4 ′ , 6 - diamidino - 2 - phenylindole ( DAPI ) . Scale bar : 100 µm .

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

E Expression of the common pluripotency markers OCT4, SOX2, and TRA-1-81, and naïve-related pluripotency markers NANOG, KLF17, and TFCP2L1 in nRH13. Nuclei were stained with 4′, 6-diamidino-2-phenylindole (DAPI). Scale bar: 100 µm.


F qRT - PCR for pluripotency markers in nRH13 in comparison to a primed hESC , pRH6 . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( t - ) . Data presented as mean ± SD ( n = 3 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 for pluripotency markers in nRH13 in comparison to a primed hESC line, pRH6. *P<0.05, **P<0.01, ***P<0.001 (t-test). Data presented as mean ± SD (n=3).


G Expression of lineage markers after embryoid ( EB ) formation spontaneous differentiation in nRH13 . * P < 0 . 05 , * * * P < 0 . 001 ( t - ) . Data presented as mean ± SD ( n = 3 ) .

G Expression of lineage markers after embryoid body (EB) formation and spontaneous differentiation in nRH13 cells. *P<0.05, ***P<0.001 (t-test). Data presented as mean ± SD (n=3).


A Pairwise Pearson correlation coefficient of transcriptome profiles ( - seq ) from 2a2iL - , pRH5 , pRH6 lines . Each of shows pairwise Pearson correlation coefficient of genes with largest expression variances in a pair of samples .

O O O O O O O O O O O O O O O O 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 Pairwise Pearson correlation coefficient heatmap of transcriptome profiles (RNA-seq) from 2a2iL-, pRH5, and pRH6 cell lines. Each cell of the heatmap shows the pairwise Pearson correlation coefficient heatmap of genes with the largest expression variances in a pair of samples.


C volcano plot shows a differential expression between 2a2iL - versus primed hPSCs lines . X - axis shows log2 fold - change , y - axis shows negative log10 scaled adjusted P - value ( Benjamini - Hochberg ) . Red blue points represent up - regulated genes in 2a2iL - primed , respectively ( absolute log - 2 fold - change > 1 , adjusted P - value < 0 . 05 ) . Gray points indicate non - significant genes between these states .

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

C The volcano plot shows a differential expression between 2a2iL- versus primed hPSCs lines. X-axis shows the log2 fold-change, and the y-axis shows the negative log10 scaled adjusted P-value (Benjamini-Hochberg). Red and blue points represent up-regulated genes in 2a2iL- and primed cells, respectively (absolute log-2 fold-change >1, adjusted P-value <0.05). Gray points indicate non-significant genes between these states.


A Position of TGF - β family members in volcano plot of differentially expressed genes in 2a2iL - primed hPSCs . X - axis shows log2 fold - change y - axis shows negative log10 adjusted P - value < 0 . 05 ( Adjusted P - value was obtained using Benjamini Hochberg correction to determine false discovery rate ) . Red blue points represent up - regulated genes in 2a2iL - primed hPSCs , respectively . Genes with no significant expression change are shown in gray .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Position of TGF-β family members in the volcano plot of differentially expressed genes in 2a2iL- and primed hPSCs. X-axis shows log2 fold-change and y-axis shows negative log10 adjusted P-value <0.05 (Adjusted P-value was obtained using the Benjamini and Hochberg correction to determine the false discovery rate). Red and blue points represent up-regulated genes in 2a2iL- and primed hPSCs, respectively. Genes with no significant expression change are shown in gray.


B qRT - PCR of several TGF - β - related genes in pRH6 2a2iL - RH6 . * * P < 0 . 01 , * * * P < 0 . 001 ( t - ) . Data presented as mean ± SD ( n = 3 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 qRT-PCR analysis of several TGF-β-related genes in pRH6 and 2a2iL-RH6. **P<0.01, ***P<0.001 (t-test). Data presented as mean ± SD (n=3).


C Inhibition of TGF - β pathway by two small molecules , SB43 A83 . Left panel : of naïve - like three days after treatment . Right panel : of treated after passaging . Scale bar : 100 µm .

O O O O O O O O O O O O B-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

C Inhibition of the TGF-β pathway by two small molecules, SB43 and A83. Left panel: morphology of naïve-like cells three days after treatment. Right panel: morphology of treated cells after passaging. Scale bar: 100 µm.


D shows expression of putative LRH - 1 target genes in this study ' s transcriptome profiles . Colors indicate log2 fold change of marker genes in naïve versus primed hPSCs , with a range from blue for lower expression to red for higher expression . E shows expression of putative RARγ target genes in this study ' s transcriptome profiles . Colors indicate log2 fold change of marker genes in naïve versus primed hPSCs , with a range from blue for lower expression to red for higher expression .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Heatmap shows expression of putative LRH-1 target genes in this study's transcriptome profiles. Colors indicate log2 fold change of marker genes in the naïve versus primed hPSCs, with a range from dark blue for lower expression to dark red for higher expression. E Heatmap shows expression of putative RARγ target genes in this study's transcriptome profiles. Colors indicate log2 fold change of marker genes in the naïve versus primed hPSCs, with a range from dark blue for lower expression to dark red for higher expression.


A qRT - PCR of ligands associated with TGF - β signaling during conversion of primed to naïve - like pluripotency by 2a2iL induction protocol at days 1 , 3 , 5 . 2iL was selected as a control group . * * * P < 0 . 0001 . One - way ANOVA . Data presented as mean ± SD ( n = 3 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ligands associated with TGF-β signaling during conversion of primed to naïve-like pluripotency by 2a2iL induction protocol at days 1, 3, and 5. 2iL was selected as a control group. ***P<0.0001. One-way ANOVA. Data presented as mean ± SD (n=3).


B Inhibition of TGF - β pathway using SB43 during conversion of primed to naïve pluripotency . Scale bar : 100 µm .

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

B Inhibition of TGF-β pathway using SB43 during conversion of primed to naïve pluripotency. Scale bar: 100 µm.


C of TGFβ2iL - treated RH6 along with ALP activity . Scale bar : 200 µm .

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

C Morphology of TGFβ2iL-treated RH6 cells along with ALP activity. Scale bar: 200 µm.


D qRT - PCR of naïve related genes in pRH6 , 2a2iL - , TGFβ2iL - RH6 . * * P < 0 . 01 , * * * P < 0 . 001 . One - way ANOVA . Data presented as mean ± SD ( n = 3 ) .

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

D qRT-PCR analysis of naïve related genes in pRH6, 2a2iL-, and TGFβ2iL-RH6. **P<0.01, ***P<0.001. One-way ANOVA. Data presented as mean ± SD (n=3).


E Bar plot representing proliferation rates of 2a2iL - TGFβ2iL - RH6 by . * * P < 0 . 01 ( t - ) . Data presented as mean ± SD ( n = 3 ) .

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

E Bar plot representing cell proliferation rates of 2a2iL- and TGFβ2iL-RH6 by cell counts. **P<0.01 (t-test). Data presented as mean ± SD (n=3).


F Bar plot of cloning efficiency of 2a2iL TGFβ2iL - RH6 on number of ALP positive colonies . * * * P < 0 . 001 ( t - ) . Data presented as mean ± SD ( n = 3 ) .

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

F Bar plot of the cloning efficiency of 2a2iL and TGFβ2iL-RH6 based on the number of ALP positive colonies. ***P<0.001 (t-test). Data presented as mean ± SD (n=3).


G Immunofluorescence show expression of naïve pluripotency markers in TGF - β2iL - RH6 . Nuclei were stained with 4 ′ , 6 - diamidino - 2 - phenylindole ( DAPI ) . Scale bar : 100 µm .

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

G Immunofluorescence images show the expression of naïve pluripotency markers in TGF-β2iL-RH6. Nuclei were stained with 4′, 6-diamidino-2-phenylindole (DAPI). Scale bar: 100 µm.


( D ) Number of putative enhancer regions per . Bar depicts mean . Diamonds represent numbers in each are color - coded according to cancer

O O 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) Number of putative enhancer regions per cell line. Bar depicts the mean. Diamonds represent individual numbers in each cell line and are color-coded according to cancer type


( E ) Heat maps of DNase I signal for enhancer regions ( rows ) . For intergenic enhancers ( left ) signal is aligned at upstream ( relative to + strand ) enhancer edge , shown from 4 kbp upstream to 5 kbp downstream , ordered by increasing of enhancer regions . For intragenic enhancers ( right ) signal was aligned at upstream downstream enhancer edges shown within ± 4 kbp . Exemplary data from LoVo colorectal cancer . ( F ) Heat maps as in ( E ) showing composite ChIP - seq peak coverage from 326 TFs

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

(E) Heat maps of DNase I signal for enhancer regions (rows). For intergenic enhancers (left) the signal is aligned at the upstream (relative to + strand) enhancer edge, shown from 4 kbp upstream to 5 kbp downstream, and ordered by increasing length of enhancer regions. For intragenic enhancers (right) the signal was aligned at the upstream and downstream enhancer edges and shown within ± 4 kbp. Exemplary data from LoVo colorectal cancer cells. (F) Heat maps as in (E) showing composite ChIP-seq peak coverage from 326 TFs


( B ) Bidirectional promoters show higher mRNA synthesis . Boxplots showing TT - seq signal for mRNAs from unidirectional ( n = 39 , 376 ) bidirectional ( n = 83 , 363 ) promoters . Bidirectional unidirectional promoters were defined for each separately then plotted together . Box limits are first third quartiles , band inside box is median . ends of whiskers extend box by 1 . 5 times interquartile range . Notches represent 95 % confidence intervals for median values . Outliers not shown . P - value by two - sided Mann - Whitney U - .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Bidirectional promoters show higher mRNA synthesis. Boxplots showing TT-seq signal for mRNAs from unidirectional (n=39,376) and bidirectional (n=83,363) promoters. Bidirectional and unidirectional promoters were defined for each cell line separately and then plotted together. Box limits are the first and third quartiles, the band inside the box is the median. The ends of the whiskers extend the box by 1.5 times the interquartile range. Notches represent 95% confidence intervals for the median values. Outliers not shown. P-value by two-sided Mann-Whitney U-test.


( A ) UCSC genome browser view of normalized TT - seq coverage on plus minus strand at TAL1 locus ( hg38 ; chr1 : 47 , 194 , 268 - 47 , 242 , 880 ; ( Kent et al . , 2002 ) ) in leukemia lines Jurkat K562 . For better visualization , TT - seq coverage is cut at 200 ( purple lines ) . eRNAs are shown between plus minus strand . Red indicates short 12 bp insertion in Jurkat , introducing novel binding sites for transcription factor

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

(A) UCSC genome browser view of normalized TT-seq coverage on the plus and minus strand at the TAL1 locus (hg38; chr1:47,194,268-47,242,880; (Kent et al., 2002)) in the leukemia cell lines Jurkat and K562. For better visualization, TT-seq coverage is cut at 200 (purple lines). eRNAs are shown between the plus and minus strand. Red line indicates the short 12 bp insertion in Jurkat cells, introducing novel binding sites for the transcription factor MYB


( B ) UCSC genome browser view of normalized TT - seq coverage on plus minus strand at 2 . 8 Mbp gene desert surrounding MYC ( hg38 ; chr8 : 127 , 042 , 406 - 129 , 779 , 109 ; ( Kent et al . , 2002 ) ) in fourteen cancer lines . eRNAs represent union from lines . Genome - wide association studies ( GWAS ) risk loci contain GWAS single nucleotide polymorphisms ( SNPs ) for prostate cancer ( PrCa ) , acute lymphocytic leukemia ( ) , breast cancer ( BrCa ) , colorectal cancer ( CRC ) , glioblastoma ( GBM ) including SNPs in linkage disequilibrium ( r2 ≥ 0 . 8 ) . TT - seq coverage is displayed between 8 - 250 to allow for better visualization of eRNA signal surrounding MYC . Dashed rectangle is shown at full scale on right . Note : TT - seq reads were mapped against hg38 reference genome we cannot rule out existence of genomic rearrangements ( e . g . translocations ) affecting shown region in used cancer clones .

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

(B) UCSC genome browser view of normalized TT-seq coverage on the plus and minus strand at the 2.8 Mbp gene desert surrounding MYC (hg38; chr8:127,042,406-129,779,109; (Kent et al., 2002)) in fourteen cancer cell lines. eRNAs represent the union from all cell lines. Genome-wide association studies (GWAS) risk loci contain GWAS single nucleotide polymorphisms (SNPs) for prostate cancer (PrCa), acute lymphocytic leukemia (ALL), breast cancer (BrCa), colorectal cancer (CRC), and glioblastoma (GBM) including SNPs in linkage disequilibrium (r2 ≥ 0.8). TT-seq coverage is displayed between 8-250 to allow for better visualization of the eRNA signal surrounding MYC. Dashed rectangle is shown at full scale on the right. Note: TT-seq reads were mapped against the hg38 reference genome and we cannot rule out the existence of genomic rearrangements (e.g. translocations) affecting the shown region in the used cancer cell line clones.


( A ) Med1 - defined enhancers ranked by Med1 ChIP - seq signal ( Yan et al . , 2013 ) in LoVo colorectal cancer . Super - enhancers ( black rectangles ) were determined as enhancer regions lying above inflection point of

O O 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

(A) Med1-defined enhancers ranked by Med1 ChIP-seq signal (Yan et al., 2013) in LoVo colorectal cancer cells. Super-enhancers (black rectangles) were determined as enhancer regions lying above the inflection point of the curve


( B ) Increased eRNA synthesis from super - enhancers . Boxplots comparing eRNA synthesis ( TT - seq signal ) at Med1 - defined typical enhancers ( n = 2 , 111 ) super - enhancers ( n = 130 ) in LoVo ( was restricted to intergenic enhancers Box limits are first third quartiles , band inside box is median . ends of whiskers extend box by 1 . 5 times interquartile range . Notches represent 95 % confidence intervals for median values . Outliers not shown . P - value by two - sided Mann - Whitney U - .

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 O O 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) Increased eRNA synthesis from super-enhancers. Boxplots comparing eRNA synthesis (TT-seq signal) at Med1-defined typical enhancers (n=2,111) and super-enhancers (n=130) in LoVo cells (analysis was restricted to intergenic enhancers Box limits are the first and third quartiles, the band inside the box is the median. The ends of the whiskers extend the box by 1.5 times the interquartile range. Notches represent 95% confidence intervals for the median values. Outliers not shown. P-value by two-sided Mann-Whitney U-test.


( C ) Super - enhancers are associated with production of long eRNAs . Boxplots showing of TT - seq - defined eRNAs originating from Med1 - defined super - enhancers ( n = 552 ) or other enhancer regions ( n = 6 , 934 ) . Median , hinges , whiskers notches are shown as in ( B ) . P - value by two - sided Mann - Whitney U - .

O 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

(C) Super-enhancers are associated with the production of long eRNAs. Boxplots showing length of TT-seq-defined eRNAs originating from Med1-defined super-enhancers (n=552) or other enhancer regions (n=6,934). Median, hinges, whiskers and notches are shown as in (B). P-value by two-sided Mann-Whitney U-test.


( D ) Barplot showing number of TT - seq enhancer regions per Med1 - defined super - enhancer region in LoVo . Super - enhancers spanning a - coding TSS ( n = 32 ) are excluded from this comparison as regions TSS ± 1 kbp were excluded from TT - seq enhancer calling .

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

(D) Barplot showing the number of TT-seq enhancer regions per Med1-defined super-enhancer region in LoVo cells. Super-enhancers spanning a protein-coding TSS (n=32) are excluded from this comparison as regions TSS ± 1 kbp were excluded from TT-seq enhancer calling.


( E ) UCSC genome browser view of normalized TT - seq coverage on plus minus strand at PHLDA1 locus ( hg38 ; chr12 : 75 , 612 , 350 - 76 , 042 , 552 ; in colorectal cancer HCT 116 . TT - seq coverage is cut at 250 ( purple lines ) to allow for better visualization . eRNAs are shown between plus minus strand . Aggregated transcription factor ChIP - seq peak regions ( TF ChIP - seq ) comprise ChIP - seq binding profiles of 19 TFs in HCT 116 from CISTROME height of signal indicates number of different TFs binding at a particular region . Binding regions for selected TFs ( TCF7l2 , YY1 , SP1 ) are shown below together with p300 polymerase II ( Pol II ) . TT - seq enhancer regions are shown at bottom together with a H3K27ac - defined super - enhancer annotation , which was downloaded from SEdb 2019 ) . At date of publication no Med1 ChIP - seq data was available for HCT 116 .

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

(E) UCSC genome browser view of normalized TT-seq coverage on the plus and minus strand at the PHLDA1 locus (hg38; chr12:75,612,350-76,042,552; in the colorectal cancer cell line HCT 116. TT-seq coverage is cut at 250 (purple lines) to allow for better visualization. eRNAs are shown between the plus and minus strand. Aggregated transcription factor ChIP-seq peak regions (TF ChIP-seq) comprise ChIP-seq binding profiles of 19 TFs in HCT 116 cells from CISTROME The height of the signal indicates the number of different TFs binding at a particular region. Binding regions for selected TFs (TCF7l2, YY1, SP1) are shown below together with p300 and RNA polymerase II (Pol II). TT-seq enhancer regions are shown at the bottom together with a H3K27ac-defined super-enhancer annotation, which was downloaded from SEdb 2019). At date of publication no Med1 ChIP-seq data was available for HCT 116.


( A ) Unsupervised clustering of pairwise Spearman correlations of normalized TT - seq signal for mRNAs ( left ) eRNAs ( right ) for union of transcripts over fourteen cancer lines Grey colored bars on top indicate gender cancer type , respectively .

O O O O O O O O O O O O O O O O O O O O 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) Unsupervised clustering of all pairwise Spearman correlations of normalized TT-seq signal for mRNAs (left) and eRNAs (right) for the union of transcripts over all fourteen cancer cell lines Grey and colored bars on top indicate gender and cancer type, respectively.


( B ) Heatmaps showing mRNAs ( left ) eRNAs ( right ) as being synthesized ( red , normalized TT - seq signal FPK ≥ 15 ) or not ( blue , normalized TT - seq signal FPK < 15 ) for fourteen cancer lines ( columns ) . Heatmaps show only transcripts ( rows ) with normalized FPK ≥ 30 in at least one of lines ( mRNAs , n = 14 , 040 ; eRNAs n = 48 , 105 ) . Heatmaps are ordered by increasing type specificity , as indicated by grey bars to right showing number of lines a transcript is observed in .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Heatmaps showing mRNAs (left) and eRNAs (right) as being synthesized (red, normalized TT-seq signal FPK ≥ 15) or not (blue, normalized TT-seq signal FPK < 15) for all fourteen cancer cell lines (columns). Heatmaps show only transcripts (rows) with normalized FPK ≥ 30 in at least one of the cell lines (mRNAs, n=14,040; eRNAs n=48,105). Heatmaps are ordered by increasing cell type specificity, as indicated by the grey bars to the right showing the number of cell lines a transcript is observed in.


( D ) Boxplots showing normalized TT - seq signal for nearest active promoters in in which enhancers are exclusively transcribed ( from left to right : n = 890 ; 500 ; 494 ; 214 ; 238 ; 239 ; 378 ; 62 ; 153 ; 879 ; 586 ; 173 ; 601 ; 232 ) compared to same promoters in other lines in which respective enhancers are not transcribed . pairwise comparisons are significantly different ( P - values < 1e - 9 , two - sided Mann - Whitney U - ) . Box limits are first third quartiles , band inside box is median . ends of whiskers extend box by 1 . 5 times interquartile range . Notches represent 95 % confidence intervals for median values . Outliers not shown .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Boxplots showing normalized TT-seq signal for nearest active promoters in the cell line in which enhancers are exclusively transcribed (from left to right: n=890; 500; 494; 214; 238; 239; 378; 62; 153; 879; 586; 173; 601; 232) compared to the same promoters in all other cell lines in which the respective enhancers are not transcribed. All pairwise comparisons are significantly different (P-values < 1e-9, two-sided Mann-Whitney U-test). Box limits are the first and third quartiles, the band inside the box is the median. The ends of the whiskers extend the box by 1.5 times the interquartile range. Notches represent 95% confidence intervals for the median values. Outliers not shown.


UCSC genome browser view of normalized TT - seq coverage on plus minus strand at BMP4 locus ( hg38 ; chr14 : 53 , 935 , 697 - 54 , 264 , 430 ; in LoVo colorectal cancer . TT - seq coverage is cut at 500 ( purple lines ) to allow for better visualization . eRNAs enhancers are shown above below minus strand , respectively . Aggregated transcription factor ChIP - seq peak regions ( TF ChIP - seq ) include ChIP - seq binding profiles of 326 TFs in LoVo height of signal indicates number of different TFs binding at a particular region . Significant promoter capture Hi - C ( CHi - C ) interactions are shown in

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

UCSC genome browser view of normalized TT-seq coverage on the plus and minus strand at the BMP4 locus (hg38; chr14:53,935,697-54,264,430; in LoVo colorectal cancer cells. TT-seq coverage is cut at 500 (purple lines) to allow for better visualization. eRNAs and enhancers are shown above and below the minus strand, respectively. Aggregated transcription factor ChIP-seq peak regions (TF ChIP-seq) include ChIP-seq binding profiles of 326 TFs in LoVo cells The height of the signal indicates the number of different TFs binding at a particular region. Significant promoter capture Hi-C (CHi-C) interactions are shown in purple


B . Western blots of MDI in wild type ( wt ) mdi1 flies , showing that mdi1 is a - null mutation . Tubulin was used as loading control .

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

B. Western blots of MDI protein in wild type (wt) and mdi1 flies, showing that mdi1 is a protein-null mutation. Tubulin was used as loading control.