B The frequency of Ch16 minichromosome loss was assayed by plating cells onto adenine limiting agar and determining the frequency of half sectored ( red - white ) colonies . At least two independently derived strain isolates were used for each genotype and the total number of colonies counted for each genotype is indicated in parenthesis .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 The frequency of Ch16 minichromosome loss was assayed by plating cells onto adenine limiting agar and determining the frequency of half sectored (red-white) colonies. At least two independently derived strain isolates were used for each genotype and the total number of colonies counted for each genotype is indicated in parenthesis.


C Heterothallic ( h90 ) strains were cultured on nitrogen limiting ( EMMG ) medium for 3 days at 25 ° C to induce mating and meiosis . The resulting asci were stained with DAPI and visualised by fluorescence microscopy . Data are representative of three biological

O O O O O O O O O O O O O O O O O O O O O O O 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 Heterothallic (h90) strains were cultured on nitrogen limiting (EMMG) medium for 3 days at 25°C to induce mating and meiosis. The resulting asci were stained with DAPI and visualised by fluorescence microscopy. Data are representative of three biological repeats


D Spore viability was measured by dissecting spores onto YE5S agar followed by incubation for 4 - 5 days at 30 ° C . Data are the mean of three independent biological repeats and error bars are ± SEM . P - value was calculated using a two - tailed unpaired t - test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Spore viability was measured by dissecting spores onto YE5S agar followed by incubation for 4-5 days at 30°C. Data are the mean of three independent biological repeats and error bars are ±SEM. P-value was calculated using a two-tailed unpaired t-test.


A ) Murine small intestines immunostained for the proliferation marker Ki67 , apoptosis marker cleaved caspase 3 ( Cas3 ) , or YAP from 4 control ( Yapfl / fl Tazfl / fl ) and 4 YAP / TAZ double conditional knockout ( Villin - CreERt Yapfl / fl Tazfl / fl ) animals treated with tamoxifen . Tissues were harvested and analyzed at day 7 after first tamoxifen injection . Loss of YAP / TAZ leads to a moderate increase in apoptosis ( Cas3 + ) in crypt base stem cells , quantified on the right , but no overall effect on the morphology of the small intestine . Arrows point to apoptotic cells . B ) Murine large intestines immunostained for proliferation marker Ki67 , apoptosis marker cleaved caspase 3 ( Cas3 ) , or YAP from control ( Yapfl / fl Tazfl / fl ) and YAP / TAZ double conditional knockout ( Villin - CreERt Yapfl / fl Tazfl / fl ) animals treated with tamoxifen . Tissues were harvested and analyzed at day 7 after first tamoxifen injection . Loss of YAP / TAZ leads to an increased number of apoptotic ( Cas3 + ) cells within the transverse folds of the ascending colon .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Murine small intestines immunostained for the proliferation marker Ki67, apoptosis marker cleaved caspase 3 (Cas3), or YAP from 4 control (Yapfl/fl Tazfl/fl) and 4 YAP/TAZ double conditional knockout (Villin-CreERt Yapfl/fl Tazfl/fl) animals treated with tamoxifen. Tissues were harvested and analyzed at day 7 after first tamoxifen injection. Loss of YAP/TAZ leads to a moderate increase in apoptosis (Cas3+) in crypt base stem cells, quantified on the right, but no overall effect on the morphology of the small intestine. Arrows point to apoptotic cells. B) Murine large intestines immunostained for proliferation marker Ki67, apoptosis marker cleaved caspase 3 (Cas3), or YAP from control (Yapfl/fl Tazfl/fl) and YAP/TAZ double conditional knockout (Villin-CreERt Yapfl/fl Tazfl/fl) animals treated with tamoxifen. Tissues were harvested and analyzed at day 7 after first tamoxifen injection. Loss of YAP/TAZ leads to an increased number of apoptotic (Cas3+) cells within the transverse folds of the ascending colon.


C ) Murine small intestines immunostained for YAP or cleaved caspase 3 ( Cas3 ) from control ( Villin - CreERt ) and YAP / TAZ double conditional knockout ( Villin - CreERt Yapfl / fl Tazfl / fl ) animals treated with tamoxifen . Tissues were harvested and analyzed at day 7 after first tamoxifen injection . Loss of YAP / TAZ leads to a mild increase in apoptosis ( Cas3 + ) in crypt base stem cells , but no overall effect on the morphology of the small intestine . Arrows point to stem cells ( YAP nuclear staining ) , while arrows point to apoptotic cells ( Cas3 staining ) . N > 10 animals per genotype . D ) Murine large intestines immunostained for YAP or cleaved caspase 3 ( Cas3 ) from control ( Villin - CreERt ) and YAP / TAZ double conditional knockout ( Villin - CreERt Yapfl / fl Tazfl / fl ) animals treated with tamoxifen . Tissues were harvested and analyzed at day 7 after first tamoxifen injection . Loss of YAP / TAZ leads to increased apopotosis ( Cas3 + ) and severe morphological defects in the transverse folds of the ascending colon , but does not affect morphology of the remaining colon regions . Quantitatively , 75 % of n = 4 animals showed this severe phenotype .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Murine small intestines immunostained for YAP or cleaved caspase 3 (Cas3) from control (Villin-CreERt) and YAP/TAZ double conditional knockout (Villin-CreERt Yapfl/fl Tazfl/fl) animals treated with tamoxifen. Tissues were harvested and analyzed at day 7 after first tamoxifen injection. Loss of YAP/TAZ leads to a mild increase in apoptosis (Cas3+) in crypt base stem cells, but no overall effect on the morphology of the small intestine. Arrows point to stem cells (YAP nuclear staining), while arrows point to apoptotic cells (Cas3 staining). N > 10 animals per genotype. D) Murine large intestines immunostained for YAP or cleaved caspase 3 (Cas3 ) from control (Villin-CreERt) and YAP/TAZ double conditional knockout (Villin-CreERt Yapfl/fl Tazfl/fl) animals treated with tamoxifen. Tissues were harvested and analyzed at day 7 after first tamoxifen injection. Loss of YAP/TAZ leads to increased apopotosis (Cas3+) and severe morphological defects in the transverse folds of the ascending colon, but does not affect morphology of the remaining colon regions. Quantitatively, 75% of n = 4 animals showed this severe phenotype.


A ) Murine small ( top ) and large ( bottom ) intestines isolated from control ( Cre negative ) Lats1flox / flox Lats2flox / flox animals and Villin - CreERt Lats1flox / flox Lats2flox / flox animals treated with tamoxifen to induce homozygous deletion of Lats1 / 2 ( dKO ) . Immunostaining for YAP and Ki67 shows a gradient of YAP expression along the crypt - villus axis in controls , with nuclear YAP and Ki67 positive cells restricted to the crypt base ( representative images from n = 5 mice ) . Tamoxifen treated ( 3 days i . p . ) Villin - CreERt Lats1flox / flox Lats2flox / flox double homozygous mouse intestines show an enlarged crypt compartment after 7 days with strongly nuclear YAP immunostaining in all epithelial cells and an expanded proliferative zone marked by Ki67 positive cells . Note the gradient of YAP expression levels is maintained along the crypt - villus axis ( representative images from n = 5 mice for each genotype ) . ( Right ) Immunostaining for the Paneth cell marker Lyz reveals loss of this marker from the crypt base . ( Bottom right ) Q - PCR analysis of Wnt pathway target genes reveals that Lats1 / 2 dKO causes a mild reduction in Lgr5 expression , with complete loss of Olfm4 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Murine small (top) and large (bottom) intestines isolated from control (Cre negative) Lats1flox/flox Lats2flox/flox animals and Villin-CreERt Lats1flox/flox Lats2flox/flox animals treated with tamoxifen to induce homozygous deletion of Lats1/2 (dKO). Immunostaining for YAP and Ki67 shows a gradient of YAP expression along the crypt-villus axis in controls, with nuclear YAP and Ki67 positive cells restricted to the crypt base (representative images from n=5 mice). Tamoxifen treated (3 days i.p.) Villin-CreERt Lats1flox/flox Lats2flox/flox double homozygous mouse intestines show an enlarged crypt compartment after 7 days with strongly nuclear YAP immunostaining in all epithelial cells and an expanded proliferative zone marked by Ki67 positive cells. Note the gradient of YAP expression levels is maintained along the crypt-villus axis (representative images from n=5 mice for each genotype). (Right) Immunostaining for the Paneth cell marker Lyz reveals loss of this marker from the crypt base. (Bottom right) Q-PCR analysis of Wnt pathway target genes reveals that Lats1/2 dKO causes a mild reduction in Lgr5 expression, with complete loss of Olfm4.


B ) Axin2 mRNA expression was measured by RNAscope ( red ) and found to be increased in both intensity and uniformity of staining along the crypt - villus axis in Lats1 / 2 dKO after 3 days post i . p . injection ( dpi ) with tamoxifen to induce the homozygous deletion of Lats1 and Lats2 . At 7dpi , Axin2 mRNA levels remain uniform along the crypt - villus axis in Lats1 / 2 dKO animals , although their total level has declined compared to 3dpi .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Axin2 mRNA expression was measured by RNAscope (red) and found to be increased in both intensity and uniformity of staining along the crypt-villus axis in Lats1/2 dKO after 3 days post i.p. injection (dpi) with tamoxifen to induce the homozygous deletion of Lats1 and Lats2. At 7dpi, Axin2 mRNA levels remain uniform along the crypt-villus axis in Lats1/2 dKO animals, although their total level has declined compared to 3dpi.


D ) Q - PCR analysis of YAP - TEAD and their target genes Ctgf and Cyr61 reveals that Lats1 / 2 dKO causes a strong induction of Ctgf and Cyr61 expression , as expected , as well as a strong induction of the common Wnt and YAP target gene CyclinD1 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Q-PCR analysis of YAP-TEAD and their target genes Ctgf and Cyr61 reveals that Lats1/2 dKO causes a strong induction of Ctgf and Cyr61 expression, as expected, as well as a strong induction of the common Wnt and YAP target gene CyclinD1.


B ) RNAscope in situ hybridisation analysis of TEAD1 / 2 / 4 mRNA expression from control ( Cre negative ) Lats1flox / flox Lats2flox / flox animals and Villin - CreERt Lats1flox / flox Lats2flox / flox animals treated with tamoxifen to induce homozygous deletion of Lats1 / 2 ( dKO ) . Tamoxifen treated ( 3 days i . p . ) Villin - CreERt Lats1flox / flox Lats2flox / flox double homozygous mouse intestines show elevated expression of TEAD1 and TEAD4 in both the small intestine ( top ) and colon ( bottom ) . Tissues were harvested and analyzed at day 7 after first tamoxifen injection . ( n = 5 animals for each genotype )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) RNAscope in situ hybridisation analysis of TEAD1/2/4 mRNA expression from control (Cre negative) Lats1flox/flox Lats2flox/flox animals and Villin-CreERt Lats1flox/flox Lats2flox/flox animals treated with tamoxifen to induce homozygous deletion of Lats1/2 (dKO). Tamoxifen treated (3 days i.p.) Villin-CreERt Lats1flox/flox Lats2flox/flox double homozygous mouse intestines show elevated expression of TEAD1 and TEAD4 in both the small intestine (top) and colon (bottom). Tissues were harvested and analyzed at day 7 after first tamoxifen injection. (n=5 animals for each genotype)


C ) Q - PCR analysis reveals a progressive increase in the YAP - TEAD target gene Ctgf in Lats1 / 2 dKO intestines at 3 - days and 7 - days post i . p . injection with tamoxifen , as well as similarly increased TEAD1 and TEAD4 expression , confirming that TEAD1 and TEAD4 are YAP - responsive genes . Notably , Axin2 and TEAD2 exhibit a mild but progressive decline over 3 to 7 days after Lats1 / 2 deletion , consistent with Axin2 being a known Wnt - specific target gene , and with Wnt signalling remaining active in Lats1 / 2 dKO intestines .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Q-PCR analysis reveals a progressive increase in the YAP-TEAD target gene Ctgf in Lats1/2 dKO intestines at 3-days and 7-days post i.p. injection with tamoxifen, as well as similarly increased TEAD1 and TEAD4 expression, confirming that TEAD1 and TEAD4 are YAP-responsive genes. Notably, Axin2 and TEAD2 exhibit a mild but progressive decline over 3 to 7 days after Lats1/2 deletion, consistent with Axin2 being a known Wnt-specific target gene, and with Wnt signalling remaining active in Lats1/2 dKO intestines.


A ) RNAscope in situ hybridisation analysis of TEAD1 / 2 / 4 mRNA expression from control ( wild - type ) and ApcMin mutant tumours from the small intestine ( top ) and colon ( bottom ) . All three genes were strongly induced within the ApcMin mutant tumours , with TEAD2 being most strongly induced in colon ApcMin tumours ( n = 3 animals with multiple tumours within the small and large intestines ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) RNAscope in situ hybridisation analysis of TEAD1/2/4 mRNA expression from control (wild-type) and ApcMin mutant tumours from the small intestine (top) and colon (bottom). All three genes were strongly induced within the ApcMin mutant tumours, with TEAD2 being most strongly induced in colon ApcMin tumours (n=3 animals with multiple tumours within the small and large intestines).


C ) Antibody staining for TEAD4 reveals a gradient of expression along the crypt - villus axis in both the small and large intestine , with uniformly high expression in colorectal adenomas , similar to SOX9 .

O O O O O O O O O O O O O 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) Antibody staining for TEAD4 reveals a gradient of expression along the crypt-villus axis in both the small and large intestine, with uniformly high expression in colorectal adenomas, similar to SOX9.


B ) YAP immunostaining of 3 - day old wild type organoids treated with DMSO ( Control ) or Porcupine inhibitor LGK974 at 5µM . Wnt signalling inhibition upon Porcupine inhibitor treatment decreases YAP levels without affecting YAP subcellular localisation . Quantification of YAP protein levels is shown below , with statistical significance in an unpaired 2 - tailed t - test .

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

B) YAP immunostaining of 3-day old wild type organoids treated with DMSO (Control) or Porcupine inhibitor LGK974 at 5µM. Wnt signalling inhibition upon Porcupine inhibitor treatment decreases YAP levels without affecting YAP subcellular localisation. Quantification of YAP protein levels is shown below, with statistical significance in an unpaired 2-tailed t-test.


C ) Wnt activation in Apc5 homozygous mutant organoids drives a drastic shift in organoid morphology from budding structures to cystic spheres and increases the YAP protein level . Scale bar is 250µm . n > 10 organoids in each experiment . Quantification of YAP protein levels from crypt and villus regions of control organoids as well as from the regions bounded by rectangles in Apc5 organoids are shown on the right , with statistical significance in an unpaired 2 - tailed t - test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Wnt activation in Apc5 homozygous mutant organoids drives a drastic shift in organoid morphology from budding structures to cystic spheres and increases the YAP protein level. Scale bar is 250µm. n > 10 organoids in each experiment. Quantification of YAP protein levels from crypt and villus regions of control organoids as well as from the regions bounded by rectangles in Apc5 organoids are shown on the right, with statistical significance in an unpaired 2-tailed t-test.


A ) Src family kinase inhibition via Dasatinib treatment in control organoids enforces YAP cytoplasmic localisation after 4h of treatment in organoids cultured for 3d . n > 10 organoids in each experiment . B ) Quantification of the percentage of nuclear YAP cells per control crypt bud as shown in panel ( A ) , error bars show 1 SD ( * * * * p < 0 . 0001 ) . n > 10 organoids per condition ( top ) . Quantification of the nuclear / cytoplasmic ratio of YAP per cell in Apc5 mutant organoids as shown in panel ( C ) . n > 10 organoids per condition ( bottom ) . Statistical analysis was performed with an unpaired student ' s t - test . C

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

A) Src family kinase inhibition via Dasatinib treatment in control organoids enforces YAP cytoplasmic localisation after 4h of treatment in organoids cultured for 3d. n > 10 organoids in each experiment. B) Quantification of the percentage of nuclear YAP cells per control crypt bud as shown in panel (A), error bars show 1 SD (**** p <0.0001). n > 10 organoids per condition (top). Quantification of the nuclear/cytoplasmic ratio of YAP per cell in Apc5 mutant organoids as shown in panel (C). n > 10 organoids per condition (bottom). Statistical analysis was performed with an unpaired student's t-test. C


C ) Src family kinase inhibition via Dasatinib or eCF506 treatment in Apc5 organoids causes relocalisation of YAP to the cytoplasm . n > 10 organoids in each experiment . Q - PCR analysis of the YAP - TEAD target gene Ctgf reveals a strong inhibition of mRNA expression upon treatment with eCF506 ( relative expression , error bars = 1 SD ) . Statistical analysis was performed with an unpaired student ' s t - test .

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 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

C) Src family kinase inhibition via Dasatinib or eCF506 treatment in Apc5 organoids causes relocalisation of YAP to the cytoplasm. n > 10 organoids in each experiment. Q-PCR analysis of the YAP-TEAD target gene Ctgf reveals a strong inhibition of mRNA expression upon treatment with eCF506 (relative expression, error bars = 1 SD). Statistical analysis was performed with an unpaired student's t-test.


A ) Mouse intestinal organoids immunostained for YAP ( green ) . Note the mostly cytoplasmic localisation of YAP at 48h in the organoid . Irradiation with 4Gy of X - rays drives strong nuclear localisation of YAP in many cells , which is reversible by treatment with the Src family kinase inhibitor Dasatinib . The Dasatinib treated organoids exhibit increased cell death after irradiation , as indicated by pyknotic nuclei ( white arrow ) . n > 10 organoids in each experiment . This experiment employs the same control batch of 2 - 3d organoids also shown in Figure 6A .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O 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) Mouse intestinal organoids immunostained for YAP (green). Note the mostly cytoplasmic localisation of YAP at 48h in the organoid. Irradiation with 4Gy of X-rays drives strong nuclear localisation of YAP in many cells, which is reversible by treatment with the Src family kinase inhibitor Dasatinib. The Dasatinib treated organoids exhibit increased cell death after irradiation, as indicated by pyknotic nuclei (white arrow). n > 10 organoids in each experiment. This experiment employs the same control batch of 2-3d organoids also shown in Figure 6A.


B ) Control VillinCreERt mouse small intestine immunostained for YAP shows a gradient of expression with mostly cytoplasmic localisation . Irradiation with 14Gy of X - rays drives strong nuclear localisation of YAP along the entire crypt - villus axis . Deletion of Src within VillinCreERt Srcflox / flox intestines prevents YAP nuclear localisation and normal regeneration after 14Gy irradiation . Arrows point to villar cells with nuclear YAP localisation . Lower panels show high - mag views of a different example than those shown in upper panels .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Control VillinCreERt mouse small intestine immunostained for YAP shows a gradient of expression with mostly cytoplasmic localisation. Irradiation with 14Gy of X-rays drives strong nuclear localisation of YAP along the entire crypt-villus axis. Deletion of Src within VillinCreERt Srcflox/flox intestines prevents YAP nuclear localisation and normal regeneration after 14Gy irradiation. Arrows point to villar cells with nuclear YAP localisation. Lower panels show high-mag views of a different example than those shown in upper panels.


A ) Control and irradiated murine small intestines analysed with RNAscope in situ hybridisation for Myc mRNA ( black arrows point to mRNA granules in the control ) and by immunostaining for Sox9 protein reveals an expansion in the expression domain of both genes . Nuclear localisation of beta - catenin is readily detectable after irradiation . n = 3 samples per condition , quantification shown in the insets , error bars = 1 SD . Statistical analysis was performed with an unpaired student ' s t - test . Similar results were obtained at 3dpi and 5dpi .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Control and irradiated murine small intestines analysed with RNAscope in situ hybridisation for Myc mRNA (black arrows point to mRNA granules in the control) and by immunostaining for Sox9 protein reveals an expansion in the expression domain of both genes. Nuclear localisation of beta-catenin is readily detectable after irradiation. n = 3 samples per condition, quantification shown in the insets, error bars = 1 SD. Statistical analysis was performed with an unpaired student's t-test. Similar results were obtained at 3dpi and 5dpi.


B ) Q - PCR analysis of YAP , TEADs and their target genes Ctgf and Cyr61 over a 4 days post irradiation time course at 12Gy . Note the strong upregulation of TEAD4 , Ctgf , and Cyr61 . In contrast , Axin2 , TEAD2 and Sox9 mRNA expression levels are only moderately altered , confirming that Wnt signalling remains active upon irradiation - induced YAP - TEAD activation . n = 4 biological replicates per time point . Statistical analysis was performed with unpaired t - test compared to the control 0Gy condition , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

B) Q-PCR analysis of YAP, TEADs and their target genes Ctgf and Cyr61 over a 4 days post irradiation time course at 12Gy. Note the strong upregulation of TEAD4, Ctgf, and Cyr61. In contrast, Axin2, TEAD2 and Sox9 mRNA expression levels are only moderately altered, confirming that Wnt signalling remains active upon irradiation-induced YAP-TEAD activation. n = 4 biological replicates per time point. Statistical analysis was performed with unpaired t-test compared to the control 0Gy condition, * p<0.05, ** p<0.01, ***p<0.001.


C ) Control and irradiated murine small intestines analysed by immunostaining for Sox9 protein reveals that treatment with Porcupine inhibitor LGK974 strongly reduces the Sox9 expression domain induced by 3 days after 12Gy irradiation . Phosphorylated Src immunostaining reveals an upregulation by 3 days after irradiation which is also lost upon treatment with Porcupine inhibitor LGK974 ( n = 4 animals in each condition ) .

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

C) Control and irradiated murine small intestines analysed by immunostaining for Sox9 protein reveals that treatment with Porcupine inhibitor LGK974 strongly reduces the Sox9 expression domain induced by 3 days after 12Gy irradiation. Phosphorylated Src immunostaining reveals an upregulation by 3 days after irradiation which is also lost upon treatment with Porcupine inhibitor LGK974 (n= 4 animals in each condition).


D ) Q - PCR analysis of Wnt and YAP target gene expression in intestinal crypts isolated at 1 day after 12Gy irradiation . Note the inhibition of Axin2 , Sox9 , YAP , Ctgf , Cyr61 , and TEAD family expression upon treatment with Porcupine inhibitor LGK974 . The mild reduction of Sox9 mRNA expression by LGK974 at 1 day after irradiation ( 1dpi ) becomes more pronounced at 3 days after irradiation ( 3dpi ) . n = 4 biological replicates per condition , statistical analysis was performed with Student ' s 2 - tailed t - test , error bars = 1 SD , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Q-PCR analysis of Wnt and YAP target gene expression in intestinal crypts isolated at 1 day after 12Gy irradiation. Note the inhibition of Axin2, Sox9, YAP, Ctgf, Cyr61, and TEAD family expression upon treatment with Porcupine inhibitor LGK974. The mild reduction of Sox9 mRNA expression by LGK974 at 1 day after irradiation (1dpi) becomes more pronounced at 3 days after irradiation (3dpi). n = 4 biological replicates per condition, statistical analysis was performed with Student's 2-tailed t-test, error bars = 1 SD, * p<0.05, ** p<0.01, ***p<0.001.


Immunofluorescence and reporter expression in spheroids derived from single WT and Hdac3 - / - naïve mESCs . DNA counterstain is Hoechst . Dashed white lines indicate the embryonic Gata6 : : mCherry negative part . Scale bar : 10 μm .

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Immunofluorescence and reporter expression in spheroids derived from single WT and Hdac3-/- naïve mESCs. DNA counterstain is Hoechst. Dashed white lines indicate the embryonic Gata6::mCherry negative part. Scale bar: 10 μm.


Nanog > GFP and Gata6 : : mCherry fluorescence intensities of WT and Hdac3 - / - Fraction of Gata6 : : mCherry positive cells in indicated genotypes and conditions after 4d Jak ( i ) blocks LIF , LDN19 BMP4 and PD03 FGF signaling . Average and SD of at least two independent clones .

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Nanog>GFP and Gata6::mCherry fluorescence intensities of WT and Hdac3-/- Fraction of Gata6::mCherry positive cells in indicated genotypes and conditions after 4d Jak(i) blocks LIF, LDN19 BMP4 and PD03 FGF signaling. Average and SD of at least two independent clones.


Quantification of spatial lineage segregation in spheroids . Representative spheroid segmentations of indicated genotypes Scale bar : 25 μm .

Quantification of spatial lineage segregation in spheroids. Representative spheroid segmentations of indicated genotypes Scale bar: 25 μm.


Scatter plots of log2 fold change ( FC ) gene expression changes ( ∆ mRNA ) between naïve Hdac3 - / - or Dax1 - / - mutants and WT mESCs ( left ) , between Nanog > GFP - expressing Hdac3 - / - or Dax1 - / - mutants and WT cells ( middle ) , and between Gata6 : : mCherry - expressing Hdac3 - / - or Dax1 - / - mutants and Nanog > GFP - expressing WT cells ( right ) . Cluster 5 genes are colored and selected PrE TFs labeled .

Scatter plots of log2 fold change (FC) gene expression changes (∆mRNA) between naïve Hdac3-/- or Dax1-/- mutants and WT mESCs (left), between Nanog>GFP-expressing Hdac3-/- or Dax1-/- mutants and WT cells (middle), and between Gata6::mCherry-expressing Hdac3-/- or Dax1-/- mutants and Nanog>GFP-expressing WT cells (right). Cluster 5 genes are colored and selected PrE TFs labeled.


Expression of PrE markers relative to Dax1 - / - cells ( B ) , and fraction of Gata6 : : mCherry positive cells after 3d in SLRA ( C ) in indicated genotypes . Average and SD of three independent clones .

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Expression of PrE markers relative to Dax1-/- cells (B), and fraction of Gata6::mCherry positive cells after 3d in SLRA (C) in indicated genotypes. Average and SD of three independent clones.


Genome - browser view of the Gata6 locus showing accessibility and chromatin mark changes in indicated mutants , and of ChIPseq signals of indicated TFs and H3K27ac in E6 . 5 VE and EPI . The grey area indicates enh - 45 .

Genome-browser view of the Gata6 locus showing accessibility and chromatin mark changes in indicated mutants, and of ChIPseq signals of indicated TFs and H3K27ac in E6.5 VE and EPI. The grey area indicates enh-45.


FC of PrE and naïve pluripotency markers relative to WT cells in 2iL of indicated genotypes after 3d in SLRA ( D ) and in 2iL ( E ) . Average and SD of at least three independent clones .

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FC of PrE and naïve pluripotency markers relative to WT cells in 2iL of indicated genotypes after 3d in SLRA (D) and in 2iL (E). Average and SD of at least three independent clones.


C - E . Logistic statistical model based on the ( C ) single cell sequencing , ( D ) ImageStream ( E ) analysis or automated FISH of RPE - 1 cells in untreated condition ( blue circles ) or treated with Auxin for 48 h ( red squares ) . Error bars represent the SEM based on the number of cells analyzed Dashed lines indicate the means of aneuploidy rates in untreated ( blue line ) or Auxin - treated ( red line ) condition . Red asterisks ( IAA ) and Blue ( Untreated ) indicate significance over the respective mean using a binomial test . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 .

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

C-E. Logistic statistical model based on the (C) single cell sequencing, (D) ImageStream (E) analysis or automated FISH of RPE-1 cells in untreated condition (blue circles) or treated with Auxin for 48 h (red squares). Error bars represent the SEM based on the number of cells analyzed Dashed lines indicate the means of aneuploidy rates in untreated (blue line) or Auxin-treated (red line) condition. Red asterisks (IAA) and Blue (Untreated) indicate significance over the respective mean using a binomial test. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.


F . Table summarizing whole - chromosome aneuploidy ( fold over the mean ) using the indicated methods to measure chromosome mis - segregation rate . Bold numbers represent a statistically significant difference from the mean for each method . Orange to dark red gradient highlights chromosomes that mis - segregate ( with at least one method ) at a significantly higher rate compared to the mean level ( weak chromosomes ) . Light green to dark green gradient highlights chromosomes that mis - segregate ( with at least one method ) at a significantly lower rate compared to the mean ( stronger chromosomes ) . Data obtained with only one method were excluded . * = centromere 4 probe with image stream was reported to lead to non - specific signal ( Worrall et al , 2018 ) . Im . St . = image stream ; Seq = single cell sequencing .

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F. Table summarizing whole-chromosome aneuploidy (fold over the mean) using the indicated methods to measure chromosome mis-segregation rate. Bold numbers represent a statistically significant difference from the mean for each method. Orange to dark red gradient highlights chromosomes that mis-segregate (with at least one method) at a significantly higher rate compared to the mean level (weak chromosomes). Light green to dark green gradient highlights chromosomes that mis-segregate (with at least one method) at a significantly lower rate compared to the mean (stronger chromosomes). Data obtained with only one method were excluded. * = centromere 4 probe with image stream was reported to lead to non-specific signal (Worrall et al, 2018). Im. St. = image stream; Seq = single cell sequencing.


B - C . ( B ) Micronuclei frequency formation by live cell imaging in untreated ( NT ) and Auxin - treated ( IAA ) condition . ( C ) Bar graph represents the type of chromosome mis - segregation ( independently of the dCas9 signal ) observed in the indicated conditions , with ( IAA ) or without ( NT ) auxin , in the cell lines expressing dCas9 mScarlet - I and sgRNA targeting chromosome 1 or 3 . Error bars represent the SEM of four independent experiments in which cells labeled for chromosome 1 and 3 were analyzed together ( n = 45 - 149 cells ) .

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 O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O 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. (B) Micronuclei frequency formation by live cell imaging in untreated (NT) and Auxin-treated (IAA) condition. (C) Bar graph represents the type of chromosome mis-segregation (independently of the dCas9 signal) observed in the indicated conditions, with (IAA) or without (NT) auxin, in the cell lines expressing dCas9 mScarlet-I and sgRNA targeting chromosome 1 or 3. Error bars represent the SEM of four independent experiments in which cells labeled for chromosome 1 and 3 were analyzed together (n = 45 - 149 cells).


D . Representative live cell imaging of RPE - 1 cells dCas9 mScarlet - I with sgRNA targeting chromosome 1 ( red dots ) starting from metaphase and showing example of correct ( untreated ) or mis - aligned chromosome ( auxin ) leading to the formation of a micronucleus . Yellow arrows mark mis - aligned - and micronucleus - containing chromosome 1 . Cells were imaged every five minutes . The numbers indicate the time point at which the presented images were taken . Cells were fixed at the end of the movie to detect Cas9 with an antibody . Scale bar represents 10 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 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

D. Representative live cell imaging of RPE-1 cells dCas9 mScarlet-I with sgRNA targeting chromosome 1 (red dots) starting from metaphase and showing example of correct (untreated) or mis-aligned chromosome (auxin) leading to the formation of a micronucleus. Yellow arrows mark mis-aligned- and micronucleus-containing chromosome 1. Cells were imaged every five minutes. The numbers indicate the time point at which the presented images were taken. Cells were fixed at the end of the movie to detect Cas9 with an antibody. Scale bar represents 10 µm.


E . Bar graph representing the proportion of chromosome mis - segregation observed in both cell lines after auxin addition . Only movies in which the dCas9 mScarlet - I dots were clearly visible during the whole division were taken into consideration for the analysis ( N = 6 - 10 cells ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 graph representing the proportion of chromosome mis-segregation observed in both cell lines after auxin addition. Only movies in which the dCas9 mScarlet-I dots were clearly visible during the whole division were taken into consideration for the analysis (N = 6 - 10 cells).


F . Representative images of mitotic errors leading to aneuploidy in fixed cells after CENP - A depletion ( 28 h auxin ) . Chromosomes are stained using whole ( X ) or centromeric ( 6 ) FISH probes . Yellow arrows mark lagging chromosomes . Scale bar represents 5 µm . G - H . Bars represent the ( G ) proportion of chromosome mis - segregation and ( H ) the type of the different chromosome mis - segregations observed . Error bars represent the SEM of two replicates . n > 66 mis - segregation events . Paired t test lagging chromosomes vs mis - alignment chromosomes , * p = 0 . 0275 .

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F. Representative images of mitotic errors leading to aneuploidy in fixed cells after CENP-A depletion (28 h auxin). Chromosomes are stained using whole (X) or centromeric (6) FISH probes. Yellow arrows mark lagging chromosomes. Scale bar represents 5 µm. G-H. Bars represent the (G) proportion of chromosome mis-segregation and (H) the type of the different chromosome mis-segregations observed. Error bars represent the SEM of two replicates. n > 66 mis-segregation events. Paired t test lagging chromosomes vs mis-alignment chromosomes, *p = 0.0275.


Bar plot showing the mean of ( B ) centromere length ( n = 4 ) as determined by whole genome sequencing . Error bars represent the SEM of four independent experiments . Acrocentric chromosomes 13 , 14 , 21 and 22 were marked by a line as we could not assign the respective reads . Dashed lines indicate the mean . Bars were labeled with asterisks according to the significance of their difference from the mean ( t - test ) . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 . Bars represent the sum of the

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

Bar plot showing the mean of (B) centromere length (n = 4) as determined by whole genome sequencing. Error bars represent the SEM of four independent experiments. Acrocentric chromosomes 13, 14, 21 and 22 were marked by a line as we could not assign the respective reads. Dashed lines indicate the mean. Bars were labeled with asterisks according to the significance of their difference from the mean (t-test). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Bars represent the sum of the length


Bar plot showing the mean of ( C ) CENP - B boxes counts ( n = 4 ) as determined by whole genome sequencing . Error bars represent the SEM of four independent experiments . Acrocentric chromosomes 13 , 14 , 21 and 22 were marked by a line as we could not assign the respective reads . Dashed lines indicate the mean . Bars were labeled with asterisks according to the significance of their difference from the mean ( t - test ) . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 . Bars represent the sum of counts of different HOR arrays CENP - B boxes counts are normalized to the average number of mapped reads in each replicate .

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Bar plot showing the mean of (C) CENP-B boxes counts (n = 4) as determined by whole genome sequencing. Error bars represent the SEM of four independent experiments. Acrocentric chromosomes 13, 14, 21 and 22 were marked by a line as we could not assign the respective reads. Dashed lines indicate the mean. Bars were labeled with asterisks according to the significance of their difference from the mean (t-test). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Bars represent the sum of counts of different HOR arrays CENP-B boxes counts are normalized to the average number of mapped reads in each replicate.


D . Schematic using representative images of the mFISH labeling followed by CENP - B box FISH method used to identify and quantify centromere specific CENP - B boxes signal in ( E ) . Scale bar represents 10 µm .

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D. Schematic using representative images of the mFISH labeling followed by CENP-B box FISH method used to identify and quantify centromere specific CENP-B boxes signal in (E). Scale bar represents 10 µm.


E . Box and whisker plots of normalized CENP - B boxes intensity over the mean on metaphase spread from three independent experiments ( n > 50 cells ) using the Tukey plot . One - way ANOVA with post - hoc Tukey ' s multiple comparison test shows high diversity between chromosomes . t - test against the mean was used to estimate the statistical significance for each chromosome . * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 . .

E. Box and whisker plots of normalized CENP-B boxes intensity over the mean on metaphase spread from three independent experiments (n>50 cells) using the Tukey plot. One-way ANOVA with post-hoc Tukey's multiple comparison test shows high diversity between chromosomes. t-test against the mean was used to estimate the statistical significance for each chromosome. **p < 0.01; ***p < 0.001; ****p < 0.0001..


Scatter plot showing a non - significant or significant negative correlation between the mean of ( F ) centromere length ( n = 4 ) and mis - segregation rate ( from Fig . 1F ) ( r = Spearman rank coefficient ) . Lines represent the linear regression with a 95 % confidence interval . Data from chr 13 , 14 , 21 and 22 were excluded from the analysis .

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

Scatter plot showing a non-significant or significant negative correlation between the mean of (F) centromere length (n = 4) and mis-segregation rate (from Fig. 1F) (r = Spearman rank coefficient). Lines represent the linear regression with a 95% confidence interval. Data from chr 13, 14, 21 and 22 were excluded from the analysis.


Scatter plot showing a non - significant or significant negative correlation between the mean of ( G ) centromere CENP - B boxes FISH signal ( n > 50 cells ) and mis - segregation rate , respectively ( r = Spearman rank coefficient ) . Lines represent the linear regression with a 95 % confidence interval . Data from chr 13 , 14 , 21 and 22 were excluded from the analysis .

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

Scatter plot showing a non-significant or significant negative correlation between the mean of (G) centromere CENP-B boxes FISH signal (n>50 cells) and mis-segregation rate, respectively (r = Spearman rank coefficient). Lines represent the linear regression with a 95% confidence interval. Data from chr 13, 14, 21 and 22 were excluded from the analysis.


B . Representative immunofluorescence FISH ( IF - FISH ) images to measure CENP - B intensity at specific chromosome in untreated cells . Scale bar represents 5 µm .

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B. Representative immunofluorescence FISH (IF-FISH) images to measure CENP-B intensity at specific chromosome in untreated cells. Scale bar represents 5 µm.


C . Bar plot showing the normalized CENP - B fluorescence intensity at every chromosome over the mean on each metaphase spread ( n > 37 per chromosome ) ± SEM . Acrocentric chromosomes 13 , 14 , 21 and 22 were not analyzed and are marked by a line . One - way ANOVA with post - hoc Tukey ' s multiple comparison test shows high diversity between chromosomes . t - test against the mean was used to estimate the statistical significance for each chromosome . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 .

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C. Bar plot showing the normalized CENP-B fluorescence intensity at every chromosome over the mean on each metaphase spread (n > 37 per chromosome) ± SEM. Acrocentric chromosomes 13, 14, 21 and 22 were not analyzed and are marked by a line. One-way ANOVA with post-hoc Tukey's multiple comparison test shows high diversity between chromosomes. t-test against the mean was used to estimate the statistical significance for each chromosome. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.


D . Scatter plot showing a significant negative correlation between the mean of CENP - B IF - FISH signal ( n > 37 ) and the mis - segregation rate ( r = Spearman rank coefficient ) . The lines represent linear regression with 95 % confidence band .

O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Scatter plot showing a significant negative correlation between the mean of CENP-B IF-FISH signal (n > 37) and the mis-segregation rate (r = Spearman rank coefficient). The lines represent linear regression with 95% confidence band.


E - F . Bar graphs report the sum of the normalized reads counts of different HOR arrays representing CENP - C binding following CUT & RUN , sequencing and centromere mapping . Cells were treated for 6 h with IAA to deplete CENP - A . Error bars represent the SEM of three independent experiments and the dashed line represents the mean . Acrocentric chromosomes 13 - 14 , 21 and 22 were marked by a line . t - test against the mean was used to estimate the statistical significance for each chromosome . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 .

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E-F. Bar graphs report the sum of the normalized reads counts of different HOR arrays representing CENP-C binding following CUT&RUN, sequencing and centromere mapping. Cells were treated for 6 h with IAA to deplete CENP-A. Error bars represent the SEM of three independent experiments and the dashed line represents the mean. Acrocentric chromosomes 13-14, 21 and 22 were marked by a line. t-test against the mean was used to estimate the statistical significance for each chromosome. *p < 0.05; **p < 0.01; ***p < 0.001.


G . Scatter plot showing a significant positive correlation between the mean of CENP - C reads ( n = 3 ) and CENP - B reads ( n = 3 ) ( r = Spearman rank coefficient ) . Data from chr 1 , 5 and 19 were excluded from the analysis in addition to chr 13 , 14 , 21 and 22 to better assess correlation without the FISH correction The lines represent linear regression with a 95 % confidence interval .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Scatter plot showing a significant positive correlation between the mean of CENP-C reads (n = 3) and CENP-B reads (n = 3) (r = Spearman rank coefficient). Data from chr 1, 5 and 19 were excluded from the analysis in addition to chr 13, 14, 21 and 22 to better assess correlation without the FISH correction The lines represent linear regression with a 95% confidence interval.


H . Scatter plot showing a significant negative correlation between the mean of CENP - C reads ( n = 3 ) and the mis - segregation rate ( r = Spearman rank coefficient ) . The lines represent linear regression with 95 % confidence interval . Data from chr 13 , 14 , 21 and 22 were excluded from the analysis . The lines represent linear regression with a 95 % confidence interval .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Scatter plot showing a significant negative correlation between the mean of CENP-C reads (n = 3) and the mis-segregation rate (r = Spearman rank coefficient). The lines represent linear regression with 95% confidence interval. Data from chr 13, 14, 21 and 22 were excluded from the analysis. The lines represent linear regression with a 95% confidence interval.


B . Representative image of centromere 11 ( red ) and centromere X ( green ) FISH on RPE - 1 CENP - AAID CENP - B KO mutant in control ( top ) or IAA treated condition ( bottom ) . Scale bar represents 5 µm . Bar graphs show automatic FISH quantification of chromosome mis - segregation in the indicated cell lines with and without IAA addition for 24 h . Error bars represent the SEM of three independent experiments ( n > 200 cells per experiment ) . Dunnett ' s multiple comparisons test was used to compare conditions . Grey lines separate independent experiments . Fold changes between grey and red bars are also indicated .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O 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. Representative image of centromere 11 (red) and centromere X (green) FISH on RPE-1 CENP-AAID CENP-B KO mutant in control (top) or IAA treated condition (bottom). Scale bar represents 5 µm. Bar graphs show automatic FISH quantification of chromosome mis-segregation in the indicated cell lines with and without IAA addition for 24 h. Error bars represent the SEM of three independent experiments (n> 200 cells per experiment). Dunnett's multiple comparisons test was used to compare conditions. Grey lines separate independent experiments. Fold changes between grey and red bars are also indicated.


C . Schematic of the centromere strength model with the microtubule destabilizer BAL27862 compound . D . Representative image of centromere 17 ( red ) and centromere X ( green ) FISH on RPE - 1 cells treated with the BAL27862 compound . Scale bar represents 5 µm .

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C. Schematic of the centromere strength model with the microtubule destabilizer BAL27862 compound. D. Representative image of centromere 17 (red) and centromere X (green) FISH on RPE-1 cells treated with the BAL27862 compound. Scale bar represents 5 µm.


E . Bars represent the frequency of chromosome mis - segregation into micronuclei after 28 h treatment with the BAL27862 drug at indicated concentration . Error bars represent the SEM of three independent experiments , dashed line represents the mean . n > 40 micronuclei were analyzed . Dunnett ' s multiple comparisons test , * : p < 0 . 05 . DMSO was used at the same concentration of BAL27862 .

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 B-CONTROLLED_VAR O

E. Bars represent the frequency of chromosome mis-segregation into micronuclei after 28 h treatment with the BAL27862 drug at indicated concentration. Error bars represent the SEM of three independent experiments, dashed line represents the mean. n > 40 micronuclei were analyzed. Dunnett's multiple comparisons test, *: p<0.05. DMSO was used at the same concentration of BAL27862.


A . Representative image of CENP - B box FISH and mFISH stained RPE - 1 metaphase spread cells . Chromosome 3 homologs are indicated by white arrows with single CENP - B box chromosomes signal magnified in insets . Scale bar represents 5 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

A. Representative image of CENP-B box FISH and mFISH stained RPE-1 metaphase spread cells. Chromosome 3 homologs are indicated by white arrows with single CENP-B box chromosomes signal magnified in insets. Scale bar represents 5 µm.


B . Representative image of CENP - B box ( red ) and centromere 3 ( green ) FISH on RPE - 1 interphase nucleus after 48 h IAA treatment . CENP - B box signal at individual homologs are magnified in insets . Scale bar represents 5 µm .

B. Representative image of CENP-B box (red) and centromere 3 (green) FISH on RPE-1 interphase nucleus after 48 h IAA treatment. CENP-B box signal at individual homologs are magnified in insets. Scale bar represents 5 µm.


C . Bar plots represent the quantification of the CENP - B box FISH intensity at the chromosome 3 homolog contained in the main nucleus versus the homolog present in the micronucleus Error bars represent the SEM of three independent experiments . n = 69 cells . Unpaired t test , * * * * p < 0 . 0001 .

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C. Bar plots represent the quantification of the CENP-B box FISH intensity at the chromosome 3 homolog contained in the main nucleus versus the homolog present in the micronucleus Error bars represent the SEM of three independent experiments. n = 69 cells. Unpaired t test, ****p < 0.0001.


E . Immunoblot shows expression of the ( ΔN ) CENP - B - dCas9 after doxycycline induction .

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E. Immunoblot shows expression of the (ΔN)CENP-B-dCas9 after doxycycline induction.


F . Representative images show a micronucleus or the main nucleus containing the Y chromosome ( red ) detected by FISH in no - doxycycline or doxycycline treated cells respectively . Chromosome X is shown in green . Scale bar represents 5 µm .

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F. Representative images show a micronucleus or the main nucleus containing the Y chromosome (red) detected by FISH in no-doxycycline or doxycycline treated cells respectively. Chromosome X is shown in green. Scale bar represents 5 µm.


G . Bar plot shows the mean ( n ≥ 3 ) frequency of micronuclei containing the chromosome Y or chromosome X ± doxycycline treatment for 72 h and IAA for 48 h ± SEM . n > 200 cells with a micronucleus . Mann - Whitney test . * p = 0 . 0173 .

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G. Bar plot shows the mean (n ≥ 3) frequency of micronuclei containing the chromosome Y or chromosome X ± doxycycline treatment for 72 h and IAA for 48 h ± SEM. n > 200 cells with a micronucleus. Mann-Whitney test. *p=0.0173.


B . Table summarizing whole - chromosome aneuploidy ( fold over the mean ) using the indicated methods to measure chromosome mis - segregation rate . Bold numbers represent significant statistical difference from the mean for each method . Orange to dark red gradient highlights chromosomes that mis - segregate ( with at least one method ) at a significantly higher rate compared to the mean level ( weak chromosomes ) . Light green to dark green gradient highlights chromosomes that mis - segregate ( with at least one method ) at a significantly lower rate compared to the mean ( stronger chromosomes ) . Data obtained with only one method were excluded . Im . St . = image stream .

B. Table summarizing whole-chromosome aneuploidy (fold over the mean) using the indicated methods to measure chromosome mis-segregation rate. Bold numbers represent significant statistical difference from the mean for each method. Orange to dark red gradient highlights chromosomes that mis-segregate (with at least one method) at a significantly higher rate compared to the mean level (weak chromosomes). Light green to dark green gradient highlights chromosomes that mis-segregate (with at least one method) at a significantly lower rate compared to the mean (stronger chromosomes). Data obtained with only one method were excluded. Im. St. = image stream.


C . Plot shows the karyotype distribution of DLD - 1 and RPE - 1 cells in untreated or Auxin - treated

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C. Plot shows the karyotype distribution of DLD-1 and RPE-1 cells in untreated or Auxin-treated condition


E . Representative image of a chromosome spreads in DLD - 1 cells following mFISH and subsequently CENP - B boxes FISH . Inset shows the supernumerary ( SN ) chromosome . Scale bar represents 10 µm . F . Bar plot shows the mean variation in CENP - B boxes in DLD - 1 cells from data from E ( n = 10 cells ) and whole - genome sequencing and centromere mapping ± SD .

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E. Representative image of a chromosome spreads in DLD-1 cells following mFISH and subsequently CENP-B boxes FISH. Inset shows the supernumerary (SN) chromosome. Scale bar represents 10 µm. F. Bar plot shows the mean variation in CENP-B boxes in DLD-1 cells from data from E (n = 10 cells) and whole-genome sequencing and centromere mapping ± SD.


G - H . Scatter plot showing a significant positive correlation between the indicated features in DLD - 1 cells ( n = 1 ) . The lines represent linear regression with 95 % confidence band . Data from chr 13 , 14 , 21 , 22 , Y and SN were excluded from the analysis .

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G-H. Scatter plot showing a significant positive correlation between the indicated features in DLD-1 cells (n = 1). The lines represent linear regression with 95% confidence band. Data from chr 13, 14, 21, 22, Y and SN were excluded from the analysis.


J . Bars show frequency of micronuclei with or without siRNA treatment . Error bars represent the SEM of three independent experiments ( n > 170 cells per replicate ) .

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J. Bars show frequency of micronuclei with or without siRNA treatment. Error bars represent the SEM of three independent experiments (n > 170 cells per replicate).


K . ( Left ) Representative image of CENP - B box ( red ) staining after siRNA against CENP - A / HJURP for 48 h . CENP - B box signals at individual micronuclei are magnified in insets . Scale bar represent 5 µm . ( Right ) Bar graph represents the mean of the CENP - B box FISH intensity contained in the main nucleus versus the one present in the micronucleus ± SEM . Color dots represent independent experiments . ( n > 43 micronuclei and 840 main nucleus ) Mann - Whitney U test : * * * p = 0 . 0001 .

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K. (Left) Representative image of CENP-B box (red) staining after siRNA against CENP-A/HJURP for 48 h. CENP-B box signals at individual micronuclei are magnified in insets. Scale bar represent 5 µm. (Right) Bar graph represents the mean of the CENP-B box FISH intensity contained in the main nucleus versus the one present in the micronucleus ± SEM. Color dots represent independent experiments. (n > 43 micronuclei and 840 main nucleus) Mann-Whitney U test: ***p = 0.0001.


A - B . Scatter plot showing ( A ) a significant positive or ( B ) a negative correlation between chromosome length and the mis - segregation rate or CENP - C ( n = 3 ) reads over chromosome length and mis - segregation rate ( r = Spearman rank coefficient ) . The lines represent linear regression with 95 % confidence interval . Data from chr 13 , 14 , 21 and 22 were excluded from the analysis . Numbers indicate chromosomes with low or high mis - segregation frequency .

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A-B. Scatter plot showing (A) a significant positive or (B) a negative correlation between chromosome length and the mis-segregation rate or CENP-C (n = 3) reads over chromosome length and mis-segregation rate (r = Spearman rank coefficient). The lines represent linear regression with 95% confidence interval. Data from chr 13, 14, 21 and 22 were excluded from the analysis. Numbers indicate chromosomes with low or high mis-segregation frequency.


A - F ( A , B , D - F ) RNA levels determined by RT - qPCR . Results are presented as mean ± SD , n = 10 . P - values are indicated on the top of each plot . ( C ) Spearman correlation suggests an in vivo positive correlation between the expressions of H19 and Igf1r in a statistically significant manner . Spearman correlation coefficient , P - values , and sample numbers are marked on the top left of the plot .

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A-F (A, B, D-F) RNA levels determined by RT-qPCR. Results are presented as mean ± SD,n = 10. P-values are indicated on the top of each plot. (C) Spearman correlation suggests an in vivo positive correlation between the expressions of H19 and Igf1r in a statistically significant manner. Spearman correlation coefficient, P-values, and sample numbers are marked on the top left of the plot.


A - D Endometrial stromal cells from patients # 166 and # 80 were each transfected with a mixture of siCon ( control siRNA ) and microRNA inhibitor control ( iCon ) , siH19 and iCon , or siH19 and iLet7 ( let - 7 - specific inhibitor ) . RNA and proteins were analyzed at 48 h post - transfection . Combined results from the two patient cells are presented .

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A-D Endometrial stromal cells from patients #166 and #80 were each transfected with a mixture of siCon (control siRNA) and microRNA inhibitor control (iCon), siH19 and iCon, or siH19 and iLet7 (let-7-specific inhibitor). RNA and proteins were analyzed at 48 h post-transfection. Combined results from the two patient cells are presented.


A - D Endometrial stromal cells from patients # 166 and # 80 were each transfected with a mixture of siCon ( control siRNA ) and microRNA inhibitor control ( iCon ) , siH19 and iCon , or siH19 and iLet7 ( let - 7 - specific inhibitor ) . Western blot gels from # 166 cells are shown in ( C ) . Quantitation of Western blots combining both patient cells are shown in ( D ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-D Endometrial stromal cells from patients #166 and #80 were each transfected with a mixture of siCon (control siRNA) and microRNA inhibitor control (iCon), siH19 and iCon, or siH19 and iLet7 (let-7-specific inhibitor). Western blot gels from #166 cells are shown in (C). Quantitation of Western blots combining both patient cells are shown in (D).


E - H Endometrial stromal cells from patients # 98 and # 212S were each transfected with empty vector or pH19 . RNA and proteins were analyzed 48 h post - transfection . Combined results from the two patient cells are presented .

O O O O O O O O O O O O O O O O O O O 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-H Endometrial stromal cells from patients #98 and #212S were each transfected with empty vector or pH19. RNA and proteins were analyzed 48 h post-transfection. Combined results from the two patient cells are presented.


E - H Endometrial stromal cells from patients # 98 and # 212S were each transfected with empty vector or pH19 . RNA and proteins were analyzed 48 h post - transfection . Combined results from the two patient cells are presented . Western blot gels from # 98 cells are shown in ( G ) . Quantitation of Western blots combining both patient cells are shown in ( H ) .

E-H Endometrial stromal cells from patients #98 and #212S were each transfected with empty vector or pH19. RNA and proteins were analyzed 48 h post-transfection. Combined results from the two patient cells are presented. Western blot gels from #98 cells are shown in (G). Quantitation of Western blots combining both patient cells are shown in (H).


A - F The indicated cells were transfected with siCon , siH19 , empty vector , or pH19 . RNA , cell viability ( as indicated by viable cell numbers ) , and caspase activity ( as a readout for apoptosis ) were analyzed 48 h post - transfection . Combined results from two patient cells in each group are presented .

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A-F The indicated cells were transfected with siCon, siH19, empty vector, or pH19. RNA, cell viability (as indicated by viable cell numbers), and caspase activity (as a readout for apoptosis) were analyzed 48 h post-transfection. Combined results from two patient cells in each group are presented.


G E2 stimulates H19 expression in endometrial stromal cells . Endometrial stromal cells # 166 and # 98 were stimulated with E2 ( + ) or vehicle ( − ) for 48 h , followed by RT - qPCR analysis of RNA extracted from the cells . Results combining two patient cells are shown .

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G E2 stimulates H19 expression in endometrial stromal cells. Endometrial stromal cells #166 and #98 were stimulated with E2 (+) or vehicle (−) for 48 h, followed by RT-qPCR analysis of RNA extracted from the cells. Results combining two patient cells are shown.


( A ) Examples of chromosome arms exhibiting features of telomeric semiconservative ( Semi ) replication , conservative ( Consrv ) replication of the entire telomere ( E ) and conservative replication of part of the telomere ( P ) . Idealized chromosome diagrams ( left ) and actual microscopy images ( right ) are shown . D - FISH , denaturing FISH ; ND - CO - FISH ( non - denaturing CO - FISH ) . ( B ) Percentages of chromosome arms exhibiting conservative ( Consrv ) replication of the entire telomere ( E ) , conservative replication of part of the telomere ( P ) and conservative replication of either the entire telomere of part of it ( E + P ) . NE , U2OS cells expressing normal levels of cyclin E ; OE , U2OS cells overexpressing cyclin E for four days . Bars represent means and standard errors of the mean from three independent experiments . Cyclin E overexpression resulted in higher percentages of conservatively replicated telomeres : P < 0 . 025 for Consrv ( P ) and P < 0 . 05 for Consrv ( E + P ) , as calculated using the paired t test .

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(A) Examples of chromosome arms exhibiting features of telomeric semiconservative (Semi) replication, conservative (Consrv) replication of the entire telomere (E) and conservative replication of part of the telomere (P). Idealized chromosome diagrams (left) and actual microscopy images (right) are shown. D-FISH, denaturing FISH; ND-CO-FISH (non-denaturing CO-FISH).(B) Percentages of chromosome arms exhibiting conservative (Consrv) replication of the entire telomere (E), conservative replication of part of the telomere (P) and conservative replication of either the entire telomere of part of it (E+P). NE, U2OS cells expressing normal levels of cyclin E; OE, U2OS cells overexpressing cyclin E for four days. Bars represent means and standard errors of the mean from three independent experiments. Cyclin E overexpression resulted in higher percentages of conservatively replicated telomeres: P<0.025 for Consrv (P) and P<0.05 for Consrv (E+P), as calculated using the paired t test.


( A ) Decrease in the fraction of chromosome arms exhibiting conservative ( Consrv ) DNA replication of the entire telomere length or part of the telomere ( E + P ) in U2OS cells after depletion of POLD3 or POLD4 by siRNA . si Ctrl , control siRNA ; NE , cells expressing normal levels of cyclin E ; OE , cells overexpressing cyclin E for four days . Bars represent means and standard errors of the mean from two independent experiments . For statistical comparisons the effects of PolD3 and Pold4 depletion were examined after grouping together the data from the NE and OE cells . PolD3 depletion reduced the fraction of conservatively replicated telomeres at a level of P = 0 . 072 ; whereas for PolD4 depletion the decrease was significant at a level of P < 0 . 025 , as calculated using the paired t test . ( B ) Decreased telomere length following PolD3 or PolD4 depletion in U2OS cells expressing normal levels of cyclin E ( NE ) or overexpressing cyclin E ( OE ) . The cells were transfected with control siRNA ( si Ctrl ) or siRNAs targeting POLD3 or POLD4 and , five days later , fixed and examined for telomere length by Q - FISH . Bars indicate means and standard error of the mean from more than 2 , 600 telomeres examined per condition . The differences between control and PolD3 or PolD4 - depleted cells were significant ( P < 10 - 6 ) for both NE and OE cells , as determined by unpaired t tests .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Decrease in the fraction of chromosome arms exhibiting conservative (Consrv) DNA replication of the entire telomere length or part of the telomere (E+P) in U2OS cells after depletion of POLD3 or POLD4 by siRNA. si Ctrl, control siRNA; NE, cells expressing normal levels of cyclin E; OE, cells overexpressing cyclin E for four days. Bars represent means and standard errors of the mean from two independent experiments. For statistical comparisons the effects of PolD3 and Pold4 depletion were examined after grouping together the data from the NE and OE cells. PolD3 depletion reduced the fraction of conservatively replicated telomeres at a level of P=0.072; whereas for PolD4 depletion the decrease was significant at a level of P<0.025, as calculated using the paired t test.(B) Decreased telomere length following PolD3 or PolD4 depletion in U2OS cells expressing normal levels of cyclin E (NE) or overexpressing cyclin E (OE). The cells were transfected with control siRNA (si Ctrl) or siRNAs targeting POLD3 or POLD4 and, five days later, fixed and examined for telomere length by Q-FISH. Bars indicate means and standard error of the mean from more than 2,600 telomeres examined per condition. The differences between control and PolD3 or PolD4-depleted cells were significant (P<10-6) for both NE and OE cells, as determined by unpaired t tests.


( C ) Decreased telomere length following PolD3 or PolD4 depletion in parental U2OS cells . The cells were transfected with control siRNA ( si Ctrl ) or siRNAs targeting POLD3 or POLD4 and , five days later , fixed and examined for telomere length by Q - FISH . Bars indicate means and standard error of the mean from more than 2 , 800 telomeres examined per condition . The differences between control and PolD3 or PolD4 - depleted cells were significant ( P < 10 - 6 ) , as determined by unpaired t tests .

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(C) Decreased telomere length following PolD3 or PolD4 depletion in parental U2OS cells. The cells were transfected with control siRNA (si Ctrl) or siRNAs targeting POLD3 or POLD4 and, five days later, fixed and examined for telomere length by Q-FISH. Bars indicate means and standard error of the mean from more than 2,800 telomeres examined per condition. The differences between control and PolD3 or PolD4-depleted cells were significant (P<10-6), as determined by unpaired t tests.


( D ) Increased frequency of chromosome end - to - end fusions following POLD3 or POLD4 depletion . U2OS cells expressing normal levels of cyclin E ( NE ) or overexpressing cyclin E for four days ( OE ) were transfected with control siRNA ( siCtrl ) os siRNAs targeting POLD3 or POLD4 . For each condition , 25 metaphases were examined ( representing about 1 , 900 high quality chromosome arms per condition ) ; the percentage of chromosome fusions was determined for each metaphase and used to calculate means and standard errors of the mean . For statistical analysis , the number of chromosome end - to - end fusions were compared by chi square tests among the various conditions . Cyclin E overexpression led to a higher number of fusions ( P < 0 . 0005 ) ; PolD3 and PolD4 depletion also led to a higher number of fusions ( P < 0 . 001 and P < 0 . 025 , 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Increased frequency of chromosome end-to-end fusions following POLD3 or POLD4 depletion. U2OS cells expressing normal levels of cyclin E (NE) or overexpressing cyclin E for four days (OE) were transfected with control siRNA (siCtrl) os siRNAs targeting POLD3 or POLD4. For each condition, 25 metaphases were examined (representing about 1,900 high quality chromosome arms per condition); the percentage of chromosome fusions was determined for each metaphase and used to calculate means and standard errors of the mean. For statistical analysis, the number of chromosome end-to-end fusions were compared by chi square tests among the various conditions. Cyclin E overexpression led to a higher number of fusions (P<0.0005); PolD3 and PolD4 depletion also led to a higher number of fusions (P<0.001 and P<0.025, respectively).


( A Total PD - L1 , PARP from colon ( Colo - 205 ) , lung ( A549 ) , pancreatic ( PANK1 ) lysates treated with indicated DR5 agonist antibodies named Lexa ( lexatumumab ) , KMTR2 GAPDH is loading

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(A Total PD-L1, PARP from colon (Colo-205), lung (A549), pancreatic (PANK1) lysates treated with indicated DR5 agonist antibodies named Lexa (lexatumumab), KMTR2 GAPDH is loading control


Total PD - L1 , PARP from triple negative breast cancer cell ( MDA - MB - 436 ) lysates treated with indicated DR5 agonist antibodies named Lexa ( lexatumumab ) , KMTR2 , BaCa , GAPDH is loading

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Total PD-L1, PARP from triple negative breast cancer cell (MDA-MB-436) lysates treated with indicated DR5 agonist antibodies named Lexa (lexatumumab), KMTR2, BaCa, GAPDH is loading control


C ) Total PD - L1 , PARP , cleaved caspase - 3 from ovarian cell ( Cavo - 3 ) lysates treated with indicated DR5 agonist antibodies named tigatuzumab . GAPDH is loading

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C) Total PD-L1, PARP, cleaved caspase-3 from ovarian cell (Cavo-3) lysates treated with indicated DR5 agonist antibodies named tigatuzumab. GAPDH is loading control


( D ) Total PD - L1 , CD47 , Calreticulin , PARP from MDA - MB - 436 cell lysates treated with indicated DR5 agonist antibodies ± caspase inhibitor Z - VAD . GAPDH is loading control .

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(D) Total PD-L1, CD47, Calreticulin, PARP from MDA-MB-436 cell lysates treated with indicated DR5 agonist antibodies ± caspase inhibitor Z-VAD. GAPDH is loading control.


( E ) Total PD - L1 western blotting signal from the lysates of OVCAR - 3 ( n = 3 ) and MDA - MB - 436 ( n = 3 ) cells after 6 hrs treatment of 100nM lexa ( DR5 agonist antibody ) was normalized to GAPDH .

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(E) Total PD-L1 western blotting signal from the lysates of OVCAR-3 (n=3) and MDA-MB-436 (n=3) cells after 6 hrs treatment of 100nM lexa (DR5 agonist antibody) was normalized to GAPDH.


( F ) Surface biotinylation of PD - L1 from indicated tumor cells after indicated DR5 agonist treatments . Cleaved caspase - 3 and PARP indicates activation of DR5 signaling . In lane 3 and 4 , KMTR2 was pre - neutralized either with recombinant DR5 ( rDR5 ) or recombinant FOLR1 ( rFOLR1 ) .

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(F) Surface biotinylation of PD-L1 from indicated tumor cells after indicated DR5 agonist treatments. Cleaved caspase-3 and PARP indicates activation of DR5 signaling. In lane 3 and 4, KMTR2 was pre-neutralized either with recombinant DR5 (rDR5) or recombinant FOLR1 (rFOLR1).


( G ) Representative flow cytometry plots of PD - L1 from two different tumor cell lines treated with indicated DR5 antibodies . Secondary alone and IgG1 control are included .

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(G) Representative flow cytometry plots of PD-L1 from two different tumor cell lines treated with indicated DR5 antibodies. Secondary alone and IgG1 control are included.


( H ) Relative surface PD - L1 % cells , after DR5 agonist treatments Relative signal is normalized to surface PD - L1 in IgG1 treated cells in corresponding tumor cells and horizontal line in samples indicated mean ( n = 3 - 4 ) .

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(H) Relative surface PD-L1 % cells, after DR5 agonist treatments Relative signal is normalized to surface PD-L1 in IgG1 treated cells in corresponding tumor cells and horizontal line in samples indicated mean (n=3-4).


( J ) Relative surface PD - L1 % cells in indicated tumors after DR5 agonist treatments Relative signal is normalized to surface PD - L1 in IgG1 treated tumors and horizontal line in samples indicated mean ( n = 2 - 6 )

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(J) Relative surface PD-L1 % cells in indicated tumors after DR5 agonist treatments Relative signal is normalized to surface PD-L1 in IgG1 treated tumors and horizontal line in samples indicated mean (n=2-6)


( K ) ER ( - ) , PR ( - ) and HER2 ( - ) UCD52 patient derived tumor tissue was xenografted in breast fat pad of NOD . Cg - Prkdcscid Il2rgtm1Wjl / SzJ mice , following by treatment with IgG1 or KMTR2 ( 50μg , 4 doses ) . Harvested tumors were analyzed for PD - L1 , CD47 and PARP in lysates .

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(K) ER (-), PR (-) and HER2 (-) UCD52 patient derived tumor tissue was xenografted in breast fat pad of NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice, following by treatment with IgG1 or KMTR2 (50μg, 4 doses). Harvested tumors were analyzed for PD-L1, CD47 and PARP in lysates.


( L ) KMTR2 treated ( 50μg , 4 doses ) UCD52 TNBC PDX tumors were stained for PD - L1 using immunohistochemistry ( IHC ) .

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(L) KMTR2 treated (50μg, 4 doses) UCD52 TNBC PDX tumors were stained for PD-L1 using immunohistochemistry (IHC).


( M ) Total PD - L1 and CD47 blotting analysis from DR5 resistant MDA - MB - 436 cell lines after indicated DR5 agonist treatment ( 50nM , 6hr ) .

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(M) Total PD-L1 and CD47 blotting analysis from DR5 resistant MDA-MB-436 cell lines after indicated DR5 agonist treatment (50nM, 6hr).


( C ) Luciferase activity of reporter lines from tumor cell - Jurkat cell co - culture assay using MDA - MB - 436 and OVCAR3 cells after treatment ( 50nM ) with indicated DR5 agonists ( tiga : tigatuzumab , AMG : AMG655 , KMTR1 , Lexa : Lexatumumab ) . The background luciferase signal from untreated cells ( due to basal surface PD - L1 ) was subtracted . Various controls treatments ( IgG1 , anti - PD - L1 , anti - EGFR ) are also shown ( n = 3 ) . ( D ) Same as ( C ) except tumor cells were pre - treated with indicated inhibitors for AKT , ERK , mTOR , MEK , STAT3 , PARP - i and NF - Kβ along with DR5 agonist KMTR2 antibody prior to co - culture .

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(C) Luciferase activity of reporter lines from tumor cell-Jurkat cell co-culture assay using MDA-MB-436 and OVCAR3 cells after treatment (50nM) with indicated DR5 agonists (tiga: tigatuzumab, AMG: AMG655, KMTR1, Lexa: Lexatumumab). The background luciferase signal from untreated cells (due to basal surface PD-L1) was subtracted. Various controls treatments (IgG1, anti-PD-L1, anti-EGFR) are also shown (n=3). (D) Same as (C) except tumor cells were pre-treated with indicated inhibitors for AKT, ERK, mTOR, MEK, STAT3, PARP-i and NF-Kβ along with DR5 agonist KMTR2 antibody prior to co-culture.


MDA - MB - 436 cells were treated with TNFα and indicated DR5 agonists for indicated times . Lysates were analyzed for PD - L1 , CSN5 GAPDH is loading control in

MDA-MB-436 cells were treated with TNFα and indicated DR5 agonists for indicated times. Lysates were analyzed for PD-L1, CSN5 GAPDH is loading control in E


MDA - MB - 436 cells were treated with TNFα and indicated DR5 agonists for indicated times . Lysates were analyzed for PD - L1 phosphorylated p65 , total p65 , cleaved caspase - 3 and PARP . Tubulin is loading control in F .

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MDA-MB-436 cells were treated with TNFα and indicated DR5 agonists for indicated times. Lysates were analyzed for PD-L1 phosphorylated p65, total p65, cleaved caspase-3 and PARP. Tubulin is loading control in F.


MDA - MB - 436 cells were treated with TNFα and indicated DR5 agonists for indicated times . Lysates were analyzed for PD - L1 , CSN5 , phosphorylated p65 and PARP . GAPDH is loading control in G .

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MDA-MB-436 cells were treated with TNFα and indicated DR5 agonists for indicated times. Lysates were analyzed for PD-L1, CSN5, phosphorylated p65 and PARP. GAPDH is loading control in G.


( H ) Flow cytometry analysis of MDA - MB - 436 cells treated with TNFα ± MG132 ( top ) and indicated DR5 agonist ± MG132 ( bottom ) .

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(H) Flow cytometry analysis of MDA-MB-436 cells treated with TNFα ± MG132 (top) and indicated DR5 agonist ± MG132 (bottom).


( I ) Total PD - L1 from OVCAR3 cell lysates was analyzed after treatment of indicated DR5 agonist ± MG132 .

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(I) Total PD-L1 from OVCAR3 cell lysates was analyzed after treatment of indicated DR5 agonist ± MG132.


( J ) Flow cytometry surface PD - L1 analysis of OVCAR - 3 cells treated with indicated DR5 agonist ± MG132 .

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(J) Flow cytometry surface PD-L1 analysis of OVCAR-3 cells treated with indicated DR5 agonist ± MG132.


( K ) MDA - MB - 436 cells were treated with MG132 for indicated times and total PD - L1 from lysates was analyzed . GAPDH is loading control .

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(K) MDA-MB-436 cells were treated with MG132 for indicated times and total PD-L1 from lysates was analyzed. GAPDH is loading control.


( L ) Immunoblotting of DR5 confirming generation of knock out ( DR5 - KO ) cell lines .

(L) Immunoblotting of DR5 confirming generation of knock out (DR5-KO) cell lines.


( M ) Cell viability analysis of DR5 - KO and DR5 - WT cells ( n = 3 ) .

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(M) Cell viability analysis of DR5-KO and DR5-WT cells (n=3).


( N ) DR5 - KO and WT cells were treated with TNFα and indicated DR5 agonist followed by flow cytometry using PD - L1 specific antibodies .

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(N) DR5-KO and WT cells were treated with TNFα and indicated DR5 agonist followed by flow cytometry using PD-L1 specific antibodies.


( O ) TNBC WT and DR5 - KO ( MDA - MB - 436 ) cells were treated with either DR5 agonist or TNFα as indicated . Lysates were analyzed for PD - L1 , S5a and DR5 . GAPDH is loading control .

(O) TNBC WT and DR5-KO (MDA-MB-436) cells were treated with either DR5 agonist or TNFα as indicated. Lysates were analyzed for PD-L1, S5a and DR5. GAPDH is loading control.


( P ) OVCAR - 3 and MDA - MB - 436 ( WT and DR5 - KO ) cells were treated with KMTR2 followed by ubiquitin and S5a immunoblotting from total lysates . GAPDH is loading control .

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(P) OVCAR-3 and MDA-MB-436 (WT and DR5-KO) cells were treated with KMTR2 followed by ubiquitin and S5a immunoblotting from total lysates. GAPDH is loading control.


ApoEVs isolated after IgG1 and KMTR2 treatment ( OVCAR - 3 ) were blotted against CD63 and PD - L1 in dot blots .

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ApoEVs isolated after IgG1 and KMTR2 treatment (OVCAR-3) were blotted against CD63 and PD-L1 in dot blots.


ApoEVs isolated after IgG1 and tigatuzumab treatment ( MDA - MB - 436 ) were run on western and blotted against CD63 and PD - L1 .

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ApoEVs isolated after IgG1 and tigatuzumab treatment (MDA-MB-436) were run on western and blotted against CD63 and PD-L1.


ApoEVs isolated after IgG1 and lexatumumab ( D ) treatment ( MDA - MB - 436 ) were run on western and blotted against CD63 and PD - L1 .

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ApoEVs isolated after IgG1 and lexatumumab (D) treatment (MDA-MB-436) were run on western and blotted against CD63 and PD-L1.


( F ) ApoEVs isolated from DR5 sensitive tumor cells grown in Met - HPG + media were added on to DR5 - KO cells ( growing in regular media ) . After 48 hrs flow cytometry analysis was carried out with HPG catalyzing dye . HPG incorporation from flow cytometry data confirms ApoEVs transfer from cells growing in Met - HPG + ( DR5 - WT ) to DR5 - KO cells .

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(F) ApoEVs isolated from DR5 sensitive tumor cells grown in Met-HPG+ media were added on to DR5-KO cells (growing in regular media). After 48 hrs flow cytometry analysis was carried out with HPG catalyzing dye. HPG incorporation from flow cytometry data confirms ApoEVs transfer from cells growing in Met-HPG+ (DR5-WT) to DR5-KO cells.


( G ) ApoEVs isolated from DR5 - WT OVCAR - 3 cells ( KMTR2 treated ) were added on to DR5 - KO ( MDA - MB - 231 ) cells for indicated times ( 24 - 96 hrs ) to analyze PD - L1 transfer kinetics from

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(G) ApoEVs isolated from DR5-WT OVCAR-3 cells (KMTR2 treated) were added on to DR5-KO (MDA-MB-231) cells for indicated times (24-96 hrs) to analyze PD-L1 transfer kinetics from ApoEVs


after addition of MDA - MB - 436 derived ApoEVs ( treated with combination of KMTR2 + lexatumumab DR5 agonists or IgG1 ) on to DR5 - KO MDA - MB - 231 cells , total lysates were immunoblotted for PD - L1 after 24 hrs . KMTR2 + lexatumumab ( n = 3 ) , IgG1 ( n = 2 ) .

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after addition of MDA-MB-436 derived ApoEVs (treated with combination of KMTR2+lexatumumab DR5 agonists or IgG1) on to DR5-KO MDA-MB-231 cells, total lysates were immunoblotted for PD-L1 after 24 hrs. KMTR2+lexatumumab (n=3), IgG1 (n=2).


( I ) Left : PD - L1 surface histogram shows direct antibody treatment on DR5 - KO cells . Right : PD - L1 surface histogram from DR5 - KO cells after treatment ( 24 hrs ) with ApoEVs isolated from DR5 sensitive cells after either Lexa or KMTR2 treatment .

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(I) Left: PD-L1 surface histogram shows direct antibody treatment on DR5-KO cells. Right: PD-L1 surface histogram from DR5-KO cells after treatment (24 hrs) with ApoEVs isolated from DR5 sensitive cells after either Lexa or KMTR2 treatment.


ApoEVs treated tumor cells were analyzed in PD - 1 reporter co - culture assays for luciferase

ApoEVs treated tumor cells were analyzed in PD-1 reporter co-culture assays for luciferase signal


( K ) MDA - MB - 436 cells were treated with KMTR2 for 6 hrs and along with ZDEVD for indicated times . - ZDEVD shows final time of KMTR2 exposure to cells in absence inhibitor . Lysates were immunoblotted for cleaved caspase - 3 , 8 , PARP , PD - L1 , S5a , ROCK1 and GAPDH ( n = 3 ) . u . c : uncleaved , c : cleaved , n . s : non - specific

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(K) MDA-MB-436 cells were treated with KMTR2 for 6 hrs and along with ZDEVD for indicated times. - ZDEVD shows final time of KMTR2 exposure to cells in absence inhibitor. Lysates were immunoblotted for cleaved caspase-3, 8, PARP, PD-L1, S5a, ROCK1 and GAPDH (n=3). u.c: uncleaved, c: cleaved, n.s: non-specific band


( L ) Relative caspase - 8 and caspase - 3 activity assays caspase - 8 maintained steady activity , while caspase - 3 activity required at least 40 ( + ) mins of DR5 agonist treatment without ZDEVD ( n = 3 ) .

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(L) Relative caspase-8 and caspase-3 activity assays caspase-8 maintained steady activity, while caspase-3 activity required at least 40 (+) mins of DR5 agonist treatment without ZDEVD (n=3).


( M ) After indicated treatments with KMTR2 ( DR5 agonist ) and ZDEVD ( caspase - 3 preferred inhibitor ) , cells were allowed to grow 24 hrs , followed by cell viability analysis .

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(M) After indicated treatments with KMTR2 (DR5 agonist) and ZDEVD (caspase-3 preferred inhibitor) , cells were allowed to grow 24 hrs, followed by cell viability analysis.


( A KMTR2 , KMTR2 + Z - VAD treated MDA - MB - 436 lysates for indicated early time points were analyzed for caspase - 8 , caspase - 3 , ROCK1 PARP as indicated .

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(A KMTR2, KMTR2+Z-VAD treated MDA-MB-436 lysates for indicated early time points were analyzed for caspase-8, caspase-3, ROCK1 PARP as indicated.


B ) lexatumumab treated OVCAR3 lysates for indicated early time points were analyzed for caspase - 3 , ROCK1 , pMLC , PARP , PD - L1 and CMTM6 etc . as indicated .

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B) lexatumumab treated OVCAR3 lysates for indicated early time points were analyzed for caspase-3, ROCK1, pMLC, PARP, PD-L1 and CMTM6 etc. as indicated.


( C ) OVCAR3 cells were treated with pMLC activating ionophore A23187 ( positive control ) and lexatumumab ± ROCK1 inhibitors or ± rDR5 or ± rFOLR1 . Lysates were later analyzed for ROCK1 , pMLC , caspase - 3 and PARP with GAPDH as loading control . Blue arrows indicate cleaved and activated ROCK1 and caspase - 3 .

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(C) OVCAR3 cells were treated with pMLC activating ionophore A23187 (positive control) and lexatumumab ± ROCK1 inhibitors or ± rDR5 or ± rFOLR1. Lysates were later analyzed for ROCK1, pMLC, caspase-3 and PARP with GAPDH as loading control. Blue arrows indicate cleaved and activated ROCK1 and caspase-3.


( D ) MDA - MB - 436 and OVCAR3 cells were treated with indicated ROCK1 inhibitors 2 hours prior to DR5 agonist KMTR2 treatment ( 20nM ) . After 6 hours flow cytometry was used to analyze surface PD - L1 . ( GSK269 : GSK269962A , GSK429 : GSK429286A )

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(D) MDA-MB-436 and OVCAR3 cells were treated with indicated ROCK1 inhibitors 2 hours prior to DR5 agonist KMTR2 treatment (20nM). After 6 hours flow cytometry was used to analyze surface PD-L1. (GSK269: GSK269962A, GSK429: GSK429286A)


( E ) MDA - MB - 436 cell survival assay after treatment with DR5 agonist antibody KMTR2 ± ROCK1 inhibitors , error bars indicate SD ( n = 2 ) . ( GSK269 : GSK269962A , GSK429 : GSK429286A )

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(E) MDA-MB-436 cell survival assay after treatment with DR5 agonist antibody KMTR2 ± ROCK1 inhibitors, error bars indicate SD (n=2). (GSK269: GSK269962A, GSK429: GSK429286A)


Cultured MDA - MB - 436 cells were treated with KMTR2 ( IgG1 - Fc ) or IgG1 control ± ROCK1i ( GSK269962 ) for 6 hrs . After 6 hrs , 500nM anti - PD - L1 avelumab ( IgG4 - Fc ) was added to the media for additional 1 hr . Cellular lysates were pulled down with anti - hu - IgG4 specific beads . ( G ) Immunoprecipitated lysates and leftover supernatant after various treatments were run at the same time followed by blotting with ROCK1 , PD - L1 and CMTM6 . Total lysates as a control were also generated in exactly similar conditions and run next to supernatant and IP samples . Blue arrows indicate cleaved and activated ROCK1 .

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Cultured MDA-MB-436 cells were treated with KMTR2 (IgG1-Fc) or IgG1 control ± ROCK1i (GSK269962) for 6 hrs. After 6 hrs, 500nM anti-PD-L1 avelumab (IgG4-Fc) was added to the media for additional 1 hr. Cellular lysates were pulled down with anti-hu-IgG4 specific beads. (G) Immunoprecipitated lysates and leftover supernatant after various treatments were run at the same time followed by blotting with ROCK1, PD-L1 and CMTM6. Total lysates as a control were also generated in exactly similar conditions and run next to supernatant and IP samples. Blue arrows indicate cleaved and activated ROCK1.