( I , J ) Cox6a2 - / - PV + interneurons showed a negative correlation between age and dendritic length and between age and full branch depth ( maximal number of branches between the soma and a terminal dendritic point ) , which contrasted with the expected positive correlation between these parameters in WT PV + interneurons . Linear regression of Pearson correlation .

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(I,J) Cox6a2-/- PV+ interneurons showed a negative correlation between age and dendritic length and between age and full branch depth (maximal number of branches between the soma and a terminal dendritic point), which contrasted with the expected positive correlation between these parameters in WT PV+ interneurons. Linear regression of Pearson correlation.


( K , L ) WT and Cox6a2 - / - PV + interneurons showed similar values for dendritic length and full branch depth at P22 - P33 . However , at P34 - P45 , Cox6a2 - / - PV + interneurons showed lower dendritic length and full branch depth than WT PV + interneurons . Two - way ANOVA followed by Tukey ' s multiple comparisons tests .

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(K,L) WT and Cox6a2-/- PV+ interneurons showed similar values for dendritic length and full branch depth at P22-P33. However, at P34-P45, Cox6a2-/- PV+ interneurons showed lower dendritic length and full branch depth than WT PV+ interneurons. Two-way ANOVA followed by Tukey's multiple comparisons tests.


( M , N ) While younger WT and Cox6a2 - / - PV + interneurons have similar dendritic tree complexities ( M ) , at the later stage of maturation Cox6a2 - / - PV + interneurons show a reduction in dendritic tree complexity in comparison to WT PV + interneurons ( N ) . Multiple t - tests , two - stage step - up method of Benjamini , Krieger and Yekutieli .

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(M,N) While younger WT and Cox6a2-/- PV+ interneurons have similar dendritic tree complexities (M), at the later stage of maturation Cox6a2-/- PV+ interneurons show a reduction in dendritic tree complexity in comparison to WT PV+ interneurons (N). Multiple t-tests, two-stage step-up method of Benjamini, Krieger and Yekutieli.


( B ) In the OFT , Cox6a2 - / - mice travelled a longer distance and moved faster than WT mice . Cox6a2 - / - mice also moved more often from the outer zone to the inner zone of the arena . Two - tailed unpaired Student ' s t - tests .

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(B) In the OFT, Cox6a2-/- mice travelled a longer distance and moved faster than WT mice. Cox6a2-/- mice also moved more often from the outer zone to the inner zone of the arena. Two-tailed unpaired Student's t-tests.


( D ) In the SIT , Cox6a2 - / - mice also travelled a longer distance with higher mean velocity than WT mice . Additionally , both Cox6a2 - / - and WT mice spent more time exploring the container with the stranger mouse than the empty container . Total distance moved , mean velocity , and f [ number of crossings ] outer to inner zone : two - tailed unpaired Student ' s t - test . Social interaction : two - way ANOVA followed by Tukey ' s multiple comparisons test .

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(D) In the SIT, Cox6a2-/- mice also travelled a longer distance with higher mean velocity than WT mice. Additionally, both Cox6a2-/- and WT mice spent more time exploring the container with the stranger mouse than the empty container. Total distance moved, mean velocity, and f [number of crossings] outer to inner zone: two-tailed unpaired Student's t-test. Social interaction: two-way ANOVA followed by Tukey's multiple comparisons test.


( F ) The startle responses were significantly lower in Cox6a2 - / - mice when the loud stimulus ( 120 dB ) was presented alone the first 5 times . Two - way ANOVA followed by Tukey ' s multiple comparisons tests .

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(F) The startle responses were significantly lower in Cox6a2-/- mice when the loud stimulus (120 dB) was presented alone the first 5 times. Two-way ANOVA followed by Tukey's multiple comparisons tests.


( G ) The PPI of the acoustic startle response was similar at all pre - pulses for Cox6a2 - / - and WT mice . Two - way ANOVA followed by Sidak ' s multiple comparisons tests .

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(G) The PPI of the acoustic startle response was similar at all pre-pulses for Cox6a2-/- and WT mice. Two-way ANOVA followed by Sidak's multiple comparisons tests.


Survival curves for control ( n = 23 biological replicates ) , Rorc ( t ) CreTg Tet1 / 3fl / fl ( n = 20 biological replicates ) , Rorc ( t ) CreTg Tet1 / 2 / 3fl / fl mice ( n = 10 biological replicates ) . Paired male or female mice were used and no gender biases associated with genotypes were observed .

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Survival curves for control (n=23 biological replicates), Rorc(t)CreTg Tet1/3fl/fl(n=20 biological replicates), Rorc(t)CreTg Tet1/2/3fl/fl mice (n=10 biological replicates). Paired male or female mice were used and no gender biases associated with genotypes were observed.


Representative H & E staining of the lungs ( top panel ) and small intestine ( bottom panel ) and histopathological scoring of 3 months old control ( n = 3 biological replicates ) and Rorc ( t ) CreTg Tet1 / 3fl / fl mice ( n = 3 biological replicates ) . Data are means + / - SEM , One - Way ANOVA with uncorrected Fisher ' s LSD test . Scale bar = 263 and 53 µm , top and bottom panels , respectively . , Paired male or female mice were used and no gender biases associated with genotypes were observed .

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Representative H&E staining of the lungs (top panel) and small intestine (bottom panel) and histopathological scoring of 3 months old control (n=3 biological replicates) and Rorc(t)CreTg Tet1/3fl/fl mice (n=3 biological replicates). Data are means +/-SEM, One-Way ANOVA with uncorrected Fisher's LSD test. Scale bar = 263 and 53 µm, top and bottom panels, respectively. , Paired male or female mice were used and no gender biases associated with genotypes were observed.


Frequency of FoxP3 + cells among CD4 + T cells in the thymus ( n = 6 or 10 biological replicates ) , peripheral lymph nodes ( n = 5 or 6 biological replicates ) , spleen ( n = 5 or 6 biological replicates ) and mesenteric lymph nodes ( n = 4 or 8 biological replicates ) pre - gated on live CD4 + TCRβ + T cells and representative flow plots ( bottom ) . Data are means + / - SEM , One - Way ANOVA with uncorrected Fisher ' s LSD test . Paired male or female mice were used and no gender biases associated with genotypes were observed .

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Frequency of FoxP3+ cells among CD4+ T cells in the thymus (n=6 or 10 biological replicates), peripheral lymph nodes (n=5 or 6 biological replicates), spleen (n=5 or 6 biological replicates) and mesenteric lymph nodes (n=4 or 8 biological replicates) pre-gated on live CD4+TCRβ+ T cells and representative flow plots (bottom). Data are means +/- SEM, One-Way ANOVA with uncorrected Fisher's LSD test. Paired male or female mice were used and no gender biases associated with genotypes were observed.


Absolute number of FoxP3 + Tregs from the thymus ( n = 5 or 7 biological replicates ) and spleen ( n = 7 or 8 biological replicates ) of control Tet1 / 3fl / fl and Rorc ( t ) CreTg Tet1 / 3fl / fl mice . Data is means + / - SEM , unpaired Mann - Whitney t test . Paired male or female mice were used and no gender biases associated with genotypes were observed .

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Absolute number of FoxP3+ Tregs from the thymus (n=5 or 7 biological replicates) and spleen (n=7 or 8 biological replicates) of control Tet1/3fl/fl and Rorc(t)CreTg Tet1/3fl/fl mice. Data is means +/- SEM, unpaired Mann-Whitney t test. Paired male or female mice were used and no gender biases associated with genotypes were observed.


Quantification of CD25 MFI expression on FoxP3 + T cells in the spleen of control Tet1 / 3fl / fl ( n = 4 biological replicates ) and Rorc ( t ) CreTg Tet1 / 3fl / fl mice ( n = 4 biological replicates ) . Data is means + / - SEM , unpaired Mann - Whitney t test . Paired male or female mice were used and no gender biases associated with genotypes were observed .

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Quantification of CD25 MFI expression on FoxP3+ T cells in the spleen of control Tet1/3fl/fl (n=4 biological replicates) and Rorc(t)CreTg Tet1/3fl/fl mice (n=4 biological replicates). Data is means +/- SEM, unpaired Mann-Whitney t test. Paired male or female mice were used and no gender biases associated with genotypes were observed.


Quantification of FoxP3 MFI expression on FoxP3 + T cells in the spleen of control Tet1 / 3fl / fl ( n = 6 biological replicates ) and Rorc ( t ) CreTg Tet1 / 3fl / fl mice ( n = 6 biological replicates ) . Data is means + / - SEM , unpaired Mann - Whitney t test . Quantification of ICOS MFI expression on FoxP3 + T cells from the mesenteric lymph nodes of control Tet1 / 3fl / fl ( n = 5 biological replicates ) and Rorc ( t ) CreTg Tet1 / 3fl / fl ( n = 7 biological replicates ) mice . Data is means + / - SEM , unpaired Mann - Whitney t test . , Paired male or female mice were used and no gender biases associated with genotypes were observed .

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Quantification of FoxP3 MFI expression on FoxP3+ T cells in the spleen of control Tet1/3fl/fl (n=6 biological replicates) and Rorc(t)CreTg Tet1/3fl/fl mice (n=6 biological replicates). Data is means +/- SEM, unpaired Mann-Whitney t test.   Quantification of ICOS MFI expression on FoxP3+ T cells from the mesenteric lymph nodes of control Tet1/3fl/fl (n=5 biological replicates) and Rorc(t)CreTg Tet1/3fl/fl (n=7 biological replicates) mice. Data is means +/- SEM, unpaired Mann-Whitney t test.   , Paired male or female mice were used and no gender biases associated with genotypes were observed.


Representative flow plots , histogram and quantification of CD4 gMFI expression in Treg cells activated as in ( A ) . Data is means + / - SEM , unpaired t - test .

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Representative flow plots, histogram and quantification of CD4 gMFI expression in Treg cells activated as in (A). Data is means+/-SEM, unpaired t-test.


Weight curve of female Rag - / - mice after T - cell transfer colitis induced via transfer of naïve WT CD45 . 1 conventional CD4 + T cells alone ( n = 5 recipients ) or in combination with Tregs isolated from Tet1 / 3fl / fl and Rorc ( t ) CreTg Tet1 / 3fl / fl littermates ( n = 5 recipients / group ) . Data is means + / - SEM , Two - Way ANOVA with Geisser - Greenhouse correction . Experiment was replicated 2 times .

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Weight curve of female Rag-/- mice after T-cell transfer colitis induced via transfer of naïve WT CD45.1 conventional CD4+ T cells alone (n=5 recipients) or in combination with Tregs isolated from Tet1/3fl/fl and Rorc(t)CreTg Tet1/3fl/fl littermates (n=5 recipients/group). Data is means +/- SEM, Two-Way ANOVA with Geisser-Greenhouse correction. Experiment was replicated 2 times.


Representative flow plots and quantification of percent Tbet + , Rorγt + and FoxP3 + of CD45 . 1 CD4 + effector cells isolated from the MLN of female Rag - / - mice 7 weeks post T cell transfer induced colitis . Respective cohorts were Naïve CD45 . 1 T cells only ( n = 5 mice ) , WT CD45 . 1 + Tet1 / 3fl / fl Tregs ( n = 5 mice ) and WT CD45 . 1 + Rorc ( t ) CreTg Tet1 / 3fl / fl Tregs ( n = 4 mice ) . Cells were pre - gated on Live CD45 . 1 + TCRβ + cells . Data is means + / - SEM , Two - Way ANOVA with Tukey ' s Multiple comparison test . Experiment was replicated 2 times .

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Representative flow plots and quantification of percent Tbet+, Rorγt+ and FoxP3+ of CD45.1 CD4+ effector cells isolated from the MLN of female Rag-/- mice 7 weeks post T cell transfer induced colitis. Respective cohorts were Naïve CD45.1 T cells only (n=5 mice), WT CD45.1+ Tet1/3fl/fl Tregs (n=5 mice) and WT CD45.1+Rorc(t)CreTg Tet1/3fl/fl Tregs (n=4 mice). Cells were pre-gated on Live CD45.1+TCRβ+ cells. Data is means+/- SEM, Two-Way ANOVA with Tukey's Multiple comparison test. Experiment was replicated 2 times.


Quantification of Live CD4 + FoxP3 + T cells absolute cell numbers in different organs of FoxP3Cre Tet1 / 3 + / + ( n = 3 biological replicates ) and FoxP3Cre Tet1 / 3 fl / fl ( n = 3 biological replicates ) male littermate mice . Data is shown as means + / - SEM , One - Way ANOVA with uncorrected Fisher ' s LSD test .

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Quantification of Live CD4+ FoxP3+ T cells absolute cell numbers in different organs of FoxP3Cre Tet1/3+/+ (n=3 biological replicates) and FoxP3Cre Tet1/3 fl/fl (n=3 biological replicates) male littermate mice. Data is shown as means+/- SEM, One-Way ANOVA with uncorrected Fisher's LSD test.


Quantification of CD25 - FoxP3lo , CD25 + FoxP3 - and CD25 + FoxP3 + cells among Live CD73 - CD4 + T cells thymocytes of control Tet3fl / fl ( n = 5 biological replicates ) and Rorc ( t ) CreTg Tet3fl / fl mice ( n = 3 biological replicates ) mice . Data is shown as means + / - SEM , Two - Way ANOVA with Sidak ' s multiple comparison test . Quantification of CD25 - FoxP3lo , CD25 + FoxP3 - and CD25 + FoxP3 + cells among Live CD73 - CD4 + T cells thymocytes of control Tet1fl / fl ( n = 6 biological replicates ) and Rorc ( t ) CreTg Tet1fl / fl mice ( n = 6 biological replicates ) . Data is shown as means + / - SEM , Two - Way ANOVA with Sidak ' s multiple comparison test . paired male or female mice were used and no gender biases associated with genotypes were observed .

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Quantification of CD25-FoxP3lo, CD25+FoxP3- and CD25+FoxP3+ cells among Live CD73-CD4+ T cells thymocytes of control Tet3fl/fl (n=5 biological replicates) and Rorc(t)CreTg Tet3fl/fl mice (n=3 biological replicates) mice. Data is shown as means+/- SEM, Two-Way ANOVA with Sidak's multiple comparison test. Quantification of CD25-FoxP3lo, CD25+FoxP3- and CD25+FoxP3+ cells among Live CD73-CD4+ T cells thymocytes of control Tet1fl/fl (n=6 biological replicates) and Rorc(t)CreTg Tet1fl/fl mice (n=6 biological replicates). Data is shown as means+/- SEM, Two-Way ANOVA with Sidak's multiple comparison test. paired male or female mice were used and no gender biases associated with genotypes were observed.


Quantification of Nur77 staining among TCRβhi CD69 + CD4 SP thymocytes from control Tet3fl / fl ( n = 5 biological replicates ) and Rorc ( t ) CreTg Tet3fl / fl mice ( n = 3 biological replicates ) . Data is as means + / - SEM , unpaired two - tailed Mann - Whitney t - test Quantification of Nur77 staining among TCRβhi CD69 + CD4 SP thymocytes from control Tet1fl / fl ( n = 4 biological replicates ) and Rorc ( t ) CreTg Te1fl / fl mice ( n = 3 biological replicates ) . Data is shown as means + / - SEM , unpaired two - tailed Mann - Whitney t - test paired male or female mice were used and no gender biases associated with genotypes were observed .

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Quantification of Nur77 staining among TCRβhi CD69+ CD4 SP thymocytes from control Tet3fl/fl (n=5 biological replicates) and Rorc(t)CreTg Tet3fl/fl mice (n=3 biological replicates). Data is as means+/- SEM, unpaired two-tailed Mann-Whitney t-test Quantification of Nur77 staining among TCRβhi CD69+ CD4 SP thymocytes from control Tet1fl/fl (n=4 biological replicates) and Rorc(t)CreTg Te1fl/fl mice (n=3 biological replicates). Data is shown as means+/- SEM, unpaired two-tailed Mann-Whitney t-test paired male or female mice were used and no gender biases associated with genotypes were observed.


Quantification of % FoxP3 + cells 72hrs post activation of sorted naïve CD4 + T cells with anti - CD3 / CD28 and 20 ng / mL TGF - β in the presence of absence of indicated amounts of rhIL - 2 for 72hrs ( n = 3 biological replicates per dose ) . Data is shown as means + / - SEM of 3 independent samples , Two - Way ANOVA with uncorrected Fisher ' s LSD test .

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Quantification of % FoxP3+ cells 72hrs post activation of sorted naïve CD4+ T cells with anti-CD3/CD28 and 20 ng/mL TGF-β in the presence of absence of indicated amounts of rhIL-2 for 72hrs (n=3 biological replicates per dose). Data is shown as means+/-SEM of 3 independent samples, Two-Way ANOVA with uncorrected Fisher's LSD test.


IL - 2 mRNA quantification by qPCR in CD4 + T cells post - activation at indicated time - points ( n = 2 biological replicates / genotype ) . Cells were stimulated with anti - CD3 / CD28 without exogenous IL - 2 or TGF - β . Data is shown as means + / - SEM , Two - Way ANOVA with Sidak ' s multiple comparison

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IL-2 mRNA quantification by qPCR in CD4+ T cells post-activation at indicated time-points (n=2 biological replicates/genotype). Cells were stimulated with anti-CD3/CD28 without exogenous IL-2 or TGF-β. Data is shown as means+/-SEM, Two-Way ANOVA with Sidak's multiple comparison test


IL - 2 quantification by ELISA in supernatants collected at 48 hours post activation of naïve CD4 + T cells with anti - CD3 / CD28 without exogenous IL - 2 and in the presence or absence of 20ng / mL TGF - β ( n = 4 biological replicates ) . Data is shown as means + / - SEM , Two - Way ANOVA with Bonferroni ' s multiple comparison

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IL-2 quantification by ELISA in supernatants collected at 48 hours post activation of naïve CD4+ T cells with anti-CD3/CD28 without exogenous IL-2 and in the presence or absence of 20ng/mL TGF-β (n=4 biological replicates). Data is shown as means+/-SEM, Two-Way ANOVA with Bonferroni's multiple comparison test


IL - 2 quantification by ELISA in supernatants collected at 5 hours post activation of naïve CD4 T cells with PMA / Ionomycin ( n = 7 ) . Data is shown as means + / - SEM , Two - Way ANOVA with Sidak ' s multiple comparison test IL - 2 quantification by ELISA in supernatants collected at 72hrs following activation of CD4 T cells with anti - CD3 / CD28 without exogenous IL - 2 or TGF - β ( n = 3 ) . Data is shown as means + / - SEM , One - Way ANOVA with Tukey ' s Multiple comparison test .

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IL-2 quantification by ELISA in supernatants collected at 5 hours post activation of naïve CD4 T cells with PMA/Ionomycin (n=7). Data is shown as means+/-SEM, Two-Way ANOVA with Sidak's multiple comparison test   IL-2 quantification by ELISA in supernatants collected at 72hrs following activation of CD4 T cells with anti-CD3/CD28 without exogenous IL-2 or TGF-β (n=3). Data is shown as means+/-SEM, One-Way ANOVA with Tukey's Multiple comparison test.


Quantification of % FoxP3 + cells among CD4 T cells 72hrs activation with anti - CD3 / CD28 and 20 ng / mL TGF - β in the presence or absence of 50U rhIL - 2 for 72hrs ( n = 3 biological replicates ) . Data is shown as means + / - SEM , One - Way ANOVA with Tukey ' s Multiple comparison test . Quantification of % FoxP3 + cells among CD4 T cells 72hrs activation with anti - CD3 / CD28 and 20 ng / mL TGF - β in the presence or absence of 50U rhIL - 2 for 72hrs ( n = 3 biological replicates ) . Data is shown as means + / - SEM , One - Way ANOVA with Tukey ' s Multiple comparison test .

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Quantification of % FoxP3+ cells among CD4 T cells 72hrs activation with anti-CD3/CD28 and 20 ng/mL TGF-β in the presence or absence of 50U rhIL-2 for 72hrs (n=3 biological replicates). Data is shown as means+/-SEM, One-Way ANOVA with Tukey's Multiple comparison test.   Quantification of % FoxP3+ cells among CD4 T cells 72hrs activation with anti-CD3/CD28 and 20 ng/mL TGF-β in the presence or absence of 50U rhIL-2 for 72hrs (n=3 biological replicates). Data is shown as means+/-SEM, One-Way ANOVA with Tukey's Multiple comparison test.


Integrated genome viewer ( IGV ) browser tracks of the Lck , Fyn gene loci highlighting differential accessible regions ( in red boxes ) in biological replicates of CD4 + T cells activated with anti - CD3 / CD28 and 20 ng / mL TGF - β + / - 100U IL - 2 .

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Integrated genome viewer (IGV) browser tracks of the Lck, Fyn gene loci highlighting differential accessible regions (in red boxes) in biological replicates of CD4+ T cells activated with anti-CD3/CD28 and 20 ng/mL TGF-β +/- 100U IL-2.


Integrated genome viewer ( IGV ) browser tracks of the Itk gene loci highlighting differential accessible regions ( in red boxes ) in biological replicates of CD4 + T cells activated with anti - CD3 / CD28 and 20 ng / mL TGF - β + / - 100U IL - 2 .

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Integrated genome viewer (IGV) browser tracks of the Itk gene loci highlighting differential accessible regions (in red boxes) in biological replicates of CD4+ T cells activated with anti-CD3/CD28 and 20 ng/mL TGF-β +/- 100U IL-2.


( G ) IGV browser track of the IL - 2 locus showing accessibility peaks at the IL - 2 promoter region and upstream of the promoter ( red arrow ) in biological replicates of CD4 + T cells activated as in ( B ) .

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(G) IGV browser track of the IL-2 locus showing accessibility peaks at the IL-2 promoter region and upstream of the promoter (red arrow) in biological replicates of CD4+ T cells activated as in (B).


B ) Analysis of puromycin incorporation in MelJuSo cells that were left untreated ( - HS ) , exposed to 43 ° C for 30 minutes ( + HS ) , and followed for 4 hours after heat shock ( HS + 4h Rec . ) . The blots were probed with antibodies against puromycin , and GAPDH as loading control .

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B) Analysis of puromycin incorporation in MelJuSo cells that were left untreated (- HS), exposed to 43°C for 30 minutes (+ HS), and followed for 4 hours after heat shock (HS + 4h Rec.). The blots were probed with antibodies against puromycin, and GAPDH as loading control.


C ) Representative images of MelJuSo cells expressing Ub - YFP , which were either left untreated ( - HS ) , exposed to 43 ° C for 30 minutes ( + HS ) , and followed for 4 hours after heat shock ( HS + 4h Rec . ) .

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C) Representative images of MelJuSo cells expressing Ub-YFP, which were either left untreated (- HS), exposed to 43 °C for 30 minutes (+ HS), and followed for 4 hours after heat shock (HS + 4h Rec.).


A ) and B ) Representative images of stress granule proficient ( siControl ) and deficient ( siG3BP1 / 2 ) MelJuSo cells expressing the nuclear UPS reporter NLS - GFP - CL1 ( A ) and the cytoplasmic UPS reporter NES - GFP - CL1 ( B ) . The cells were left untreated ( - HS ) , exposed to 43 ° C for 30 minutes ( HS ) , or exposed to 43 ° C for 30 minutes and followed by 4 hours recovery ( HS + 4h Rec . ) . Nucleolar accumulation of NLS - GFP - CL1 during the recovery is indicated by arrows in A ) . C ) and D ) Quantifications of the mean cellular GFP fluorescence intensities of A ) and B ) normalized to untreated control cells ( siControl - HS ) . The frequency and distribution of the relative fluorescence intensities per cell are shown as violin plots . The solid lines in each distribution represent the median , and dash lines represent the upper and lower interquartile range limits . ( n = 3 independent experiments , > 1000 cells analyzed per condition , Kruskal - Wallis test , * * P < 0 . 01 , ns : not significant ) .

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A) and B) Representative images of stress granule proficient (siControl) and deficient (siG3BP1/2) MelJuSo cells expressing the nuclear UPS reporter NLS-GFP-CL1 (A) and the cytoplasmic UPS reporter NES-GFP-CL1 (B). The cells were left untreated (- HS), exposed to 43°C for 30 minutes (HS), or exposed to 43°C for 30 minutes and followed by 4 hours recovery (HS + 4h Rec.). Nucleolar accumulation of NLS-GFP-CL1 during the recovery is indicated by arrows in A). C) and D) Quantifications of the mean cellular GFP fluorescence intensities of A) and B) normalized to untreated control cells (siControl - HS). The frequency and distribution of the relative fluorescence intensities per cell are shown as violin plots. The solid lines in each distribution represent the median, and dash lines represent the upper and lower interquartile range limits. (n=3 independent experiments, >1000 cells analyzed per condition, Kruskal-Wallis test, ** P<0.01, ns: not significant).


E ) Representative confocal images of immunofluorescent staining of the nucleolar marker nucleolin and the nuclear UPS reporter NLS - GFP - CL1 in control ( siControl , left panel ) and G3BP1 / 2 - depleted ( siGRBP1 / 2 , right panel ) MelJuSo cells exposed to a 30 min 43 ° C heat shock , followed by 4 hours at 37 ° C ( Recovery ) . Nucleolar accumulation of NLS - GFP - CL1 is indicated by arrows . F ) Quantification of the nucleolus / nucleoplasm ratios of NLS - GFP - CL1 intensities in images of experiment shown in E ) . The frequency and distribution of the ratio per cell are shown as violin plots . The solid lines in each distribution represent the median , and dash lines represent the upper and lower interquartile range limits . ( n = 3 independent experiments , 100 cells analyzed per condition , Kruskal - Wallis test , * * P < 0 . 01 ) .

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E) Representative confocal images of immunofluorescent staining of the nucleolar marker nucleolin and the nuclear UPS reporter NLS-GFP-CL1 in control (siControl, left panel) and G3BP1/2-depleted (siGRBP1/2, right panel) MelJuSo cells exposed to a 30 min 43°C heat shock, followed by 4 hours at 37°C (Recovery). Nucleolar accumulation of NLS-GFP-CL1 is indicated by arrows. F) Quantification of the nucleolus/nucleoplasm ratios of NLS-GFP-CL1 intensities in images of experiment shown in E). The frequency and distribution of the ratio per cell are shown as violin plots. The solid lines in each distribution represent the median, and dash lines represent the upper and lower interquartile range limits. (n=3 independent experiments, 100 cells analyzed per condition, Kruskal-Wallis test, ** P<0.01).


A ) Representative confocal images of immunofluorescent staining of the stress granule marker TIA1 and puromycin - labelled proteins in parental and G3BP1 / 2 knock - out U2OS cells . Cells were subjected to 43 ° C heat shock . B ) Quantification of the nucleus / cytoplasm ratios of puromycin intensities in images from A ) . The frequency and distribution of the ratio per cell are shown as violin plots . The solid lines in each distribution represent the median , and dash lines represent the upper and lower interquartile range limits ( n = 3 independent experiments , > 50 cells analyzed per condition , Kruskal - Wallis test , * * P < 0 . 01 ) .

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A) Representative confocal images of immunofluorescent staining of the stress granule marker TIA1 and puromycin-labelled proteins in parental and G3BP1/2 knock-out U2OS cells. Cells were subjected to 43°C heat shock. B) Quantification of the nucleus/cytoplasm ratios of puromycin intensities in images from A). The frequency and distribution of the ratio per cell are shown as violin plots. The solid lines in each distribution represent the median, and dash lines represent the upper and lower interquartile range limits (n=3 independent experiments, >50 cells analyzed per condition, Kruskal-Wallis test, ** P<0.01).


E ) Representative confocal images of immunofluorescent staining of the stress granule marker TIA1 and puromycin - labelled proteins in control ( siControl ) and Hsp70 - depleted ( siHsp70 ) U2OS cells . Cells were subjected to 43 ° C heat shock F ) Quantification of the numbers of puromycin positive TIA dots per cell in images from E ) . The numbers of positive foci per cell are shown as violin plots . The solid lines in each distribution represent the median , and dash lines represent the upper and lower interquartile range limits ( n = 3 independent experiments , > 50 cells analyzed per condition , Kruskal - Wallis test , * * P < 0 . 01 ) .

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E) Representative confocal images of immunofluorescent staining of the stress granule marker TIA1 and puromycin-labelled proteins in control (siControl) and Hsp70-depleted (siHsp70) U2OS cells. Cells were subjected to 43°C heat shock F) Quantification of the numbers of puromycin positive TIA dots per cell in images from E). The numbers of positive foci per cell are shown as violin plots. The solid lines in each distribution represent the median, and dash lines represent the upper and lower interquartile range limits (n=3 independent experiments, >50 cells analyzed per condition, Kruskal-Wallis test, ** P<0.01).


G ) Representative confocal images of immunofluorescent staining of the nucleolar marker nucleolin and puromycin - labelled proteins in control ( siControl ) and Hsp70 - depleted ( siHsp70 ) G3BP1 / 2 knock - out U2OS cells . Cells were subjected to 43 ° C heat shock . H ) Quantification of the nucleolus / nucleoplasm ratio of puromycin intensities in images from G ) . The frequency and distribution of the ratio per cell are shown as violin plots . The solid lines in each distribution represent the median , and dash lines represent the upper and lower interquartile range limits ( n = 3 independent experiments , > 50 cells analyzed per condition , Mann - Whitney test , * * P < 0 . 01 ) .

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G) Representative confocal images of immunofluorescent staining of the nucleolar marker nucleolin and puromycin-labelled proteins in control (siControl) and Hsp70-depleted (siHsp70) G3BP1/2 knock-out U2OS cells. Cells were subjected to 43°C heat shock. H) Quantification of the nucleolus/nucleoplasm ratio of puromycin intensities in images from G). The frequency and distribution of the ratio per cell are shown as violin plots. The solid lines in each distribution represent the median, and dash lines represent the upper and lower interquartile range limits (n=3 independent experiments, >50 cells analyzed per condition, Mann-Whitney test, ** P<0.01).


B ) Representative confocal images of immunofluorescent staining of HSF1 in parental and G3BP1 / 2 knock - out U2OS cells directly after heat shock ( + HS ) and 2 hours after 2 hours recovery ( HS + 2h Recovery ) . C ) Quantification of the number of HSF1 foci per cell in images from B ) , shown as box plot with median and 5 - 95 percentiles ( n = 3 independent experiments , > 300 cells analyzed per condition , Kruskal - Wallis test , * * P < 0 . 01 ) .

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B) Representative confocal images of immunofluorescent staining of HSF1 in parental and G3BP1/2 knock-out U2OS cells directly after heat shock (+ HS) and 2 hours after 2 hours recovery (HS + 2h Recovery). C) Quantification of the number of HSF1 foci per cell in images from B), shown as box plot with median and 5-95 percentiles (n=3 independent experiments, >300 cells analyzed per condition, Kruskal-Wallis test, ** P<0.01).


D ) Western blot analysis of parental and G3BP1 / 2 knock - out U2OS cells that were left untreated ( - HS ) , heat shocked ( HS ) , and followed for 1 , 2 , and 4 hours after heat shock ( Rec ) . The blots were probed with antibodies against HSF1 and GAPDH ( * indicated phosphorylated HSF1 , and * * indicates unphosphorylated HSF1 ) .

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D) Western blot analysis of parental and G3BP1/2 knock-out U2OS cells that were left untreated (- HS), heat shocked (HS), and followed for 1, 2, and 4 hours after heat shock (Rec). The blots were probed with antibodies against HSF1 and GAPDH (* indicated phosphorylated HSF1, and ** indicates unphosphorylated HSF1).


A ) Representative confocal images of immunofluorescent staining of the nucleolar marker nucleolin and SUMO2 / 3 in parental and G3BP1 / 2 knock - out U2OS cells . Cells were subjected to a heat shock followed by 2 hours recovery . B ) Quantification of the nucleolus / nucleoplasm ratio of SUMO2 / 3 intensities in images from A ) . The frequency and distribution of the ratio per cell are shown as violin plots . The solid lines in each distribution represent the median , and dash lines represent the upper and lower interquartile range limits ( n = 3 independent experiments , > 50 cells analyzed per condition , Mann - Whitney test , * * P < 0 . 01 ) .

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A) Representative confocal images of immunofluorescent staining of the nucleolar marker nucleolin and SUMO2/3 in parental and G3BP1/2 knock-out U2OS cells. Cells were subjected to a heat shock followed by 2 hours recovery. B) Quantification of the nucleolus/nucleoplasm ratio of SUMO2/3 intensities in images from A). The frequency and distribution of the ratio per cell are shown as violin plots. The solid lines in each distribution represent the median, and dash lines represent the upper and lower interquartile range limits (n=3 independent experiments, >50 cells analyzed per condition, Mann-Whitney test, ** P<0.01).


C ) Representative confocal images of immunofluorescent staining of PML bodies ( PML ) and SUMO2 / 3 in parental and G3BP1 / 2 knock - out U2OS cells . Cells were heat shocked followed by 2 hours recovery . Arrows indicate dots positive for PML and SUMO2 / 3 staining . D ) Quantification of SUMO2 / 3 positive PML puncta in images from C ) . Data represent the mean ± SD ( n = 3 independent experiments , > 50 cells analyzed per condition , Mann - Whitney test , * * P < 0 . 01 ) .

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C) Representative confocal images of immunofluorescent staining of PML bodies (PML) and SUMO2/3 in parental and G3BP1/2 knock-out U2OS cells. Cells were heat shocked followed by 2 hours recovery. Arrows indicate dots positive for PML and SUMO2/3 staining. D) Quantification of SUMO2/3 positive PML puncta in images from C). Data represent the mean ± SD (n=3 independent experiments, >50 cells analyzed per condition, Mann-Whitney test, ** P<0.01).


E ) Analysis of SUMO2 / 3 conjugates in parental and G3BP1 / 2 knock - out U2OS cells that were left untreated ( - HS ) , exposed to a heat shock ( + HS ) , and followed for 2 hours ( 2h Rec . ) with or without 100 nM proteasome inhibitor epoxomicin ( EPX ) .

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E) Analysis of SUMO2/3 conjugates in parental and G3BP1/2 knock-out U2OS cells that were left untreated (- HS), exposed to a heat shock (+ HS), and followed for 2 hours (2h Rec.) with or without 100 nM proteasome inhibitor epoxomicin (EPX).


A ) Representative confocal images of immunofluorescent staining of SUMO2 / 3 and TDP43 in parental and G3BP1 / 2 knock - out U2OS cells . Cells were exposed to a heat shocked followed by 2 hours recovery . Arrows indicate dots positive for TDP - 43 and SUMO2 / 3 staining . B ) Quantification of the percentage of SUMO2 / 3 positive TDP43 puncta in images from experiment shown in A ) . . Data represent the mean ± SD ( n = 3 independent experiments , > 50 cells analyzed per condition , Mann - Whitney test , * * P < 0 . 01 ) .

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A) Representative confocal images of immunofluorescent staining of SUMO2/3 and TDP43 in parental and G3BP1/2 knock-out U2OS cells. Cells were exposed to a heat shocked followed by 2 hours recovery. Arrows indicate dots positive for TDP-43 and SUMO2/3 staining. B) Quantification of the percentage of SUMO2/3 positive TDP43 puncta in images from experiment shown in A)..Data represent the mean ± SD (n=3 independent experiments, >50 cells analyzed per condition, Mann-Whitney test, ** P<0.01).


D ) Analysis of detergent SDS and NP40 soluble fractions of TDP43 in parental and G3BP1 / 2 knock - out U2OS cells that were left untreated ( - HS ) , exposed to 43 ° C ( + HS ) followed by 2 hours recovery ( 2h Rec . ) in absence or presence of 100 nM proteasome inhibitor epoxomicin ( EPX ) . E ) Quantification of the TDP43 band densities in the SDS - soluble fraction . Data represent the mean ± SD ( n = 3 independent experiments , Student ' s unpaired t - test , * P < 0 . 05 ) .

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D) Analysis of detergent SDS and NP40 soluble fractions of TDP43 in parental and G3BP1/2 knock-out U2OS cells that were left untreated (-HS), exposed to 43°C (+HS) followed by 2 hours recovery (2h Rec.) in absence or presence of 100 nM proteasome inhibitor epoxomicin (EPX). E) Quantification of the TDP43 band densities in the SDS-soluble fraction. Data represent the mean ± SD (n=3 independent experiments, Student's unpaired t-test, * P<0.05).


A ) Analysis of SUMO2 / 3 conjugates in control ( siControl ) or RNF4 - depleted ( siRNF4 ) G3BP1 / 2 knock - out U2OS cells . Cells were left untreated ( - HS ) , exposed to 43 ° C ( + HS ) , followed by 2 hours recovery ( HS + 2h Rec . ) in the absence or presence of 100 nM proteasome inhibitor epoxomicin ( EPX ) . B ) Quantification of the total SUMO2 / 3 band densities . Data represent the mean ± SD ( n = 3 independent experiments , * P < 0 . 05 ) .

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A) Analysis of SUMO2/3 conjugates in control (siControl) or RNF4-depleted (siRNF4) G3BP1/2 knock-out U2OS cells. Cells were left untreated (-HS), exposed to 43°C (+ HS), followed by 2 hours recovery (HS + 2h Rec.) in the absence or presence of 100 nM proteasome inhibitor epoxomicin (EPX). B) Quantification of the total SUMO2/3 band densities. Data represent the mean ± SD (n=3 independent experiments, *P<0.05).


E ) Representative confocal images of control ( siControl ) , RNF4 - depleted ( siRNF4 ) , G3BP1 / 2 - depleted ( siG3BP1 / 2 ) and G3BP1 / 2 + RNF4 - depleted ( siG3BP1 / 2 + siRNF4 ) MelJuSo cells expressing the nuclear UPS reporter NLS - GFP - CL1 . Cells were exposed to heat shock followed by 4 hours recovery in the absence or presence of the proteasome inhibitor epoxomicin ( EPX ) . The nucleolar marker nucleolin was stained by immunofluorescence .

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E) Representative confocal images of control (siControl), RNF4-depleted (siRNF4), G3BP1/2-depleted (siG3BP1/2) and G3BP1/2+RNF4-depleted (siG3BP1/2 + siRNF4) MelJuSo cells expressing the nuclear UPS reporter NLS-GFP-CL1. Cells were exposed to heat shock followed by 4 hours recovery in the absence or presence of the proteasome inhibitor epoxomicin (EPX). The nucleolar marker nucleolin was stained by immunofluorescence.


A ) Representative confocal images of immunofluorescent staining of FLAG - tagged ubiquitin ( FLAG - Ub ) and mutant ataxin1 ( ATX1 ) in parental and G3BP1 / 2 knock - out U2OS cells . Cells were subjected to heat shock followed by 4 hours recovery . B ) Frequency distribution of ataxin - 1 inclusions per cell from three independent experiments .

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A) Representative confocal images of immunofluorescent staining of FLAG-tagged ubiquitin (FLAG-Ub) and mutant ataxin1 (ATX1) in parental and G3BP1/2 knock-out U2OS cells. Cells were subjected to heat shock followed by 4 hours recovery. B) Frequency distribution of ataxin-1 inclusions per cell from three independent experiments.


Viability of single and double mutants of RNAPII and rad52Δ , mre11Δ and cdc44 ‐ 8 . Tenfold serial dilutions of double mutants obtained by genetic crosses were tested for growth in plain SC medium or supplemented with 10 mM HU or 0 . 005 % MMS .

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Viability of single and double mutants of RNAPII and rad52Δ, mre11Δ and cdc44‐8. Tenfold serial dilutions of double mutants obtained by genetic crosses were tested for growth in plain SC medium or supplemented with 10 mM HU or 0.005% MMS.


Tetrad analysis of rpb1 ‐ S751F , rad52Δ and mre11Δ crosses . Δ indicates double mutants that fail to grow .

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Tetrad analysis of rpb1‐S751F, rad52Δ and mre11Δ crosses. Δ indicates double mutants that fail to grow.


Tetrad analysis of rpb1 ‐ S751F crossed by pol32Δ and rad51Δ . Δ indicates triple mutants that fail to grow .

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Tetrad analysis of rpb1‐S751F crossed by pol32Δ and rad51Δ. Δ indicates triple mutants that fail to grow.


HU and MMS sensitivity of different mutant combinations of rpb1 ‐ 1 , rpb1 ‐ S751F , pol32Δ and rad51Δ .

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HU and MMS sensitivity of different mutant combinations of rpb1‐1, rpb1‐S751F, pol32Δ and rad51Δ.


Western blot against the phosphorylated form of histone H2A in the presence of 100 mM HU in asynchronously growing WT and RNAPII mutant cells . Ponceau staining is shown as a loading control .

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Western blot against the phosphorylated form of histone H2A in the presence of 100 mM HU in asynchronously growing WT and RNAPII mutant cells. Ponceau staining is shown as a loading control.


Rad52 foci formation at 30 and 37°C.


Recombination frequencies between inverted repeats in WT and RNAPII mutants measured with the plasmid ‐ born pTINV system . Recombinants were selected as Leu + .

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Recombination frequencies between inverted repeats in WT and RNAPII mutants measured with the plasmid‐born pTINV system. Recombinants were selected as Leu+.


Recombination frequencies using the direct ‐ repeat chromosomal system leu2 ‐ k : : ADE2 ‐ URA3 : : leu2 ‐ k in WT and RNAPII mutants . Deletion events were detected and quantified in media containing 5 ‐ fluorotic acid ( FOA ) and could be directly visualized as red sectors .

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Recombination frequencies using the direct‐repeat chromosomal system leu2‐k::ADE2‐URA3::leu2‐k in WT and RNAPII mutants. Deletion events were detected and quantified in media containing 5‐fluorotic acid (FOA) and could be directly visualized as red sectors.


Recombination frequencies between plasmid ‐ born direct repeats in WT and RNAPII mutants . The frequency of Leu + recombination was determined in the systems L ‐ lacZ and GL ‐ lacZ . Recombination frequencies are plotted as a function of the transcription levels . Low transcription refers to the GL ‐ lacZ systems in strains cultured in 2 % glucose ; medium refers to L ‐ lacZ in 2 % glucose , and high to GL ‐ lacZ in 2 % galactose .

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Recombination frequencies between plasmid‐born direct repeats in WT and RNAPII mutants. The frequency of Leu+ recombination was determined in the systems L‐lacZ and GL‐lacZ. Recombination frequencies are plotted as a function of the transcription levels. Low transcription refers to the GL‐lacZ systems in strains cultured in 2% glucose; medium refers to L‐lacZ in 2% glucose, and high to GL‐lacZ in 2% galactose.


Mitotic stability of centromeric plasmid pGAL ‐ lacZ in RNAPII mutants . Stability was analyzed in galactose after 23 generations . A small diagram of each system ( not drawn to scale ) is shown .

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

Mitotic stability of centromeric plasmid pGAL‐lacZ in RNAPII mutants. Stability was analyzed in galactose after 23 generations. A small diagram of each system (not drawn to scale) is shown.


Recombination frequencies measured with the pTINV system in WT , rpb1 ‐ 1 , mre11Δ and rpb1 ‐ 1 mre11Δ .

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

Recombination frequencies measured with the pTINV system in WT, rpb1‐1, mre11Δ and rpb1‐1 mre11Δ.


Rad52 focus formation in asynchronously growing cells at 30 ° C .

Rad52 focus formation in asynchronously growing cells at 30°C.


Viability after different times in 10 mM HU . The same cultures without HU were used as a control . The average values and standard deviations from three independent experiments are shown .

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

Viability after different times in 10 mM HU. The same cultures without HU were used as a control. The average values and standard deviations from three independent experiments are shown.


Rad52 focus formation in asynchronously growing cells expressing normal or high levels of RNH1 .

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

Rad52 focus formation in asynchronously growing cells expressing normal or high levels of RNH1.


FACS analysis of cell cycle progression in rpb mutants . Cells were synchronized in G1 with α ‐ factor and monitored at different times after release in the presence of 40 mM HU .

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

FACS analysis of cell cycle progression in rpb mutants. Cells were synchronized in G1 with α‐factor and monitored at different times after release in the presence of 40 mM HU.


Slowdown of RF progression through a chromosomal region . 2D gel electrophoresis of replication intermediates of 4 . 3 ‐ kb PstI DNA fragments from WT and RNAPII mutants in the presence of 40 mM HU at different times after G1 release . A black arrow indicates accumulation of long Y molecules , and a white one indicates the specific accumulation of X molecules .

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

Slowdown of RF progression through a chromosomal region. 2D gel electrophoresis of replication intermediates of 4.3‐kb PstI DNA fragments from WT and RNAPII mutants in the presence of 40 mM HU at different times after G1 release. A black arrow indicates accumulation of long Y molecules, and a white one indicates the specific accumulation of X molecules.


Analysis of replication by BrdU incorporation . Cells were synchronized in G1 with α ‐ factor and released in the presence of BrdU . Samples were taken at the indicated times and processed to measure BrdU incorporation by ChIP analysis . Replication was analyzed in two DNA regions , one covering the early origin ARS508 and the SPF1 gene and the other the late origin ARS603 and the YFL034W gene .

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

Analysis of replication by BrdU incorporation. Cells were synchronized in G1 with α‐factor and released in the presence of BrdU. Samples were taken at the indicated times and processed to measure BrdU incorporation by ChIP analysis. Replication was analyzed in two DNA regions, one covering the early origin ARS508 and the SPF1 gene and the other the late origin ARS603 and the YFL034W gene.


DNA combing analysis of replication . Graphs indicate the distribution of BrdU track length ( kb ) at 60 and 120 min after α ‐ factor release in the presence of 40 mM HU , the replication speed ( kb / min ) determined with the two time points analyzed and the inter ‐ origin distance ( kb ) at 60 min after release . The median test was applied .

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

DNA combing analysis of replication. Graphs indicate the distribution of BrdU track length (kb) at 60 and 120 min after α‐factor release in the presence of 40 mM HU, the replication speed (kb/min) determined with the two time points analyzed and the inter‐origin distance (kb) at 60 min after release. The median test was applied.


Dephosphorylation of Rad53 after exposure to HU . WT , rpb1 ‐ 1 and rpb1 ‐ S751F cells were synchronized in G1 with α ‐ factor . After G1 release , cells were exposed to 100 mM HU for 1 h , washed to remove HU and cultured in HU ‐ free medium for the indicated periods . Western blot against Rad53 , signal quantification ( ratio Rad53 ‐ P / total Rad53 protein ) and FACS profiles are shown .

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

Dephosphorylation of Rad53 after exposure to HU. WT, rpb1‐1 and rpb1‐S751F cells were synchronized in G1 with α‐factor. After G1 release, cells were exposed to 100 mM HU for 1 h, washed to remove HU and cultured in HU‐free medium for the indicated periods. Western blot against Rad53, signal quantification (ratio Rad53‐P/total Rad53 protein) and FACS profiles are shown.


DNA repair of MMS damage . Chromosome VII species revealed by hybridization with ADE5 , 7 probes in pulsed ‐ field gel electrophoresis ( PFGE ) . Asynchronously growing WT and rpb1 cells were exposed to 0 . 1 % MMS for 30 min , washed to remove MMS and cultured in MMS ‐ free medium for the indicated periods to allow repair . Repair was calculated as the percentage of FLC appearing after MMS treatment . The FLC signal was quantified with respect to the total signal of each lane . Data show the mean and SD of 3 independent experiments . NLC , nonlinear chromosome . FLC , full ‐ length linear chromosome . CF , chromosome fragment resulting from DNA breaks .

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

DNA repair of MMS damage. Chromosome VII species revealed by hybridization with ADE5,7 probes in pulsed‐field gel electrophoresis (PFGE). Asynchronously growing WT and rpb1 cells were exposed to 0.1% MMS for 30 min, washed to remove MMS and cultured in MMS‐free medium for the indicated periods to allow repair. Repair was calculated as the percentage of FLC appearing after MMS treatment. The FLC signal was quantified with respect to the total signal of each lane. Data show the mean and SD of 3 independent experiments. NLC, nonlinear chromosome. FLC, full‐length linear chromosome. CF, chromosome fragment resulting from DNA breaks.


Retention of RNAPII in the actively transcribed PMA1 gene in RNAPII mutants . ChIP analysis of RNAPII in asynchronously growing WT , rpb1 ‐ 1 , rpb1 ‐ S751F , rpb2 ‐ 10 and rpb9Δ cells at the PMA1 gene . The scheme of the gene and the PCR ‐ amplified fragments are shown . Recruitment values were calculated from the amount of DNA present in each region , normalizing IP / input ratios , and relative to the amount of DNA of the intergenic region . Error bars indicate the standard deviation ( SD ) .

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

Retention of RNAPII in the actively transcribed PMA1 gene in RNAPII mutants. ChIP analysis of RNAPII in asynchronously growing WT, rpb1‐1, rpb1‐S751F, rpb2‐10 and rpb9Δ cells at the PMA1 gene. The scheme of the gene and the PCR‐amplified fragments are shown. Recruitment values were calculated from the amount of DNA present in each region, normalizing IP/input ratios, and relative to the amount of DNA of the intergenic region. Error bars indicate the standard deviation (SD).


Northern blot analysis of the expression of PMA1 gene . RNA was isolated from mid ‐ log phase cultures grown in YPD ‐ rich medium . As 32P ‐ labeled DNA probes , an internal 776 ‐ bp PMA1 fragment and an internal 513 ‐ bp SCR1 fragment obtained by PCR were used . Bars indicate the SD of 2 independent experiments .

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Northern blot analysis of the expression of PMA1 gene. RNA was isolated from mid‐log phase cultures grown in YPD‐rich medium. As 32P‐labeled DNA probes, an internal 776‐bp PMA1 fragment and an internal 513‐bp SCR1 fragment obtained by PCR were used. Bars indicate the SD of 2 independent experiments.


Analysis of Rrm3 recruitment in WT and rpb1 ‐ 1 . Rrm3 IP histogram bars in the y ‐ axis show the average signal log2 ratio of loci enriched in the immunoprecipitated fraction along the indicated regions in the x ‐ axis . When the ratio fulfills the P ‐ value criteria ( P 0 . 01 ) it is shown in red .

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

Analysis of Rrm3 recruitment in WT and rpb1‐1. Rrm3 IP histogram bars in the y‐axis show the average signal log2 ratio of loci enriched in the immunoprecipitated fraction along the indicated regions in the x‐axis. When the ratio fulfills the P‐value criteria (P 0.01) it is shown in red.


Composite profile of Rrm3 occupancy across the average ORF plotted as Rrm3 percentage of significant ChIP hits per segment . Each gene was divided into 10 equivalent segments from the start and end coordinates ( segments 2 - 10 ) . Two additional segments of the same size were considered upstream ( 5 ′ ) and downstream ( 3 ′ ) of each ORF ( segments 1 and 12 , respectively ) . The average signal log2 ratio for Rrm3 significant hits that map on each segment was plotted .

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

Composite profile of Rrm3 occupancy across the average ORF plotted as Rrm3 percentage of significant ChIP hits per segment. Each gene was divided into 10 equivalent segments from the start and end coordinates (segments 2-10). Two additional segments of the same size were considered upstream (5′) and downstream (3′) of each ORF (segments 1 and 12, respectively). The average signal log2 ratio for Rrm3 significant hits that map on each segment was plotted.


Composite profile of Rrm3 occupancy across the average ARS plotted as Rrm3 percentage of significant ChIP hits per segment . Each ARS was divided into 11 equivalent segments from the start and end coordinates . The average signal log2 ratio for Rrm3 significant hits that map on each segment was plotted .

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

Composite profile of Rrm3 occupancy across the average ARS plotted as Rrm3 percentage of significant ChIP hits per segment. Each ARS was divided into 11 equivalent segments from the start and end coordinates. The average signal log2 ratio for Rrm3 significant hits that map on each segment was plotted.


B - F Jejunal spheroids were cultured in differentiation medium containing 0 - 1 , 000 nM dmPGE2 or Iloprost . ( B ) Representative bright - field images of spheroids treated with dmPGE2 . Scale bars , 200 µm .

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B-F Jejunal spheroids were cultured in differentiation medium containing 0-1,000 nM dmPGE2 or Iloprost. (B) Representative bright-field images of spheroids treated with dmPGE2. Scale bars, 200 µm.


B - F Jejunal spheroids were cultured in differentiation medium containing 0 - 1 , 000 nM dmPGE2 or Iloprost . ( C ) Quantification of average spheroid area ± s . e . m . relative to spheroids treated with 0 nM dmPGE2 ( average area was 3761 µm2 for 0 nM group ; n = 4 images from two independent experiments ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-F Jejunal spheroids were cultured in differentiation medium containing 0-1,000 nM dmPGE2 or Iloprost. (C) Quantification of average spheroid area ± s.e.m. relative to spheroids treated with 0 nM dmPGE2 (average area was 3761 µm2 for 0 nM group; n = 4 images from two independent experiments).


B - F Jejunal spheroids were cultured in differentiation medium containing 0 - 1 , 000 nM dmPGE2 or Iloprost . ( D ) Representative histological sections of spheroids treated with 1 µM dmPGE2 or an equivalent volume of DMSO and stained for hematoxylin . Scale bars , 100 µm .

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B-F Jejunal spheroids were cultured in differentiation medium containing 0-1,000 nM dmPGE2 or Iloprost. (D) Representative histological sections of spheroids treated with 1 µM dmPGE2 or an equivalent volume of DMSO and stained for hematoxylin. Scale bars, 100 µm.


B - F Jejunal spheroids were cultured in differentiation medium containing 0 - 1 , 000 nM dmPGE2 or Iloprost . ( E ) Quantification of the average expression ± s . e . m . of Cldn4mRNA relative to the 0 nM treatment group ( n = 3 independent experiments ) .

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

B-F Jejunal spheroids were cultured in differentiation medium containing 0-1,000 nM dmPGE2 or Iloprost. (E) Quantification of the average expression ± s.e.m. of Cldn4mRNA relative to the 0 nM treatment group (n = 3 independent experiments).


B - F Jejunal spheroids were cultured in differentiation medium containing 0 - 1 , 000 nM dmPGE2 or Iloprost . ( F ) Quantification of average spheroid area ± s . e . m . relative to spheroids treated with 0 nM Iloprost ( n = 4 images from two independent experiments ) .

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B-F Jejunal spheroids were cultured in differentiation medium containing 0-1,000 nM dmPGE2 or Iloprost. (F) Quantification of average spheroid area ± s.e.m. relative to spheroids treated with 0 nM Iloprost (n = 4 images from two independent experiments).


A Quantification of the average expression ± s . e . m . of Ptger1 , Ptger2 , Ptger3 , Ptger4 , and Ptgir mRNAs in whole - thickness lung , ileum or colon tissues or in jejunal spheroids cultured in stem cell ( Stem ) , WAE ( dmPGE2 ) or enterocyte ( EP4i ) medium ( n = 3 independent experiments ) . Data are presented as fold change compared to lung tissue . n . d . , not detected .

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

A Quantification of the average expression ± s.e.m. of Ptger1, Ptger2, Ptger3, Ptger4, and Ptgir mRNAs in whole-thickness lung, ileum or colon tissues or in jejunal spheroids cultured in stem cell (Stem), WAE (dmPGE2) or enterocyte (EP4i) medium (n = 3 independent experiments). Data are presented as fold change compared to lung tissue. n.d., not detected.


B - D Jejunal spheroids were cultured in differentiation medium with DMSO only or with 1 µM dmPGE2 and pharmacological inhibitors of EP1 ( EP1i , SC 51322 ) , EP2 ( EP2i , PF 04418948 ) , EP3 ( EP3i , L - 798 , 106 ) or EP4 ( EP4i , L - 161 , 982 ) at a concentration of 10 µM or an equivalent volume of DMSO vehicle . * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 compared to the DMSO only group by one - way ANOVA and Dunnett ' s post - test . ( B ) Representative bright field images . Scale bars , 200 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-D Jejunal spheroids were cultured in differentiation medium with DMSO only or with 1 µM dmPGE2 and pharmacological inhibitors of EP1 (EP1i, SC 51322), EP2 (EP2i, PF 04418948), EP3 (EP3i, L-798,106) or EP4 (EP4i, L-161,982) at a concentration of 10 µM or an equivalent volume of DMSO vehicle. **p<0.01, ***p<0.001, ****p<0.0001 compared to the DMSO only group by one-way ANOVA and Dunnett's post-test. (B) Representative bright field images. Scale bars, 200 µm.


B - D Jejunal spheroids were cultured in differentiation medium with DMSO only or with 1 µM dmPGE2 and pharmacological inhibitors of EP1 ( EP1i , SC 51322 ) , EP2 ( EP2i , PF 04418948 ) , EP3 ( EP3i , L - 798 , 106 ) or EP4 ( EP4i , L - 161 , 982 ) at a concentration of 10 µM or an equivalent volume of DMSO vehicle . * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 compared to the DMSO only group by one - way ANOVA and Dunnett ' s post - test . ( C ) Quantification of average spheroid area ± s . e . m . relative to spheroids treated with DMSO alone ( n = 4 images from two independent experiments ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-D Jejunal spheroids were cultured in differentiation medium with DMSO only or with 1 µM dmPGE2 and pharmacological inhibitors of EP1 (EP1i, SC 51322), EP2 (EP2i, PF 04418948), EP3 (EP3i, L-798,106) or EP4 (EP4i, L-161,982) at a concentration of 10 µM or an equivalent volume of DMSO vehicle. **p<0.01, ***p<0.001, ****p<0.0001 compared to the DMSO only group by one-way ANOVA and Dunnett's post-test. (C) Quantification of average spheroid area ± s.e.m. relative to spheroids treated with DMSO alone (n = 4 images from two independent experiments).


B - D Jejunal spheroids were cultured in differentiation medium with DMSO only or with 1 µM dmPGE2 and pharmacological inhibitors of EP1 ( EP1i , SC 51322 ) , EP2 ( EP2i , PF 04418948 ) , EP3 ( EP3i , L - 798 , 106 ) or EP4 ( EP4i , L - 161 , 982 ) at a concentration of 10 µM or an equivalent volume of DMSO vehicle . * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 compared to the DMSO only group by one - way ANOVA and Dunnett ' s post - test . ( D ) Quantification of the average expression ± s . e . m . of Cldn4 mRNA relative to stem cellspheroids ( n = 3 independent experiments ) .

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

B-D Jejunal spheroids were cultured in differentiation medium with DMSO only or with 1 µM dmPGE2 and pharmacological inhibitors of EP1 (EP1i, SC 51322), EP2 (EP2i, PF 04418948), EP3 (EP3i, L-798,106) or EP4 (EP4i, L-161,982) at a concentration of 10 µM or an equivalent volume of DMSO vehicle. **p<0.01, ***p<0.001, ****p<0.0001 compared to the DMSO only group by one-way ANOVA and Dunnett's post-test. (D) Quantification of the average expression ± s.e.m. of Cldn4 mRNA relative to stem cellspheroids (n = 3 independent experiments).


E , F Tat - Cre mediated recombination of Ptger4flox / flox ( fl / fl ) jejunal spheroids to generate Ptger4flox / Δ ( fl / Δ ) and Ptger4Δ / Δ ( Δ / Δ ) spheroid lines . ( E ) Schematic and representative PCR genotyping results . M , marker lane . bp , base pairs . ( F ) Quantification of the average expression ± s . e . m . of Ptger4 mRNA in spheroids cultured in stem cell media relative to fl / fl genotype ( n = 3 independent experiments ) . * * p < 0 . 01 , * * * * p < 0 . 0001 by 2 - way ANOVA and Dunnett ' s multiple comparisons post - test with the EP4i - treated fl / fl group set as the control .

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

E, F Tat-Cre mediated recombination of Ptger4flox/flox (fl/fl) jejunal spheroids to generate Ptger4flox/Δ (fl/Δ) and Ptger4Δ/Δ (Δ/Δ) spheroid lines. (E) Schematic and representative PCR genotyping results. M, marker lane. bp, base pairs. (F) Quantification of the average expression ± s.e.m. of Ptger4 mRNA in spheroids cultured in stem cell media relative to fl/fl genotype (n = 3 independent experiments). **p<0.01, ****p<0.0001 by 2-way ANOVA and Dunnett's multiple comparisons post-test with the EP4i-treated fl/fl group set as the control.


G - I Ptger4flox / flox , Ptger4flox / Δ and Ptger4Δ / Δ spheroids were cultured in differentiation medium with 1 µM dmPGE2 or 10 µM EP4i . * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 by 1 - way ANOVA and Tukey ' s post - test . ( G ) Representative bright - field images . Scale bars , 200 µm . ( H ) Quantification of average spheroid area ± s . e . m . ( n = 4 images per group from two independent experiments ) relative to EP4i - treated Ptger4flox / flox spheroids .

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

G-I Ptger4flox/flox, Ptger4flox/Δ and Ptger4Δ/Δ spheroids were cultured in differentiation medium with 1 µM dmPGE2 or 10 µM EP4i. *p<0.05, **p<0.01, ****p<0.0001 by 1-way ANOVA and Tukey's post-test. (G) Representative bright-field images. Scale bars, 200 µm. (H) Quantification of average spheroid area ± s.e.m. (n = 4 images per group from two independent experiments) relative to EP4i-treated Ptger4flox/flox spheroids.


G - I Ptger4flox / flox , Ptger4flox / Δ and Ptger4Δ / Δ spheroids were cultured in differentiation medium with 1 µM dmPGE2 or 10 µM EP4i . * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 by 1 - way ANOVA and Tukey ' s post - test . ( I ) average expression ± s . e . m . of Cldn4 mRNA ( n = 3 independent experiments ) relative to EP4i - treated Ptger4flox / flox spheroids .

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

G-I Ptger4flox/flox, Ptger4flox/Δ and Ptger4Δ/Δ spheroids were cultured in differentiation medium with 1 µM dmPGE2 or 10 µM EP4i. *p<0.05, **p<0.01, ****p<0.0001 by 1-way ANOVA and Tukey's post-test. (I) average expression ± s.e.m. of Cldn4 mRNA (n = 3 independent experiments) relative to EP4i-treated Ptger4flox/flox spheroids.


J - L Humanileal spheroids were cultured in differentiation medium with 1 µM dmPGE2 or 10 µM EP4i . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 by 1 - way ANOVA and Tukey ' s post - test . ( J ) Representative bright - field images . Scale bars , 200 µm . ( K ) Quantification of average spheroid area ± s . e . m . ( n = 4 images per group from 3 independent donor lines examined over 3 passages each ) relative to stem spheroids .

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

J-L Humanileal spheroids were cultured in differentiation medium with 1 µM dmPGE2 or 10 µM EP4i. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 by 1-way ANOVA and Tukey's post-test. (J) Representative bright-field images. Scale bars, 200 µm. (K) Quantification of average spheroid area ± s.e.m. (n = 4 images per group from 3 independent donor lines examined over 3 passages each) relative to stem spheroids.


J - L Humanileal spheroids were cultured in differentiation medium with 1 µM dmPGE2 or 10 µM EP4i . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 by 1 - way ANOVA and Tukey ' s post - test . ( L ) the average expression ± s . e . m . of CLDN4 mRNA ( n = 3 independent donor lines ) relative to stem spheroids .

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

J-L Humanileal spheroids were cultured in differentiation medium with 1 µM dmPGE2 or 10 µM EP4i. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 by 1-way ANOVA and Tukey's post-test. (L) the average expression ± s.e.m. of CLDN4 mRNA (n = 3 independent donor lines) relative to stem spheroids.


B Heatmap showing unsupervised hierarchical clustering of genes that were significantly ( FDR - adjusted p < 0 . 05 ) and differentially expressed between the three groups ( n = 4 samples per treatment from two independent experiments ) . Each column is one sample and each row is one gene . Red and blue indicate genes with enriched or de - enriched expression in a particular group , respectively . The dendrogram for the gene clustering is colored to highlight major gene clusters 1 - 6 . The number of probes in each cluster is in parentheses .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Heatmap showing unsupervised hierarchical clustering of genes that were significantly (FDR-adjusted p<0.05) and differentially expressed between the three groups (n = 4 samples per treatment from two independent experiments). Each column is one sample and each row is one gene. Red and blue indicate genes with enriched or de-enriched expression in a particular group, respectively. The dendrogram for the gene clustering is colored to highlight major gene clusters 1-6. The number of probes in each cluster is in parentheses.


E , F Spheroids were cultured as indicated for 24 hours followed by a 1 - hr pulse with EdU to mark the cells undergoing DNA synthesis . ( E ) Representative images of EdU staining ( red ) . Nuclei are visualized with bisbenzimide ( blue ) . Scale bars , 20 µm . ( F ) Quantification of EdU - positive nuclei as a percent of the total nuclei ( n = minimum of 15 spheroids counted per sample from 2 independent experiments ) . * * * * p < 0 . 0001 by 1 - way ANOVA and Tukey ' s post - 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 O O O O O O O O O O O O O O O O O O O O O O O

E, F Spheroids were cultured as indicated for 24 hours followed by a 1-hr pulse with EdU to mark the cells undergoing DNA synthesis. (E) Representative images of EdU staining (red). Nuclei are visualized with bisbenzimide (blue). Scale bars, 20 µm. (F) Quantification of EdU-positive nuclei as a percent of the total nuclei (n = minimum of 15 spheroids counted per sample from 2 independent experiments). ****p<0.0001 by 1-way ANOVA and Tukey's post-test.


G Graph of the fold change in background - subtracted luminescence ± s . e . m . ( relative to 0 hr measurement ) of Cdc25A - CBRLuc spheroids ( n = 3 independent experiments with 4 technical replicates ) . * * * * p < 0 . 0001 for dmPGE2 - and EP4i - treated spheroids compared to stem cells by repeated measures 2 - way ANOVA ( variable = treatment ) . p < 0 . 001 at the 16 hr , 20 hr and 24 hr time points by Dunnett ' s post - test comparing dmPGE2 - treated and EP4i - treated spheroids to the stem cell control .

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

G Graph of the fold change in background-subtracted luminescence ± s.e.m. (relative to 0 hr measurement) of Cdc25A-CBRLuc spheroids (n = 3 independent experiments with 4 technical replicates). ****p<0.0001 for dmPGE2- and EP4i-treated spheroids compared to stem cells by repeated measures 2-way ANOVA (variable = treatment). p<0.001 at the 16 hr, 20 hr and 24 hr time points by Dunnett's post-test comparing dmPGE2-treated and EP4i-treated spheroids to the stem cell control.


H Quantification of the average expression ± s . e . m . of Lgr5 and Mki67 mRNAs in jejunal spheroids cultured in stem cell ( Stem ) or in differentiation medium with the indicated supplements relative to the stem cell group ( n = 3 independent experiments ) . * * p < 0 . 01 , * * * * p < 0 . 0001 by one - way ANOVA and Dunnett ' s post - test . n . d . , not detected .

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

H Quantification of the average expression ± s.e.m. of Lgr5 and Mki67 mRNAs in jejunal spheroids cultured in stem cell (Stem) or in differentiation medium with the indicated supplements relative to the stem cell group (n = 3 independent experiments). **p<0.01, ****p<0.0001 by one-way ANOVA and Dunnett's post-test. n.d., not detected.


D Representative images of spheroids stained for Cldn4 ( red ) . Nuclei are visualized with bisbenzimide ( blue ) ( n = 2 independent experiments ) . Scale bars , 20 µm .

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

D Representative images of spheroids stained for Cldn4 (red). Nuclei are visualized with bisbenzimide (blue) (n = 2 independent experiments). Scale bars, 20 µm.


E , F Jejunal spheroids were cultured as in Figure 2B - D . Quantification of the average expression ± s . e . m . of Dpcr1 ( E ) and Cd55b mRNAs relative to DMSO group ( n = 3 independent experiments ) . * p < 0 . 05 , * * * * p < 0 . 0001 by one - way ANOVA and Dunnett ' s post - 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 B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E, F Jejunal spheroids were cultured as in Figure 2B-D. Quantification of the average expression ± s.e.m. of Dpcr1 (E) and Cd55b mRNAs relative to DMSO group (n = 3 independent experiments). *p<0.05, ****p<0.0001 by one-way ANOVA and Dunnett's post-test.


G - J Representative transmission electron microscopy ( TEM ) images of spheroids or an in vivo WAE cell from a biopsy - injured mousecolon . ( G ) The basal plasma membranes are outlined in orange solid lines , lateral plasma membranes are indicated with orange arrowheads , and nuclei are outlined with wide yellow dashed lines . Insets show a magnified view of the apical cell surface .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-J Representative transmission electron microscopy (TEM) images of spheroids or an in vivo WAE cell from a biopsy-injured mousecolon. (G) The basal plasma membranes are outlined in orange solid lines, lateral plasma membranes are indicated with orange arrowheads, and nuclei are outlined with wide yellow dashed lines. Insets show a magnified view of the apical cell surface.


G - J Representative transmission electron microscopy ( TEM ) images of spheroids or an in vivo WAE cell from a biopsy - injured mousecolon . Quantification of cytoplasmic : nuclear ratio ( H ) and microvillar length ( I ) from the TEM images ( n = minimum of 5 images per group ) . * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 by 1 - way ANOVA and Tukey ' s post - 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 O O O O O O O

G-J Representative transmission electron microscopy (TEM) images of spheroids or an in vivo WAE cell from a biopsy-injured mousecolon. Quantification of cytoplasmic:nuclear ratio (H) and microvillar length (I) from the TEM images (n = minimum of 5 images per group). *p<0.05, **p<0.01, ****p<0.0001 by 1-way ANOVA and Tukey's post-test.


G - J Representative transmission electron microscopy ( TEM ) images of spheroids or an in vivo WAE cell from a biopsy - injured mousecolon . ( J ) Higher power image of the cytoplasm . Single mitochondria are outlined with a narrow blue dashed line , vacuole structures are indicated with red asterisks . Scale bars , 1 µm .

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

G-J Representative transmission electron microscopy (TEM) images of spheroids or an in vivo WAE cell from a biopsy-injured mousecolon. (J) Higher power image of the cytoplasm. Single mitochondria are outlined with a narrow blue dashed line, vacuole structures are indicated with red asterisks. Scale bars, 1 µm.


K Representative image of a spheroid stained for β - catenin ( green ) and F - actin ( red ) . Nuclei are visualized with bisbenzimide ( blue ) . A similarly stained section of a small intestinal ulcer is shown for comparison . Arrowheads indicate the apical cell membrane . Scale bars , 50 µm .

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

K Representative image of a spheroid stained for β-catenin (green) and F-actin (red). Nuclei are visualized with bisbenzimide (blue). A similarly stained section of a small intestinal ulcer is shown for comparison. Arrowheads indicate the apical cell membrane. Scale bars, 50 µm.


A Representative image of a spheroid stained for β - catenin ( green ) and F - actin ( red ) . Nuclei are visualized with bisbenzimide ( blue ) . A similarly stained section of a mouse villus is shown for comparison . Arrowheads indicate the apical cell membrane . Scale bars , 50 µm .

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

A Representative image of a spheroid stained for β-catenin (green) and F-actin (red). Nuclei are visualized with bisbenzimide (blue). A similarly stained section of a mouse villus is shown for comparison. Arrowheads indicate the apical cell membrane. Scale bars, 50 µm.


B , C Jejunal spheroids were cultured as indicated . ( B ) Quantification of the average expression ± s . e . m . of Fabp1 , Ace2 , and Maoa mRNAs ( n = 3 independent experiments ) . * p < 0 . 05 , * * p < 0 . 01 by one - way ANOVA and Tukey ' s post - 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 B-MEASURED_VAR O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B, C Jejunal spheroids were cultured as indicated. (B) Quantification of the average expression ± s.e.m. of Fabp1, Ace2, and Maoa mRNAs (n = 3 independent experiments). *p<0.05, **p<0.01 by one-way ANOVA and Tukey's post-test.


B , C Jejunal spheroids were cultured as indicated . ( C ) Representative images of spheroids stained for Ace2 ( green ) and β - catenin ( red ) . Nuclei are visualized with bisbenzimide ( blue ) . Scale bars , 20 µm .

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

B, C Jejunal spheroids were cultured as indicated. (C) Representative images of spheroids stained for Ace2 (green) and β-catenin (red). Nuclei are visualized with bisbenzimide (blue). Scale bars, 20 µm.


F , G Representative TEM images of EP4i - treated spheroids . ( F ) The basal plasma membranes are outlined in orange solid lines , lateral plasma membranes are indicated with orange arrowheads , and nuclei are outlined with wide yellow dashed lines . Insets show a magnified view of the apical cell surface . ( G ) Higher power image of the cytoplasm . Single mitochondria are outlined with a narrow blue dashed line , vacuole structures are indicated with red asterisks . Scale bars , 1 µm .

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

F, G Representative TEM images of EP4i-treated spheroids. (F) The basal plasma membranes are outlined in orange solid lines, lateral plasma membranes are indicated with orange arrowheads, and nuclei are outlined with wide yellow dashed lines. Insets show a magnified view of the apical cell surface. (G) Higher power image of the cytoplasm. Single mitochondria are outlined with a narrow blue dashed line, vacuole structures are indicated with red asterisks. Scale bars, 1 µm.


H Graph of spheroid oxygen consumption rate ( OCR ) to extracellular acidification rate ( ECAR ) ratio expressed as mean ± s . e . m . ( n = 3 independent experiments ) . * * p < 0 . 01by 1 - way ANOVA and Tukey ' s post - 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

H Graph of spheroid oxygen consumption rate (OCR) to extracellular acidification rate (ECAR) ratio expressed as mean ± s.e.m. (n = 3 independent experiments). **p<0.01by 1-way ANOVA and Tukey's post-test.


A - D Quantification of the average expression ± s . e . m . of Dpcr1 and Cd55b mRNAs ( A , C , E ) or Fabp1 and Ace2 mRNAs ( B , D , F ) in jejunal spheroids cultured in differentiation medium containing EP4i or dmPGE2 . ( A , B ) Gene expression analyzed 2 , 6 , 12 or 24 hours after the start of treatment ( n = 3 independent experiments ) . * * * p < 0 . 001 , * * * * p < 0 . 0001 comparing the two treatment groups and † p < 0 . 05 , † † † p < 0 . 001 , † † † † p < 0 . 0001 compared to the 2 hr time point of the same medium by 2 - way ANOVA and Sidak ' s multiple comparisons test . ( C , D ) Gene expression was analyzed after culturing spheroids in EP4i ( E ) or dmPGE2 ( P ) for the first 12 hours followed by wash out and re - feeding with EP4i or dmPGE2 for the second 12 hours as shown ( n = 3 independent experiments ) . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 and * * * * p < 0 . 0001 by one - way ANOVA and Dunnett ' s post - test .

O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Quantification of the average expression ± s.e.m. of Dpcr1 and Cd55b mRNAs (A, C, E) or Fabp1 and Ace2 mRNAs (B, D, F) in jejunal spheroids cultured in differentiation medium containing EP4i or dmPGE2. (A, B) Gene expression analyzed 2, 6, 12 or 24 hours after the start of treatment (n = 3 independent experiments). ***p<0.001, ****p<0.0001 comparing the two treatment groups and †p<0.05, †††p<0.001, ††††p<0.0001 compared to the 2 hr time point of the same medium by 2-way ANOVA and Sidak's multiple comparisons test. (C, D) Gene expression was analyzed after culturing spheroids in EP4i (E) or dmPGE2 (P) for the first 12 hours followed by wash out and re-feeding with EP4i or dmPGE2 for the second 12 hours as shown (n = 3 independent experiments). *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001 by one-way ANOVA and Dunnett's post-test.


E , F Gene expression was analyzed in Ptger4flox / flox , Ptger4flox / Δ and Ptger4Δ / Δspheroids and expressed as fold change relative to EP4i - treated Ptger4flox / flox ( n = 3 independent experiments ) . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 by two - way ANOVA and Tukey ' s post - test comparing the genotypes within a treatment group . † † † p < 0 . 001 , † † † † p < 0 . 0001 by two - way ANOVA and Sidak ' s post - test comparing the effects of the treatments within a genotype .

O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, F Gene expression was analyzed in Ptger4flox/flox, Ptger4flox/Δ and Ptger4Δ/Δspheroids and expressed as fold change relative to EP4i-treated Ptger4flox/flox (n = 3 independent experiments). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 by two-way ANOVA and Tukey's post-test comparing the genotypes within a treatment group. †††p<0.001, ††††p<0.0001 by two-way ANOVA and Sidak's post-test comparing the effects of the treatments within a genotype.


A , B Jejunal spheroids were cultured in differentiation medium containing 10 µM of Forskolin or an equivalent volume of DMSO . ( A ) Representative bright - field images . Scale bars , 200 µm .

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

A, B Jejunal spheroids were cultured in differentiation medium containing 10 µM of Forskolin or an equivalent volume of DMSO. (A) Representative bright-field images. Scale bars, 200 µm.


A , B Jejunal spheroids were cultured in differentiation medium containing 10 µM of Forskolin or an equivalent volume of DMSO . ( B ) Quantification of the average expression ± s . e . m . of Cldn4 mRNA relative to the DMSO treatment group ( n = 3 independent experiments ) . * p < 0 . 05 compared to DMSO group by 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 B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A, B Jejunal spheroids were cultured in differentiation medium containing 10 µM of Forskolin or an equivalent volume of DMSO. (B) Quantification of the average expression ± s.e.m. of Cldn4 mRNA relative to the DMSO treatment group (n = 3 independent experiments). *p<0.05 compared to DMSO group by unpaired t test.


C , D Representative Western blots for β - catenin detected in nuclear ( Nuc ) and cytoplasmic ( Cyt ) protein lysates . Lamin A / C and Actin were used as loading controls for the nuclear and cytoplasmic fractions , respectively ( n = 3 independent experiments ) . ( C ) Spheroids were cultured in stem , enterocyte ( EP4i ) or WAE ( dmPGE2 ) media . ( D ) Spheroids were cultured in enterocyte medium with DMSO or 10 µM CHIR 99021 .

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

C, D Representative Western blots for β-catenin detected in nuclear (Nuc) and cytoplasmic (Cyt) protein lysates. Lamin A/C and Actin were used as loading controls for the nuclear and cytoplasmic fractions, respectively (n = 3 independent experiments). (C) Spheroids were cultured in stem, enterocyte (EP4i) or WAE (dmPGE2) media. (D) Spheroids were cultured in enterocyte medium with DMSO or 10 µM CHIR 99021.


E , F Spheroids were cultured in enterocyte medium with the indicated concentrations of CHIR 99021 . Quantification of the average expression ± s . e . m . of Cldn4 ( E ) and Fabp1 ( F ) mRNAs shown as fold change relative to 0 µM group ( n = 3 independent experiments ) . * * p < 0 . 01 , * * * * p < 0 . 0001 as determined by one - way ANOVA and Dunnett ' s post - 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 B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E, F Spheroids were cultured in enterocyte medium with the indicated concentrations of CHIR 99021. Quantification of the average expression ± s.e.m. of Cldn4 (E) and Fabp1 (F) mRNAs shown as fold change relative to 0 µM group (n = 3 independent experiments). **p<0.01, ****p<0.0001 as determined by one-way ANOVA and Dunnett's post-test.


G Representative images of Ptger4Δ / Δspheroids cultured in differentiation medium with DMSO or 10 µM CHIR 99021 and stained for β - catenin ( green ) and F - actin ( red ) . Nuclei are visualized with bisbenzimide ( blue ) . Scale bars , 20 µm .

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

G Representative images of Ptger4Δ/Δspheroids cultured in differentiation medium with DMSO or 10 µM CHIR 99021 and stained for β-catenin (green) and F-actin (red). Nuclei are visualized with bisbenzimide (blue). Scale bars, 20 µm.


H Graph of TOPflash ( TOP ) to FOPflash ( FOP ) luciferase reporter ratios ± s . e . m . ( n = 3 independent experiments ) in transfected 293FT cells treated with conditioned mediums collected from L - WRN cells and diluted as indicated . L - WRN CM had been produced in the presence or absence of the porcupine inhibitor C59 ( 10 - fold dilutions , 10 µM to 1 pM ) . * p < 0 . 05 , * * * p < 0 . 001 , * * * * p < 0 . 0001 by 1 - way ANOVA and Dunnett ' s post - test compared to 0 % L - WRN CM group .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Graph of TOPflash (TOP) to FOPflash (FOP) luciferase reporter ratios ± s.e.m. (n = 3 independent experiments) in transfected 293FT cells treated with conditioned mediums collected from L-WRN cells and diluted as indicated. L-WRN CM had been produced in the presence or absence of the porcupine inhibitor C59 (10-fold dilutions, 10 µM to 1 pM). *p<0.05, ***p<0.001, ****p<0.0001 by 1-way ANOVA and Dunnett's post-test compared to 0% L-WRN CM group.