Tg ( kdrl : eGFP ) zebrafish were injected with control - or cep41 - MOs and subjected to immunostaining with phospho - AURKA - specific antibodies ( red ) and DAPI at 26 hpf . The insets indicate the representative area from each immunostaining experiment and the red dots indicate EC aurka activation . Scale bars , 40 μm . Quantification of the phospho - aurka - positive ECs ( H ) in equivalent fields of view for each MO - injected zebrafish in images in ( G ) are the result of three independent experiments with ≥ 10 embryos per condition . The top and bottom whiskers represent the maximum and minimum values , respectively . * * P < 0 . 01 ( Unpaired Student ' s t - test with Welch ' s correction ) .

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

Tg(kdrl:eGFP) zebrafish were injected with control- or cep41-MOs and subjected to immunostaining with phospho-AURKA-specific antibodies (red) and DAPI at 26 hpf. The insets indicate the representative area from each immunostaining experiment and the red dots indicate EC aurka activation. Scale bars, 40 μm. Quantification of the phospho-aurka-positive ECs (H) in equivalent fields of view for each MO-injected zebrafish in images in (G) are the result of three independent experiments with ≥ 10 embryos per condition. The top and bottom whiskers represent the maximum and minimum values, respectively. **P < 0.01 (Unpaired Student's t-test with Welch's correction).


CEP41 - depleted hTERT - RPE1 cells were transfected with vectors encoding nothing ( MOCK ) , AURKA - T288D , or AURKA - K162R and then immunostained with ARL13b - specific antibodies after serum starvation or serum retrieval . Images were taken from the results of staining done 18 h after serum retrieval . Rectangles indicate the representative cells that appear in the magnified images in the right panels . Scale bars , 20 μm . A quantification of ARL13b - labeled ciliated cells under the indicated conditions in ( J ) . These are the results of three independent experiments with ≥ 200 cells per condition ( mean ± SD ) . * P < 0 . 05 , * * * P < 0 . 01 , ns : non - significant ( Two - way ANOVA with Tukey ' s post hoc test ) .

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CEP41-depleted hTERT-RPE1 cells were transfected with vectors encoding nothing (MOCK), AURKA-T288D, or AURKA-K162R and then immunostained with ARL13b-specific antibodies after serum starvation or serum retrieval. Images were taken from the results of staining done 18 h after serum retrieval. Rectangles indicate the representative cells that appear in the magnified images in the right panels. Scale bars, 20 μm. A quantification of ARL13b-labeled ciliated cells under the indicated conditions in (J). These are the results of three independent experiments with ≥ 200 cells per condition (mean ± SD). *P < 0.05, ***P < 0.01, ns: non-significant (Two-way ANOVA with Tukey's post hoc test).


Control and CEP41 - deficient HUVECs were transfected with expression vectors encoding nothing ( MOCK ) , AURKA , AURKA - T288D , or AURKA - K162R and subjected to an in vitro angiogenesis assay for 18 h under static ( K , L ) or shear stress ( M , N ) states . Quantification of tube node number in ( K , M ) and tube length in ( L , N ) from data examined within equivalent fields of view at each time point using the ImageJ angiogenesis analyzer . Data are shown as mean ± SD of five independent experiments with ≥ 5 tubulogenesis regions per condition . Statistical significance was determined using the two - way ANOVA followed by Tukey ' s post hoc test ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , ns : non - significant ) .

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

Control and CEP41-deficient HUVECs were transfected with expression vectors encoding nothing (MOCK), AURKA, AURKA-T288D, or AURKA-K162R and subjected to an in vitro angiogenesis assay for 18 h under static (K, L) or shear stress (M, N) states. Quantification of tube node number in (K, M) and tube length in (L, N) from data examined within equivalent fields of view at each time point using the ImageJ angiogenesis analyzer. Data are shown as mean ± SD of five independent experiments with ≥ 5 tubulogenesis regions per condition. Statistical significance was determined using the two-way ANOVA followed by Tukey's post hoc test (*P < 0.05, **P < 0.01, ***P < 0.001, ns: non-significant).


The control or CEP41 siRNA - transfected HUVECs were cultivated under hypoxia ( 1 % O2 ) and their whole cell lysates were used for immunoblot assays of CEP41 , phospho - AURKA , and AURKA . The resulting data were also compared to those of normoxic cells . Protein levels were normalized against β - ACTIN in the same blots .

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The control or CEP41 siRNA-transfected HUVECs were cultivated under hypoxia (1% O2) and their whole cell lysates were used for immunoblot assays of CEP41, phospho-AURKA, and AURKA. The resulting data were also compared to those of normoxic cells. Protein levels were normalized against β-ACTIN in the same blots.


VEGFA ( B ) and VEGFR2 ( C ) mRNA levels were measured by qRT - PCR using cDNA from normoxic and hypoxic controls or CEP41 - depleted cells . The expression of GAPDH was quantified for the normalization of those qRT - PCR results .

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VEGFA (B) and VEGFR2 (C) mRNA levels were measured by qRT-PCR using cDNA from normoxic and hypoxic controls or CEP41-depleted cells. The expression of GAPDH was quantified for the normalization of those qRT-PCR results.


Tg ( kdrl : eGFP ) zebrafish were injected with control - or cep41 - MOs and then incubated under hypoxia at 28 hpf ( a stage of high shear stress ) for 2 h . They were subjected to immunostaining with phospho - AURKA - specific antibodies ( red ) and DAPI at 30 hpf . The insets indicate the representative areas from each immunostaining and red dots indicate activated aurka within ECs . Scale bars , 40 μm . Quantification of phospho - aurka - positive ECs ( E ) in equivalent fields of view for each MO - injected zebrafish in ( D ) are the result of three independent experiments with ≥ 20 embryos per condition . The top and bottom whiskers represent the maximum and minimum values , respectively . * P < 0 . 05 ( Unpaired Student ' s t - test ) .

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

Tg(kdrl:eGFP) zebrafish were injected with control- or cep41-MOs and then incubated under hypoxia at 28 hpf (a stage of high shear stress) for 2 h. They were subjected to immunostaining with phospho-AURKA-specific antibodies (red) and DAPI at 30 hpf. The insets indicate the representative areas from each immunostaining and red dots indicate activated aurka within ECs. Scale bars, 40 μm. Quantification of phospho-aurka-positive ECs (E) in equivalent fields of view for each MO-injected zebrafish in (D) are the result of three independent experiments with ≥ 20 embryos per condition. The top and bottom whiskers represent the maximum and minimum values, respectively. *P < 0.05 (Unpaired Student's t-test).


Zebrafish vegfa mRNA levels were quantified by qRT - PCR in eGFP - positive ECs of control - or cep41 - MO - injected Tg ( kdrl : eGFP ) zebrafish subjected to either normoxia or hypoxia at 30 hpf . The expression of zebrafish β - actin was quantified for the normalization of these qRT - PCR results .

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Zebrafish vegfa mRNA levels were quantified by qRT-PCR in eGFP-positive ECs of control- or cep41-MO-injected Tg(kdrl:eGFP) zebrafish subjected to either normoxia or hypoxia at 30 hpf. The expression of zebrafish β-actin was quantified for the normalization of these qRT-PCR results.


vegfr2 ( G ) mRNA levels were quantified by qRT - PCR in eGFP - positive ECs of control - or cep41 - MO - injected Tg ( kdrl : eGFP ) zebrafish subjected to either normoxia or hypoxia at 30 hpf . The expression of zebrafish β - actin was quantified for the normalization of these qRT - PCR results .

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vegfr2 (G) mRNA levels were quantified by qRT-PCR in eGFP-positive ECs of control- or cep41-MO-injected Tg(kdrl:eGFP) zebrafish subjected to either normoxia or hypoxia at 30 hpf. The expression of zebrafish β-actin was quantified for the normalization of these qRT-PCR results.


Control and CEP41 - deficient HUVECs , transfected with expression vectors encoding nothing ( MOCK ) , AURKA , AURKA - T288D , HIF1α , or HIF1α with CEP41 were cultivated under hypoxia and subjected to an in vitro angiogenesis assay for 18 h . Scale bars , 400 μm .

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

Control and CEP41-deficient HUVECs, transfected with expression vectors encoding nothing (MOCK), AURKA, AURKA-T288D, HIF1α, or HIF1α with CEP41 were cultivated under hypoxia and subjected to an in vitro angiogenesis assay for 18 h. Scale bars, 400 μm.


Control and CEP41 - deficient HUVECs , transfected with expression vectors encoding nothing ( MOCK ) , AURKA , AURKA - T288D , HIF1α , or HIF1α with CEP41 were cultivated under hypoxia and subjected to an in vitro angiogenesis assay for 18 h . Scale bars , 400 μm . Quantification of tube node number in ( I ) and tube length in ( J ) from data examined within equivalent fields of view at each time point using the ImageJ angiogenesis analyzer . Data are shown as mean ± SD of five independent experiments with ≥ 5 tubulogenesis regions per condition .

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

Control and CEP41-deficient HUVECs, transfected with expression vectors encoding nothing (MOCK), AURKA, AURKA-T288D, HIF1α, or HIF1α with CEP41 were cultivated under hypoxia and subjected to an in vitro angiogenesis assay for 18 h. Scale bars, 400 μm. Quantification of tube node number in (I) and tube length in (J) from data examined within equivalent fields of view at each time point using the ImageJ angiogenesis analyzer. Data are shown as mean ± SD of five independent experiments with ≥ 5 tubulogenesis regions per condition.


HUVECs transfected with control or CEP41 siRNAs were cultivated under hypoxia and their whole cell lysates were used for immunoblot assays of HIF1α . The HIF1α expression of hypoxic cells was compared to that of normoxic cells and the protein levels were normalized against β - ACTIN in the same blot .

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HUVECs transfected with control or CEP41 siRNAs were cultivated under hypoxia and their whole cell lysates were used for immunoblot assays of HIF1α. The HIF1α expression of hypoxic cells was compared to that of normoxic cells and the protein levels were normalized against β-ACTIN in the same blot.


Co - immunoprecipitation assays using HIF1α - , and CEP41 - specific antibodies were performed in HEK 293T cells expressing GFP - CEP41 under normoxia or hypoxia . An IP using IgG - specific antibodies was performed as a negative control . NOR , normoxia ; HYP , hypoxia .

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Co-immunoprecipitation assays using HIF1α-, and CEP41-specific antibodies were performed in HEK 293T cells expressing GFP-CEP41 under normoxia or hypoxia. An IP using IgG-specific antibodies was performed as a negative control. NOR, normoxia; HYP, hypoxia.


The CEP41 - depleted HUVECs were transfected with expression vectors encoding nothing ( MOCK ) , CEP41 , or HIF1α and cultivated under hypoxia . Whole cell lysates were used for immunoblot assays for phospho - AURKA and AURKA , and the resulting data were compared to those of normoxic and hypoxic control cells . Protein levels were normalized against β - ACTIN in the same blots . NOR , normoxia ; HYP , hypoxia .

O B-CONTROLLED_VAR 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 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

The CEP41-depleted HUVECs were transfected with expression vectors encoding nothing (MOCK), CEP41, or HIF1α and cultivated under hypoxia. Whole cell lysates were used for immunoblot assays for phospho-AURKA and AURKA, and the resulting data were compared to those of normoxic and hypoxic control cells. Protein levels were normalized against β-ACTIN in the same blots. NOR, normoxia; HYP, hypoxia.


The CEP41 - deficient HUVECs were transfected with expression vectors encoding nothing ( MOCK ) , CEP41 , HIF1α , or HIF1α with CEP41 and exposed to hypoxia . VEGFA ( E ) and VEGFR2 ( F ) mRNA levels were analyzed by qRT - PCR using cDNA from transfected cells . Data are shown as mean ± SD of three independent experiments . Statistical significance was determined using the one - way ANOVA followed by Tukey ' s post hoc test ( * * * P < 0 . 001 , ns : non - significant ) . NOR , normoxia ; HYP , hypoxia .

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

The CEP41-deficient HUVECs were transfected with expression vectors encoding nothing (MOCK), CEP41, HIF1α, or HIF1α with CEP41 and exposed to hypoxia. VEGFA (E) and VEGFR2 (F) mRNA levels were analyzed by qRT-PCR using cDNA from transfected cells. Data are shown as mean ± SD of three independent experiments. Statistical significance was determined using the one-way ANOVA followed by Tukey's post hoc test (***P < 0.001, ns: non-significant). NOR, normoxia; HYP, hypoxia.


Control or CEP41 - depleted HUVECs were transfected with expression vectors encoding nothing ( MOCK ) or HIF1α and treated with MG132 or not , and then the cells were cultivated under hypoxia . Whole cell lysates were used for immunoblot assays for HIF1α , phospho - AURKA , and AURKA , and the protein levels were normalized against β - ACTIN in the same blots . NOR , normoxia ; HYP , hypoxia .

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

Control or CEP41-depleted HUVECs were transfected with expression vectors encoding nothing (MOCK) or HIF1α and treated with MG132 or not, and then the cells were cultivated under hypoxia. Whole cell lysates were used for immunoblot assays for HIF1α, phospho-AURKA, and AURKA, and the protein levels were normalized against β-ACTIN in the same blots. NOR, normoxia; HYP, hypoxia.


Quantification of tube node number in ( H ) and tube length in ( I ) from in vitro angiogenesis assays with HIF1α # 1 or # 2 siRNAs - transfected HUVECs overexpressing nothing ( MOCK ) or CEP41 under hypoxia . Data are shown as mean ± SD of five independent experiments with ≥ 5 tubulogenesis regions per condition .

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

Quantification of tube node number in (H) and tube length in (I) from in vitro angiogenesis assays with HIF1α #1 or #2 siRNAs-transfected HUVECs overexpressing nothing (MOCK) or CEP41 under hypoxia. Data are shown as mean ± SD of five independent experiments with ≥ 5 tubulogenesis regions per condition.


VEGFA ( J ) and VEGFR2 ( K ) mRNA levels were analyzed by qRT - PCR using cDNA from hypoxic HIF1α - depleted HUVECs transfected with control or CEP41 expression vectors . The results were compared to those of control and normoxic HIF1α - depleted cells . Data are shown as mean ± SD of three independent experiments .

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

VEGFA (J) and VEGFR2 (K) mRNA levels were analyzed by qRT-PCR using cDNA from hypoxic HIF1α-depleted HUVECs transfected with control or CEP41 expression vectors. The results were compared to those of control and normoxic HIF1α-depleted cells. Data are shown as mean ± SD of three independent experiments.


( A ) Analysis of the single - cell transcriptomics dataset for the mouse visual cortex ( Tasic et al , 2016 ) revealed that the expression of the metabolism - associated gene Cox6a2 is highly restricted to PV + interneurons . Cox6a2 is expressed in all subtypes of PV + interneurons ( marked in blue color ) but rarely detected in other neuronal subtypes from the mouse visual cortex . Cox6a1 , which encodes the other protein isoform of COX6A , is expressed in all subtypes .

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(A) Analysis of the single-cell transcriptomics dataset for the mouse visual cortex (Tasic et al, 2016) revealed that the expression of the metabolism-associated gene Cox6a2 is highly restricted to PV+ interneurons. Cox6a2 is expressed in all subtypes of PV+ interneurons (marked in blue color) but rarely detected in other neuronal subtypes from the mouse visual cortex. Cox6a1, which encodes the other protein isoform of COX6A, is expressed in all subtypes.


( B RT - PCR showing expression of Cox6a2 in the brain and heart .

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(B RT-PCR showing expression of Cox6a2 in the brain and heart.


C ) Western - blot showing expression of Cox6a2 in the brain and heart .

C) Western-blot showing expression of Cox6a2 in the brain and heart.


( D ) Western - blot analysis showed that COX6A2 is first detected in the brain at postnatal day 0 ( P0 ) . Then , COX6A2 protein levels gradually increase until P60 .

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(D) Western-blot analysis showed that COX6A2 is first detected in the brain at postnatal day 0 (P0). Then, COX6A2 protein levels gradually increase until P60.


COX6A2 immunoreactivity is detected in PV + interneurons in different brain areas of adult mice , including the cortex ( general overview in E , and single PV + interneuron magnification in F ) White arrowheads indicate PV / Cox6a2 double positive cells .

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COX6A2 immunoreactivity is detected in PV+ interneurons in different brain areas of adult mice, including the cortex (general overview in E, and single PV+ interneuron magnification in F) White arrowheads indicate PV/Cox6a2 double positive cells.


COX6A2 immunoreactivity is detected in PV + interneurons in hippocampus ( G ) and striatum ( H ) . White arrowheads indicate PV / Cox6a2 double positive cells .

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COX6A2 immunoreactivity is detected in PV+ interneurons in hippocampus (G) and striatum (H). White arrowheads indicate PV/Cox6a2 double positive cells.


( I , J ) Quantifications showing almost 100 % of co - localization of COX6A2 + cells and PV + cells in the somatosensory ( SS ) and motor ( MT ) cortices , and the hippocampus ( HI ) of adult mice ( n animals = 4 , 2 months old , n sections = 24 SS , 16 MT , 8 HI ) .

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(I,J) Quantifications showing almost 100% of co-localization of COX6A2+ cells and PV+ cells in the somatosensory (SS) and motor (MT) cortices, and the hippocampus (HI) of adult mice (n animals=4, 2 months old, n sections=24 SS, 16 MT, 8 HI).


( K ) COX6A2 immunoreactivity in the cortex at P9 ( n animals = 2 , n sections = 12 SS , 8 MT , 4 HI ) . White arrowheads indicate Cox6a2 positive cells .

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(K) COX6A2 immunoreactivity in the cortex at P9 (n animals=2, n sections=12 SS, 8 MT, 4 HI). White arrowheads indicate Cox6a2 positive cells.


( L - N ) As soon as parvalbumin immunoreactivity becomes visible at P13 , almost 100 % of PV + cells are co - labelled with COX6A2 immunoreactivity in the SS and MT cortices , and the HI of P13 mice ( n = 2 ) .

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(L-N) As soon as parvalbumin immunoreactivity becomes visible at P13, almost 100% of PV+ cells are co-labelled with COX6A2 immunoreactivity in the SS and MT cortices, and the HI of P13 mice (n=2).


( A , B ) COX6A2 immunoreactivity is virtually restricted to PV + interneurons in the somatosensory ( SS ) and motor ( MT ) cortices , and the hippocampus ( HI ) of adult rat ( n animals = 1 , 2 . 5 months old , n sections = 12 SS , 8 MT , 4 HI ) . White arrowheads indicate PV / Cox6a2 double positive cells .

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(A,B) COX6A2 immunoreactivity is virtually restricted to PV+ interneurons in the somatosensory (SS) and motor (MT) cortices, and the hippocampus (HI) of adult rat (n animals=1, 2.5 months old, n sections=12 SS, 8 MT, 4 HI). White arrowheads indicate PV/Cox6a2 double positive cells.


( C , D ) In the SS of adult rhesus monkey , COX6A2 immunoreactivity is present exclusively in PV + cells ( n animals = 1 , 1 - year - old , n sections = 6 ) . White arrowheads indicate PV / Cox6a2 double positive cells .

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(C,D) In the SS of adult rhesus monkey, COX6A2 immunoreactivity is present exclusively in PV+ cells (n animals=1, 1-year-old, n sections=6). White arrowheads indicate PV/Cox6a2 double positive cells.


( E , F ) In adult humans , all cortical PV + interneurons from Brodmann areas 9 and 10 ( BA9 and BA10 ) are co - labelled with COX6A2 immunoreactivity . However , PV + interneurons only represent 60 % of the cells in which COX6A2 is detected ( n human subjects = 2 , 59 - and 69 - year - old , n sections = 6 BA9 , 6 BA10 ) . White arrowheads indicate PV / Cox6a2 double positive cells .

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(E,F) In adult humans, all cortical PV+ interneurons from Brodmann areas 9 and 10 (BA9 and BA10) are co-labelled with COX6A2 immunoreactivity. However, PV+ interneurons only represent 60% of the cells in which COX6A2 is detected (n human subjects=2, 59- and 69-year-old, n sections=6 BA9, 6 BA10). White arrowheads indicate PV/Cox6a2 double positive cells.


( A ) Images showing absence of COX6A2 immunoreactivity in the cortex of Cox6a2 - / - mice .

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(A) Images showing absence of COX6A2 immunoreactivity in the cortex of Cox6a2-/- mice.


( B , C ) Parvalbumin immunoreactivity in PV + interneurons was significantly decreased in the somatosensory cortex of Cox6a2 - / - mice compared to wild - types ( n = 4 WT , 4 Cox6a2 - / - ; 2 - month - old ) . C : two - tailed unpaired Student ' s t - test .

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(B,C) Parvalbumin immunoreactivity in PV+ interneurons was significantly decreased in the somatosensory cortex of Cox6a2-/- mice compared to wild-types (n=4 WT, 4 Cox6a2-/-; 2-month-old). C: two-tailed unpaired Student's t-test.


( D ) In Cox6a2 - / - mice , cortical PV + interneurons showed a fewer number of synapses innervating principal neurons ( n = 3 WT , 3 Cox6a2 - / - ; 2 - month - old ) . Two - tailed unpaired Student ' s t - test .

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(D) In Cox6a2-/- mice, cortical PV+ interneurons showed a fewer number of synapses innervating principal neurons (n=3 WT, 3 Cox6a2-/-; 2-month-old). Two-tailed unpaired Student's t-test.


( E ) To analyze changes in oxidative stress in PV + interneurons , the somatosensory and motor cortices of WT and Cox6a2 - / - mice were stained with an antibody against 8 - OHdG , a marker of oxidative DNA damage . ( F ) 8 - OHdG immunoreactivity was significantly higher in PV + null in both cortices in Cox6a2 - / - mice ( n = 4 WT , 4 Cox6a2 - / - ; 2 - month - old ) . Two - tailed unpaired Mann - Whitney tests .

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(E) To analyze changes in oxidative stress in PV+ interneurons, the somatosensory and motor cortices of WT and Cox6a2-/- mice were stained with an antibody against 8-OHdG, a marker of oxidative DNA damage. (F) 8-OHdG immunoreactivity was significantly higher in PV+ null in both cortices in Cox6a2-/- mice (n=4 WT, 4 Cox6a2-/-; 2-month-old). Two-tailed unpaired Mann-Whitney tests.


( G ) The density of PNNs around PV + interneurons residing in the somatosensory and motor cortices was analyzed by measuring the immunoreactivity of WFA that stains PNNs . ( H ) WFA immunoreactivity around PV + cells was lower in both cortices in Cox6a2 - / - mice ( n = 4 WT , 4 Cox6a2 - / - ; 2 - month - old ) . Two - tailed unpaired Mann - Whitney tests .

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(G) The density of PNNs around PV+ interneurons residing in the somatosensory and motor cortices was analyzed by measuring the immunoreactivity of WFA that stains PNNs. (H) WFA immunoreactivity around PV+ cells was lower in both cortices in Cox6a2-/- mice (n=4 WT, 4 Cox6a2-/-; 2-month-old). Two-tailed unpaired Mann-Whitney tests.


( A ) PV + interneurons were sorted from cortices of PV - EGFP WT and Cox6a2 - / - mice at P15 and processed for bulk RNA sequencing ( n = 4 WT , 6 Cox6a2 - / - ; P15 ) . Heatmap shows expression for top down - ( red ) and upregulated ( blue ) genes in Cox6a2 - / - PV + interneurons relative to their WT counterparts .

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(A) PV+ interneurons were sorted from cortices of PV-EGFP WT and Cox6a2-/- mice at P15 and processed for bulk RNA sequencing (n= 4 WT, 6 Cox6a2-/-; P15). Heatmap shows expression for top down- (red) and upregulated (blue) genes in Cox6a2-/- PV+ interneurons relative to their WT counterparts.


( B ) Cox6a2 expression was effectively abolished in Cox6a2 - / - PV + interneurons . In addition , Cox6a2 knockout led to a decreased expression of Gad1 and Gad2 genes ( n = 4 WT , 6 Cox6a2 - / - ) . Two - tailed unpaired Mann - Whitney tests .

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(B) Cox6a2 expression was effectively abolished in Cox6a2-/- PV+ interneurons. In addition, Cox6a2 knockout led to a decreased expression of Gad1 and Gad2 genes (n= 4 WT, 6 Cox6a2-/-). Two-tailed unpaired Mann-Whitney tests.


( C ) Changes in expression of oxidative stress - related genes ( green ) , genes coding for AMPA receptors ( orange ) , sodium channels ( red ) , potassium channels ( magenta ) , and internal calcium signaling proteins ( blue ) . Color scale bars show normalized read counts .

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(C) Changes in expression of oxidative stress-related genes (green), genes coding for AMPA receptors (orange), sodium channels (red), potassium channels (magenta), and internal calcium signaling proteins (blue). Color scale bars show normalized read counts.


( D ) Representative images of PV + interneurons from PV - CRE and PV - CRE ; Cox6a2fl / fl mice showing PercevalHR fluorescence after excitation at 930 nm and 840 nm . White arrowheads indicate PercevalHR expressing cells . Scale bar : 20 μm .

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(D) Representative images of PV+ interneurons from PV-CRE and PV-CRE;Cox6a2fl/fl mice showing PercevalHR fluorescence after excitation at 930 nm and 840 nm. White arrowheads indicate PercevalHR expressing cells. Scale bar: 20 μm.


( E ) PV + interneurons from PV - CRE ; Cox6a2fl / fl mice exhibited significantly lower ATP - to - ADP ratios than PV + interneurons from PV - CRE mice throughout the entire duration of the recordings ( 23 cells from 4 PV - CRE mice ; 15 cells from 6 PV - CRE ; Cox6a2 - / - mice ) .

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(E) PV+ interneurons from PV-CRE;Cox6a2fl/fl mice exhibited significantly lower ATP-to-ADP ratios than PV+ interneurons from PV-CRE mice throughout the entire duration of the recordings (23 cells from 4 PV-CRE mice; 15 cells from 6 PV-CRE;Cox6a2-/- mice).


( A , B ) The resting membrane potential and amplitude of action potentials in WT and Cox6a2 - / - PV + interneurons , respectively . Two - tailed unpaired Student ' s t - tests .

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(A,B) The resting membrane potential and amplitude of action potentials in WT and Cox6a2-/- PV+ interneurons, respectively. Two-tailed unpaired Student's t-tests.


( C , D ) The threshold for action potentials increased over maturation in WT but not in Cox6a2 - / - PV + interneurons . As a result , the threshold for action potentials was significantly lower in P34 - 45 Cox6a2 - / - PV + interneurons when compared to their WT counterparts . D : two - tailed unpaired Student ' s t - tests .

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(C,D) The threshold for action potentials increased over maturation in WT but not in Cox6a2-/- PV+ interneurons. As a result, the threshold for action potentials was significantly lower in P34-45 Cox6a2-/- PV+ interneurons when compared to their WT counterparts. D: two-tailed unpaired Student's t-tests.


( E , F ) Trains of action potentials were evoked by 2 sec depolarizing current pulses . The amplitude difference ( difference between the mean amplitude of 1st - 10th action potentials and 190th - 200th action potentials ) was significantly higher in P34 - 45 Cox6a2 - / - compared to WT PV + interneurons . In addition , the amplitude difference decreased with maturation in WT but not in Cox6a2 - / - PV + interneurons . F : two - tailed unpaired Mann - Whitney tests .

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(E,F) Trains of action potentials were evoked by 2 sec depolarizing current pulses. The amplitude difference (difference between the mean amplitude of 1st-10th action potentials and 190th-200th action potentials) was significantly higher in P34-45 Cox6a2-/- compared to WT PV+ interneurons. In addition, the amplitude difference decreased with maturation in WT but not in Cox6a2-/- PV+ interneurons. F: two-tailed unpaired Mann-Whitney tests.


( G ) While in WT PV + interneurons the maximal firing frequency increased during maturation , such increase was abolished by Cox6a2 knockout . Thus , maximal firing frequency reached during 2 sec depolarizing current pulses was lower in P34 - 45 Cox6a2 - / - compared to WT PV + interneurons . Two - tailed unpaired Student ' s t - tests .

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(G) While in WT PV+ interneurons the maximal firing frequency increased during maturation, such increase was abolished by Cox6a2 knockout. Thus, maximal firing frequency reached during 2 sec depolarizing current pulses was lower in P34-45 Cox6a2-/- compared to WT PV+ interneurons. Two-tailed unpaired Student's t-tests.


( H ) Recorded PV + interneurons were filled with biocytin and their dendritic morphology was reconstructed using Imaris software . Scale bars : 30 μm .

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(H) Recorded PV+ interneurons were filled with biocytin and their dendritic morphology was reconstructed using Imaris software. Scale bars: 30 μm.


( 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 .

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

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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 ) .

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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 .

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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.