C . SEC profile of a sample containing an equimolar mixture of purified His - GFP - spMis18C - term - α His - spMis18MeDiY ( upper panel ) SDS - PAGE of SEC - fractions ( bottom panel ) . His - GFP - spMis18C - term - α His - spMis18MeDiY eluted separately at 12 . 3 15 . 3 ml , respectively , demonstrating inability of these domains to interact with each other .

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C. SEC profile of a sample containing an equimolar mixture of purified His-GFP-spMis18C-term-α and His-spMis18MeDiY (upper panel) and SDS-PAGE analysis of SEC-fractions (bottom panel). His-GFP-spMis18C-term-α and His-spMis18MeDiY eluted separately at 12.3 and 15.3 ml, respectively, demonstrating the inability of these domains to interact with each other.


D . SEC - MALS of His - GFP - spMis18C - term - α shows that it exists as a homotrimer with a measured molecular of 119 . 8 kDa . ( Theoretically calculated MW of a monomer is 38 . 3 kDa ) .

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D. SEC-MALS analysis of His-GFP-spMis18C-term-α shows that it exists as a homotrimer with a measured molecular weight of 119.8 kDa. (Theoretically calculated MW of a monomer is 38.3 kDa).


A . spMis18fl - Myc co - immunoprecipitates with both GFP - tagged spMis18fl spMis18MeDiY , but shows reduced association when MeDiY - mediated dimerization is disrupted in spMis18flI31A . asterisk ( * ) in top panel denotes IgG heavy chain .

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A. spMis18fl-Myc co-immunoprecipitates with both GFP-tagged spMis18fl and spMis18MeDiY, but shows reduced association when MeDiY-mediated dimerization is disrupted in spMis18flI31A. The asterisk (*) in the top panel denotes the IgG heavy chain.


B C . Dimer - II interface mutations I31A Y114A affect ability of ectopically expressed spMis18fl to rescue sensitivity of mis18 - 818 mis18 - 262 , while expression of spMis18MeDiY alone confers a dominant negative effect on growth in a MeDiY dimerization - dependent manner . Five - fold serial dilutions of expressing indicated spMis18 constructs integrated at leu1 locus in genome , spotted on complete PMG + Phloxine B media supplemented with ( repressed ) or without ( expressed ) thiamine , incubated at indicated temperatures ; dead stain pink .

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B C. Dimer-II interface mutations I31A and Y114A affect the ability of ectopically expressed spMis18fl to rescue the temperature sensitivity of mis18-818 and mis18-262 cells, while expression of spMis18MeDiY alone confers a dominant negative effect on growth in a MeDiY dimerization-dependent manner. Five-fold serial dilutions of cells expressing the indicated spMis18 constructs integrated at the leu1 locus in the genome, spotted on complete PMG + Phloxine B media supplemented with (repressed) or without (expressed) thiamine, and incubated at the indicated temperatures; dead cells stain dark pink.


D . Mutations that disrupt MeDiY dimerization lead to reduced of spMis18fl association with centromeres . qChIP analyses of spMis18fl - GFP association with centromere 2 ( cc2 ) in indicated strains when grown in complete PMG media supplemented with ( repressed ) or without ( expressed ) thiamine . Error bars represent standard deviation between at least three biological replicates .

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D. Mutations that disrupt MeDiY dimerization lead to reduced levels of spMis18fl association with centromeres. qChIP analyses of spMis18fl-GFP association with centromere 2 (cc2) in the indicated strains when grown in complete PMG media supplemented with (repressed) or without (expressed) thiamine. Error bars represent standard deviation between at least three biological replicates.


A - B HT ‑ 29 were transfected with non - silencing control siRNA ( sictr ) or siRNAs against USP22 ( siUSP22 ) for 48 h at 20 nM . After transfection , were treated with 20 µM zVAD . fmk , 0 . 5 µM BV6 1 ng / ml TNFα either for 6 h analyzed by Western blotting ( A ) or for 18 h , of PI - positive was assessed by - PI ( B ) . Vinculin served as a loading control .

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A-B HT‑29 cells were transfected with non-silencing control siRNA (sictr) or siRNAs against USP22 (siUSP22) for 48 h at 20 nM. After transfection, cells were treated with 20 µM zVAD.fmk, 0.5 µM BV6 and 1 ng/ml TNFα either for 6 h and analyzed by Western blotting (A) or for 18 h, and the percentage of PI-positive cells was assessed by fluorescence-based PI staining (B). Vinculin served as a loading control.


C HT ‑ 29 CRISPR / Cas9 control ( ctr ) USP22 KO were analyzed by Western blotting for USP22 expression . GAPDH served as loading control .

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C HT‑29 CRISPR/Cas9 control (ctr) and USP22 KO cells were analyzed by Western blotting for USP22 expression. GAPDH served as loading control.


D HT ‑ 29 control USP22 KO were treated with 20 µM zVAD . fmk , 0 . 5 µM BV6 1 ng / ml TNFα after pre - incubation with 30 µM Nec1 - s 20 µM GSK ' 872 for 1 h incubated for 18 h before - of PI - positive .

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D HT‑29 control and USP22 KO cells were treated with 20 µM zVAD.fmk, 0.5 µM BV6 and 1 ng/ml TNFα after pre-incubation with 30 µM Nec1-s and 20 µM GSK'872 for 1 h and incubated for 18 h before fluorescence-based quantification of PI-positive cells.


E HT ‑ 29 control USP22 KO , generated with 2 guide RNAs ( 2g ) , expressing empty vector ( EV ) or PAM mutated 3xFLAG - HA - USP22 WT ( USP22 PAM ) , C185S ( USP22 PAM C185S ) or C185A ( USP22 PAM C185A ) were analyzed by Western blotting for USP22 expression . β - Actin was used as a loading control .

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E HT‑29 control and USP22 KO cells, generated with 2 guide RNAs (2g), expressing empty vector (EV) or PAM mutated 3xFLAG-HA-USP22 WT (USP22 PAM), C185S (USP22 PAM C185S) or C185A (USP22 PAM C185A) were analyzed by Western blotting for USP22 expression levels. β-Actin was used as a loading control.


F HT - 29 control USP22 KO , generated with 2 guide RNAs ( 2g ) , expressing empty vector ( EV ) or PAM mutated 3xFLAG - HA - USP22 WT ( USP22 PAM ) , C185S ( USP22 PAM C185S ) or C185A ( USP22 PAM C185A ) were stimulated with 20 µM zVAD . fmk , 0 . 5 µM BV6 , 1 ng / ml TNFα for 18 h . of PI - positive was assessed by - PI .

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F HT-29 control and USP22 KO cells, generated with 2 guide RNAs (2g), expressing empty vector (EV) or PAM mutated 3xFLAG-HA-USP22 WT (USP22 PAM), C185S (USP22 PAM C185S) or C185A (USP22 PAM C185A) were stimulated with 20 µM zVAD.fmk, 0.5 µM BV6, 1 ng/ml TNFα for 18 h. The percentage of PI-positive cells was assessed by fluorescence-based PI staining.


A HT ‑ 29 control USP22 KO were stimulated with 20 µM zVAD . fmk , 0 . 5 µM BV6 1 ng / ml TNFα for indicated time points . Detection of indicated was carried out by Western blotting . GAPDH served as a loading control .

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A HT‑29 control and USP22 KO cells were stimulated with 20 µM zVAD.fmk, 0.5 µM BV6 and 1 ng/ml TNFα for the indicated time points. Detection of indicated proteins was carried out by Western blotting. GAPDH served as a loading control.


B HT ‑ 29 control USP22 KO were stimulated with 20 µM zVAD . fmk , 0 . 5 µM BV6 1 ng / ml TNFα for 4 h . Detection of indicated was carried out by Western blotting . β - Actin served as a loading control .

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B HT‑29 control and USP22 KO cells were stimulated with 20 µM zVAD.fmk, 0.5 µM BV6 and 1 ng/ml TNFα for 4 h. Detection of indicated proteins was carried out by Western blotting. β-Actin served as a loading control.


C HT ‑ 29 control USP22 KO were incubated with 30 µM Nec1 - s or 20 µM Dab for 18 h , as indicated . were stimulated with 20 µM zVAD . fmk , 0 . 5 µM BV6 1 ng / ml TNFα for 5 h . 100 μg of each lysate were incubated with 400 U / μl λ - phosphatase for 30 min at 30 ° C . expression of RIPK3 was monitored by Western blotting . β - Actin was used as loading control . High molecular RIPK3 ' smears ' were quantified after λ - phosphatase treatment normalized to total RIPK3 β - Actin .

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C HT‑29 control and USP22 KO cells were incubated with 30 µM Nec1-s or 20 µM Dab for 18 h, as indicated. Cell were stimulated with 20 µM zVAD.fmk, 0.5 µM BV6 and 1 ng/ml TNFα for 5 h. 100 μg of each lysate were incubated with 400 U/μl λ-phosphatase for 30 min at 30 °C. Protein expression of RIPK3 was monitored by Western blotting. β-Actin was used as loading control. High molecular weight RIPK3 'smears' were quantified after λ-phosphatase treatment and normalized to total RIPK3 and β-Actin levels.


A HeLa TRex RIPK3 CRISPR / Cas9 control ( ctr ) USP22 KO were treated with 1 µg / ml Dox overnight . expression of induced Strep - RIPK3 was analyzed by Western blotting . GAPDH served as loading control . asterisk marks an unspecific band .

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A HeLa TRex RIPK3 CRISPR/Cas9 control (ctr) and USP22 KO cells were treated with 1 µg/ml Dox overnight. Protein expression of induced Strep-RIPK3 was analyzed by Western blotting. GAPDH served as loading control. The asterisk marks an unspecific band.


B HeLa TRex RIPK3 control USP22 KO were incubated with 1 µg / ml Dox for 18 h before pre - treatment with 20 µM zVAD . fmk , 5 µM BV6 for 1 h . After pre - treatment , 10 ng / ml TNFα was added was measured after 4 5 h by of PI - positive nuclei .

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B HeLa TRex RIPK3 control and USP22 KO cells were incubated with 1 µg/ml Dox for 18 h before pre-treatment with 20 µM zVAD.fmk, 5 µM BV6 for 1 h. After pre-treatment, 10 ng/ml TNFα was added and cell death was measured after 4 and 5 h by analysis of PI-positive nuclei.


C HeLa TRex RIPK3 control USP22 KO were pre - treated with 20 µM zVAD . fmk , 5 µM BV6 for 1 h . After pre - treatment , 10 ng / ml TNFα were added for 1 , 2 , 3 , 4 5 h . expression of phosphorylated RIPK1 , total RIPK1 , total RIPK3 , phosphorylated MLKL , total MLKL USP22 , without ( left ) or with ( right ) 1 µg / ml Dox treatment overnight , was monitored by Western blotting . GAPDH was used as a loading control .

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C HeLa TRex RIPK3 control and USP22 KO cells were pre-treated with 20 µM zVAD.fmk, 5 µM BV6 for 1 h. After pre-treatment, 10 ng/ml TNFα were added for 1, 2, 3, 4 and 5 h. Protein expression of phosphorylated RIPK1, total RIPK1, total RIPK3, phosphorylated MLKL, total MLKL and USP22, without (left) or with (right) 1 µg/ml Dox treatment overnight, was monitored by Western blotting. GAPDH was used as a loading control.


D HT - 29 control RIPK3 KO re - expressing PAM - mutated Dox - inducible RIPK3 WT were incubated overnight with 1 µg / ml Dox . were pre - treated with 20 µM zVAD . fmk , 5 µM BV6 for 1 h . After pre - treatment , 10 ng / ml TNFα were added for 2 h , as indicated . Strep - RIPK3 was immunoprecipitated using anti ‑ Strep - beads indicated co - immunoprecipitated were analyzed by Western blotting . β - Actin served as a loading control .

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D HT-29 control cells and RIPK3 KO cells re-expressing PAM-mutated Dox-inducible RIPK3 WT were incubated overnight with 1 µg/ml Dox. Cells were pre-treated with 20 µM zVAD.fmk, 5 µM BV6 for 1 h. After pre-treatment, 10 ng/ml TNFα were added for 2 h, as indicated. Strep-RIPK3 was immunoprecipitated using anti‑Strep-beads and the indicated co-immunoprecipitated proteins were analyzed by Western blotting. β-Actin served as a loading control.


E USP22 KO HT - 29 USP22 KO re - expressing PAM - mutated 3xFLAG - HA - USP22 were pre - treated with 20 µM zVAD . fmk , 5 µM BV6 for 1 h . After pre - treatment , 10 ng / ml TNFα was added for 2 h , as indicated . 3xFLAG - HA - USP22 was immunoprecipitated using anti - HA - beads indicated co - immunoprecipitated were analyzed by Western blotting . β - Actin served as a loading control .

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E USP22 KO HT-29 cells and USP22 KO cells re-expressing PAM-mutated 3xFLAG-HA-USP22 were pre-treated with 20 µM zVAD.fmk, 5 µM BV6 for 1 h. After pre-treatment, 10 ng/ml TNFα was added for 2 h, as indicated. 3xFLAG-HA-USP22 was immunoprecipitated using anti-HA-beads and the indicated co-immunoprecipitated proteins were analyzed by Western blotting. β-Actin served as a loading control.


A HT - 29 control , USP22 KO USP22 KO re - expressing PAM - mutated 3xFLAG - HA - USP22 WT or C185S were stimulated with 20 µM zVAD . fmk , 0 . 5 µM BV6 1 ng / ml TNFα for 4 h . Poly - ubiquitinated were enriched by GST - TUBE pull - down , followed by incubation with catalytic domain of USP2 , as indicated . RIPK3 USP22 expression of ubiquitinated RIPK3 were monitored using Western blotting with indicated antibodies . β - Actin served as loading control . Ponceau - was used to confirm equal loading of GST - TUBE .

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A HT-29 control, USP22 KO and USP22 KO cells re-expressing PAM-mutated 3xFLAG-HA-USP22 WT or C185S were stimulated with 20 µM zVAD.fmk, 0.5 µM BV6 and 1 ng/ml TNFα for 4 h. Poly-ubiquitinated proteins were enriched by GST-TUBE pull-down, followed by incubation with the catalytic domain of USP2, as indicated. RIPK3 and USP22 expression and levels of ubiquitinated RIPK3 were monitored using Western blotting with the indicated antibodies. β-Actin served as loading control. Ponceau-staining was used to confirm equal loading of GST-TUBE.


B HT - 29 control USP22 KO were transfected with His - ubiquitin for 24 h , as indicated . were pre ‑ stimulated with 20 µM zVAD . fmk , 0 . 5 µM BV6 for 1 h . Following pre - treatment , 1 ng / ml TNFα was added for 4 h . His - ubiquitin was immunoprecipitated using Ni - NTA beads detection of indicated was performed by Western blotting . β - Actin served as loading control for input , whereas His - ubiquitin served as loading control for immunoprecipitated ubiquitin .

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B HT-29 control and USP22 KO cells were transfected with His-ubiquitin for 24 h, as indicated. Cells were pre‑stimulated with 20 µM zVAD.fmk, 0.5 µM BV6 for 1 h. Following pre-treatment, 1 ng/ml TNFα was added for 4 h. His-ubiquitin was immunoprecipitated using Ni-NTA beads and detection of indicated proteins was performed by Western blotting. β-Actin served as loading control for the input, whereas His-ubiquitin levels served as loading control for immunoprecipitated ubiquitin.


C HeLa TRex RIPK3 control USP22 KO were incubated with 1 µg / ml Dox transfected with HA - ubiquitin for 24 h , as indicated . were pre - stimulated with 20 µM zVAD . fmk , 5 µM BV6 for 1 h . Following pre - treatment , 10 ng / ml TNFα were added for 3 h . HA - ubiquitin was immunoprecipitated using anti - HA - beads detection of indicated was performed by Western blotting . β - Actin served as loading control for input , whereas HA - served as loading control for immunoprecipitated ubiquitin .

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C HeLa TRex RIPK3 control and USP22 KO cells were incubated with 1 µg/ml Dox and transfected with HA-ubiquitin for 24 h, as indicated. Cells were pre-stimulated with 20 µM zVAD.fmk, 5 µM BV6 for 1 h. Following pre-treatment, 10 ng/ml TNFα were added for 3 h. HA-ubiquitin was immunoprecipitated using anti-HA-beads and detection of indicated proteins was performed by Western blotting. β-Actin served as loading control for the input, whereas HA-levels served as loading control for immunoprecipitated ubiquitin.


B Bar graphs demonstrating number of up ‐ , non ‐ , downregulated ubiquitination sites in USP22 KO HT - 29 versus control under TBZ - treatment .

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B Bar graphs demonstrating the number of up‐, non‐, and downregulated ubiquitination sites in USP22 KO HT-29 cells versus control under TBZ-treatment.


D HeLa expressing Dox - inducible RIPK3 WT , D160N , K42R , K351 , K518R , 2xKR or 3xKR were pre ‑ incubated with 1 µg / ml Dox , 20 µM Dab , or 10 µM NSA overnight , followed by pre - treatment with 20 µM zVAD . fmk , 5 µM BV6 for 1 h . After pre - treatment , 10 ng / ml TNFα were added for 4 h . was measured by of PI - positive nuclei .

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D HeLa cells expressing Dox-inducible RIPK3 WT, D160N, K42R, K351, K518R, 2xKR or 3xKR were pre‑incubated with 1 µg/ml Dox, 20 µM Dab, or 10 µM NSA overnight, followed by pre-treatment with 20 µM zVAD.fmk, 5 µM BV6 for 1 h. After pre-treatment, 10 ng/ml TNFα were added for 4 h. Cell death was measured by quantification of PI-positive nuclei.


E HeLa expressing Dox - inducible RIPK3 mutants were treated with 1 µg / ml Dox overnight . of inducible RIPK3 expression were analyzed by Western blotting . GAPDH served as loading control .

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E HeLa cells expressing Dox-inducible RIPK3 mutants were treated with 1 µg/ml Dox overnight. Protein levels of inducible RIPK3 expression were analyzed by Western blotting. GAPDH served as loading control.


A HeLa expressing Dox - inducible RIPK3 WT , K518R or 3xKR were treated with 1 µg / ml Dox / or 10 µM NSA 20 µM Dab overnight . of inducible RIPK3 expression phosphorylated MLKL were examined by Western blotting . β - Actin was used as a loading control .

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A HeLa cells expressing Dox-inducible RIPK3 WT, K518R or 3xKR were treated with 1 µg/ml Dox and/or 10 µM NSA and 20 µM Dab overnight. Protein levels of inducible RIPK3 expression and phosphorylated MLKL were examined by Western blotting. β-Actin was used as a loading control.


B HeLa expressing Dox - inducible RIPK3 WT or 3xKR were treated with 1 µg / ml Dox overnight before pre - treatment with 20 µM zVAD . fmk , 5 µM BV6 for 1 h . After pre - treatment , 10 ng / ml TNFα were added for 1 , 2 , 3 4 h . of inducible RIPK3 expression phosphorylated MLKL were analyzed by Western blotting . β - Actin served as loading control .

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B HeLa cells expressing Dox-inducible RIPK3 WT or 3xKR were treated with 1 µg/ml Dox overnight before pre-treatment with 20 µM zVAD.fmk, 5 µM BV6 for 1 h. After pre-treatment, 10 ng/ml TNFα were added for 1, 2, 3 and 4 h. Protein levels of inducible RIPK3 expression and phosphorylated MLKL were analyzed by Western blotting. β-Actin served as loading control.


C HeLa expressing Dox - inducible RIPK3 WT , 2xKR or 3xKR were treated with 1 µg / ml Dox overnight before pre - treatment with 20 µM zVAD . fmk , 5 µM BV6 for 1 h . After pre - treatment , 10 ng / ml TNFα were added for 2 h . of inducible RIPK3 expression were analyzed by Western blotting . β - Actin served as loading control . D HeLa expressing Dox - inducible RIPK3 WT , K518R or 3xKR were treated with 1 µg / ml Dox overnight before pre - treatment with 20 µM zVAD . fmk , 5 µM BV6 for 1 h . After pre - treatment , 10 ng / ml TNFα were added for 2 h . Whole lysates were generated using RIPA lysis buffer containing 2 % SDS . of inducible RIPK3 expression were analyzed by Western blotting . β - Actin served as loading control .

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C HeLa cells expressing Dox-inducible RIPK3 WT, 2xKR or 3xKR were treated with 1 µg/ml Dox overnight before pre-treatment with 20 µM zVAD.fmk, 5 µM BV6 for 1 h. After pre-treatment, 10 ng/ml TNFα were added for 2 h. Protein levels of inducible RIPK3 expression were analyzed by Western blotting. β-Actin served as loading control. D HeLa cells expressing Dox-inducible RIPK3 WT, K518R or 3xKR were treated with 1 µg/ml Dox overnight before pre-treatment with 20 µM zVAD.fmk, 5 µM BV6 for 1 h. After pre-treatment, 10 ng/ml TNFα were added for 2 h. Whole cell lysates were generated using RIPA lysis buffer containing 2% SDS. Protein levels of inducible RIPK3 expression were analyzed by Western blotting. β-Actin served as loading control.


E HeLa expressing Dox - inducible RIPK3 WT , D160N , K518R or 3xKR were incubated overnight with 1 µg / ml Dox pre - treated with 20 µM zVAD . fmk , 5 µM BV6 for 1 h . After pre - treatment , 10 ng / ml TNFα were added for 1 2 h . Strep - RIPK3 was immunoprecipitated by using anti - Strep - beads . Co - immunoprecipitated phosphorylated MLKL RIPK1 , as well as expression of indicated were analyzed by Western blotting . β - Actin served as a loading control .

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E HeLa cells expressing Dox-inducible RIPK3 WT, D160N, K518R or 3xKR were incubated overnight with 1 µg/ml Dox and pre-treated with 20 µM zVAD.fmk, 5 µM BV6 for 1 h. After pre-treatment, 10 ng/ml TNFα were added for 1 and 2 h. Strep-RIPK3 was immunoprecipitated by using anti-Strep-beads. Co-immunoprecipitated phosphorylated MLKL and RIPK1, as well as protein expression of indicated proteins were analyzed by Western blotting. β-Actin served as a loading control.


F HeLa expressing Dox - inducible RIPK3 WT , K518R or 3xKR were incubated with 1 µg / ml Dox transfected with HA - ubiquitin for 24 h , as indicated . were pre - treated with 20 µM zVAD . fmk , 5 µM BV6 for 1 h . After pre - treatment , 10 ng / ml TNFα were added for 2 h . HA - ubiquitin was immunoprecipitated using anti - HA - beads detection of indicated was performed by Western blotting . β - Actin served as loading control for input , whereas HA - ubiquitin served as loading control for immunoprecipitated ubiquitin .

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F HeLa cells expressing Dox-inducible RIPK3 WT, K518R or 3xKR were incubated with 1 µg/ml Dox and transfected with HA-ubiquitin for 24 h, as indicated. Cells were pre-treated with 20 µM zVAD.fmk, 5 µM BV6 for 1 h. After pre-treatment, 10 ng/ml TNFα were added for 2 h. HA-ubiquitin was immunoprecipitated using anti-HA-beads and detection of indicated proteins was performed by Western blotting. β-Actin served as loading control for the input, whereas HA-ubiquitin levels served as loading control for immunoprecipitated ubiquitin.


A HT ‑ 29 RIPK3 KO re - expressing Dox - inducible WT RIPK3 or indicated RIPK3 mutants were incubated with 1 µg / ml Dox overnight . were treated with 20 µM zVAD . fmk , 5 µM BV6 10 ng / ml TNFα for 4 h . was measured by of PI - positive nuclei .

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A HT‑29 RIPK3 KO cells re-expressing Dox-inducible WT RIPK3 or the indicated RIPK3 mutants were incubated with 1 µg/ml Dox overnight. Cells were treated with 20 µM zVAD.fmk, 5 µM BV6 and 10 ng/ml TNFα for 4 h. Cell death was measured by analysis of PI-positive nuclei.


B HT ‑ 29 RIPK3 KO re - expressing Dox - inducible WT RIPK3 or indicated RIPK3 mutants were incubated with 1 µg / ml Dox overnight , as indicated . were treated with 20 µM zVAD . fmk , 5 µM BV6 10 ng / ml TNFα for 2 h analyzed by Western blotting for RIPK3 phosphorylated MLKL expression . β - Actin served as a loading control .

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B HT‑29 RIPK3 KO cells re-expressing Dox-inducible WT RIPK3 or the indicated RIPK3 mutants were incubated with 1 µg/ml Dox overnight, as indicated. Cells were treated with 20 µM zVAD.fmk, 5 µM BV6 and 10 ng/ml TNFα for 2 h and analyzed by Western blotting for RIPK3 and phosphorylated MLKL expression levels. β-Actin served as a loading control.


presence of GFP - PSF - 1 on mitotic ( indicated by arrows ) was monitored by spinning disk confocal microscopy ( see Materials Methods ) , in GFP - psf - 1 mCherry - Histone H2B worms that were fed on containing a single plasmid expressing indicated RNAi treatments ( ' Control ' = empty vector ) . NEB = envelope breakdown . scalebars correspond to 5 µm .

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The presence of GFP-PSF-1 on mitotic chromatin (indicated by white arrows) was monitored by spinning disk confocal microscopy (see Materials and Methods), in GFP-psf-1 mCherry-Histone H2B worms that were fed on bacteria containing a single plasmid expressing the indicated RNAi treatments ('Control' = empty vector). NEB = nuclear envelope breakdown. The scalebars correspond to 5 µm.


Ubiquitylation of CMG - MCM - 7 was reconstituted with indicated factors as described in Materials Methods . ' Neddylation ' indicates addition of C . elegans ULA - 1 _ RFL - 1 E1 enzyme , UBC - 12 E2 enzyme , DCN - 1 E3 enzyme NED - 8 .

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Ubiquitylation of CMG-MCM-7 was reconstituted with the indicated factors as described in Materials and Methods. 'Neddylation' indicates addition of the C. elegans ULA-1_RFL-1 E1 enzyme, the UBC-12 E2 enzyme, the DCN-1 E3 enzyme and NED-8.


Reactions containing CMG either CUL - 2LRR - 1 or CUL - 2VHL - 1 were performed in presence or absence of other replisome factors as indicated . CMG was then isolated by immunoprecipitation of SLD - 5 indicated factors were monitored by immunoblotting .

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Reactions containing CMG and either CUL-2LRR-1 or CUL-2VHL-1 were performed in the presence or absence of other replisome factors as indicated. CMG was then isolated by immunoprecipitation of SLD-5 and the indicated factors were monitored by immunoblotting.


Reconstituted CMG - MCM - 7 ubiquitylation reactions were performed as above in presence of indicated factors . ' Neddylation ' indicates addition of C . elegans ULA - 1 _ RFL - 1 E1 enzyme , UBC - 12 E2 enzyme , DCN - 1 E3 enzyme NED - 8 .

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Reconstituted CMG-MCM-7 ubiquitylation reactions were performed as above in the presence of the indicated factors. 'Neddylation' indicates addition of the C. elegans ULA-1_RFL-1 E1 enzyme, the UBC-12 E2 enzyme, the DCN-1 E3 enzyme and NED-8.


CMG was isolated by immunoprecipitation of SLD - 5 , after ubiquitylation in presence of TIM - 1 _ TIPN - 1 , before incubation with indicated factors . ' Neddylation ' indicates addition of C . elegans ULA - 1 _ RFL - 1 E1 enzyme , UBC - 12 E2 enzyme , DCN - 1 E3 enzyme NED - 8 . CMG disassembly was monitored by release of CDC - 45 or MCM - 2 - 7 into supernatant , whilst SLD - 5 other GINS subunits remained on beads . Asterisks indicate non - specific bands in anti - MCM - 7 CDC - 45 immunoblots .

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CMG was isolated by immunoprecipitation of SLD-5, after ubiquitylation in the presence of TIM-1_TIPN-1, before incubation with the indicated factors. 'Neddylation' indicates addition of the C. elegans ULA-1_RFL-1 E1 enzyme, the UBC-12 E2 enzyme, the DCN-1 E3 enzyme and NED-8. CMG disassembly was monitored by release of CDC-45 or MCM-2-7 proteins into the supernatant, whilst SLD-5 and other GINS subunits remained on the beads. Asterisks indicate non-specific bands in the anti-MCM-7 and CDC-45 immunoblots.


TAP - psf - 1 worms were fed on containing a single plasmid expressing indicated RNAi treatments , before preparation of embryonic extracts isolation of TAP - PSF - 1 by immunoprecipitation . bound material was released from beads by cleavage of TAP tag with TEV protease , before detection of indicated factors by immunoblotting .

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TAP-psf-1 worms were fed on bacteria containing a single plasmid expressing the indicated RNAi treatments, before preparation of embryonic cell extracts and isolation of TAP-PSF-1 by immunoprecipitation. The bound material was released from beads by cleavage of the TAP tag with TEV protease, before detection of the indicated factors by immunoblotting.


Similar experiment comparing impact of RNAi inactivation of lrr - 1 , tim - 1 tipn - 1 upon CMG - MCM - 7 ubiquitylation association of CUL - 2LRR - 1 with worm replisome . Asterisks indicate non - specific bands in immunoblots .

Similar experiment comparing the impact of RNAi inactivation of lrr-1, tim-1 and tipn-1 upon CMG-MCM-7 ubiquitylation and the association of CUL-2LRR-1 with the worm replisome. Asterisks indicate non-specific bands in the immunoblots.


Embryonic viability was monitored as described in Materials Methods , after feeding worms on indicated proportions of expressing RNAi ( ' % RNAi ' ) to denoted genes , mixed with containing empty vector ( to make a total of 100 % ) .

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Embryonic viability was monitored as described in Materials and Methods, after feeding worms on the indicated proportions of bacteria expressing RNAi ('% RNAi') to the denoted genes, mixed with bacteria containing empty vector (to make a total of 100%).


presence of GFP - PSF - 1 on mitotic ( indicated by arrows ) was monitored by spinning disk confocal microscopy ( see Materials Methods ) , in derived from worms that were fed on containing a single plasmid expressing indicated RNAi ( ' Control ' = empty vector ) . scalebars correspond to 5 µm .

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The presence of GFP-PSF-1 on mitotic chromatin (indicated by white arrows) was monitored by spinning disk confocal microscopy (see Materials and Methods), in embryos derived from worms that were fed on bacteria containing a single plasmid expressing the indicated RNAi ('Control' = empty vector). The scalebars correspond to 5 µm.


Analogous experiment to that in ( A ) , but with worms fed on indicated proportions of expressing ubxn - 3 RNAi , in combination with 20 % expressing tim - 1 RNAi specific for tim - 1 , plus required remainder of containing empty vector ( to make a total of 100 % ) .

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Analogous experiment to that in (A), but with worms fed on the indicated proportions of bacteria expressing ubxn-3 RNAi, in combination with 20% bacteria expressing tim-1 RNAi specific for tim-1, plus the required remainder of bacteria containing empty vector (to make a total of 100%).


B SDS - PAGE of soluble ( Supernatant ) insoluble ( Pellet ) from human neurons after treatment for one hour with a mixture of RNase A RNase T1 ( A / T1 ) , or vehicle ( Ve - ) .

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B SDS-PAGE analysis of soluble (Supernatant) and insoluble proteins (Pellet) from human neurons after treatment for one hour with a mixture of RNase A and RNase T1 (A/T1), or vehicle (Ve-).


C aggregation ( Pellet ) after incubation with different ribonucleases or DNase I in presence of either EDTA or Mg2 + . Ribonucleases used were RNase A ( A ) , RNase T1 ( T1 ) , a mixture of RNase A RNase T1 ( A / T1 ) , RNase 1f ( 1f ) , RNase V1 ( V1 ) .

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C Protein aggregation (Pellet) after incubation with different ribonucleases or DNase I in the presence of either EDTA or Mg2+. Ribonucleases used were RNase A (A), RNase T1 (T1), a mixture of RNase A and RNase T1 (A/T1), RNase 1f (1f), and RNase V1 (V1).


Western blot of soluble aggregated after RNase treatment of lysate from human neurons ( A )

Western blot analysis of soluble and aggregated proteins after RNase treatment of lysate from human neurons (A)


Western blot of soluble aggregated after RNase treatment of lysate from HEK293 expressing GFP - Aβ or GFP ( B ) .

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Western blot analysis of soluble and aggregated proteins after RNase treatment of lysate from HEK293 cells expressing GFP-Aβ or GFP (B).


C Effect of an RNase A inhibitor ( RNasin ) on RNase A mediated aggregation in neuronal lysate .

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C Effect of an RNase A inhibitor (RNasin) on RNase A mediated protein aggregation in neuronal cell lysate.


B Effect of / on aggregation after renaturing .

B Effect of RNA/protein ratio on protein aggregation after renaturing.


C Coomassie - stained gel of soluble ( Sup 1 / 2 ) aggregated ( Pel 1 / 2 ) after removal of GuHCl in presence ( + ) or absence ( - ) of total . Asterix ( * ) denotes added RNase A .

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C Coomassie-stained gel of soluble (Sup 1/2) and aggregated (Pel 1/2) proteins after removal of GuHCl in the presence (+) or absence (-) of total RNA. Asterix (*) denotes added RNase A.


soluble ( Sup 1 / 2 ) aggregated ( Pel 1 / 2 ) after removal of GuHCl in presence ( + ) or absence ( - ) of total . D As in ( c ) but analysed by Western blot .

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soluble (Sup 1/2) and aggregated (Pel 1/2) proteins after removal of GuHCl in the presence (+) or absence (-) of total RNA. D As in (c) but analysed by Western blot.


E ATP - hydrolysing activity of renatured with total or buffer ( Ve - ) after removal of GuHCl .

E ATP-hydrolysing activity of proteins renatured with total RNA or buffer (Ve-) after removal of GuHCl.


A Genomic attributes of associated with renatured NF - H , Tau , or Aβ . Inset shows which fractions of peaks that derive from exons or introns have their origin in coding or non - coding transcripts , respectively .

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A Genomic attributes of RNA associated with renatured NF-H, Tau, or Aβ. Inset shows which fractions of the peaks that derive from exons or introns have their origin in coding or non-coding transcripts, respectively.


B Characterization of associated with soluble , renatured Aβ or ( Total ) , captured either by immunoprecipitation ( Aβ ) or absorption to membranes ( Total ) .

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B Characterization of DNA associated with soluble, renatured Aβ or all proteins (Total), captured either by immunoprecipitation (Aβ ) or absorption to membranes (Total).


A Proportion of aggregated after renaturation with either single - stranded ( Forward or Reverse ) or complementary strands ( For / Rev ) of selected M1x4 or control oligonucleotide , M2x4 .

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A Proportion of aggregated proteins after renaturation with either single-stranded (Forward or Reverse) or complementary strands (For/Rev) of the selected M1x4 or the control oligonucleotide, M2x4.


B Western blot of renatured with either single - stranded or double - stranded M1x4 or M2x4 oligonucleotides .

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B Western blot analysis of proteins renatured with either single-stranded or double-stranded M1x4 or M2x4 DNA oligonucleotides.


C effect on re - aggregation from varying number of motif repeats when complementary oligonucleotides are given together .

C The effect on protein re-aggregation from varying the number of DNA motif repeats when complementary oligonucleotides are given together.


D aggregation after renaturation with M1x4 oligonucleotides pre - annealed with 100 mM NaCl or vehicle ( None ) .

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D Protein aggregation after renaturation with the M1x4 DNA oligonucleotides pre-annealed with 100 mM NaCl or vehicle (None).


B Proportion of aggregation following renaturing with ds / ss / ds oligonucleotides shown in ( A ) .

B Proportion of protein aggregation following renaturing with the ds/ss/ds oligonucleotides shown in (A).


C Renaturing capacity of structurally different oligonucleotides . diagrams on right show a theoretical structure of each oligonucleotide . oligonucleotides , except 3x - loop 3x - bulges , contain a stretch of 30 Ts in single - stranded region same sequences ( 15 nucleotides each ) in double stranded regions . 3x - loops bulges oligonucleotides have 3 stretches of 9 Ts same in ds - regions .

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C Renaturing capacity of structurally different oligonucleotides. The diagrams on the right show a theoretical structure of each oligonucleotide. All oligonucleotides, except the 3x-loop and 3x-bulges, contain a stretch of 30 Ts in the single-stranded region and the same sequences (15 nucleotides each) in the double stranded regions. The 3x-loops and bulges oligonucleotides have 3 stretches of 9 Ts and the same sequence in the ds-regions.


D Proportion of aggregation in Jurkat lysate supplemented with various amounts configurations of M1x4 or M2x4 oligonucleotides .

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D Proportion of protein aggregation in Jurkat cell lysate supplemented with various amounts and configurations of the M1x4 or M2x4 DNA oligonucleotides.


e Insoluble from two ALS brain tissues were chemically denatured in guanidine hydrochloride treated with either buffer ( Vehicle ) , total from Jurkat or complementary strands of M1x4 oligonucleotides ( M1 F / R ) ( Pellet 1 , top panel ) . After removal of GuHCl , soluble fraction from these samples was treated with RNase any aggregated ( Pellet 2 ) , analysed by western blot ( middle panel ) . Remaining supernatants were then treated with Benzonase to degrade any remaining nucleic aggregated collected by centrifugation analysed by western blot ( bottom panel ) . aggregated by enzymatic degradation in Jurkat lysates , which do not contain NF - H , were used as a control .

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e Insoluble proteins from two ALS brain tissues were chemically denatured in guanidine hydrochloride and treated with either buffer (Vehicle), total RNA from Jurkat cells or the complementary strands of the M1x4 DNA oligonucleotides (M1 F/R) (Pellet 1, top panel). After removal of GuHCl, the soluble fraction from these samples was treated with RNase and any aggregated proteins (Pellet 2), analysed by western blot (middle panel). Remaining supernatants were then treated with Benzonase to degrade any remaining nucleic acids and aggregated proteins collected by centrifugation and analysed by western blot (bottom panel). Proteins aggregated by enzymatic RNA degradation in Jurkat cell lysates, which do not contain NF-H, were used as a control.


( A ) HEK transfected with STIM1 - CFP , Orai1 - mCherry , with ( red ) or without ( black ) untagged ANO8 were used to measure CRAC current with pipette solution ( buffer ) containing 3 mM EGTA . columns show averaged effect of ANO8 on current density on slop of slow Ca2 + - dependent inactivation ( SCDI ) . results are mean ± s . e . m difference were analyzed by unpaired t . Data information : first number in parenthesis indicates number of similar experiments performed second number is number of analyzed . results are given as mean ± s . e . m of indicated number of experiments or analyzed .

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(A) HEK cells transfected with STIM1-CFP, Orai1-mCherry, and with (red) or without (black) untagged ANO8 were used to measure CRAC current with pipette solution (cytoplasmic buffer) containing 3 mM EGTA. The columns show the averaged effect of ANO8 on current density and on the slop of slow Ca2+-dependent inactivation (SCDI). The results are mean±s.e.m and difference were analyzed by unpaired t test. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed.


( B ) Fura2 - loaded HEK transfected with YFP ( control , black ) or ANO8 - YFP ( red ) were used to measure store - mediated Ca2 + influx . Stores were depleted by treatment with SERCA inhibitor CPA Ca2 + influx was measured by Ca2 + add - back . results are mean ± s . e . m difference were analyzed by unpaired t . Data information : first number in parenthesis indicates number of similar experiments performed second number is number of analyzed . results are given as mean ± s . e . m of indicated number of experiments or analyzed .

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(B) Fura2-loaded HEK cells transfected with YFP (control, black) or ANO8-YFP (red) were used to measure store-mediated Ca2+ influx. Stores were depleted by treatment with the SERCA inhibitor CPA and Ca2+ influx was measured by Ca2+ add-back. The results are mean±s.e.m and difference were analyzed by unpaired t test. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed.


( C ) FRET efficiency was measured with HEK transfected with STIM1 - CFP , ANO8 - YFP , with ( red ) without ( black ) Orai1 - HA before after store depletion . Here other FRET measurements , representative of FRET signal under each condition are shown next to traces . Data information : first number in parenthesis indicates number of similar experiments performed second number is number of analyzed . results are given as mean ± s . e . m of indicated number of experiments or analyzed .

O O O 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

(C) FRET efficiency was measured with HEK cells transfected with STIM1-CFP, ANO8-YFP, and with (red) and without (black) Orai1-HA before and after store depletion. Here and all other FRET measurements, representative images of the FRET signal under each condition are shown next to the traces. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed.


( D ) Summary of FRET efficiency measurements at indicated conditions . results are mean ± s . e . m difference were analyzed by unpaired t . Data information : first number in parenthesis indicates number of similar experiments performed second number is number of analyzed . results are given as mean ± s . e . m of indicated number of experiments or analyzed .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Summary of FRET efficiency measurements at the indicated conditions. The results are mean±s.e.m and difference were analyzed by unpaired t test. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed.


( E ) FRET efficiency was measured with HEK transfected with STIM1 - CFP , STIM1 - YFP ( blue ) , co - transfected with untagged ANO8 ( green ) or treated with siANO8 ( red ) before after store depletion . Data information : first number in parenthesis indicates number of similar experiments performed second number is number of analyzed . results are given as mean ± s . e . m of indicated number of experiments or analyzed .

O O O 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

(E) FRET efficiency was measured with HEK cells transfected with STIM1-CFP, STIM1-YFP (blue), and co-transfected with untagged ANO8 (green) or treated with siANO8 (red) before and after store depletion. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed.


( F ) Resting ( R ) or store - depleted ( S ) HEK transfected with STIM1 - YFP myc - STIM1 with or without ANO8 or treated with siANO8 , were used to immunoprecipitate ( IP ) STIM1 - YFP blot ( B ) for mys - STIM1 . inputs ( In ) are with anti - myc , anti - YFP or anti - ANO8 . Data information : first number in parenthesis indicates number of similar experiments performed second number is number of analyzed . results are given as mean ± s . e . m of indicated number of experiments or analyzed .

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

(F) Resting (R) or store-depleted cells (S) HEK cells transfected with STIM1-YFP and myc-STIM1 and with or without ANO8 or treated with siANO8, were used to immunoprecipitate (IP) STIM1-YFP and blot (B) for mys-STIM1. The inputs (In) are with anti-myc, anti-YFP or anti-ANO8. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed.


( G ) Confocal bright field of basal ( upper ) store depleted ( lower ) of HEK transfected with ANO8 - YFP alone . Data information : first number in parenthesis indicates number of similar experiments performed second number is number of analyzed . results are given as mean ± s . e . m of indicated number of experiments or analyzed . experiments represent at least 4 separate experiments with similar results .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Confocal and bright field images of basal (upper) and store depleted cells (lower) of HEK cells transfected with ANO8-YFP alone. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed. The experiments represent at least 4 separate experiments with similar results.


( H ) TIRF ( upper ) TIRF - Z scan ( lower ) of same resting store depleted HEK transfected with ANO8 - YFP alone . Data information : first number in parenthesis indicates number of similar experiments performed second number is number of analyzed . results are given as mean ± s . e . m of indicated number of experiments or analyzed . experiments represent at least 4 separate experiments with similar results .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) TIRF images (upper) and TIRF-Z scan (lower) of the same resting and store depleted cells HEK cells transfected with ANO8-YFP alone. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed. The experiments represent at least 4 separate experiments with similar results.


( I ) : Confocal of resting transfected with ANO8 - YFP , STIM1 - CFP Orai1 - mCherry . ( J ) Confocal of store depleted transfected with ANO8 - YFP , STIM1 - CFP Orai1 - mCherry . Data information : first number in parenthesis indicates number of similar experiments performed second number is number of analyzed . results are given as mean ± s . e . m of indicated number of experiments or analyzed . experiments represent at least 4 separate experiments with similar results .

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

(I): Confocal images of resting cells transfected with ANO8-YFP, STIM1-CFP and Orai1-mCherry. (J) Confocal images of store depleted cells transfected with ANO8-YFP, STIM1-CFP and Orai1-mCherry. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed. The experiments represent at least 4 separate experiments with similar results.


( K ) TIRF - Z scan of resting ( upper ) store depleted ( lower ) transfected with ANO8 - YFP STIM1 - mCherry demonstrating relationship between STIM1 ANO8 at TIRF field . surface is on top . Data information : first number in parenthesis indicates number of similar experiments performed second number is number of analyzed . results are given as mean ± s . e . m of indicated number of experiments or analyzed . experiments represent at least 4 separate experiments with similar results .

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

(K) TIRF-Z scan of resting (upper) and store depleted (lower) cells transfected with ANO8-YFP and STIM1-mCherry demonstrating the relationship between STIM1 and ANO8 puncta at the TIRF field. Cell surface is on top. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed. The experiments represent at least 4 separate experiments with similar results.


( A ) FRET was measured with HEK transfected with STIM1 - CFP , Orai1 - YFP ( black ) co - transfected with untagged ANO8 ( red ) before after store depletion with 25 µM CPA in Ca2 + - free solution . Data information : first number in parenthesis indicates number of similar experiments performed second number is number of analyzed . results are given as mean ± s . e . m of indicated number of experiments or analyzed .

O 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

(A) FRET ratio was measured with HEK cells transfected with STIM1-CFP, Orai1-YFP (black) and co-transfected with untagged ANO8 (red) before and after store depletion with 25 µM CPA in Ca2+-free solution. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed.


TIRF microscopy was used to quantify formation at TIRF field in transfected with STIM1 - CFP , ANO8 - YFP Orai1 - mCherry before after store depletion . ( A ) Example . ( B ) Time course of STIM1 formation in transfected with STIM1 Orai1 alone ( black ) or together with ANO8 ( red ) . Data information : first number in parenthesis indicates number of similar experiments performed second number is number of analyzed . results are given as mean ± s . e . m of indicated number of experiments or analyzed .

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

TIRF microscopy was used to quantify puncta formation at the TIRF field in cells transfected with STIM1-CFP, ANO8-YFP and Orai1-mCherry before and after store depletion. (A) Example images. (B) Time course of STIM1 puncta formation in cells transfected with STIM1 and Orai1 alone (black) or together with ANO8 (red). Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed.


( C - E ) Effect of ANO8 on rate of STIM1 - STIM1 clustering ( C ) , number of STIM1 ( D ) number of Orai1 ( E ) . results are mean ± s . e . m difference were analyzed by unpaired t . Data information : first number in parenthesis indicates number of similar experiments performed second number is number of analyzed . results are given as mean ± s . e . m of indicated number of experiments or analyzed .

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

(C-E) Effect of ANO8 on the rate of STIM1-STIM1 clustering (C), number of STIM1 puncta (D) and number of Orai1 puncta (E). The results are mean±s.e.m and difference were analyzed by unpaired t test. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed.


( F ) HEK were used to immunoprecipitate ( IP ) native ANO8 ( A8 ) or Orai1 ( O1 ) blotted ( B ) for STIM1 . R = resting , S = stimulated . Data information : first number in parenthesis indicates number of similar experiments performed second number is number of analyzed . results are given as mean ± s . e . m of indicated number of experiments or analyzed .

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

(F) HEK cells were used to immunoprecipitate (IP) the native ANO8 (A8) or Orai1 (O1) and blotted (B) for STIM1. R=resting, S=stimulated. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed.


( G ) HEK treated with scrambled ( siScr ) or siANO8 ( siA8 ) were used to IP native Orai1 ( O1 ) or STIM1 ( S1 ) blotted ( B ) for native STIM1 , Orai1 or ANO8 , as indicated . columns show mean ± s . e . m of 4 independent experiments . results are mean ± s . e . m difference were analyzed by unpaired t . Data information : first number in parenthesis indicates number of similar experiments performed second number is number of analyzed . results are given as mean ± s . e . m of indicated number of experiments or analyzed .

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 B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HEK cells treated with scrambled (siScr) or siANO8 (siA8) were used to IP the native Orai1 (O1) or STIM1 (S1) and blotted (B) for the native STIM1, Orai1 or ANO8, as indicated. The columns show the mean±s.e.m of 4 independent experiments. The results are mean±s.e.m and difference were analyzed by unpaired t test. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed.


( H ) Co - IP of myc - STIM1 Orai1 - HA , myc - STIM1 ANO8 - YFP , Orai1 - HA ANO8 - YFP was measured in transfected HEK before ( R , resting ) in response to store depletion ( S , stimulated ) . Data information : first number in parenthesis indicates number of similar experiments performed second number is number of analyzed . results are given as mean ± s . e . m of indicated number of experiments or analyzed .

O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O B-MEASURED_VAR I-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 O O O O O O O O O O O O O O O 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) Co-IP of myc-STIM1 and Orai1-HA, myc-STIM1 and ANO8-YFP, and Orai1-HA and ANO8-YFP was measured in transfected HEK cells before (R, resting) and in response to store depletion (S, stimulated). Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed.


( I ) Effect of ANO8 - YFP on myc - STIM1 Orai1 - HA at ER / PM junction . Data information : first number in parenthesis indicates number of similar experiments performed second number is number of analyzed . results are given as mean ± s . e . m of indicated number of experiments or analyzed .

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

(I) Effect of ANO8-YFP on myc-STIM1 and Orai1-HA at the ER/PM junction. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed.


( J - L ) Junctional Orai1 ( J ) , STIM1 ( K ) ANO8 ( L ) in resting ( R ) store - depleted ( S ) . results are mean ± s . e . m of 4 experiments difference were analyzed by unpaired t . Data information : first number in parenthesis indicates number of similar experiments performed second number is number of analyzed . results are given as mean ± s . e . m of indicated number of experiments or analyzed .

O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Junctional Orai1 (J), STIM1 (K) and ANO8 (L) in resting (R) and store-depleted cells (S). The results are mean±s.e.m of 4 experiments and difference were analyzed by unpaired t test. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed.


( M ) Example EM recorded from HEK transfected with empty vector ( left ) or ANO8 ( right ) . Red arrows mark ER / PM junctions . Data information : first number in parenthesis indicates number of similar experiments performed second number is number of analyzed . results are given as mean ± s . e . m of indicated number of experiments or analyzed .

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

(M) Example EM images recorded from HEK cells transfected with empty vector (left) or ANO8 (right). Red arrows mark ER/PM junctions. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed.


( N , O ) ( N ) density ( O ) of ER / PM junctions in vector - transfected ( control ) ANO8 - transfected . Data information : first number in parenthesis indicates number of similar experiments performed second number is number of analyzed . results are given as mean ± s . e . m of indicated number of experiments or analyzed .

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

(N, O) The size (N) and density (O) of ER/PM junctions in vector-transfected (control) and ANO8-transfected cells. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed.


( A , B ) Knockdown of ANO8 ( siA8 ) reduced CRAC current in transfected with Orai1 ( O1 ) STIM1 ( S1 ) buffered with 3 mM EGTA . Data information : first number in parenthesis indicates number of similar experiments performed second number is number of analyzed . results are given as mean ± s . e . m of indicated number of experiments or analyzed difference were analyzed by unpaired t .

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

(A, B) Knockdown of ANO8 (siA8) reduced CRAC current in cells transfected with Orai1 (O1) and STIM1 (S1) and buffered with 3 mM EGTA. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed and difference were analyzed by unpaired t test.


( C ) Knockdown of ANO8 reduces native store - dependent Ca2 + influx measured in store depleted by Ca2 + add - back . Data information : first number in parenthesis indicates number of similar experiments performed second number is number of analyzed . results are given as mean ± s . e . m of indicated number of experiments or analyzed difference were analyzed by unpaired t .

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

(C) Knockdown of ANO8 reduces the native store-dependent Ca2+ influx measured in store depleted cells by Ca2+ add-back. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed and difference were analyzed by unpaired t test.


( D , E ) Knockdown of ANO8 reduced number of store - dependent STIM1 at TIRF plane in expressing STIM1 Orai1 . Panel ( D ) shows representative ( E ) is summary of 7 experiments . Data information : first number in parenthesis indicates number of similar experiments performed second number is number of analyzed . results are given as mean ± s . e . m of indicated number of experiments or analyzed difference were analyzed by unpaired t .

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

(D,E) Knockdown of ANO8 reduced the number of store-dependent STIM1 puncta at the TIRF plane in cells expressing STIM1 and Orai1. Panel (D) shows representative images and (E) is the summary of 7 experiments. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed and difference were analyzed by unpaired t test.


( F , G ) Current was measured with pipette solution contacting fast strong Ca2 + buffer 10 mM BAPTA in HEK transfected with STIM1 , Orai1 , with ( red ) or without ANO8 ( black ) . Panel ( G ) shows increase in current density at peak current . Note prominent current inactivation in presence of ANO8 . Data information : first number in parenthesis indicates number of similar experiments performed second number is number of analyzed . results are given as mean ± s . e . m of indicated number of experiments or analyzed difference were analyzed by unpaired t .

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

(F, G) Current was measured with pipette solution contacting the fast and strong Ca2+ buffer 10 mM BAPTA in HEK cells transfected with STIM1, Orai1, and with (red) or without ANO8 (black). Panel (G) shows the increase in current density at peak current. Note the prominent current inactivation in the presence of ANO8. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed and difference were analyzed by unpaired t test.


( H ) Knockdown of SARAF ( red ) in wild - type had no effect on current inactivation in presence of 10 mM BAPTA . ( I ) Knockdown of SARAF did not prevent ANO8 - dependent current inactivation in presence of 10 mM BAPTA . Data information : first number in parenthesis indicates number of similar experiments performed second number is number of analyzed . results are given as mean ± s . e . m of indicated number of experiments or analyzed difference were analyzed by unpaired t .

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 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

(H) Knockdown of SARAF (red) in wild-type cells had no effect on current inactivation in the presence of 10 mM BAPTA. (I) Knockdown of SARAF did not prevent the ANO8-dependent current inactivation in the presence of 10 mM BAPTA. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed and difference were analyzed by unpaired t test.


( A , B ) Depletion of plasma PI ( 4 , 5 ) P2 with FRB / FKBP system prevented ANO8 - mediated increase in STIM1 - Orai1 current measured in pipette solutions containing 3 mM EGTA ( A ) or 10 mM BAPTA ( B ) . Data information : first number in parenthesis indicates number of similar experiments performed second number is number of analyzed . results are given as mean ± s . e . m of indicated number of experiments or analyzed .

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

(A, B) Depletion of plasma membrane PI(4,5)P2 with the FRB/FKBP system prevented the ANO8-mediated increase in STIM1-Orai1 current measured in pipette solutions containing 3 mM EGTA (A) or 10 mM BAPTA (B). Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed.


( C ) HEK transfected with ANO8 - YFP , STIM1 - CFP Orai1 - HA FRB mCherry - FKBP PI ( 4 , 5 ) P2 depleting constructs were treated with 0 . 2 µM rapamycin ( rapa ) for 5 min then with 25 µM CPA for 10 min before fixation imaging by confocal microscopy . Data information : first number in parenthesis indicates number of similar experiments performed second number is number of analyzed . results are given as mean ± s . e . m of indicated number of experiments or analyzed .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HEK cells transfected with ANO8-YFP, STIM1-CFP Orai1-HA and FRB and mCherry-FKBP PI(4,5)P2 depleting constructs were treated with 0.2 µM rapamycin (rapa) for 5 min and then with 25 µM CPA for 10 min before fixation and imaging by confocal microscopy. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed.


( D ) Mutating ANO8 R948 , R950 , R951 in RPRRP , a region that is predicted to include an ANO8 PI ( 4 , 5 ) P2 binding site , prevented increased STIM1 - Orai1 current inactivation . Data information : first number in parenthesis indicates number of similar experiments performed second number is number of analyzed . results are given as mean ± s . e . m of indicated number of experiments or analyzed .

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 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

(D) Mutating ANO8 R948, R950, R951 in RPRRP, a region that is predicted to include an ANO8 PI(4,5)P2 binding site, prevented increased STIM1-Orai1 current and inactivation. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed.


( E ) Mutating ANO8 R949 , 950 , 951 to Q reduced translocation of ANO8 . Data information : first number in parenthesis indicates number of similar experiments performed second number is number of analyzed . results are given as mean ± s . e . m of indicated number of experiments or analyzed .

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

(E) Mutating ANO8 R949,950,951 to Q reduced translocation of ANO8. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed.


( A ) Plot of slope of inactivation as a function of current density in presence ( red ) absence of ANO8 ( black ) . Increasing current density was obtained by varying external Ca2 + between 2 - 50 mM . results are mean ± s . e . m difference were analyzed by unpaired t . Data information : first number in parenthesis indicates number of similar experiments performed second number is number of analyzed . results are given as mean ± s . e . m of indicated number of experiments or analyzed .

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 B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Plot of the slope of inactivation as a function of current density in the presence (red) and absence of ANO8 (black). Increasing current density was obtained by varying external Ca2+ between 2-50 mM. The results are mean±s.e.m and difference were analyzed by unpaired t test. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed.


( B , C ) CRAC current was measured in HEK expressing STIM1 Orai1 in pipette solution containing 3 mM EGTA ( b ) or 10 mM BAPTA ( c ) . SERCA pump was inhibited with 25 µM CPA in red traces where indicated 100 µM IP3 was included in pipette solution ( green in c ) . Data information : first number in parenthesis indicates number of similar experiments performed second number is number of analyzed . results are given as mean ± s . e . m of indicated number of experiments or analyzed .

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

(B, C) CRAC current was measured in HEK cells expressing STIM1 and Orai1 and in pipette solution containing 3 mM EGTA (b) or 10 mM BAPTA (c). SERCA pump was inhibited with 25 µM CPA in the red traces where indicated and 100 µM IP3 was included in the pipette solution (green in c). Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed.


( D , E ) Experimental protocol conditions except that were also transfected with ANO8 . Data information : first number in parenthesis indicates number of similar experiments performed second number is number of analyzed . results are given as mean ± s . e . m of indicated number of experiments or analyzed .

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

(D, E) Experimental protocol and conditions except that cells were also transfected with ANO8. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed.


( F , G ) HEK expressing STIM1 , Orai1 , ANO8 were used to measure current in pipette solution containing 10 mM BAPTA with ( red ) or without ( black ) 100 µM IP3 or 10 mM of ER Ca2 + chelator TPEN ( green ) . current density ( F ) normalized current ( G ) are shown , illustrating delay in current inactivation by IP3 TPEN . Data information : first number in parenthesis indicates number of similar experiments performed second number is number of analyzed . results are given as mean ± s . e . m of indicated number of experiments or analyzed .

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

(F, G) HEK cells expressing STIM1, Orai1, and ANO8 were used to measure current in pipette solution containing 10 mM BAPTA and with (red) or without (black) 100 µM IP3 or 10 mM of the ER Ca2+ chelator TPEN (green). The current density (F) and the normalized current (G) are shown, illustrating the delay in current inactivation by IP3 and TPEN. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed.


( A , B ) : ER Ca2 + content was measured with ER - GECO1 in periphery ( A ) center ( B ) or transfected with M3 receptors with ( red , green ) or without ( black , blue ) ANO8 . Receptor mediated store depletion was initiated by stimulating with 0 . 5 mM carbachol in Ca2 + - free solution . stimulation was terminated with 10 µM atropine ER Ca2 + uptake was initiated by perfusing with a solution containing 5 mM Ca2 + . was measured as F / F0 normalized to initial in presence of ANO8 . Panel ( C ) shows traces of ER Ca2 + uptake at expanded time scale in periods marked by rectangular in ( A B ) averaged slopes of ER Ca2 + influx ( D ) shows averaged slope of Ca2 + release at periphery ( Peri ) center ( Center ) . In ( C ) traces were aligned along Y axis to better show difference in uptake rate . Data information : first number in parenthesis indicates number of similar experiments performed second number is number of analyzed . results are given as mean ± s . e . m of indicated number of experiments or analyzed , difference were analyzed by unpaired t .

O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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): ER Ca2+ content was measured with ER-GECO1 in the periphery (A) and the center (B) or cells transfected with M3 receptors and with (red, green) or without (black, blue) ANO8. Receptor mediated store depletion was initiated by stimulating the cells with 0.5 mM carbachol in Ca2+-free solution. Cell stimulation was terminated with 10 µM atropine and ER Ca2+ uptake was initiated by perfusing the cells with a solution containing 5 mM Ca2+. The fluorescence was measured as F/F0 and normalized to the initial fluorescence in the presence of ANO8. Panel (C) shows traces of ER Ca2+ uptake at expanded time scale in the periods marked by rectangular in (A and B) and the averaged slopes of ER Ca2+ influx and (D) shows the averaged slope of Ca2+ release at the cell periphery (Peri) and cell center (Center). In (C) the traces were aligned along the Y axis to better show the difference in uptake rate. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed, and difference were analyzed by unpaired t test.


( E ) Enhancement by ANO8 of Co - IP of expressed STIM1 with PMCA4a SERCA2 of Orai1 with SERCA2 in resting store depleted . Data information : first number in parenthesis indicates number of similar experiments performed second number is number of analyzed . results are given as mean ± s . e . m of indicated number of experiments or analyzed , difference were analyzed by unpaired t .

O O O O O B-CONTROLLED_VAR O O O O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Enhancement by ANO8 of the Co-IP of expressed STIM1 with PMCA4a and SERCA2 and of Orai1 with SERCA2 in resting and store depleted cells. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed, and difference were analyzed by unpaired t test.


( F ) Effect of ANO8 expression on Co - IP of native SERCA2 STIM1 in resting store depleted . columns are mean ± s . e . m of 3 experiments . Data information : first number in parenthesis indicates number of similar experiments performed second number is number of analyzed . results are given as mean ± s . e . m of indicated number of experiments or analyzed , difference were analyzed by unpaired t .

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

(F) Effect of ANO8 expression on the Co-IP of the native SERCA2 and STIM1 in resting and store depleted cells. The columns are the mean±s.e.m of 3 experiments. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed, and difference were analyzed by unpaired t test.


( G ) FRET between STIM1 SERCA2 in response to store depletion its enhancement by ANO8 . Data information : first number in parenthesis indicates number of similar experiments performed second number is number of analyzed . results are given as mean ± s . e . m of indicated number of experiments or analyzed , difference were analyzed by unpaired t .

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

(G) FRET ratio between STIM1 and SERCA2 in response to store depletion and its enhancement by ANO8. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed, and difference were analyzed by unpaired t test.


( H , I ) Simultaneous measurements of ER Ca2 + with ER - GECO1 PI ( 4 , 5 ) P2 hydrolysis with PHPLCδ - GFP in response to stimulation of M3 receptors . Traces are shown in ( h ) average slopes are in ( i ) . Data information : first number in parenthesis indicates number of similar experiments performed second number is number of analyzed . results are given as mean ± s . e . m of indicated number of experiments or analyzed , difference were analyzed by unpaired t .

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

(H, I) Simultaneous measurements of ER Ca2+ with ER-GECO1 and PI(4,5)P2 hydrolysis with PHPLCδ-GFP in response to cell stimulation of M3 receptors. Traces are shown in (h) and the average slopes are in (i). Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed, and difference were analyzed by unpaired t test.


( J ) Effect of ANO8 on Co - IP of native STIM1 IP3Rs . columns are mean ± s . e . m of 3 experiments . Data information : first number in parenthesis indicates number of similar experiments performed second number is number of analyzed . results are given as mean ± s . e . m of indicated number of experiments or analyzed , difference were analyzed by unpaired t .

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

(J) Effect of ANO8 on the Co-IP of the native STIM1 and IP3Rs. The columns are the mean±s.e.m of 3 experiments. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed, and difference were analyzed by unpaired t test.


( K ) FRET between expressed STIM1 IP3R3 in response to store depletion its enhancement by ANO8 . Data information : first number in parenthesis indicates number of similar experiments performed second number is number of analyzed . results are given as mean ± s . e . m of indicated number of experiments or analyzed , difference were analyzed by unpaired t .

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

(K) FRET ratio between expressed STIM1 and IP3R3 in response to store depletion and its enhancement by ANO8. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed, and difference were analyzed by unpaired t test.


( L ) FRET between expressed STIM1 PMCA4 in response to store depletion its enhancement by ANO8 . Data information : first number in parenthesis indicates number of similar experiments performed second number is number of analyzed . results are given as mean ± s . e . m of indicated number of experiments or analyzed , difference were analyzed by unpaired t .

(L) FRET ratio between expressed STIM1 and PMCA4 in response to store depletion and its enhancement by ANO8. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed, and difference were analyzed by unpaired t test.


( A - D ) HeLa transfected with GFP ( black ) or ANO8 ( red ) were stimulated with 0 . 5 then 1 µM ATP to activate native P2Y2 receptors . oscillations were analyzed in terms of % of responding ( B ) , oscillation frequency ( C ) , amplitude of Ca2 + signal ( D ) . Data information : results are given as mean ± s . e . m of indicated number of experiments or analyzed , difference were analyzed by unpaired t .

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

(A-D) HeLa cells transfected with GFP (black) or ANO8 (red) were stimulated with 0.5 and then 1 µM ATP to activate the native P2Y2 receptors. The oscillations were analyzed in terms of the % of responding cells (B), oscillation frequency (C), and the amplitude of the Ca2+ signal (D). Data information: All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed, and difference were analyzed by unpaired t test.


( E - H ) : HeLa were treated with scrambled siRNA ( black ) or siANO8 ( red ) were stimulated with 1 then 5 µM ATP . oscillations were analyzed in terms of % of responding ( F ) , oscillations frequency ( G ) , amplitude of Ca2 + signal ( H ) . Data information : results are given as mean ± s . e . m of indicated number of experiments or analyzed , difference were analyzed by unpaired t .

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

(E-H): HeLa cells were treated with scrambled siRNA (black) or siANO8 (red) and were stimulated with 1 and then 5 µM ATP. The oscillations were analyzed in terms of the % of responding cells (F), oscillations frequency (G), and the amplitude of the Ca2+ signal (H). Data information: All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed, and difference were analyzed by unpaired t test.


( A ) Total lysates from indicated lines cultured in regular conditions at a steady state were subjected to western blotting . s . e . , short exposure ; l . e . , long exposure . major Merlin band is indicated by an asterisk . of mono - ubiquitinated to native Merlin in each lane of short exposure blot was quantified by Image J is shown under blot . same lysates were also used in Figure EV1A . Asterisks indicate native Merlin , arrows show ubiquitinated Merlin .

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

(A) Total lysates from the indicated cell lines cultured in regular conditions at a steady state were subjected to western blotting. s.e., short exposure; l.e., long exposure. The major Merlin band is indicated by an asterisk. The ratio of mono-ubiquitinated to native Merlin in each lane of the short exposure blot was quantified by Image J and is shown under the blot. The same lysates were also used in Figure EV1A. Asterisks indicate native Merlin, arrows show ubiquitinated Merlin.


( B ) Total lysates from LN229 in which Merlin was or was not knocked out ( KO ) were subjected to western blotting .

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) Total lysates from LN229 cells in which Merlin was or was not knocked out (KO) were subjected to western blotting.


LN229 ( C ) were detached ( denoted by sus . , referring to suspension ) by trypsinization ( see Methods ) reseeded ( attach ) for two ( LN229 ) hours . Total lysates from these were subjected to western blotting . of mono - ubiquitinated to native Merlin in each lane was quantified by Image J is shown under blot . Ubiquitinated Merlin is marked with arrows .

O O O O O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O O O O O

LN229 (C) cells were detached (denoted by sus., referring to suspension) by trypsinization (see Methods) and reseeded (attach) for two (LN229) hours. Total lysates from these cells were subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot. Ubiquitinated Merlin is marked with arrows.


Met5 - A ( D ) were detached ( denoted by sus . , referring to suspension ) by trypsinization ( see Methods ) reseeded ( attach ) for four ( Met5 - A ) hours . Total lysates from these were subjected to western blotting . of mono - ubiquitinated to native Merlin in each lane was quantified by Image J is shown under blot . Ubiquitinated Merlin is marked with arrows .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O O O O O

Met5-A (D) cells were detached (denoted by sus., referring to suspension) by trypsinization (see Methods) and reseeded (attach) for four (Met5-A) hours. Total lysates from these cells were subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot. Ubiquitinated Merlin is marked with arrows.