( C , D , E , F ) WT ( Pml + / + ) and Pml - / - mice were treated with DMSO or Nec - 1s ( 6 μg / g ) or MK2 inhibitor PF - 3644022 ( 75 μg / mouse ) by intraperitoneal injection 15 min before and 60 min after intravenous TNF ( 1 . 5 μg / g body weight ) injection . Body temperature ( C , E ) and survival ( D , F ) were monitored . Experiments C - F were conducted concurrently . Mean ± S . E . M is shown ( C , E ) .

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

(C, D, E, F) WT (Pml+/+) and Pml-/- mice were treated with DMSO or Nec-1s (6 μg/g) or MK2 inhibitor PF-3644022 (75 μg/mouse) by intraperitoneal injection 15 min before and 60 min after intravenous TNF (1.5 μg/g body weight) injection. Body temperature (C, E) and survival (D, F) were monitored. Experiments C-F were conducted concurrently. Mean ± S.E.M is shown (C, E).


( A , B ) WT and Mk2 - / - BMDMs were treated with TNF ( 100 ng / ml ) + zVAD ( 20 μM ) ( A ) or with zVAD ( 20 μM ) + AT - 406 ( 1 μM ) ( B ) , and then the contents of p - RIPK1 [ S321 ] , RIPK1 , p - p38 , p38 was determined at the indicated time - points .

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

(A, B) WT and Mk2-/- BMDMs were treated with TNF (100 ng/ml) + zVAD (20 μM) (A) or with zVAD (20 μM) + AT-406 (1 μM) (B), and then the contents of p-RIPK1[S321], RIPK1, p-p38, p38 was determined at the indicated time-points.


( C ) WT , Pml - / - , Mk2 - / - and Pml - / - Mk2 - / - BMDMs were treated with zVAD ( 20 μM ) , AT - 406 ( 1 μM ) , Nec - 1 ( 40 μM ) and GSK872 ( 10 μM ) , as indicated , for 18 h . Cell viability was determined by ATP release . Data show mean ± SD of three biological replicates . * P < 0 . 05 for multiple t - tests according to the Holm - Sidak method . ( D ) WT , Pml - / - , Mk2 - / - and Pml - / - Mk2 - / - BMDMs were treated with TNF ( 100 ng / ml ) , zVAD ( 20 μM ) , Nec - 1 ( 40 μM ) and GSK872 ( 10 μM ) , as indicated , for 16 h . Cell viability was determined by ATP release . Data show mean ± SD of three biological replicates . * P < 0 . 05 for multiple t - tests according to the Holm - Sidak method .

O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O 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) WT, Pml-/-, Mk2-/- and Pml-/-Mk2 -/- BMDMs were treated with zVAD (20 μM), AT-406 (1 μM), Nec-1 (40 μM) and GSK872 (10 μM), as indicated, for 18 h. Cell viability was determined by ATP release. Data show mean ± SD of three biological replicates. *P < 0.05 for multiple t-tests according to the Holm-Sidak method. (D) WT, Pml-/-, Mk2-/- and Pml-/-Mk2 -/- BMDMs were treated with TNF (100 ng/ml), zVAD (20 μM), Nec-1 (40 μM) and GSK872 (10 μM), as indicated, for 16 h. Cell viability was determined by ATP release. Data show mean ± SD of three biological replicates. *P < 0.05 for multiple t-tests according to the Holm-Sidak method.


( F ) WT , Pml - / - , Mk2 - / - and Pml - / - Mk2 - / - mice were injected i . v . with TNF ( 1 . 5 mg / kg ) , and survival was monitored for 24 h . Data represent a combination of two independent experiments ( n = 10 ) . * P < 0 . 05 for Log - rank ( Mantel - Cox ) test .

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

(F) WT, Pml-/-, Mk2-/- and Pml-/-Mk2 -/- mice were injected i.v. with TNF (1.5 mg/kg), and survival was monitored for 24 h. Data represent a combination of two independent experiments (n=10). *P < 0.05 for Log-rank (Mantel-Cox) test.


( D ) WT and Pml - / - BMDMs were treated with TNF ( 100 ng / ml ) + zVAD ( 20 μM ) for the indicated times . p38 MAPK was immunoprecipitated , and its association with MK2 was then analysed .

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

(D) WT and Pml-/- BMDMs were treated with TNF (100 ng/ml) + zVAD (20 μM) for the indicated times. p38 MAPK was immunoprecipitated, and its association with MK2 was then analysed.


( E ) Recombinant Flag - PML - I , GST - MK2 ( 60 ng ) and His - p38 ( 60 ng ) were incubated , as indicated , at 4 ° C for 2 h . p38 was pulled down by anti - p38 and then PML , MK2 and p38 contents in the precipitate and reaction mixture ( input ) were detected by immunoblotting .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Recombinant Flag-PML-I, GST-MK2 (60 ng) and His-p38 (60 ng) were incubated, as indicated, at 4 °C for 2 h. p38 was pulled down by anti-p38 and then PML, MK2 and p38 contents in the precipitate and reaction mixture (input) were detected by immunoblotting.


( F ) Recombinant Flag - PML - I , GST - MK2 ( 100 ng ) and His - p38 ( 200 ng ) were incubated , as indicated , at 30 ° C in in vitro kinase assay buffer ( 20 mM HEPES , 10 mM MgCl2 , 100 nM ATP , 1 mM DTT ) for 20 min . The levels of p - MK2 , MK2 , p38 , and PML ( FLAG ) in the reaction mixture were then determined .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Recombinant Flag-PML-I, GST-MK2 (100 ng) and His-p38 (200 ng) were incubated, as indicated, at 30 °C in in vitro kinase assay buffer (20 mM HEPES, 10 mM MgCl2, 100 nM ATP, 1 mM DTT) for 20 min. The levels of p-MK2, MK2, p38, and PML (FLAG) in the reaction mixture were then determined.


( B ) ARSA activity quantification ( nmol / h / mg ) after treatment of MSDi cells with a selection of four positive hit drugs at a final concentration of 10 µM on MSDi cells in 25cm2 cell culture flasks for three days . Data represent mean ± SD of 7 independent experiments ( biological replicates ) . One - way ANOVA followed by Tukey ' s test for multiple comparisons . Difference against negative

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) ARSA activity quantification (nmol/h/mg) after treatment of MSDi cells with a selection of four positive hit drugs at a final concentration of 10 µM on MSDi cells in 25cm2 cell culture flasks for three days. Data represent mean ±SD of 7 independent experiments (biological replicates). One-way ANOVA followed by Tukey's test for multiple comparisons. Difference against negative control


( E ) Analysis of different retinoids ' potential to restore ARSA activity in MSDi cells in comparison to tazarotene after treatment for 3 days at a final concentration 10 µM of each drug ( Adapalene 1 µM ) . Data represent mean ± SD of 3 independent experiments ( biological replicates ) . One - way ANOVA followed by Tukey ' s test for multiple comparisons . Difference against DMSO control : * *

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

(E) Analysis of different retinoids' potential to restore ARSA activity in MSDi cells in comparison to tazarotene after treatment for 3 days at a final concentration 10 µM of each drug (Adapalene 1 µM). Data represent mean ±SD of 3 independent experiments (biological replicates). One-way ANOVA followed by Tukey's test for multiple comparisons. Difference against DMSO control: **


( D ) Analysis and quantification of a time dependent increase of ARSA activity in MSD primary fibroblasts ( variant FGE Gly247Arg homozygous ) simultaneously treated with 10 and 20 µM tazarotene and bexarotene , respectively , up to 21 days . Data represent mean ± SD of 3 - 6 independent experiments ( biological replicates ) . One - way ANOVA followed by Tukey ' s test for multiple comparisons . Displayed are significance levels for the next significant difference between adjacent treatment times . # # # # p < 0 . 0001 . Difference against 0 days control : * p < 0 . 05 , * * * * p < 0 . 0001 .

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

(D) Analysis and quantification of a time dependent increase of ARSA activity in MSD primary fibroblasts (variant FGE Gly247Arg homozygous) simultaneously treated with 10 and 20 µM tazarotene and bexarotene, respectively, up to 21 days. Data represent mean ±SD of 3-6 independent experiments (biological replicates). One-way ANOVA followed by Tukey's test for multiple comparisons. Displayed are significance levels for the next significant difference between adjacent treatment times. #### p<0.0001. Difference against 0 days control: * p<0.05, **** p<0.0001.


( E ) Analysis and quantification of increased sulfatase activities different to ARSA , namely ARSB , GALNS , and STS in MSD primary fibroblasts ( variant FGE Gly247Arg homozygous ) after 6 days of simultaneous treatment with tazarotene / bexarotene 10 / 20 µM . Data represent mean ± SD of 3 - 6 independent experiments ( biological replicates ) . One - way ANOVA followed by Tukey ' s test for multiple comparisons . * * * p < 0 . 001 , * * * * p < 0 . 0001 .

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

(E) Analysis and quantification of increased sulfatase activities different to ARSA, namely ARSB, GALNS, and STS in MSD primary fibroblasts (variant FGE Gly247Arg homozygous) after 6 days of simultaneous treatment with tazarotene/bexarotene 10/20 µM. Data represent mean ±SD of 3-6 independent experiments (biological replicates). One-way ANOVA followed by Tukey's test for multiple comparisons. *** p<0.001, **** p<0.0001.


( F ) Quantification of ARSA activities in MSD primary fibroblasts with different homozygous SUMF1 mutations ( FGE Gly247Arg , FGE Gly263Val , FGE Ala279Val , FGE Arg349Trp ) after 6 days of simultaneous treatment with tazarotene / bexarotene 10 / 20 µM . Data represent mean ± SD of 3 independent experiments ( biological replicates ) . One - way ANOVA followed by Tukey ' s test for multiple comparisons . * * * * p < 0 . 0001 .

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

(F) Quantification of ARSA activities in MSD primary fibroblasts with different homozygous SUMF1 mutations (FGE Gly247Arg, FGE Gly263Val, FGE Ala279Val, FGE Arg349Trp) after 6 days of simultaneous treatment with tazarotene/bexarotene 10/20 µM. Data represent mean ±SD of 3 independent experiments (biological replicates). One-way ANOVA followed by Tukey's test for multiple comparisons. ****p<0.0001.


( G ) Quantification of ARSA activity in MSD patient - derived iPSCs differentiated into NPCs and unaffected control NPCs controls . Simultaneous treatment with 5 µM tazarotene and 5 µM bexarotene for 4 days . Data represent mean ± SD of 6 independent experiments ( biological replicates ) . Unpaired t - test . * * * * p < 0 . 0001 . ( H ) Quantification of SGSH activity in MSD patient - derived iPSCs differentiated into NPCs and unaffected control NPCs controls . Simultaneous treatment with 5 µM tazarotene and 5 µM bexarotene for 4 days . Data represent mean ± SD of 3 independent experiments ( biological replicates ) . Unpaired t - test . * p < 0 . 05 , * * * p < 0 . 001 .

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

(G) Quantification of ARSA activity in MSD patient-derived iPSCs differentiated into NPCs and unaffected control NPCs controls. Simultaneous treatment with 5 µM tazarotene and 5 µM bexarotene for 4 days. Data represent mean ±SD of 6 independent experiments (biological replicates). Unpaired t-test. **** p<0.0001. (H) Quantification of SGSH activity in MSD patient-derived iPSCs differentiated into NPCs and unaffected control NPCs controls. Simultaneous treatment with 5 µM tazarotene and 5 µM bexarotene for 4 days. Data represent mean ±SD of 3 independent experiments (biological replicates). Unpaired t-test. * p<0.05, *** p<0.001.


( A ) Representative confocal images of control and MSD fibroblasts with either tazarotene / bexarotene ( 10 / 20 µM , 6 days ) . Labelling with anti - LAMP1 antibody ( green fluorescence ) and DAPI ( nuclei , blue ) . ( B ) Representative confocal images of control and MSD fibroblasts with either tazarotene / bexarotene ( 10 / 20 µM , 6 days ) . Labelling with anti - LAMP1 antibody ( green fluorescence ) and DAPI ( nuclei , blue ) . ( C ) Quantification of total intensity of LAMP1 - green fluorescence . N = 20 images and 13 z - series optical sections per condition with a step - size of 0 . 26 microns , displayed at maximum extension and automated equalization of brightness . Data represent mean ± SD of 3 independent experiments ( biological replicates ) . One - way ANOVA followed by Tukey ' s test for multiple comparisons . * * * p < 0 . 001 ( DMSO treated MSD cells compared to DMSO treated control cells ) , # p < 0 . 05 ( MSD DMSO vs MSD treated ) . RFU : relative fluorescence units . ( D ) Quantification of LAMP1 - green fluorescence vesicle size ( µm ) . N = 20 images and 13 z - series optical sections per condition with a step - size of 0 . 26 microns , displayed at maximum extension and automated equalization of brightness . Data represent mean ± SD of 3 independent experiments ( biological replicates ) . One - way ANOVA followed by Tukey ' s test for multiple comparisons . * * * p < 0 . 001 ( DMSO treated MSD cells compared to DMSO treated control cells ) , # # p < 0 . 01 ( MSD DMSO vs MSD treated ) .

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

(A) Representative confocal images of control and MSD fibroblasts with either tazarotene/bexarotene (10/20 µM, 6 days). Labelling with anti-LAMP1 antibody (green fluorescence) and DAPI (nuclei, blue). (B) Representative confocal images of control and MSD fibroblasts with either tazarotene/bexarotene (10/20 µM, 6 days). Labelling with anti-LAMP1 antibody (green fluorescence) and DAPI (nuclei, blue). (C) Quantification of total intensity of LAMP1- green fluorescence. N = 20 images and 13 z-series optical sections per condition with a step-size of 0.26 microns, displayed at maximum extension and automated equalization of brightness. Data represent mean ±SD of 3 independent experiments (biological replicates). One-way ANOVA followed by Tukey's test for multiple comparisons. *** p<0.001 (DMSO treated MSD cells compared to DMSO treated control cells), # p<0.05 (MSD DMSO vs MSD treated). RFU: relative fluorescence units. (D) Quantification of LAMP1- green fluorescence vesicle size (µm). N = 20 images and 13 z-series optical sections per condition with a step-size of 0.26 microns, displayed at maximum extension and automated equalization of brightness. Data represent mean ±SD of 3 independent experiments (biological replicates). One-way ANOVA followed by Tukey's test for multiple comparisons. *** p<0.001 (DMSO treated MSD cells compared to DMSO treated control cells), ## p<0.01 (MSD DMSO vs MSD treated).


( A ) ARSA activity quantification upon treatment of MSDi cells with 10 µM tazarotene , 20 µM bexarotene , and 10 µM tazarotene and 20 µM bexarotene in combination with increasing concentrations of the pan - RAR receptor antagonist AGN 193109 ( 24 h pretreatment ) for 72 hours . Data represent mean ± SD of 3 - 11 independent experiments ( biological replicates ) . Two - way ANOVA followed by Tukey ' s test for multiple comparisons . Displayed are significance levels for the next significant difference between adjacent concentrations / conditions . # p < 0 . 05 , # # p < 0 . 01 , # # # p < 0 . 001 . Difference against 0 µM DMSO control ( B ) ARSA activity quantification upon treatment of MSDi cells with 10 µM tazarotene , 20 µM bexarotene , and 10 µM tazarotene and 20 µM bexarotene in combination with increasing concentrations of the pan - RXR receptor antagonist HX 531 ( 24 h pretreatment ) for 72 hours . Data represent mean ± SD of 4 - 14 independent experiments ( biological replicates ) . Two - way ANOVA followed by Tukey ' s test for multiple comparisons . Displayed are significance levels for the next significant difference between adjacent concentrations / conditions . # p < 0 . 05 , # # p < 0 . 01 . Difference against 0 µM DMSO control : * * * * p < 0 . 0001 .

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 B-CONTROLLED_VAR 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 O O O O O O O O O O O O O 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 B-CONTROLLED_VAR 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 O 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) ARSA activity quantification upon treatment of MSDi cells with 10 µM tazarotene, 20 µM bexarotene, and 10 µM tazarotene and 20 µM bexarotene in combination with increasing concentrations of the pan-RAR receptor antagonist AGN 193109 (24 h pretreatment) for 72 hours. Data represent mean ±SD of 3-11 independent experiments (biological replicates). Two-way ANOVA followed by Tukey's test for multiple comparisons. Displayed are significance levels for the next significant difference between adjacent concentrations/conditions. # p<0.05, ## p<0.01, ### p<0.001. Difference against 0 µM DMSO control (B) ARSA activity quantification upon treatment of MSDi cells with 10 µM tazarotene, 20 µM bexarotene, and 10 µM tazarotene and 20 µM bexarotene in combination with increasing concentrations of the pan-RXR receptor antagonist HX 531 (24 h pretreatment) for 72 hours. Data represent mean ±SD of 4-14 independent experiments (biological replicates). Two-way ANOVA followed by Tukey's test for multiple comparisons. Displayed are significance levels for the next significant difference between adjacent concentrations/conditions. # p<0.05, ## p<0.01. Difference against 0 µM DMSO control: **** p<0.0001.


( A ) ARSA protein amount quantification after treatment of MSD primary fibroblasts ( variant FGE Gly247Arg homozygous ) with tazarotene , bexarotene , and tazarotene / bexarotene in combination for 6 days referred to β - actin amounts and normalization of ARSA activity based on ARSA protein amount ( specific ARSA activity ) . Data represent mean ± SD of 3 independent experiments ( biological replicates ) . One - way ANOVA followed by Tukey ' s test for multiple comparisons . Displayed are significance levels for the next significant difference between adjacent concentrations . # p < 0 . 05 . Difference against 0 / 0 µM control : * p < 0 . 05 , * * * p < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O 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 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) ARSA protein amount quantification after treatment of MSD primary fibroblasts (variant FGE Gly247Arg homozygous) with tazarotene, bexarotene, and tazarotene/bexarotene in combination for 6 days referred to β- actin amounts and normalization of ARSA activity based on ARSA protein amount (specific ARSA activity). Data represent mean ±SD of 3 independent experiments (biological replicates). One-way ANOVA followed by Tukey's test for multiple comparisons. Displayed are significance levels for the next significant difference between adjacent concentrations. # p<0.05. Difference against 0/0 µM control: * p<0.05, *** p<0.001.


( C ) Quantification of ARSA activities in CRISPR / Cas9 generated ARPE19 SUMF1 - / - cells and appropriate controls ( ARPE19 wild type , MSD primary fibroblasts ( variant FGE Gly247Arg homozygous ) ) after 6 days of simultaneous treatment with tazarotene / bexarotene 10 / 20 µM for up to 21 days . Data represent mean ± SD of 3 independent experiments ( biological replicates ) . One - way ANOVA followed by Tukey ' s test for multiple comparisons . Displayed are significance levels for the next significant difference between adjacent concentrations . # # p < 0 . 01 . Difference against 0 days control : * * * * p < 0 . 0001 .

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

(C) Quantification of ARSA activities in CRISPR/Cas9 generated ARPE19 SUMF1 -/- cells and appropriate controls (ARPE19 wild type, MSD primary fibroblasts (variant FGE Gly247Arg homozygous)) after 6 days of simultaneous treatment with tazarotene/bexarotene 10/20 µM for up to 21 days. Data represent mean ±SD of 3 independent experiments (biological replicates). One-way ANOVA followed by Tukey's test for multiple comparisons. Displayed are significance levels for the next significant difference between adjacent concentrations. ## p<0.01. Difference against 0 days control: **** p<0.0001.


( A ) Pulse - chase - experiment in HT1080 FGE Ser155Pro cells after pre - treatment with tazarotene / bexarotene and DMSO ( control ) for 3 days . Upper panel : representative autoradiogram of intracellular ( C ) 35S isotope labelled FGE protein in either condition with a chase time of 4 hours . Lower panel : quantification of 35S isotope labelled intracellular FGE protein amounts . Data represent mean ± SD of 3 independent experiments ( biological replicates ) . One - way ANOVA followed by Tukey ' s test for multiple comparisons . * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Pulse-chase-experiment in HT1080 FGE Ser155Pro cells after pre-treatment with tazarotene/bexarotene and DMSO (control) for 3 days. Upper panel: representative autoradiogram of intracellular (C) 35S isotope labelled FGE protein in either condition with a chase time of 4 hours. Lower panel: quantification of 35S isotope labelled intracellular FGE protein amounts. Data represent mean ±SD of 3 independent experiments (biological replicates). One-way ANOVA followed by Tukey's test for multiple comparisons. ** p<0.01, *** p<0.001.


( B ) Pull - Down of GST - tagged α11 . 2 segments ( top immunoblot ; IB ) by immobilized MBP - β2AR C - terminus ( residues 326 - 413 of human β2AR ) . GST fusion proteins were detected by an anti - GST antibody and MBP fusion proteins by an anti - MBP antibody . Middle IB shows that comparable amounts of the various GST fusion proteins had been added to the resin samples and bottom IB illustrates equal loading of all amylose resin samples with MBP - β2AR . ( C ) Quantification of ( B ) ( * * * p < 0 . 001 , ANOVA ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Pull-Down of GST-tagged α11.2 segments (top immunoblot; IB) by immobilized MBP-β2AR C-terminus (residues 326-413 of human β2AR). GST fusion proteins were detected by an anti-GST antibody and MBP fusion proteins by an anti-MBP antibody. Middle IB shows that comparable amounts of the various GST fusion proteins had been added to the resin samples and bottom IB illustrates equal loading of all amylose resin samples with MBP-β2AR.(C) Quantification of (B) (***p<0.001, ANOVA).


( D ) Pull - Down of GST - CTC but not GST - CTD or GST - CTE ( middle IB ) by immobilized MBP - β2AR C - terminus ( left ) but not MBP alone ( right ) , all of which were present at comparable amounts ( bottom and top IBs , respectively ) . GST fusion proteins were detected by an anti - GST antibody and MBP fusion proteins by an anti - MBP antibody . ( E ) Quantification of ( D ) ( * * * p < 0 . 001 , ANOVA ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Pull-Down of GST-CTC but not GST-CTD or GST-CTE (middle IB) by immobilized MBP-β2AR C-terminus (left) but not MBP alone (right), all of which were present at comparable amounts (bottom and top IBs, respectively). GST fusion proteins were detected by an anti-GST antibody and MBP fusion proteins by an anti-MBP antibody.(E) Quantification of (D) (***p<0.001, ANOVA).


( F ) IP of β2AR in the presence of 10 μM GST ( Control ; Ctr ) or GST - tagged C - terminal fragments as indicated . CT4 and CTC specifically displaced α11 . 2 ( top of IB ) but not GluA1 ( middle of same IB ) from β2AR ( bottom of same IB ) . Use of non - specific IgG ( left lane in right panels ) indicates specificity of IP . ( G , H ) . For quantification of coIPs , α11 . 2 and GluA1 immunosignals were normalized to β2AR signals ( * * * p < 0 . 001 , ANOVA ) . ( I ) Representative IB showing amounts of the GST fusion proteins that were added in ( F ) , as detected by anti - GST antibodies .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) IP of β2AR in the presence of 10 μM GST (Control; Ctr) or GST-tagged C-terminal fragments as indicated. CT4 and CTC specifically displaced α11.2 (top of IB) but not GluA1 (middle of same IB) from β2AR (bottom of same IB). Use of non-specific IgG (left lane in right panels) indicates specificity of IP.(G, H). For quantification of coIPs, α11.2 and GluA1 immunosignals were normalized to β2AR signals (***p<0.001, ANOVA).(I) Representative IB showing amounts of the GST fusion proteins that were added in (F), as detected by anti-GST antibodies.


( A ) Titration of fluorescence polarization of FITC - peptides ( 100 nM ) spanning α11 . 2 aa 1906 - 1959 with purified MBP - β2AR C - terminus . Kd value was obtained by fitting a non - linear direct binding curve to Pep2 .

O O O O O O O O O O O O O O O O O O O O O O O O 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) Titration of fluorescence polarization of FITC-peptides (100 nM) spanning α11.2 aa 1906-1959 with purified MBP-β2AR C-terminus. Kd value was obtained by fitting a non-linear direct binding curve to Pep2.


( B ) IP of β2AR in the absence ( Control ) or presence of 10 μM peptides , as indicated . Pep2 specifically displaced α11 . 2 ( top of IB ) but not GluA1 ( middle , same IB ) from β2AR ( bottom , same IB ) . Use of non - specific IgG ( left lane ) indicates specificity of coIPs . ( C , D ) . For quantification , α11 . 2 and GluA1IB signals were normalized to β2AR signals ( * * * p < 0 . 001 , ANOVA ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) IP of β2AR in the absence (Control) or presence of 10 μM peptides, as indicated. Pep2 specifically displaced α11.2 (top of IB) but not GluA1 (middle, same IB) from β2AR (bottom, same IB). Use of non-specific IgG (left lane) indicates specificity of coIPs.(C, D). For quantification, α11.2 and GluA1IB signals were normalized to β2AR signals (***p<0.001, ANOVA).


( E ) IP of β2AR in the absence ( Control ) or presence of 10 μM Pep2 or Pep2 with S1928 being phosphorylated ( phPep2 ) , which did not displaced α11 . 2 . ( F ) α11 . 2 and GluA1 IB signals were normalized to β2AR signals ( * * * p < 0 . 001 , ANOVA ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) IP of β2AR in the absence (Control) or presence of 10 μM Pep2 or Pep2 with S1928 being phosphorylated (phPep2), which did not displaced α11.2.(F) α11.2 and GluA1 IB signals were normalized to β2AR signals (***p<0.001, ANOVA).


Forebrain slices from WT ( A - F ) and S1928A KI mice ( G - L ) were treated with vehicle ( water ) or 10 M isoproterenol ( ISO ) for 0 . 5 - 10 min before solubilization , ultracentrifugation , IP of α11 . 2 ( A - C , G - I ) or β2AR ( D - F , J - L ) , and sequential IB for pS1928 , pS1700 , and α11 . 2 , for GluA1 , or for β2AR , of corresponding regions of the blots , as indicated . All the α11 . 2 IPs in A - C and G - I were from the same samples ( which were split in half for parallel IP ) as the β2AR IPs in D - F and J - L , respectively ( for quantification of coIP of β2AR with α11 . 2 see Figure EV1F , G ) . ( A - F ) In WT , the time - dependent increase in S1928 and S1700 phosphorylation ( A - C ) paralleled the decrease in coIP of β2AR with α11 . 2 ( A bottom , Figure EV1F ) and of α11 . 2 with β2AR ( D - F ) . ( G - L ) In S1928A KI mice , ISO induced S1700 phosphorylation ( G , I ) but did not disrupt the α11 . 2 - β2AR interaction ( G bottom , J - L , Figure EV1G ) . ( B , C , H , I ) For quantification of α11 . 2 phosphorylation , pS1928 and pS1700 signals were normalized to α11 . 2 ( * * p < 0 . 01 , * * * p < 0 . 001 , ANOVA ) .

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

Forebrain slices from WT (A-F) and S1928A KI mice (G-L) were treated with vehicle (water) or 10 M isoproterenol (ISO) for 0.5 - 10 min before solubilization, ultracentrifugation, IP of α11.2 (A-C, G-I) or β2AR (D-F, J-L), and sequential IB for pS1928, pS1700, and α11.2, for GluA1, or for β2AR, of corresponding regions of the blots, as indicated. All the α11.2 IPs in A-C and G-I were from the same samples (which were split in half for parallel IP) as the β2AR IPs in D-F and J-L, respectively (for quantification of coIP of β2AR with α11.2 see Figure EV1F,G).(A-F) In WT, the time-dependent increase in S1928 and S1700 phosphorylation (A-C) paralleled the decrease in coIP of β2AR with α11.2 (A bottom, Figure EV1F) and of α11.2 with β2AR (D-F).(G-L) In S1928A KI mice, ISO induced S1700 phosphorylation (G,I) but did not disrupt the α11.2 - β2AR interaction (G bottom, J-L, Figure EV1G).(B,C,H,I) For quantification of α11.2 phosphorylation, pS1928 and pS1700 signals were normalized to α11.2 (**p<0.01, ***p<0.001, ANOVA).


Forebrain slices from WT ( A - F ) and S1928A KI mice ( G - L ) were treated with vehicle ( water ) or 10 M isoproterenol ( ISO ) for 0 . 5 - 10 min before solubilization , ultracentrifugation , IP of α11 . 2 ( A - C , G - I ) or β2AR ( D - F , J - L ) , and sequential IB for pS1928 , pS1700 , and α11 . 2 , for GluA1 , or for β2AR , of corresponding regions of the blots , as indicated . All the α11 . 2 IPs in A - C and G - I were from the same samples ( which were split in half for parallel IP ) as the β2AR IPs in D - F and J - L , respectively ( for quantification of coIP of β2AR with α11 . 2 see Figure EV1F , G ) . ( A - F ) In WT , the time - dependent increase in S1928 and S1700 phosphorylation ( A - C ) paralleled the decrease in coIP of β2AR with α11 . 2 ( A bottom , Figure EV1F ) and of α11 . 2 with β2AR ( D - F ) . ( G - L ) In S1928A KI mice , ISO induced S1700 phosphorylation ( G , I ) but did not disrupt the α11 . 2 - β2AR interaction ( G bottom , J - L , Figure EV1G ) . ( B , C , H , I ) For quantification of α11 . 2 phosphorylation , pS1928 and pS1700 signals were normalized to α11 . 2 ( * * p < 0 . 01 , * * * p < 0 . 001 , ANOVA ) . ( E , F , K , L ) For quantification of coIP , α11 . 2 and GluA1 signals were normalized to β2AR ( * * * p < 0 . 001 , One Way ANOVA ) .

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

Forebrain slices from WT (A-F) and S1928A KI mice (G-L) were treated with vehicle (water) or 10 M isoproterenol (ISO) for 0.5 - 10 min before solubilization, ultracentrifugation, IP of α11.2 (A-C, G-I) or β2AR (D-F, J-L), and sequential IB for pS1928, pS1700, and α11.2, for GluA1, or for β2AR, of corresponding regions of the blots, as indicated. All the α11.2 IPs in A-C and G-I were from the same samples (which were split in half for parallel IP) as the β2AR IPs in D-F and J-L, respectively (for quantification of coIP of β2AR with α11.2 see Figure EV1F,G).(A-F) In WT, the time-dependent increase in S1928 and S1700 phosphorylation (A-C) paralleled the decrease in coIP of β2AR with α11.2 (A bottom, Figure EV1F) and of α11.2 with β2AR (D-F).(G-L) In S1928A KI mice, ISO induced S1700 phosphorylation (G,I) but did not disrupt the α11.2 - β2AR interaction (G bottom, J-L, Figure EV1G).(B,C,H,I) For quantification of α11.2 phosphorylation, pS1928 and pS1700 signals were normalized to α11.2 (**p<0.01, ***p<0.001, ANOVA).(E,F,K,L) For quantification of coIP, α11.2 and GluA1 signals were normalized to β2AR (***p<0.001, One Way ANOVA).


Hippocampal cultures were transfected with FLAG - β2AR and α11 . 2 - HA at 6 days in vitro ( DIV ) , treated with vehicle ( water ) or 1 M isoproterenol ( ISO ) for 5 min at 18 DIV , fixed and surface labeled for HA and FLAG . ( A , B ) Representative immunofluorescence microscopy images obtained by wide - field microscopy at lower and higher resolutions ( scale bar in A is 5 m ) . ( C ) Quantification of distance between centers of HA and FLAG puncta ( * * p < 0 . 001 , Mann - Whitney Rank Sum Test ) .

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

Hippocampal cultures were transfected with FLAG-β2AR and α11.2-HA at 6 days in vitro (DIV), treated with vehicle (water) or 1 M isoproterenol (ISO) for 5 min at 18 DIV, fixed and surface labeled for HA and FLAG.(A,B) Representative immunofluorescence microscopy images obtained by wide-field microscopy at lower and higher resolutions (scale bar in A is 5 m).(C) Quantification of distance between centers of HA and FLAG puncta (**p<0.001, Mann-Whitney Rank Sum Test).


Forebrain slices from WT mice were treated with vehicle ( water ; lanes 1 , 5 ; numbers on bottom ) or 10 M ISO for 5 min , followed , if indicated , by 1 , 3 , or 20 min wash out of ISO ( lanes 3 - 7 ) and a second application of ISO for 5 min ( lane 6 ) , before solubilization and ultracentrifugation . ( A ) β2AR was IPed before IB for α11 . 2 ( top part of IB ) , GluA1 ( middle part of IB ) , and β2AR ( bottom part of IB ) , as indicated . ( B , C ) For quantification , α11 . 2 and GluA1 IB signals were normalized to β2AR ( * * p < 0 . 01 , ANOVA ) . ISO - induced displacement of the β2AR from α11 . 2 ( A , lanes 2 - 6 ) lasted at least 3 min but not 20 min ( compare lanes 6 and 7 ) .

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

Forebrain slices from WT mice were treated with vehicle (water; lanes 1,5; numbers on bottom) or 10 M ISO for 5 min, followed, if indicated, by 1, 3, or 20 min wash out of ISO (lanes 3-7) and a second application of ISO for 5 min (lane 6), before solubilization and ultracentrifugation.(A) β2AR was IPed before IB for α11.2 (top part of IB), GluA1 (middle part of IB), and β2AR (bottom part of IB), as indicated.(B,C) For quantification, α11.2 and GluA1 IB signals were normalized to β2AR (**p<0.01, ANOVA).ISO-induced displacement of the β2AR from α11.2 (A, lanes 2-6) lasted at least 3 min but not 20 min (compare lanes 6 and 7).


Forebrain slices from WT mice were treated with vehicle ( water ; lanes 1 , 5 ; numbers on bottom ) or 10 M ISO for 5 min , followed , if indicated , by 1 , 3 , or 20 min wash out of ISO ( lanes 3 - 7 ) and a second application of ISO for 5 min ( lane 6 ) , before solubilization and ultracentrifugation . ( D ) α11 . 2 and GluA1 were concurrently IPed from same samples as in ( A ) by simultaneous addition of anti - α11 . 2 and - GluA1 antibodies before probing and stripping / re - probing upper part of IB for pS1928 , pS1700 , and total α11 . 2 ( top three panels ) and middle part for pS845 , pS831 , and total GluA1 ( bottom three panels ) . ( E - H ) For quantification , pS1928 and pS1700 IB signals were normalized to total α11 . 2 and pS845 and pS831 signals to total GluA1 ( * * p < 0 . 01 , ANOVA ) . ISO - induced displacement of the β2AR from α11 . 2 ( see A ) rendered α11 . 2 ( but not GluA1 ) refractory to re - phosphorylation of S1928 and S1700 upon a second ISO application of α11 . 2 ( D , compare lanes 5 and 6 ) .

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

Forebrain slices from WT mice were treated with vehicle (water; lanes 1,5; numbers on bottom) or 10 M ISO for 5 min, followed, if indicated, by 1, 3, or 20 min wash out of ISO (lanes 3-7) and a second application of ISO for 5 min (lane 6), before solubilization and ultracentrifugation.(D) α11.2 and GluA1 were concurrently IPed from same samples as in (A) by simultaneous addition of anti-α11.2 and -GluA1 antibodies before probing and stripping/re-probing upper part of IB for pS1928, pS1700, and total α11.2 (top three panels) and middle part for pS845, pS831, and total GluA1 (bottom three panels).(E-H) For quantification, pS1928 and pS1700 IB signals were normalized to total α11.2 and pS845 and pS831 signals to total GluA1 (**p<0.01, ANOVA).ISO-induced displacement of the β2AR from α11.2 (see A) rendered α11.2 (but not GluA1) refractory to re-phosphorylation of S1928 and S1700 upon a second ISO application of α11.2 (D, compare lanes 5 and 6).


Forebrain slices from S1928A KI mice were treated with vehicle ( lanes 1 , 5 ) or 10 M ISO for 5 min , followed , if indicated , by 1 , 3 , or 20 min wash out of ISO ( lanes 3 - 7 ) and a second application of ISO for 5 min ( lane 6 ) , before solubilization and ultracentrifugation . ( A ) α11 . 2 and GluA1 were concurrently IPed from same samples by simultaneous addition of anti - α11 . 2 and - GluA1 antibodies before probing and stripping / re - probing upper part of IB for pS1928 , pS1700 , and total α11 . 2 and middle part for pS845 , pS831 , and total GluA1 . In S1928A KI mice , S1700 re - phosphorylation after a 3 min wash out of ISO was not blunted ( lane 6 ) in contrast to WT mice but rather augmented ( compare to lane 5 ) . ( B - E ) For quantification , pS1928 ( virtually absent ) and pS1700 IB signals were normalized to total α11 . 2 and pS845 and pS831 signals to total GluA1 ( * * p < 0 . 01 , * * * p < 0 . 001 , ANOVA ) .

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

Forebrain slices from S1928A KI mice were treated with vehicle (lanes 1,5) or 10 M ISO for 5 min, followed, if indicated, by 1, 3, or 20 min wash out of ISO (lanes 3-7) and a second application of ISO for 5 min (lane 6), before solubilization and ultracentrifugation.(A) α11.2 and GluA1 were concurrently IPed from same samples by simultaneous addition of anti-α11.2 and -GluA1 antibodies before probing and stripping/re-probing upper part of IB for pS1928, pS1700, and total α11.2 and middle part for pS845, pS831, and total GluA1. In S1928A KI mice, S1700 re-phosphorylation after a 3 min wash out of ISO was not blunted (lane 6) in contrast to WT mice but rather augmented (compare to lane 5).(B-E) For quantification, pS1928 (virtually absent) and pS1700 IB signals were normalized to total α11.2 and pS845 and pS831 signals to total GluA1 (**p<0.01, ***p<0.001, ANOVA).


( A ) Representative single channel recordings from hippocampal neurons at 7 - 14 DIV with 500 nM BayK - 8644 , 1 µM ω - conotoxin GVIA , and 1 µM MVIIC in patch pipette upon depolarization from - 80 to - 30 , - 20 , - 10 , and 0 mV . The right panel depicts the single - channel current - voltage relationship . Mean amplitude of unitary currents for different membrane potential studied are - 2 . 07 pA ( - 30 mV ) , - 1 . 75 ( - 20 mV ) , - 1 . 53 ( - 10 mV ) and - 1 . 25 ( 0 mV ; n = 5 patches per test potential ) . Solid line represents best - fit of data using a linear equation ( R2 = 0 . 92 ) revealing a slope conductance for these channels of 27 ± 2 pS .

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

(A) Representative single channel recordings from hippocampal neurons at 7-14 DIV with 500 nM BayK-8644, 1 µM ω-conotoxin GVIA, and 1 µM MVIIC in patch pipette upon depolarization from -80 to -30, -20, -10, and 0 mV. The right panel depicts the single-channel current-voltage relationship. Mean amplitude of unitary currents for different membrane potential studied are -2.07 pA (-30 mV), -1.75 (-20 mV), -1.53 (-10 mV) and -1.25 (0 mV; n=5 patches per test potential). Solid line represents best-fit of data using a linear equation (R2=0.92) revealing a slope conductance for these channels of 27 ± 2 pS.


( B - D ) Representative single channel traces and summary plot upon depolarization from - 80 to 0 mV under control conditions and in the presence of ISO in the patch pipette . Cultures were pre - incubated for 15 min with 10 M 11R - PKI if indicated . The patch pipette contained either vehicle for control , nifedipine ( nif ; 1M ) , ISO ( 1M ) , or ISO plus nifedipine , which blocked all currents . The ISO - induced increase in NPo was prevented by 11R - PKI .

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

(B-D) Representative single channel traces and summary plot upon depolarization from -80 to 0 mV under control conditions and in the presence of ISO in the patch pipette. Cultures were pre-incubated for 15 min with 10 M 11R-PKI if indicated. The patch pipette contained either vehicle for control, nifedipine (nif; 1M), ISO (1M), or ISO plus nifedipine, which blocked all currents. The ISO-induced increase in NPo was prevented by 11R-PKI.


( E - H ) Representative single channel currents upon depolarization from - 80 to 0 mV and summary plot after pretreatment of whole cultures with ISO . Cultures were pre - incubated with vehicle ( H2O , mock wash , E ) or 1 M ISO for 5 min ( F , G ) and washed for 3 ( E , F ) or 10 min ( G ) before forming the cell - attached patch with ISO present in the patch pipette . The upregulation of NPo to ~ 0 . 4 ( cf . C , D ) occurred only if neurons were pretreated with vehicle instead of ISO ( E , H ; mock wash ) or if ISO wash out duration was 10 min ( G , H ) but not if wash out was only 3 min ( F , H ) .

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

(E-H) Representative single channel currents upon depolarization from -80 to 0 mV and summary plot after pretreatment of whole cultures with ISO. Cultures were pre-incubated with vehicle (H2O, mock wash, E) or 1 M ISO for 5 min (F,G) and washed for 3 (E,F) or 10 min (G) before forming the cell-attached patch with ISO present in the patch pipette. The upregulation of NPo to ~0.4 (cf. C,D) occurred only if neurons were pretreated with vehicle instead of ISO (E,H; mock wash) or if ISO wash out duration was 10 min (G,H) but not if wash out was only 3 min (F,H).


( A - F ) Forebrain slices were pre - incubated for 30 min with vehicle ( water ) or 10 μM Myr - Pep2 or Myr - DSPL . Like Myr - DSPL ( Joiner et al . , 2010 ) , Pep2 was myristoylated at its N terminus ( Myr - Pep2 ) to make it membrane permeant . Slices were then treated with ISO ( 10 M , 5 min ) or vehicle ( water ) before solubilization , ultracentrifugation , and IP of β2AR ( A - C ) or simultaneously α11 . 2 and GluA1 with a combination of corresponding antibodies within same samples ( D - F ) . ( A ) Myr - Pep 2 displaced α11 . 2 ( lane 5 vs . 3 , top of blot ) but not GluA1 ( middle , same blot ) from β2AR ( bottom , same blot ) ; the inverse was true for Myr - DSPL ( lane 1 vs . 3 ) . ( B , C ) For quantification , α11 . 2 and GluA1 immunosignals were normalized to β2AR signals ( * * p < 0 . 01 , * * * p < 0 . 001 , One Way ANOVA ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-F) Forebrain slices were pre-incubated for 30 min with vehicle (water) or 10 μM Myr-Pep2 or Myr-DSPL. Like Myr-DSPL (Joiner et al., 2010), Pep2 was myristoylated at its N terminus (Myr-Pep2) to make it membrane permeant. Slices were then treated with ISO (10 M, 5 min) or vehicle (water) before solubilization, ultracentrifugation, and IP of β2AR (A-C) or simultaneously α11.2 and GluA1 with a combination of corresponding antibodies within same samples (D-F).(A) Myr-Pep 2 displaced α11.2 (lane 5 vs. 3, top of blot) but not GluA1 (middle, same blot) from β2AR (bottom, same blot); the inverse was true for Myr-DSPL (lane 1 vs. 3).(B,C) For quantification, α11.2 and GluA1 immunosignals were normalized to β2AR signals (**p<0.01, ***p<0.001, One Way ANOVA).


( A - F ) Forebrain slices were pre - incubated for 30 min with vehicle ( water ) or 10 μM Myr - Pep2 or Myr - DSPL . Like Myr - DSPL ( Joiner et al . , 2010 ) , Pep2 was myristoylated at its N terminus ( Myr - Pep2 ) to make it membrane permeant . Slices were then treated with ISO ( 10 M , 5 min ) or vehicle ( water ) before solubilization , ultracentrifugation , and IP of β2AR ( A - C ) or simultaneously α11 . 2 and GluA1 with a combination of corresponding antibodies within same samples ( D - F ) . ( D ) Myr - Pep2 blunted ISO - induced phosphorylation of α11 . 2 S1700 ( lane 6 vs . 4 , top of blot ) but not GluA1 S845 ( middle , same blot ) ; the inverse was true for Myr - DSPL ( lane 2 vs . 4 ) . ( E , F ) For quantification , pS1700 and pS845 immunosignals were normalized to α11 . 2 and GluA1 signals , respectively ( * * * p < 0 . 001 , One Way ANOVA ) .

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

(A-F) Forebrain slices were pre-incubated for 30 min with vehicle (water) or 10 μM Myr-Pep2 or Myr-DSPL. Like Myr-DSPL (Joiner et al., 2010), Pep2 was myristoylated at its N terminus (Myr-Pep2) to make it membrane permeant. Slices were then treated with ISO (10 M, 5 min) or vehicle (water) before solubilization, ultracentrifugation, and IP of β2AR (A-C) or simultaneously α11.2 and GluA1 with a combination of corresponding antibodies within same samples (D-F).(D) Myr-Pep2 blunted ISO-induced phosphorylation of α11.2 S1700 (lane 6 vs. 4, top of blot) but not GluA1 S845 (middle, same blot); the inverse was true for Myr-DSPL (lane 2 vs. 4).(E,F) For quantification, pS1700 and pS845 immunosignals were normalized to α11.2 and GluA1 signals, respectively (***p<0.001, One Way ANOVA).


( G ) Representative cell - attached recordings from hippocampal neurons as in Figure 7 . In interleafed experiments cultures were pre - incubated for 30 min with 10 M Myr - Pep2 or scrambled Myr - Pep2 ( Myr - Pep2scr ) . The patch pipette contained either vehicle ( H2O ; control ) or 1 µM ISO . The ISO induced increase in NPo was prevented by Myr - Pep2 but not Myr - Pep2scr . Arrows indicate the 0 - current level ( i . e . closed channel ) . ( H ) Summary plot for ( G ) . For statistical analysis , the NPo value was determined for each recording and pooled under each condition for comparison ( n = 10 - 12 patches ; * p < 0 . 05 , One Way ANOVA with Tukey post - hoc test ) .

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

(G) Representative cell-attached recordings from hippocampal neurons as in Figure 7. In interleafed experiments cultures were pre-incubated for 30 min with 10 M Myr-Pep2 or scrambled Myr-Pep2 (Myr-Pep2scr). The patch pipette contained either vehicle (H2O; control) or 1 µM ISO. The ISO induced increase in NPo was prevented by Myr-Pep2 but not Myr-Pep2scr. Arrows indicate the 0-current level (i.e. closed channel).(H) Summary plot for (G). For statistical analysis, the NPo value was determined for each recording and pooled under each condition for comparison (n=10-12 patches; *p<0.05, One Way ANOVA with Tukey post-hoc test).


Graphs depict fEPSP initial slopes recorded from hippocampal CA1 before and after either a 5Hz / 3 min ( A - C ) or 100Hz / 1sec tetanus ( D - F ) . Arrowheads mark onset of tetani and bars perfusion with 1 µM ISO . Inserts show sample traces immediately before ( left ) and ~ 30 min after ( right ) tetani . ( A - C ) Litter - matched WT but not conditional Cav1 . 2 KO mice showed PTT - LTP ( * * p < 0 . 001 , one - way ANOVA ) . ( D - F ) WT as well as Cav1 . 2 KO mice showed NMDAR - dependent 100 Hz LTP .

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

Graphs depict fEPSP initial slopes recorded from hippocampal CA1 before and after either a 5Hz/3 min (A-C) or 100Hz/1sec tetanus (D-F). Arrowheads mark onset of tetani and bars perfusion with 1 µM ISO. Inserts show sample traces immediately before (left) and ~30 min after (right) tetani.(A-C) Litter-matched WT but not conditional Cav1.2 KO mice showed PTT-LTP (**p < 0.001, one-way ANOVA).(D-F) WT as well as Cav1.2 KO mice showed NMDAR-dependent 100 Hz LTP.


Graphs depict fEPSP initial slopes recorded from hippocampal CA1 before and after either a 5Hz / 3 min ( A - C , G - I ) or 100Hz / 1sec tetanus ( D - F ) . Arrowheads mark onset of tetani and bars perfusion with 1 µM ISO and 10 µM of Myr - Pep2 or Myr - Pep2scr . Inserts show sample traces immediately before ( left ) and ~ 30 min after ( right ) tetani . ( G - I ) Myr - Pep2 but not Myr - Pep2scr blocked PTT - LTP ( * * p < 0 . 001 , one - way ANOVA ) .

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

Graphs depict fEPSP initial slopes recorded from hippocampal CA1 before and after either a 5Hz/3 min (A-C, G-I) or 100Hz/1sec tetanus (D-F). Arrowheads mark onset of tetani and bars perfusion with 1 µM ISO and 10 µM of Myr-Pep2 or Myr-Pep2scr. Inserts show sample traces immediately before (left) and ~30 min after (right) tetani.(G-I) Myr-Pep2 but not Myr-Pep2scr blocked PTT-LTP (**p < 0.001, one-way ANOVA).


( A - C ) Distribution of spindle pole factors in centrosomal and acentrosomal spindles . DMSO - treated control mitotic spindles ( 2 - centrosomes ) and centrinone - treated acentrosomal spindles ( 0 - centrosome ) of HeLa cells . ( A ) Red , gray , green , and blue represent NuMA , α - tubulin , Cep152 , and DNA , respectively . Z - projections of eight sections , every 0 . 3 μm . Scale bar , 5 μm . ( B ) Red , gray , and blue represent NuMA , Katanin p60 , and DNA , respectively . Z - projections of five sections , every 0 . 3 μm . Scale bar , 5 μm . ( C ) Red , gray , and blue represent p150glued , ASPM , and DNA , respectively . Z - projections of 10 ( 2 - centrosomes ) or five ( 0 - centrosome ) sections , every 0 . 3 μm . Scale bar , 5 μm .

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

(A-C) Distribution of spindle pole factors in centrosomal and acentrosomal spindles. DMSO-treated control mitotic spindles (2-centrosomes) and centrinone-treated acentrosomal spindles (0-centrosome) of HeLa cells. (A) Red, gray, green, and blue represent NuMA, α-tubulin, Cep152, and DNA, respectively. Z-projections of eight sections, every 0.3 μm. Scale bar, 5 μm. (B) Red, gray, and blue represent NuMA, Katanin p60, and DNA, respectively. Z-projections of five sections, every 0.3 μm. Scale bar, 5 μm. (C) Red, gray, and blue represent p150glued, ASPM, and DNA, respectively. Z-projections of 10 (2-centrosomes) or five (0-centrosome) sections, every 0.3 μm. Scale bar, 5 μm.


( D ) The structure of NuMA and microtubules in centrinone - treated acentrosomal spindles of HeLa cells . Two - way arrows indicate the bipolarity of elongated NuMA structures . Gray , red , green and blue represent α - tubulin , NuMA , Cep152 and DNA , respectively . Scale bar , 5 μm .

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

(D) The structure of NuMA and microtubules in centrinone-treated acentrosomal spindles of HeLa cells. Two-way arrows indicate the bipolarity of elongated NuMA structures. Gray, red, green and blue represent α-tubulin, NuMA, Cep152 and DNA, respectively. Scale bar, 5 μm.


( E ) The localization of p150glued in centrinone - treated acentrosomal spindles of HeLa cells . Red , gray , green and blue represent NuMA , p150glued , Cep152 and DNA , respectively . Scale bar , 5 μm .

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

(E) The localization of p150glued in centrinone-treated acentrosomal spindles of HeLa cells. Red, gray, green and blue represent NuMA, p150glued, Cep152 and DNA, respectively. Scale bar, 5 μm.


( F ) NuMA distribution in 3D . Images of NuMA ( Red ) and chromosomes ( blue ) were reconstructed in 3D and surface - rendered .

O O O O 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) NuMA distribution in 3D. Images of NuMA (Red) and chromosomes (blue) were reconstructed in 3D and surface-rendered.


( G ) Representative images of NuMA ( arrowheads ) in centrosomal and acentrosomal cells before and after photobleaching . DMSO - or centrinone - treated HCT116 TetOsTIR1 NuMA - mAID - mClover - FLAG cells were observed . Time after photobleaching ( sec ) is indicated . Scale bar , 5 μm .

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

(G) Representative images of NuMA (arrowheads) in centrosomal and acentrosomal cells before and after photobleaching. DMSO- or centrinone-treated HCT116 TetOsTIR1 NuMA-mAID-mClover-FLAG cells were observed. Time after photobleaching (sec) is indicated. Scale bar, 5 μm.


( H ) Normalized fluorescence intensities of NuMA - mClover at centrosomal poles ( control , 2 - centrosomes ) or at acentrosomal spindle pole ( 0 - centrosome ) during the FRAP time course ( N = 20 cells for each condition ) . The first three time points are before photobleaching . Error bars show standard deviation ( SD ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Normalized fluorescence intensities of NuMA-mClover at centrosomal poles (control, 2-centrosomes) or at acentrosomal spindle pole (0-centrosome) during the FRAP time course (N=20 cells for each condition). The first three time points are before photobleaching. Error bars show standard deviation (SD).


( I ) Mobile fractions from the normalized recovery curves of NuMA - mClover at centrosomal spindle poles ( control , 2 - centrosomes ) or at acentrosomal spindle poles ( 0 - centrosome ) . Mobile fractions ( roughly corresponds to normalized fluorescence intensities at the last time point were determined by single exponential fitting . Line and error bars represent the median and interquartile range ( N = 20 cells for each condition ) . The Mann - Whitney U test ( two - tailed ) was used to obtain a P value .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Mobile fractions from the normalized recovery curves of NuMA-mClover at centrosomal spindle poles (control, 2-centrosomes) or at acentrosomal spindle poles (0-centrosome). Mobile fractions (roughly corresponds to normalized fluorescence intensities at the last time point were determined by single exponential fitting. Line and error bars represent the median and interquartile range (N=20 cells for each condition). The Mann-Whitney U test (two-tailed) was used to obtain a P value.


( A ) Time - lapse observation of the establishment of bipolarity in acentrosomal cells . HeLa cells expressing EGFP - centrin1 and mCherry - NuMA were observed with a 63 × objective . Magenta and green represent mCherry - NuMA and EGFP - centrin1 , respectively . Arrowheads indicate the assembling NuMA after NEBD . Two - way arrows indicate the bipolarity . Z - projections of 20 sections , every 1 . 2 μm . Scale bar , 10 μm . Time zero corresponds to NEBD .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Time-lapse observation of the establishment of bipolarity in acentrosomal cells. HeLa cells expressing EGFP-centrin1 and mCherry-NuMA were observed with a 63× objective. Magenta and green represent mCherry-NuMA and EGFP-centrin1, respectively. Arrowheads indicate the assembling NuMA after NEBD. Two-way arrows indicate the bipolarity. Z-projections of 20 sections, every 1.2 μm. Scale bar, 10 μm. Time zero corresponds to NEBD.


( B ) Time - lapse observation of the dynamics of NuMA and chromosomes . Centrinone - treated HeLa cells expressing mCover - NuMA were observed with a 63 × objective . Magenta and green represent SiR - DNA and mClover - NuMA , respectively . Arrowheads and two - way arrows indicate the assembling NuMA after NEBD and bipolarity , respectively . Z - projections of 20 sections , every 1 . 2 μm . Scale bar , 10 μm . Time zero corresponds to NEBD .

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

(B) Time-lapse observation of the dynamics of NuMA and chromosomes. Centrinone-treated HeLa cells expressing mCover-NuMA were observed with a 63× objective. Magenta and green represent SiR-DNA and mClover-NuMA, respectively. Arrowheads and two-way arrows indicate the assembling NuMA after NEBD and bipolarity, respectively. Z-projections of 20 sections, every 1.2 μm. Scale bar, 10 μm. Time zero corresponds to NEBD.


( C ) The timing of bipolarity establishment in the acentrosomal pole , chromosome alignment , and anaphase onset Each plot shows the cumulative percentage of each event at individual time points ( N = 49 cells from two independent experiments ) .

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

(C) The timing of bipolarity establishment in the acentrosomal pole, chromosome alignment, and anaphase onset Each plot shows the cumulative percentage of each event at individual time points (N=49 cells from two independent experiments).


( D ) The distribution of kinetochores during acentrosomal spindle pole separation was confirmed in fixed cells . Two - way arrow indicates the bipolarity of elongated NuMA structures . Red , gray , green , and blue represent NuMA , CENP - C , GT335 , and DNA , respectively . Scale bar , 5 μm .

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

(D) The distribution of kinetochores during acentrosomal spindle pole separation was confirmed in fixed cells. Two-way arrow indicates the bipolarity of elongated NuMA structures. Red, gray, green, and blue represent NuMA, CENP-C, GT335, and DNA, respectively. Scale bar, 5 μm.


( E , F ) Time - lapse observation of the structure of NuMA and microtubules upon centrosome removal . Centrinone - treated HCT116 TetOsTIR1 NuMA - mAID - mClover - FLAG cells were observed with a 60 × objective . Red and green represent NuMA and SiR - tubulin , respectively . Z - projections of 17 sections , every 1 μm . Scale bar , 5 μm . Time zero corresponds to NEBD . ( F ) An example of NuMA formed several asters ( arrowheads ) at the time of NEBD .

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

(E, F) Time-lapse observation of the structure of NuMA and microtubules upon centrosome removal. Centrinone-treated HCT116 TetOsTIR1 NuMA-mAID-mClover-FLAG cells were observed with a 60× objective. Red and green represent NuMA and SiR-tubulin, respectively. Z-projections of 17 sections, every 1 μm. Scale bar, 5 μm. Time zero corresponds to NEBD. (F) An example of NuMA formed several asters (arrowheads) at the time of NEBD.


( A ) NuMA structure upon microtubule depolymerization . Nocodazole - treated HCT116 CMV - OsTIR1 CEP152 - mClover - mAID cells were treated with 100 ng / ml nocodazole . Gray , red , green and blue represent α - tubulin , NuMA , CEP152 ( mClover ) and DNA , respectively . Scale bar , 5 μm .

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

(A) NuMA structure upon microtubule depolymerization. Nocodazole-treated HCT116 CMV-OsTIR1 CEP152-mClover-mAID cells were treated with 100 ng/ml nocodazole. Gray, red, green and blue represent α-tubulin, NuMA, CEP152 (mClover) and DNA, respectively. Scale bar, 5 μm.


( B ) Immunostaining of DHC1 and NuMA in centrinone - treated HCT116 TetOsTIR1 DHC1 - 3X - mAID - mClover cells . Gray , green , red and blue represent DHC1 ( mClover ) , GT335 , NuMA , and DNA , respectively . Scale bar , 5 μm .

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

(B) Immunostaining of DHC1 and NuMA in centrinone-treated HCT116 TetOsTIR1 DHC1-3X-mAID-mClover cells. Gray, green, red and blue represent DHC1 (mClover), GT335, NuMA, and DNA, respectively. Scale bar, 5 μm.


( D ) NuMA structure upon DHC1 depletion . Centrinone - treated HCT116 TetOsTIR1 DHC1 - 3X - mAID - mClover cells were treated with 1 μg / ml doxycycline ( Dox ) and 500 μM indole - 3 - acetic acid ( IAA ) . Arrowhead indicates the spindle pole . Green , red , gray and blue represent DHC1 ( mClover ) , NuMA , GT335 and DNA , respectively . Scale bar , 5 μm . ( E ) Frequency of acentrosomal pole patterns upon DHC1 depletion in ( D ) . Values are mean percentages ± SD . from three independent experiments ( N ≧ 24 spindles in each experiment ) . (

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

(D) NuMA structure upon DHC1 depletion. Centrinone-treated HCT116 TetOsTIR1 DHC1-3X-mAID-mClover cells were treated with 1 μg/ml doxycycline (Dox) and 500 μM indole-3-acetic acid (IAA). Arrowhead indicates the spindle pole. Green, red, gray and blue represent DHC1 (mClover), NuMA, GT335 and DNA, respectively. Scale bar, 5 μm. (E) Frequency of acentrosomal pole patterns upon DHC1 depletion in (D). Values are mean percentages ± SD. from three independent experiments (N≧24 spindles in each experiment). (


( F ) The distribution of NuMA upon DHC1 depletion . Arrowheads indicate the presence of NuMA on microtubules . Green , red , and blue represent α - tubulin , NuMA , and DNA , respectively . Scale bars , 5 μm .

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

(F) The distribution of NuMA upon DHC1 depletion. Arrowheads indicate the presence of NuMA on microtubules. Green, red, and blue represent α-tubulin, NuMA, and DNA, respectively. Scale bars, 5 μm.


( G ) Structure of acentrosomal spindle poles upon replacement of endogenous NuMA - mAID - mClover - FLAG with either mCherry - NuMA WT or 5A - 3 . Arrowheads indicate the assembled NuMA structure . Green , red , gray and blue represent endogenous NuMA ( mClover ) , expressed NuMA ( mCherry ) , GT335 and DNA , respectively . Z - projections of 21 sections , every 1 μm . Scale bar , 5 μm . ( H ) The number of NuMA structure in ( G ) . Values are mean percentages ± SD . from four independent experiments ( N ≧ 15 spindles in each experiment ) . One - way ANOVA with Tukey ' s multiple comparisons test was used to obtain a P value .

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

(G) Structure of acentrosomal spindle poles upon replacement of endogenous NuMA-mAID-mClover-FLAG with either mCherry-NuMA WT or 5A-3. Arrowheads indicate the assembled NuMA structure. Green, red, gray and blue represent endogenous NuMA (mClover), expressed NuMA (mCherry), GT335 and DNA, respectively. Z-projections of 21 sections, every 1 μm. Scale bar, 5 μm. (H) The number of NuMA structure in (G). Values are mean percentages ± SD. from four independent experiments (N≧15 spindles in each experiment). One-way ANOVA with Tukey's multiple comparisons test was used to obtain a P value.


( A , B ) Structure of the spindle upon NuMA depletion . Centrinone - treated HCT116 TetOsTIR1 NuMA - mAID - mClover - FLAG cells were treated with 1 μg / ml doxycycline ( Dox ) and 500 μM IAA . Two - way arrow indicates the bipolarity . Gray , green , red , and blue represent α - tubulin , NuMA ( mClover ) , GT335 , and DNA , respectively . Z - projections of 10 sections , every 0 . 3 μm . Scale bar , 5 μm . ( C ) Frequency of spindle structure patterns upon NuMA depletion in ( A , B ) . Values are mean percentages ± SD . from three independent experiments ( N = 30 spindles in each experiment ) . (

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

(A, B) Structure of the spindle upon NuMA depletion. Centrinone-treated HCT116 TetOsTIR1 NuMA-mAID-mClover-FLAG cells were treated with 1 μg/ml doxycycline (Dox) and 500 μM IAA. Two-way arrow indicates the bipolarity. Gray, green, red, and blue represent α-tubulin, NuMA (mClover), GT335, and DNA, respectively. Z-projections of 10 sections, every 0.3 μm. Scale bar, 5 μm. (C) Frequency of spindle structure patterns upon NuMA depletion in (A, B). Values are mean percentages ± SD. from three independent experiments (N=30 spindles in each experiment). (


( D ) The localization of Eg5 in centrinone - treated acentrosomal HeLa cells . Red , gray , green and blue represent NuMA , Eg5 , GT335 and DNA , respectively . Scale bar , 5 μm .

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(D) The localization of Eg5 in centrinone-treated acentrosomal HeLa cells. Red, gray, green and blue represent NuMA, Eg5, GT335 and DNA, respectively. Scale bar, 5 μm.


( E ) The localization of Eg5 in the spindle upon NuMA depletion . Centrinone - treated HCT116 TetOsTIR1 NuMA - mAID - mClover - FLAG cells were treated with Dox and 500 μM IAA . Gray , green , red , and blue represent Eg5 , NuMA ( mClover ) , CP110 , and DNA , respectively . Z - projections of 5 sections , every 0 . 3 μm . Scale bar , 5 μm .

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

(E) The localization of Eg5 in the spindle upon NuMA depletion. Centrinone-treated HCT116 TetOsTIR1 NuMA-mAID-mClover-FLAG cells were treated with Dox and 500 μM IAA. Gray, green, red, and blue represent Eg5, NuMA (mClover), CP110, and DNA, respectively. Z-projections of 5 sections, every 0.3 μm. Scale bar, 5 μm.


( F ) Structure of the spindle upon Eg5 inhibition . Centrinone - treated HCT116 TetOsTIR1 NuMA - mAID - mClover - FLAG cells were treated with 30 μM monastrol . Gray , green , red , and blue represent α - tubulin , NuMA ( mClover ) , GT335 , and DNA , respectively . Z - projections of 5 sections , every 0 . 3 μm . Scale bar , 5 μm . ( G ) Frequency of spindle structure patterns upon Eg5 inhibition in ( F ) . Values are mean percentages ± SD . from three independent experiments ( N ≧ 23 spindles in each experiment ) .

O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Structure of the spindle upon Eg5 inhibition. Centrinone-treated HCT116 TetOsTIR1 NuMA-mAID-mClover-FLAG cells were treated with 30 μM monastrol. Gray, green, red, and blue represent α-tubulin, NuMA (mClover), GT335, and DNA, respectively. Z-projections of 5 sections, every 0.3 μm. Scale bar, 5 μm. (G) Frequency of spindle structure patterns upon Eg5 inhibition in (F). Values are mean percentages ± SD. from three independent experiments (N≧23 spindles in each experiment).


( A ) Time - lapse observation of the establishment of bipolarity within the acentrosomal spindle pole upon inhibition of Eg5 . HeLa cells expressing EGFP - centrin1 and mCherry - NuMA were observed in the presence of 50 μM of monastrol with a 63 × objective . Magenta and green represent mCherry - NuMA and EGFP - centrin1 , respectively . Arrowheads and two - way arrows indicate the monopolar and bipolar states , respectively . Z - projections of 20 sections , every 1 . 2 μm . Scale bar , 10 μm . Time zero corresponds to NEBD . ( B ) The time of bipolarity establishment and being stuck in the monopolar - like state of acentrosomal spindle poles in ( A ) . Each plot shows the cumulative percentage of each event at each time point ( N = total 75 cells from two independent experiments ) . (

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

(A) Time-lapse observation of the establishment of bipolarity within the acentrosomal spindle pole upon inhibition of Eg5. HeLa cells expressing EGFP-centrin1 and mCherry-NuMA were observed in the presence of 50 μM of monastrol with a 63× objective. Magenta and green represent mCherry-NuMA and EGFP-centrin1, respectively. Arrowheads and two-way arrows indicate the monopolar and bipolar states, respectively. Z-projections of 20 sections, every 1.2 μm. Scale bar, 10 μm. Time zero corresponds to NEBD. (B) The time of bipolarity establishment and being stuck in the monopolar-like state of acentrosomal spindle poles in (A). Each plot shows the cumulative percentage of each event at each time point (N=total 75 cells from two independent experiments). (


( C ) Time - lapse observation of the dynamics of mClover - NuMA and chromosomes . HeLa cells expressing mClover - NuMA were observed in the presence of SiR - DNA with a 63 × objective . Magenta and green represent SiR - DNA and mClover - NuMA , respectively . Arrowheads indicate initial bipolarity in the acentrosomal spindle pole ( 8 min ) , dispersed chromosomes ( 328 min ) , and the monopolar - like state of the acentrosomal spindle pole ( 332 min ) , respectively . Z - projections of 20 sections , every 1 . 2 μm . Scale bar , 10 μm . Time zero corresponds to NEBD . ( D ) The time of bipolarity establishment and being stuck in the monopolar - like state of acentrosomal spindle poles in ( C ) . Each plot shows the cumulative percentage of each event at each time point ( N = total 35 cells from three independent experiments ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Time-lapse observation of the dynamics of mClover-NuMA and chromosomes. HeLa cells expressing mClover-NuMA were observed in the presence of SiR-DNA with a 63× objective. Magenta and green represent SiR-DNA and mClover-NuMA, respectively. Arrowheads indicate initial bipolarity in the acentrosomal spindle pole (8 min), dispersed chromosomes (328 min), and the monopolar-like state of the acentrosomal spindle pole (332 min), respectively. Z-projections of 20 sections, every 1.2 μm. Scale bar, 10 μm. Time zero corresponds to NEBD. (D) The time of bipolarity establishment and being stuck in the monopolar-like state of acentrosomal spindle poles in (C). Each plot shows the cumulative percentage of each event at each time point (N=total 35 cells from three independent experiments).


( A ) Structure of the spindle upon NuMA and Eg5 inhibition . HCT116 TetOsTIR1 NuMA - mAID - mClover - FLAG cells with 2 - centrosomes were treated with 1 μg / ml doxycycline ( Dox ) , 500 μM IAA and monastrol . Green , red , and blue represent NuMA ( mClover ) , α - tubulin , and DNA , respectively . Z - projections of 10 sections , every 0 . 3 μm . Scale bar , 5 μm . ( B ) Frequency of spindle structure patterns upon NuMA depletion and Eg5 inhibition in ( A ) . Values are mean percentages ± SD . from three independent experiments ( N ≧ 22 spindles in each experiment ) . (

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

(A) Structure of the spindle upon NuMA and Eg5 inhibition. HCT116 TetOsTIR1 NuMA-mAID-mClover-FLAG cells with 2-centrosomes were treated with 1 μg/ml doxycycline (Dox), 500 μM IAA and monastrol. Green, red, and blue represent NuMA (mClover), α-tubulin, and DNA, respectively. Z-projections of 10 sections, every 0.3 μm. Scale bar, 5 μm. (B) Frequency of spindle structure patterns upon NuMA depletion and Eg5 inhibition in (A). Values are mean percentages ± SD. from three independent experiments (N≧22 spindles in each experiment). (


( C ) The localization of Eg5 in the spindle upon NuMA depletion . HCT116 TetOsTIR1 NuMA - mAID - mClover - FLAG cells with 2 - centrosomes were treated with 1 μg / ml doxycycline Dox and 500 μM IAA . Gray , green , red , and blue represent α - tubulin , NuMA ( mClover ) , Eg5 and DNA , respectively . Z - projections of 5 sections , every 0 . 3 μm . Scale bar , 5 μm .

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

(C) The localization of Eg5 in the spindle upon NuMA depletion. HCT116 TetOsTIR1 NuMA-mAID-mClover-FLAG cells with 2-centrosomes were treated with 1 μg/ml doxycycline Dox and 500 μM IAA. Gray, green, red, and blue represent α-tubulin, NuMA (mClover), Eg5 and DNA, respectively. Z-projections of 5 sections, every 0.3 μm. Scale bar, 5 μm.


( D ) Time - lapse observation of the establishment of spindle bipolarity upon NuMA depletion . HCT116 TetOsTIR1 NuMA - mAID - mClover - FLAG cells with 2 - centrosomes were treated with 1 μg / ml Dox and 500 μM IAA and observed with a 40 × objective . Red and green represent SiR - tubulin and NuMA , respectively . Z - projections of 14 sections , every 2 μm . Scale bar , 10 μm . Time zero corresponds to NEBD . ( E ) The time required for the initial establishment of spindle bipolarity in ( D ) . Line and error bars represent the mean and SD . ( N ≧ 60 cells from two independent experiments ) . The Mann - Whitney U test ( two - tailed ) was used to obtain a P value .

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

(D) Time-lapse observation of the establishment of spindle bipolarity upon NuMA depletion. HCT116 TetOsTIR1 NuMA-mAID-mClover-FLAG cells with 2-centrosomes were treated with 1 μg/ml Dox and 500 μM IAA and observed with a 40× objective. Red and green represent SiR-tubulin and NuMA, respectively. Z-projections of 14 sections, every 2 μm. Scale bar, 10 μm. Time zero corresponds to NEBD. (E) The time required for the initial establishment of spindle bipolarity in (D). Line and error bars represent the mean and SD. (N≧60 cells from two independent experiments). The Mann-Whitney U test (two-tailed) was used to obtain a P value.


A - C . CHO cells expressing PD - L1 alone ( A ) or co - expressing CD80 ( B ) or CD86 ( C ) were stained with CD80 Ig , PD - 1 Ig , CTLA - 4 Ig or anti - PD - L1 Ab clones MIH1 , MIH3 , 29E . 2A3 ) at 1µg / ml for 30 min at 37 ° C . Representative FACS plots show staining plotted against total PD - L1 mCherry levels .

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A-C. CHO cells expressing PD-L1 alone (A) or co-expressing CD80 (B) or CD86 (C) were stained with CD80 Ig, PD-1 Ig, CTLA-4 Ig or anti-PD-L1 Ab clones MIH1, MIH3, 29E.2A3) at 1µg/ml for 30 min at 37°C. Representative FACS plots show staining plotted against total PD-L1 mCherry levels.


E . Western blot analysis of an immunoprecipitation by anti - mCherry antibody ( PDL1 ) from indicated DG - 75 cell lysates , with and without the BS3 crosslinker , and probed for mCherry ( PD - L1 ) or GFP ( CD80 ) .

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E. Western blot analysis of an immunoprecipitation by anti-mCherry antibody (PDL1) from indicated DG-75 cell lysates, with and without the BS3 crosslinker, and probed for mCherry (PD-L1) or GFP (CD80).


A . Transendocytosis assays were carried out overnight using CHO donor cells ( CD80GFP alone or CD80GFP with PD - L1mCherry co - expression ) incubated with CTLA - 4 recipient cells at a ratio of 1 : 1 . Recipient cells either lacked CTLA - 4 ( No CTLA - 4 ) or expressed CTLA - 4 . FACS plots show CD80GFP ligand loss from donor cells highlighted in blue quadrants in the presence of CTLA - 4 + recipients .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Transendocytosis assays were carried out overnight using CHO donor cells (CD80GFP alone or CD80GFP with PD-L1mCherry co-expression) incubated with CTLA-4 recipient cells at a ratio of 1:1. Recipient cells either lacked CTLA-4 (No CTLA-4) or expressed CTLA-4. FACS plots show CD80GFP ligand loss from donor cells highlighted in blue quadrants in the presence of CTLA-4+ recipients.


C - F . Transendocytosis assays were performed at a ratio of 2 donor : 1 CTLA - 4 recipient for the times indicated . ( C ) , Representative FACS plots showing CD80GFP levels and ( D ) full kinetic analysis of CD80 or CD86 downregulation on donor cells quantified as a percentage relative to no CTLA - 4 control ( mean ± SEM , 3 independent experiments , ns - not significant : two - way ANOVA with Tukey ' s multiple comparisons test ) . ( E ) Representative FACS plots showing PD - L1mCherry level at time points indicated . ( F ) Full kinetic analysis of PD - L1mCherry level on donor cells ( mean ± SEM , 3 independent experiments ) .

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C-F. Transendocytosis assays were performed at a ratio of 2 donor:1 CTLA-4 recipient for the times indicated. (C), Representative FACS plots showing CD80GFP levels and (D) full kinetic analysis of CD80 or CD86 downregulation on donor cells quantified as a percentage relative to no CTLA-4 control (mean ± SEM, 3 independent experiments, ns - not significant: two-way ANOVA with Tukey's multiple comparisons test). (E) Representative FACS plots showing PD-L1mCherry level at time points indicated. (F) Full kinetic analysis of PD-L1mCherry level on donor cells (mean ± SEM, 3 independent experiments).


D . Levels of CD86GFP and PD - L1mCherry remaining on donor cells over a 24h transendocytosis period with CTLA - 4 WT or Del36 plotted relative to no CTLA - 4 control ( mean ± SEM , 6 independent experiments ) . * * * * P ≤ 0 . 0001 , RM one - way ANOVA .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Levels of CD86GFP and PD-L1mCherry remaining on donor cells over a 24h transendocytosis period with CTLA-4 WT or Del36 plotted relative to no CTLA-4 control (mean ± SEM, 6 independent experiments). ****P≤0.0001, RM one-way ANOVA.


A - B . APC - labelled PD - 1 Ig ( 0 . 75µg / ml ) was used to detect PD - L1 in DG - 75 cells co - expressing CD80GFP ( A ) or CD86GFP ( B ) . Staining was carried out following transendocytosis using Jurkat cells expressing no CTLA - 4 , CTLA - 4 WT or CTLA - 4 Del36 ( at a ratio of 1 : 1 ) for the indicated durations . ( A ) shows representative FACS plots of CD80GFP vs . PD - 1 Ig at the time points indicated , with full kinetic data plotted below ( mean ± SEM , 3 independent experiments , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 : two - way ANOVA with Tukey ' s multiple comparisons test ) . ( B ) As for ( A ) except using CD86 - PD - L1 expressing cells .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. APC-labelled PD-1 Ig (0.75µg/ml) was used to detect PD-L1 in DG-75 cells co-expressing CD80GFP (A) or CD86GFP (B). Staining was carried out following transendocytosis using Jurkat cells expressing no CTLA-4, CTLA-4 WT or CTLA-4 Del36 (at a ratio of 1:1) for the indicated durations. (A) shows representative FACS plots of CD80GFP vs. PD-1 Ig at the time points indicated, with full kinetic data plotted below (mean ± SEM, 3 independent experiments, ***P≤0.001, ****P≤0.0001: two-way ANOVA with Tukey's multiple comparisons test). (B) As for (A) except using CD86-PD-L1 expressing cells.


A . Staining of free PD - L1 with APC - conjugated PD - 1 Ig in DG - 75 cells expressing only PD - L1mCherry , using cell lines with increasing ratios of CD80GFP : PD - L1mCherry compared to CD80GFP only . FACS plots are representative of 4 independent experiments .

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

A. Staining of free PD-L1 with APC-conjugated PD-1 Ig in DG-75 cells expressing only PD-L1mCherry, using cell lines with increasing ratios of CD80GFP: PD-L1mCherry compared to CD80GFP only. FACS plots are representative of 4 independent experiments.


A . DG - 75 B cells expressing CD80GFP or CD86GFP ( green ) and PD - L1mCherry ( red ) were incubated with Jurkat cells ( nuclei shown in grey ) expressing CTLA - 4 WT or mutant CTLA - 4 Del36 for the indicated durations and analysed by confocal microscopy . 2 hours prior to assay endpoint , APC - labelled PD - 1 Ig ( 0 . 75µg / ml ) was added . CD80 / CD86 and PD - L1 co - localisation at the immune synapse is shown in yellow with PD - 1 Ig binding shown in purple . Scale bar , 10µm .

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A. DG-75 B cells expressing CD80GFP or CD86GFP (green) and PD-L1mCherry (red) were incubated with Jurkat cells (nuclei shown in grey) expressing CTLA-4 WT or mutant CTLA-4 Del36 for the indicated durations and analysed by confocal microscopy. 2 hours prior to assay endpoint, APC-labelled PD-1 Ig (0.75µg/ml) was added. CD80/CD86 and PD-L1 co-localisation at the immune synapse is shown in yellow with PD-1 Ig binding shown in purple. Scale bar, 10µm.


B . Flow cytometry plots of CD80GFP levels on DG - 75 B - cells ( gated on CTFR + cells ) at 24h with graphical representation of the MFI in each condition ( RH graph ) . The Jreg cells used , and the anti - PD - 1 status is indicated above the plot . C . Flow cytometry plots of CD28 levels indicating contact between the CD80 + APC and the PD - 1 + CD28 + responder Jurkat T - cells , following CD80 transendocytosis . Graphical representation of the CD28 MFI in each condition is shown on the right . D . Flow cytometry of CD69 levels on responder Jurkat T - cells and graphical representation of CD69 MFI in each condition . E . Flow cytometry of CD69 levels on CD28KO ( PD - 1 + ) responder Jurkat T - cells and graphical representation of the CD69 + cells MFI in each condition . Cartoons on the left of B - E illustrate the cell type analysed . Data information : Data is representative of 3 independent experiments showing mean ± SEM . * P ≤ 0 . 05 , * * P ≤ 0 . 01 , ns - not significant : two - way ANOVA with Tukey ' s multiple comparisons test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Flow cytometry plots of CD80GFP levels on DG-75 B-cells (gated on CTFR+ cells) at 24h with graphical representation of the MFI in each condition (RH graph). The Jreg cells used, and the anti-PD-1 status is indicated above the plot. C. Flow cytometry plots of CD28 levels indicating contact between the CD80+ APC and the PD-1+ CD28+ responder Jurkat T-cells, following CD80 transendocytosis. Graphical representation of the CD28 MFI in each condition is shown on the right. D. Flow cytometry of CD69 levels on responder Jurkat T-cells and graphical representation of CD69 MFI in each condition. E. Flow cytometry of CD69 levels on CD28KO (PD-1+) responder Jurkat T-cells and graphical representation of the CD69+ cells MFI in each condition. Cartoons on the left of B-E illustrate the cell type analysed. Data information: Data is representative of 3 independent experiments showing mean ± SEM. *P≤0.05, **P≤0.01, ns - not significant: two-way ANOVA with Tukey's multiple comparisons test.


B . Titration of Abatacept staining ( left hand panel ) and EC50 values of Abatacept ( right hand panel ) for CD80 only or PD - L1 / CD80 co - expressing DG - 75 . Data information : Data is representative of 3 independent experiments showing mean ± SEM . * P ≤ 0 . 05 , * * P ≤ 0 . 01 , ns - not significant : paired t - test ( B )

O O O O B-MEASURED_VAR 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

B. Titration of Abatacept staining (left hand panel) and EC50 values of Abatacept (right hand panel) for CD80 only or PD-L1/CD80 co-expressing DG-75. Data information: Data is representative of 3 independent experiments showing mean ± SEM. *P≤0.05, **P≤0.01, ns - not significant: paired t-test (B)


D . Comparison of PD - L1 detection using PD - 1 - Ig or anti - PD - L1 antibodies ( MIH3 or 29E . 2A3 clone ) with and without prior incubation with 10µg / ml of abatacept , based on data from ( C ) . E . Graphs showing PD - L1 detection under conditions used in ( C ) following different Abatacept incubation periods ( 30mins , 3h or 6h ) . Data information : Data is representative of 3 independent experiments showing mean ± SEM . * P ≤ 0 . 05 , * * P ≤ 0 . 01 , ns - not significant : RM one - way ANOVA ( D ) .

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

D. Comparison of PD-L1 detection using PD-1-Ig or anti-PD-L1 antibodies (MIH3 or 29E.2A3 clone) with and without prior incubation with 10µg/ml of abatacept, based on data from (C). E. Graphs showing PD-L1 detection under conditions used in (C) following different Abatacept incubation periods (30mins, 3h or 6h). Data information: Data is representative of 3 independent experiments showing mean ± SEM. *P≤0.05, **P≤0.01, ns - not significant: RM one-way ANOVA (D).


D Western blot of Sec63 , mCherry and Pgk1 from WT cells ( SSY1404 ) , cells harboring the estradiol - inducible system ( SSY1405 ) or ∆ opi1 cells ( SSY1607 ) , all of which expressed Sec63 - mNeon and Rtn1 - mCherry . Cells were untreated or treated with 800 nM estradiol for 6 h . Pgk1 served as a loading control .

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D Western blot of Sec63, mCherry and Pgk1 from WT cells (SSY1404), cells harboring the estradiol-inducible system (SSY1405) or ∆opi1 cells (SSY1607), all of which expressed Sec63-mNeon and Rtn1-mCherry. Cells were untreated or treated with 800 nM estradiol for 6 h. Pgk1 served as a loading control.


E Quantification of ER size in estradiol - treated cells harboring the inducible system ( SSY1405 ) , untreated ∆ opi1 cells ( SSY1607 ) and WT cells ( SSY1404 ) treated with 8 mM DTT for 1 h . Bars represent mean + s . e . m . , n = 3 biological replicates . Asterisks indicate statistical significance compared with 0 nM estradiol or ∆ opi1 cells , as judged by a two - tailed Student ' s t - test assuming equal variance . * , P < 0 . 05 ; * * , P < 0 . 01 ; n . s . , not significant .

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E Quantification of ER size in estradiol-treated cells harboring the inducible system (SSY1405), untreated ∆opi1 cells (SSY1607) and WT cells (SSY1404) treated with 8 mM DTT for 1 h. Bars represent mean + s.e.m., n = 3 biological replicates. Asterisks indicate statistical significance compared with 0 nM estradiol or ∆opi1 cells, as judged by a two-tailed Student's t-test assuming equal variance. *, P < 0.05; **, P < 0.01; n.s., not significant.


A Sec63 - mNeon images of cells of the indicated genotypes harboring the inducible system . Cells were treated with 800 nM estradiol for 6 h . Two examples of each phenotypic class are shown .

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A Sec63-mNeon images of cells of the indicated genotypes harboring the inducible system. Cells were treated with 800 nM estradiol for 6 h. Two examples of each phenotypic class are shown.


C Sec63 - mNeon and Rtn1 - mCherry images of ∆ lnp1 and ∆ sey1 cells harboring the inducible system and treated with 800 nM estradiol for 6 h .

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C Sec63-mNeon and Rtn1-mCherry images of ∆lnp1 and ∆sey1 cells harboring the inducible system and treated with 800 nM estradiol for 6 h.