E , F . Spontaneous differentiation of 3KI mESC leading to coexistence of belonging to three germ layers . Bar : 100 μm .

O O O O O B-EXP_ASSAY O O B-CELL_TYPE O O O O O O O O O O O O O O O

E, F. Spontaneous differentiation of the 3KI mESC line leading to the coexistence of cells belonging to the three germ layers. Bar: 100 μm.


B . Differentiation of Atf1 - / - , Fos - / - Jun - / - , Foxj2 - / - , Meis3 - / - , Nr5a2 - / - , Sp1 + / - Zfp354c - / - mESCs in 3KI background to ectoderm . C . Differentiation of Atf1 - / - , Fos - / - Jun - / - , Foxj2 - / - , Meis3 - / - , Nr5a2 - / - , Sp1 + / - Zfp354c - / - mESCs in 3KI background to mesendoderm . D . Differentiation of Atf1 - / - , Fos - / - Jun - / - , Foxj2 - / - , Meis3 - / - , Nr5a2 - / - , Sp1 + / - Zff354c - / - mESCs in 3KI background to definitive endoderm .

O O B-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O O O B-CELL_TYPE O O O O B-TISSUE O O O B-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O O O B-CELL_TYPE O O O O B-TISSUE O O O B-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O O O B-CELL_TYPE O O O O O B-TISSUE O

B. Differentiation of Atf1-/-, Fos-/-Jun-/-, Foxj2-/-, Meis3-/-, Nr5a2-/-, Sp1+/- and Zfp354c-/- mESCs in the 3KI background to ectoderm. C. Differentiation of Atf1-/-, Fos-/-Jun-/-, Foxj2-/-, Meis3-/-, Nr5a2-/-, Sp1+/- and Zfp354c-/- mESCs in the 3KI background to mesendoderm. D. Differentiation of Atf1-/-, Fos-/-Jun-/-, Foxj2-/-, Meis3-/-, Nr5a2-/-, Sp1+/- and Zff354c-/- mESCs in the 3KI background to definitive endoderm.


F . Differentiation of wild type ( WT ) , Sp1 + / - , Nr5a2 - / - Fos - / - Jun - / - mESCs in 3KI background to definitive endoderm .

O O B-EXP_ASSAY O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O B-CELL_TYPE O O O O O B-TISSUE O

F. Differentiation of wild type (WT), Sp1+/-, Nr5a2-/- and Fos-/-Jun-/- mESCs in the 3KI background to definitive endoderm.


( B ) microscopy of LGCs , FBGCs osteoclasts stained with Giemsa ( upper panel ) . Representative Hoechst dye acquired by ImageStream ( lower panel ) of sorted showing mononuclear ( mono ) multinucleated ( multi ) populations for each type .

O O O B-EXP_ASSAY O B-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-CELL_TYPE O O O B-CELL_TYPE O O O O O O O O

(B) Light microscopy images of LGCs, FBGCs and osteoclasts stained with Giemsa staining (upper panel). Representative Hoechst dye images acquired by ImageStream (lower panel) of sorted cells showing mononuclear (mono) and multinucleated (multi) populations for each cell type.


( C ) - seq volcano plots highlighting top 15 differentially expressed genes between mononuclear multinucleated populations for each type .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O

(C) RNA-seq volcano plots highlighting the top 15 differentially expressed genes between mononuclear and multinucleated populations for each cell type.


( C ) MRC1 surface marker expression ( red ) acquired by ImageStream in mononuclear ( mono ) multinucleated ( multi ) LGCs , FBGCs osteoclasts stained for Hoechst . Bar graphs ( lower panel ) represent normalized MRC1 mean ( MFI ) , measured by ImageStream ; n = 4 donors .

O O O B-GENEPROD O O B-EXP_ASSAY O O O O O O O B-CELL_TYPE O O O B-CELL_TYPE O O O B-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O

(C) MRC1 surface marker expression (red) acquired by ImageStream in mononuclear (mono) and multinucleated (multi) LGCs, FBGCs and osteoclasts stained for Hoechst. Bar graphs (lower panel) represent normalized MRC1 mean fluorescence intensity (MFI), measured by ImageStream; n=4 donors.


( E ) CSF1R , MRC1 , TLR2 FOS relative expression measured by qRT - PCR for LGCs ( upper ) , FBGCs ( middle ) osteoclasts ( lower ) , following fusion regulator DC - STAMP knockdown . si - Ctrl , scrambled siRNA ; si - DC - STAMP , DC - STAMP siRNA ; n = 7 donors .

O O O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE O O O O B-CELL_TYPE O O O B-CELL_TYPE O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O

(E) CSF1R, MRC1, TLR2 and FOS relative expression measured by qRT-PCR for LGCs (upper), FBGCs (middle) and osteoclasts (lower), following the cell membrane fusion regulator DC-STAMP knockdown. si-Ctrl, scrambled siRNA; si-DC-STAMP, DC-STAMP siRNA; n=7 donors.


( E ) effect of lysosomotropic agents ( hydroxychloroquine , hydroxy ; ammonium chloride , NH4Cl ) v - ATPase inhibitors ( Bafilomycin A1 , Baf A1 ; Concanamycin A , Con A ) on fusion multinucleation in LGCs ( upper panel ) , FBGCs ( middle panel ) osteoclasts ( lower panel ) . To effect of lysosome dysfunction on iron , FeCl3 was supplemented . Error bars are median with interquartile range ; significance tested by one - way Anova followed by Sídák ' s multiple comparisons on log transformed data ; n = 4 donors ; * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * P < 0 . 001 ; * * * * , P < 0 . 0001 .

O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O B-GENEPROD I-GENEPROD I-GENEPROD O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE O O O O O B-CELL_TYPE O O O O B-CELL_TYPE O O O O O O O O B-SUBCELLULAR O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) The effect of lysosomotropic agents (hydroxychloroquine, hydroxy; ammonium chloride, NH4Cl) and v-ATPase inhibitors (Bafilomycin A1, Baf A1; Concanamycin A, Con A) on fusion and multinucleation in LGCs (upper panel), FBGCs (middle panel) and osteoclasts (lower panel). To test the effect of lysosome dysfunction on cellular iron, FeCl3 was supplemented. Error bars are median with interquartile range; significance tested by one-way Anova followed by Sídák's multiple comparisons on log transformed data; n=4 donors; *, P < 0.05; **, P < 0.01; *** P < 0.001; ****, P < 0.0001.


( E ) B7 - H3 surface marker expression ( red ) acquired by ImageStream in mononuclear multinucleated LGCs , FBGCs osteoclasts stained for Hoechst . Bar graphs ( lower panel ) show normalized B7 - H3 mean ( MFI ) , measured by ImageStream ; n = 4 donors . Error bars are mean ± SD ; significance tested by paired t - ; * * , P < 0 . 01 ; ns , non - significant .

O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-EXP_ASSAY O O O O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O 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) B7-H3 surface marker expression (red) acquired by ImageStream in mononuclear and multinucleated LGCs, FBGCs and osteoclasts stained for Hoechst. Bar graphs (lower panel) show normalized B7-H3 mean fluorescence intensity (MFI), measured by ImageStream; n=4 donors. Error bars are mean ± SD; significance tested by paired t-test; **, P < 0.01; ns, non-significant.


( C ) Phagocytosis quantified by ImageStream in LGCs , FBGCs osteoclasts . Representative showing uptake of S . Aureus bioparticles ( yellow ) in mononuclear ( mono ) multinucleated ( multi ) . Bar graphs ( lower panel ) show normalized S . Aureus bioparticles mean ( MFI ) measured by ImageStream ; n = 5 donors . Error bars are mean ± SD ; significance tested by paired t - ; * * , P < 0 . 01 ; ns , non - significant .

O O O B-EXP_ASSAY O O O O B-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O B-CELL_TYPE O O O B-CELL_TYPE O O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O O O O 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) Phagocytosis quantified by ImageStream analysis in LGCs, FBGCs and osteoclasts. Representative images showing the uptake of S. Aureus bioparticles (yellow) in mononuclear (mono) and multinucleated (multi) cells. Bar graphs (lower panel) show normalized S. Aureus bioparticles mean fluorescence intensity (MFI) measured by ImageStream; n=5 donors. Error bars are mean ± SD; significance tested by paired t-test; **, P < 0.01; ns, non-significant.


( C ) TRAP immunohistochemistry ( upper panel ) hydroxyapatite resorption ( lower panel ) in LGCs , FBGCs osteoclasts . Note presence of TRAP + MGCs in types hydroxyapatite resorption only in osteoclasts .

O O O B-GENEPROD B-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE O O O O B-GENEPROD O B-CELL_TYPE O O B-EXP_ASSAY I-EXP_ASSAY O O B-CELL_TYPE O

(C) TRAP immunohistochemistry (upper panel) and hydroxyapatite resorption (lower panel) in LGCs, FBGCs and osteoclasts. Note the presence of TRAP+ MGCs in all cell types and hydroxyapatite resorption only in osteoclasts.


( D ) Oxygen consumption rate ( OCR , upper panel ) , its related readout ( middle panel ) extracellular acidification rate ( ECAR , lower panel ) measured by extracellular flux in sorted mononuclear multinucleated osteoclasts ; n = 4 donors . ( E ) Extracellular acidification rate ( ECAR , left ) glycolysis ( right ) in mononuclear multinucleated osteoclasts ; n = 4 donors .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O B-EXP_ASSAY O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O

(D) Oxygen consumption rate (OCR, upper panel), its related readout (middle panel) and extracellular acidification rate (ECAR, lower panel) measured by extracellular flux analysis in sorted mononuclear and multinucleated osteoclasts; n=4 donors. (E) Extracellular acidification rate (ECAR, left) and glycolysis (right) in mononuclear and multinucleated osteoclasts; n=4 donors.


( F ) Mitochondria copy number measured by qPCR in mononuclear multinucleated osteoclasts ; n = 3 donors .

O O O B-SUBCELLULAR B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O

(F) Mitochondria copy number measured by qPCR in mononuclear and multinucleated osteoclasts; n=3 donors.


( G ) Relative expression of mitochondrial transcripts in sorted mononuclear multinucleated osteoclasts after normalisation by mitochondrial copy number ; n = 6 donors .

O O O O B-EXP_ASSAY O B-SUBCELLULAR O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O B-SUBCELLULAR B-EXP_ASSAY I-EXP_ASSAY O O O O O O

(G) Relative expression of mitochondrial transcripts in sorted mononuclear and multinucleated osteoclasts after normalisation by mitochondrial copy number; n=6 donors.


A Western blot of GFUS . Expression of GFUS was analyzed by Western blotting on a 15 % SDS PAGE with fractions derived from control fibroblasts . revealed a significant decrease of GFUS . Data were obtained from fibroblasts ; n = 4 ; experiment was independently repeated 4 times , for statistics an unpaired t was performed . Data information : * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 . Bars error bars represent mean ± SD .

O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-EXP_ASSAY O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-CELL_TYPE O O O O O O B-GENEPROD O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Western blot analysis and quantification of GFUS. Expression of GFUS was analyzed by Western blotting on a 15% SDS PAGE with cytosolic fractions derived from control and patient fibroblasts. Quantification revealed a significant decrease of GFUS protein. Data were obtained from fibroblasts; n = 4; experiment was independently repeated 4 times, for statistics an unpaired t test was performed. Data information: *p < 0.05; **p < 0.01; ***p<0.001. Bars and error bars represent mean ± SD.


B mRNA expression studies . qRT - PCR studies showed a significant decrease in mRNA level of GFUS by 40 . 0 % ( ± 9 . 3 % , * * p = 0 . 0010 ) normalized to a control . Also , expression of SLC35C1 SLC35C2 were significantly decreased in - derived fibroblasts . Data were obtained from fibroblasts ; n = 10 ; experiment was independently repeated 3 times , for statistics an one - way ANOVA was performed . Data information : * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 . Bars error bars represent mean ± SD .

O O B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-GENEPROD O O O O O 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-GENEPROD I-GENEPROD O O O O O O B-CELL_TYPE O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 mRNA expression studies. qRT-PCR studies showed a significant decrease in mRNA level of GFUS by 40.0% (± 9.3%, **p = 0.0010) normalized to a control. Also, expression of SLC35C1 and SLC35C2 were significantly decreased in patient-derived fibroblasts. Data were obtained from fibroblasts; n = 10; experiment was independently repeated 3 times, for statistics an one-way ANOVA was performed. Data information: *p < 0.05; **p < 0.01; ***p<0.001. Bars and error bars represent mean ± SD.


C Conversion of GDP - D - mannose into GDP - L - fucose . conversion GDP - D - [ 14C ] mannose to GDP - L - [ 14C ] fucose was analyzed in an in - vitro . After 1 h nearly half of GDP - D - mannose was transformed to GDP - fucose in control , whereas ´ s fibroblasts were nearly unable to catalyze reaction . Data were obtained from fibroblasts ; n = 1 .

C Conversion of GDP-D-mannose into GDP-L-fucose. The conversion GDP-D-[14C]mannose to GDP-L-[14C]fucose was analyzed in an in-vitro assay. After 1 h nearly half of GDP-D-mannose was transformed to GDP-fucose in the control cell line, whereas the patient´s fibroblasts were nearly unable to catalyze the reaction. Data were obtained from fibroblasts; n = 1.


D Lectin binding studies in fibroblasts . Aleuria aurantia lectin ( AAL ) was used to address fucosylation level in fibroblasts . AAL was performed before after treatment of with 100 µM L - fucose . Before sugar supplementation a significant loss of AAL signal strength was measured , whereas after treatment a significant re - fucosylation was detected . Data were obtained from fibroblasts ; n = 4 ; experiment was independently repeated 4 times , for statistics an unpaired t was performed . Data information : * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 . Bars error bars represent mean ± SD .

O B-GENEPROD B-EXP_ASSAY O O B-CELL_TYPE O B-ORGANISM I-ORGANISM B-GENEPROD O B-GENEPROD O O O O O B-EXP_ASSAY O O B-CELL_TYPE O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O B-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Lectin binding studies and quantification in fibroblasts. Aleuria aurantia lectin (AAL) was used to address the fucosylation level in fibroblasts. AAL staining was performed before and after treatment of the patient cell line with 100 µM L-fucose. Before sugar supplementation a significant loss of AAL signal strength was measured, whereas after treatment a significant re-fucosylation was detected. Data were obtained from fibroblasts; n = 4; experiment was independently repeated 4 times, for statistics an unpaired t test was performed. Data information: *p < 0.05; **p < 0.01; ***p<0.001. Bars and error bars represent mean ± SD.


E Complementation study with - derived fibroblasts . Viral infection was used to introduce an empty cloning vector wild - type GFUS cDNA in , respectively . For lysates of a control - , a control - supplemented with 100 µM fucose , , supplemented with 100 µM fucose , transfected with an empty vector transfected with wild type GFUS were analyzed by AAL clearly indicating disease - causing influence of defective GFUS in . Western blot against GFUS further showed expression of wildtype - GFUS in infected derived fibroblasts . Data were obtained from fibroblasts ; n = 4 ; experiment was independently repeated 2 times , for statistics an one - way ANOVA was performed . Data information : * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 . Bars error bars represent mean ± SD .

O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O B-GENEPROD O O O B-CELL_TYPE O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Complementation study with patient-derived fibroblasts. Viral infection was used to introduce an empty cloning vector and the wild-type GFUS cDNA in patient cells, respectively. For analysis lysates of a control cell-line, a control cell-line supplemented with 100 µM fucose, patient cells, patient cells supplemented with 100 µM fucose, patient cells transfected with an empty vector and patient cells transfected with wild type GFUS were analyzed by AAL staining clearly indicating the disease-causing influence of the defective GFUS in the patient. Western blot analysis against GFUS further showed expression of wildtype-GFUS protein in the infected patient derived fibroblasts. Data were obtained from fibroblasts; n = 4; experiment was independently repeated 2 times, for statistics an one-way ANOVA was performed. Data information: *p < 0.05; **p < 0.01; ***p<0.001. Bars and error bars represent mean ± SD.


A Aleuria aurantia lectin ( AAL ) was used to address fucosylation level in sera of controls ( left ) which revealed significant loss of fucose residues in case of . Data were obtained from serum ; n = 6 ; experiment was independently repeated 3 times , for statistics an unpaired t was performed . Data information : * * p < 0 . 01 . Bars error bars represent mean ± SD . Exact p ‑ values are reported in results part .

O B-ORGANISM I-ORGANISM B-GENEPROD O B-GENEPROD O O O O O B-EXP_ASSAY O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O 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 Aleuria aurantia lectin (AAL) was used to address the fucosylation level in sera of controls and the patient (left) which revealed significant loss of fucose residues in case of the patient. Data were obtained from serum; n = 6; experiment was independently repeated 3 times, for statistics an unpaired t test was performed. Data information: **p < 0.01. Bars and error bars represent mean ± SD. Exact p‑values are reported in the results part.


B FACS of PBMCs . Results of flow cytometry of ( red ) control ( green ) PBMCs stained with 1 µg / ml FAA lectin without ( ) or with 200 mM L - fucose ( ) before start of fucose therapy . C FACS of PBMCs . Results of flow cytometry of ( red ) control ( green ) PBMCs stained with 1 µg / ml FAA lectin without ( ) or with 200 mM L - fucose ( ) 8 after therapy start

O B-EXP_ASSAY O B-CELL_TYPE O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-CELL_TYPE B-EXP_ASSAY O O O O O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O B-EXP_ASSAY O B-CELL_TYPE O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-CELL_TYPE B-EXP_ASSAY O O O O O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O

B FACS analysis of PBMCs. Results of flow cytometry analysis of patient (red) and control (green) PBMCs stained with 1 µg/ml FAA lectin without (dark) or with 200 mM L-fucose (light) before the start of fucose therapy. C FACS analysis of PBMCs. Results of flow cytometry analysis of patient (red) and control (green) PBMCs stained with 1 µg/ml FAA lectin without (dark) or with 200 mM L-fucose (light) 8 weeks after therapy start


C . SDS - PAGE showing a representative fraction of purified spMis18 N - terminal Yippee - like globular domain ( acid residues 1 - 120 ; spMis181 - 120 ) .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O

C. SDS-PAGE showing a representative fraction of the purified spMis18 N-terminal Yippee-like globular domain (amino acid residues 1-120; spMis181-120).


D . Cartoon representation of crystal structure of spMis181 - 120 in two different orientations , highlighting key residues within putative substrate - binding pocket .

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D. Cartoon representation of the crystal structure of spMis181-120 in two different orientations, highlighting key residues within the putative substrate-binding pocket.


E . Substrate - binding pocket mutations Y90A Y74A / Y90A / T105A / S107K affect ability of ectopically expressed spMis18fl to rescue sensitivity of mis18 - 262 to varying degrees . Five - fold serial dilutions of mis18 - 262 transformed with plasmids harbouring indicated spMis18fl constructs , spotted on PMG - uracil + Phloxine B media supplemented with ( repressed ) or without ( expressed ) thiamine , incubated at indicated temperatures ; dead stain pink .

O 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-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O

E. Substrate-binding pocket mutations Y90A and Y74A/Y90A/T105A/S107K affect the ability of ectopically expressed spMis18fl to rescue the temperature sensitivity of mis18-262 cells to varying degrees. Five-fold serial dilutions of mis18-262 cells transformed with plasmids harbouring the indicated spMis18fl constructs, spotted on PMG - uracil + Phloxine B media supplemented with (repressed) or without (expressed) thiamine, and incubated at the indicated temperatures; dead cells stain dark pink.


A . Cartoon representation of assembly of spMis18 Yippee - like domains ( spMis181 - 120 ) as observed in crystal asymmetric unit . Close up views highlight acid composition interactions stabilizing dimeric arrangements . Interface - II is composed of conserved residues an extensive binding surface as compared to Interface - I ( see text for details ) , suggesting Dimer - II to be a physiologically relevant dimer . Residues mutated in this study are highlighted with circles .

O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Cartoon representation of the assembly of spMis18 Yippee-like domains (spMis181-120) as observed in the crystal asymmetric unit. Close up views highlight the amino acid composition and interactions stabilizing the dimeric arrangements. Interface-II is composed of conserved residues and an extensive binding surface as compared to Interface-I (see text for details), suggesting Dimer-II to be a physiologically relevant dimer. Residues mutated in this study are highlighted with circles.


B . Exclusion Chromatography ( SEC ) SEC - MALS ( SEC combined with Multi Angle scattering ) analyses of recombinant wild - type ( wt ) dimer disrupting mutant ( I31A ) of spMis181 - 120 . Measured molecular weights from SEC - MALS confirm that spMis181 - 120 wt is a dimer in solution spMis181 - 120 I31A is a monomer .

O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O 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-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O

B. Size Exclusion Chromatography (SEC) and SEC-MALS (SEC combined with Multi Angle Light scattering) analyses of the recombinant wild-type (wt) and dimer disrupting mutant (I31A) of spMis181-120. Measured molecular weights from SEC-MALS confirm that spMis181-120 wt is a dimer in solution and spMis181-120 I31A is a monomer.


C . Evaluation of ability of Yippee - like domains of human Mis18 ( hsMis18α77 - 187 hsMis18β56 - 183 ) to form a heterodimer . SEC SEC - MALS analyses demonstrate that while hsMis18α77 - 187 elutes at 15 . 5 ml as a dimer ( 31 . 3 kDa ) , hsMis18β56 - 183 elutes at 17 . 3 ml as a monomer ( 15 kDa ) . Purified hsMis18α77 - 187 hsMis18β56 - 183 when mixed together elute at 15 . 4 ml as a heterodimer ( 27 . 5 kDa ) .

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C. Evaluation of the ability of Yippee-like domains of human Mis18 proteins (hsMis18α77-187 and hsMis18β56-183) to form a heterodimer. SEC and SEC-MALS analyses demonstrate that while hsMis18α77-187 elutes at 15.5 ml as a dimer (31.3 kDa), hsMis18β56-183 elutes at 17.3 ml as a monomer (15 kDa). Purified hsMis18α77-187 and hsMis18β56-183 when mixed together elute at 15.4 ml as a heterodimer (27.5 kDa).


D . Cartoon representation of structure of homology - modelled human Mis18α77 - 187 - Mis18β56 - 183 heterodimer using crystal structure of spMis181 - 120 reported here as a template . Residues mutated in this study are highlighted with circles . Modelling was carried out using Phyre2 web server ( www . sbg . bio . ic . ac . uk / phyre2 / ) .

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D. Cartoon representation of the structure of the homology-modelled human Mis18α77-187 - Mis18β56-183 heterodimer using the crystal structure of spMis181-120 reported here as a template. Residues mutated in this study are highlighted with circles. Modelling was carried out using Phyre2 web server (www.sbg.bio.ic.ac.uk/phyre2/).


E . SDS - PAGE of GST - pull down where wt dimer disrupting mutants of hsMis18α77 - 187 hsMis18β56 - 183 were co - expressed as His - GST - tagged , respectively in E . coli . While wt GST - hsMis18β56 - 183 showed interaction with wt His - hsMis18α77 - 187 , hsMis18 harbouring dimer - disrupting mutations , GST - hsMis18β56 - 183 V77E / Y172D His - hsMis18α77 - 187 V82E / Y176D did not show noticeable interaction . corresponding Ni - NTA pull downs showing expression of His - tagged are shown in Fig EV2D .

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E. SDS-PAGE analysis of the GST-pull down assay where wt and dimer disrupting mutants of hsMis18α77-187 and hsMis18β56-183 were co-expressed as His- and GST-tagged proteins, respectively in E. coli. While wt GST-hsMis18β56-183 showed interaction with wt His-hsMis18α77-187, hsMis18 proteins harbouring dimer-disrupting mutations, GST-hsMis18β56-183 V77E/Y172D and His-hsMis18α77-187 V82E/Y176D did not show noticeable interaction. The corresponding Ni-NTA pull downs showing the expression of His-tagged proteins are shown in Fig EV2D.


A B . SEC - MALS of His - GFP - spMis18fl His - spMis18ΔC , respectively . Refractive index ( RI , left Y - axis ) molecular mass ( kDa , right Y - axis ) profiles show that both His - GFP - spMis18fl ( predicted MW of a monomer is 51 . 97 kDa ) His - spMis18ΔC ( Predicted MW of a monomer 21 . 8 kDa ) predominantly exist as tetramers ( 187 . 6 kDa 78 . 6 kDa , respectively ) with a small population of dimer ( 128 . 5 kDa 45 . 8 kDa , respectively ) .

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A B. SEC-MALS analysis of His-GFP-spMis18fl and His-spMis18ΔC, respectively. Refractive index (RI, left Y-axis) and molecular mass (kDa, right Y-axis) profiles show that both His-GFP-spMis18fl (predicted MW of a monomer is 51.97 kDa) and His-spMis18ΔC (Predicted MW of a monomer 21.8 kDa) predominantly exist as tetramers (187.6 kDa and 78.6 kDa, respectively) with a small population of dimer (128.5 kDa and 45.8 kDa, respectively).


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-GENEPROD O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O

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


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

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


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

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


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

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


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

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


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

O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-GENEPROD O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O

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


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

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


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

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


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

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


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

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


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

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


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

O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD B-EXP_ASSAY O B-EXP_ASSAY B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD O O O

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


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

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


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

O B-CELL_LINE I-CELL_LINE B-GENEPROD O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O B-SMALL_MOLECULE O B-EXP_ASSAY O O O B-GENEPROD O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-GENEPROD O O O O O O B-EXP_ASSAY B-SMALL_MOLECULE O

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


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

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


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

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


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

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


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

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


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

O B-CELL_LINE O B-SMALL_MOLECULE O O B-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O

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


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

O B-CELL_LINE O B-SMALL_MOLECULE O O B-GENEPROD O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD B-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-CELL_LINE O B-SMALL_MOLECULE O O B-GENEPROD O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-GENEPROD B-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O

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


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

O B-CELL_LINE O B-SMALL_MOLECULE O O B-GENEPROD O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O B-EXP_ASSAY O O O O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY B-GENEPROD I-GENEPROD O O O O B-EXP_ASSAY O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O

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


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

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


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

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


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

O B-CELL_LINE I-CELL_LINE I-CELL_LINE B-GENEPROD O O O O B-SMALL_MOLECULE O O O B-GENEPROD O O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD B-EXP_ASSAY B-GENEPROD B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O

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


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

O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-SUBCELLULAR O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

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


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

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


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

O B-SUBCELLULAR O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O B-EXP_ASSAY O O O O B-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O

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


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

B-SUBCELLULAR O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O B-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-EXP_ASSAY O O O O B-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O B-SUBCELLULAR O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-SUBCELLULAR O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O

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


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

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


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

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


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

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


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

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


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

O O O O O O O O O O O B-ORGANISM O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O

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


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

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


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

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


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

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


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

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


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

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


B Effect of / on aggregation after renaturing .

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

O O O O B-DISEASE B-TISSUE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O B-CELL_LINE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-EXP_ASSAY O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-GENEPROD O O O O O B-EXP_ASSAY O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-EXP_ASSAY O O O O B-CELL_LINE O O O O O O O O O O O O O O O O

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


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

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


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

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


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

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


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

O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

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


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

O O O O O O O O O O O O O O B-CELL_LINE O O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

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


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

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


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

O O O O B-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O B-EXP_ASSAY O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

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


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

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


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

B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O O O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O O O O B-EXP_ASSAY O B-GENEPROD O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O O B-CELL_LINE O O O B-EXP_ASSAY O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-EXP_ASSAY O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O O B-CELL_LINE O O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-EXP_ASSAY O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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